Abstract: It is much more difficult for tubulin from plant sources to polymerize in vitro than tubulin from animal sources. Taxol, a most widely used reagent in microtubule studies, enhances plant microtubule assembly...Abstract: It is much more difficult for tubulin from plant sources to polymerize in vitro than tubulin from animal sources. Taxol, a most widely used reagent in microtubule studies, enhances plant microtubule assembly, but hinders microtubule dynamics. Dimethyl sulfoxide (DMSO), a widely used reagent in animal microtubule studies, is a good candidate for the investigation of plant microtubule assembly in vitro. However, proper investigation is lacking about the effects of DMSO on plant microtubule assembly in vitro. In the present study, DMSO was used to establish optimal conditions for the polymerization of plant tubulin. Tubulin, purified from lily pollen, polymerizes into microtubules at a critical concentration of 1.2 mg/mL in the presence of 10% DMSO. The polymers appear to have a normal microtubule structure, as revealed by electron microscopy. In the presence of 10% DMSO, microtubule polymerization decreases when the pH of the medium is increased from 6.5 to 7.4. Both the polymerization rate and the mass of the polymers increase as temperature increases from 25 to 40 °C. Tubulin polymerizes and depolymerizes along with cycling of temperature, from 37 to 4 °C, or following the addition to or the removal of Ca2+ from the medium. When incubated with nuclei isolated from tobacco BY-2 suspension cells, tubulin assembles onto the nuclear surface in the presence of 10% DMSO. Labeling lily pollen tubulin with 5- (and 6-) carboxytetramethyl-rhodamine succinimidyl ester (NHS-rhodamine) was performed successfully in the presence of 10% DMSO. Labeled tubulin assembles into a radial structure on the surface of BY-2 nuclei. The polymerization of lily pollen tubulin is also enhanced by microtubule-associated proteins from animal sources in the presence of 10% DMSO. All the experimental results indicate that plant tubulin functions normally in the presence of DMSO. Therefore, DMSO is an appropriate reagent for plant tubulin polymerization and investigation of plant microtubules in vitro.展开更多
The thermal properties of porcine brain tubulin polymerization and denaturation and the efftcts of taxol were studied from 4 ℃ to 115 ℃ with a new highly-sensitive differential scanming calorimeter (Micro DSC Ⅲ, Se...The thermal properties of porcine brain tubulin polymerization and denaturation and the efftcts of taxol were studied from 4 ℃ to 115 ℃ with a new highly-sensitive differential scanming calorimeter (Micro DSC Ⅲ, Setaram,France). The DSC results indicated that microtubule polymerization can proceed uia an endothermic process and that taxol could alter the process.A large aggregation peak was found at about 85 ℃ in the course of the denaturation and it disap pcared with adding taxol. The heat flow oscillation was observed during the controlled-temperature experiments and taxol could stabilize the structure of the microtubules so that the oscillation disappezred.展开更多
The use of radio frequency energy is an established technology for certain oncology therapies. Direct inputs of radio frequency (RF) energy as thermal energy are applied to ablate tumors or catalyze secondary reaction...The use of radio frequency energy is an established technology for certain oncology therapies. Direct inputs of radio frequency (RF) energy as thermal energy are applied to ablate tumors or catalyze secondary reactions in adjunct treatments against certain tumor types. Yet, other applications are being developed which take advantage of properties of RFs that impinge on biological proteins and cells without thermal effects. Here we report a proof-of-concept application of specific, digitally encoded, low power (non-thermal) radio frequency energy in an in vitro preparation of a tubulin polymerization assay. The radio frequency energy signal, designated M2(3), was applied to the tubulin polymerization assay samples during spectrophotometric measurements to assess the effectiveness for enhancing tubulin polymerization. A commercially available taxane (paclitaxel) that promotes tubulin polymerization was used as a control to assess the effectiveness of the M2(3) radio frequency energy signal on tubulin polymerization rates. A low power, specific, digital radio frequency energy signal is capable of promoting tubulin polymerization as effectively as a commercially available taxane.展开更多
Objective: The aim of this study was to investigate the impact of beta-elemene injection on the growth and beta-tubulin of human hepatocarcinoma HepG2 cells. Methods: Cell proliferation was assessed by MTT assay. Cell...Objective: The aim of this study was to investigate the impact of beta-elemene injection on the growth and beta-tubulin of human hepatocarcinoma HepG2 cells. Methods: Cell proliferation was assessed by MTT assay. Cell cycle distribution was detected by flow cytometry(FCM). The mRNA expression of beta-tubulin was measured by RT-PCR. Western blot analysis was used to determine protein expression of beta-tubulin and the polymerization of beta-tubulin. Results: Beta-elemene injection inhibited HepG2 cells proliferation in a dose- and time-dependent manner; FCM analysis indicated beta-elemene injection induced cell cycle arrested at S phase. RT-PCR and western-blot analysis showed that beta-elemene injection down-regulated beta-tubulin expression at both mRNA and protein levels, presenting a dose-dependent manner. Moreover, beta-elemene injection reduced the polymerization of microtubules in a dose-dependent manner. Conclusion: Beta-elemene injection can inhibit the proliferation of hepatoma HepG2 cells, the mechanism might be partly related to the down-regulation of beta-tubulin and inhibition of microtubular polymerization.展开更多
A series of new combretastatin-A4 analogs were synthesized, in which a six-membered ring connects the linking bridge and A ring, and their tumor cell growth and tubulin-polymerization inhibitory activity were evaluate...A series of new combretastatin-A4 analogs were synthesized, in which a six-membered ring connects the linking bridge and A ring, and their tumor cell growth and tubulin-polymerization inhibitory activity were evaluated. These compounds appear to be potential tubulin-polymerization inhibitors, Compounds 1b with amino substituted on position 3 of B ring conferred optimal bioactivity, higher than that of the lead compound 22b and equivalent to that of CA-4. The binding modes of these compounds to tuhulin were obtained by molecular docking, which can explain the structure-activity relationship. The studies presented here provide a new structural type for the development of novel antitumor agents.展开更多
文摘Abstract: It is much more difficult for tubulin from plant sources to polymerize in vitro than tubulin from animal sources. Taxol, a most widely used reagent in microtubule studies, enhances plant microtubule assembly, but hinders microtubule dynamics. Dimethyl sulfoxide (DMSO), a widely used reagent in animal microtubule studies, is a good candidate for the investigation of plant microtubule assembly in vitro. However, proper investigation is lacking about the effects of DMSO on plant microtubule assembly in vitro. In the present study, DMSO was used to establish optimal conditions for the polymerization of plant tubulin. Tubulin, purified from lily pollen, polymerizes into microtubules at a critical concentration of 1.2 mg/mL in the presence of 10% DMSO. The polymers appear to have a normal microtubule structure, as revealed by electron microscopy. In the presence of 10% DMSO, microtubule polymerization decreases when the pH of the medium is increased from 6.5 to 7.4. Both the polymerization rate and the mass of the polymers increase as temperature increases from 25 to 40 °C. Tubulin polymerizes and depolymerizes along with cycling of temperature, from 37 to 4 °C, or following the addition to or the removal of Ca2+ from the medium. When incubated with nuclei isolated from tobacco BY-2 suspension cells, tubulin assembles onto the nuclear surface in the presence of 10% DMSO. Labeling lily pollen tubulin with 5- (and 6-) carboxytetramethyl-rhodamine succinimidyl ester (NHS-rhodamine) was performed successfully in the presence of 10% DMSO. Labeled tubulin assembles into a radial structure on the surface of BY-2 nuclei. The polymerization of lily pollen tubulin is also enhanced by microtubule-associated proteins from animal sources in the presence of 10% DMSO. All the experimental results indicate that plant tubulin functions normally in the presence of DMSO. Therefore, DMSO is an appropriate reagent for plant tubulin polymerization and investigation of plant microtubules in vitro.
文摘The thermal properties of porcine brain tubulin polymerization and denaturation and the efftcts of taxol were studied from 4 ℃ to 115 ℃ with a new highly-sensitive differential scanming calorimeter (Micro DSC Ⅲ, Setaram,France). The DSC results indicated that microtubule polymerization can proceed uia an endothermic process and that taxol could alter the process.A large aggregation peak was found at about 85 ℃ in the course of the denaturation and it disap pcared with adding taxol. The heat flow oscillation was observed during the controlled-temperature experiments and taxol could stabilize the structure of the microtubules so that the oscillation disappezred.
文摘The use of radio frequency energy is an established technology for certain oncology therapies. Direct inputs of radio frequency (RF) energy as thermal energy are applied to ablate tumors or catalyze secondary reactions in adjunct treatments against certain tumor types. Yet, other applications are being developed which take advantage of properties of RFs that impinge on biological proteins and cells without thermal effects. Here we report a proof-of-concept application of specific, digitally encoded, low power (non-thermal) radio frequency energy in an in vitro preparation of a tubulin polymerization assay. The radio frequency energy signal, designated M2(3), was applied to the tubulin polymerization assay samples during spectrophotometric measurements to assess the effectiveness for enhancing tubulin polymerization. A commercially available taxane (paclitaxel) that promotes tubulin polymerization was used as a control to assess the effectiveness of the M2(3) radio frequency energy signal on tubulin polymerization rates. A low power, specific, digital radio frequency energy signal is capable of promoting tubulin polymerization as effectively as a commercially available taxane.
基金Supported by grants from the National Natural Science Foundation of China(No.81173615)the Scientific Research Foundation for the Returned Overseas Chinese Scholars and State Education Ministrythe Specialized Research Fund for the Doctoral Program of Higher Education(No.20102105120002)
文摘Objective: The aim of this study was to investigate the impact of beta-elemene injection on the growth and beta-tubulin of human hepatocarcinoma HepG2 cells. Methods: Cell proliferation was assessed by MTT assay. Cell cycle distribution was detected by flow cytometry(FCM). The mRNA expression of beta-tubulin was measured by RT-PCR. Western blot analysis was used to determine protein expression of beta-tubulin and the polymerization of beta-tubulin. Results: Beta-elemene injection inhibited HepG2 cells proliferation in a dose- and time-dependent manner; FCM analysis indicated beta-elemene injection induced cell cycle arrested at S phase. RT-PCR and western-blot analysis showed that beta-elemene injection down-regulated beta-tubulin expression at both mRNA and protein levels, presenting a dose-dependent manner. Moreover, beta-elemene injection reduced the polymerization of microtubules in a dose-dependent manner. Conclusion: Beta-elemene injection can inhibit the proliferation of hepatoma HepG2 cells, the mechanism might be partly related to the down-regulation of beta-tubulin and inhibition of microtubular polymerization.
基金supported by the National Natural Science Foundation of China(Nos.21172260 and 30901859)Shanghai Natural Science Foundation(No.09ZR1438800)"Chen Guang" Project supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation(No.12CG42)
文摘A series of new combretastatin-A4 analogs were synthesized, in which a six-membered ring connects the linking bridge and A ring, and their tumor cell growth and tubulin-polymerization inhibitory activity were evaluated. These compounds appear to be potential tubulin-polymerization inhibitors, Compounds 1b with amino substituted on position 3 of B ring conferred optimal bioactivity, higher than that of the lead compound 22b and equivalent to that of CA-4. The binding modes of these compounds to tuhulin were obtained by molecular docking, which can explain the structure-activity relationship. The studies presented here provide a new structural type for the development of novel antitumor agents.
