为了优化裂殖壶菌油脂胞内转乙酯化工艺条件,以DHA酯化率为指标,在单因素实验基础上采用Box-Behnken响应面实验设计,考察了无水乙醇用量、浓硫酸用量、反应温度和反应时间对裂殖壶菌油脂胞内转乙酯化的影响。结果表明:针对0.5 g干菌粉,...为了优化裂殖壶菌油脂胞内转乙酯化工艺条件,以DHA酯化率为指标,在单因素实验基础上采用Box-Behnken响应面实验设计,考察了无水乙醇用量、浓硫酸用量、反应温度和反应时间对裂殖壶菌油脂胞内转乙酯化的影响。结果表明:针对0.5 g干菌粉,得到最优的胞内转乙酯化工艺条件为无水乙醇用量11.76 m L、浓硫酸用量0.41 m L、反应温度75.5℃、反应时间2.24 h,在此条件下DHA酯化率达到85.19%。展开更多
Background &Aims: Secreted immunoglobulins play an integral role in host defense at mucosal surfaces, and recent evidence shows that IgG can participate in antigen sampling from the intestinal lumen. We examined w...Background &Aims: Secreted immunoglobulins play an integral role in host defense at mucosal surfaces, and recent evidence shows that IgG can participate in antigen sampling from the intestinal lumen. We examined whether IgE also could facilitate transepithelial antigen sampling. Methods: Stool samples from food-allergic patients undergoing oral food challenge were analyzed for CD23 and food-specific IgE. CD23 isoform expression on primary human intestinal epithelial cells (IEC) was analyzed by polymerase chain reaction. The role of CD23 isoforms in transcytosis of antigen and IgE-antigen complexes was assessed using polarized human T84 cells retrovirally transfected with CD23a or CD23b. Results: CD23 was expressed constitutively on IECs, and food-allergic patients had increased levels of soluble CD23 and food-specific IgE in the stool after challenge. CD23a, but not CD23b, was expressed by primary human IECs. We show in transcytosis assays that CD23a, but not CD23b, acts as a bidirectional transporter of IgE. In addition, specific IgE facilitated the uptake of antigen from the apical surface of an epithelial monolayer by diverting antigen from delivery to lysosomes. Finally, delivery of antigen-IgE complexes across the epithelial barrier could induce the degranulation of rat basophil leukemia cells transfected with the human high-affinity IgE receptor. Conclusions: These studies show that CD23a is expressed normally on human IECs, and in the presence of IgE can function as an antigen-sampling mechanism capable of activating subepithelial mast cells. IgE may serve as a secretory immunoglobulin that in concert with CD23 participates in food-induced pathophysiology of the gastrointestinal tract.展开更多
BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene ma...BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.展开更多
Objective In kinesin-3,the neck coil correlates with the following segments to form an extended neck that contains a characteristic hinge diverse from a proline in KIF13B to a long flexible linker in KIF1A.The functio...Objective In kinesin-3,the neck coil correlates with the following segments to form an extended neck that contains a characteristic hinge diverse from a proline in KIF13B to a long flexible linker in KIF1A.The function of this neck hinge for controlling processive movement,however,remains unclear.Methods We made a series of modifications to the neck hinges of KIF13B and KIF1A and tested their movement using a single-molecule motility assay.Results In KIF13B,the insertion of flexible residues before or after the proline differentially impacts the processivity or velocity,while the removal of this proline increases the both.In KIF1A,the deletion of entire flexible neck hinge merely enhances the processivity.The engineering of these hinge-truncated necks of kinesin-3 into kinesin-1 similarly boosts the processive movement of kinesin-1.Conclusion The neck hinge in kinesin-3 controls its processive movement and proper modifications tune the motor motility,which provides a novel strategy to reshape the processive movement of kinesin motors.展开更多
[Objective] The aim of the research was to study the effect of the ice bath time after heat shock and the incubation time on the transformation efficacy,and to establish a simple and quick transformation method.[Metho...[Objective] The aim of the research was to study the effect of the ice bath time after heat shock and the incubation time on the transformation efficacy,and to establish a simple and quick transformation method.[Method]Competent cells were prepared with two buffer solutions;with the ice bath time after heat shock and the recovery time as the variables,the relationship between these two factors and transformation efficacy was studied.[Result]The transformation efficacy was the best when the ice bath time was 2 min and the recovery time was 30 or 40 min;when the ice bath time and the recovery time was 0 min,a certain amount of transformants still could be obtained.[Conclusion]The ice bath time after heat shock and the recovery time had certain impact on transformation efficacy,but they were not the decisive factors.Therefore,in the general transformation experiment,these two steps could be omitted.展开更多
文摘为了优化裂殖壶菌油脂胞内转乙酯化工艺条件,以DHA酯化率为指标,在单因素实验基础上采用Box-Behnken响应面实验设计,考察了无水乙醇用量、浓硫酸用量、反应温度和反应时间对裂殖壶菌油脂胞内转乙酯化的影响。结果表明:针对0.5 g干菌粉,得到最优的胞内转乙酯化工艺条件为无水乙醇用量11.76 m L、浓硫酸用量0.41 m L、反应温度75.5℃、反应时间2.24 h,在此条件下DHA酯化率达到85.19%。
文摘Background &Aims: Secreted immunoglobulins play an integral role in host defense at mucosal surfaces, and recent evidence shows that IgG can participate in antigen sampling from the intestinal lumen. We examined whether IgE also could facilitate transepithelial antigen sampling. Methods: Stool samples from food-allergic patients undergoing oral food challenge were analyzed for CD23 and food-specific IgE. CD23 isoform expression on primary human intestinal epithelial cells (IEC) was analyzed by polymerase chain reaction. The role of CD23 isoforms in transcytosis of antigen and IgE-antigen complexes was assessed using polarized human T84 cells retrovirally transfected with CD23a or CD23b. Results: CD23 was expressed constitutively on IECs, and food-allergic patients had increased levels of soluble CD23 and food-specific IgE in the stool after challenge. CD23a, but not CD23b, was expressed by primary human IECs. We show in transcytosis assays that CD23a, but not CD23b, acts as a bidirectional transporter of IgE. In addition, specific IgE facilitated the uptake of antigen from the apical surface of an epithelial monolayer by diverting antigen from delivery to lysosomes. Finally, delivery of antigen-IgE complexes across the epithelial barrier could induce the degranulation of rat basophil leukemia cells transfected with the human high-affinity IgE receptor. Conclusions: These studies show that CD23a is expressed normally on human IECs, and in the presence of IgE can function as an antigen-sampling mechanism capable of activating subepithelial mast cells. IgE may serve as a secretory immunoglobulin that in concert with CD23 participates in food-induced pathophysiology of the gastrointestinal tract.
文摘BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.
文摘Objective In kinesin-3,the neck coil correlates with the following segments to form an extended neck that contains a characteristic hinge diverse from a proline in KIF13B to a long flexible linker in KIF1A.The function of this neck hinge for controlling processive movement,however,remains unclear.Methods We made a series of modifications to the neck hinges of KIF13B and KIF1A and tested their movement using a single-molecule motility assay.Results In KIF13B,the insertion of flexible residues before or after the proline differentially impacts the processivity or velocity,while the removal of this proline increases the both.In KIF1A,the deletion of entire flexible neck hinge merely enhances the processivity.The engineering of these hinge-truncated necks of kinesin-3 into kinesin-1 similarly boosts the processive movement of kinesin-1.Conclusion The neck hinge in kinesin-3 controls its processive movement and proper modifications tune the motor motility,which provides a novel strategy to reshape the processive movement of kinesin motors.
基金Supported by Foundation for Returned Scholars of Hebei Province(2010)Research Fund for the Doctoral Program of Hebei Normal University(L2009B13)~~
文摘[Objective] The aim of the research was to study the effect of the ice bath time after heat shock and the incubation time on the transformation efficacy,and to establish a simple and quick transformation method.[Method]Competent cells were prepared with two buffer solutions;with the ice bath time after heat shock and the recovery time as the variables,the relationship between these two factors and transformation efficacy was studied.[Result]The transformation efficacy was the best when the ice bath time was 2 min and the recovery time was 30 or 40 min;when the ice bath time and the recovery time was 0 min,a certain amount of transformants still could be obtained.[Conclusion]The ice bath time after heat shock and the recovery time had certain impact on transformation efficacy,but they were not the decisive factors.Therefore,in the general transformation experiment,these two steps could be omitted.
