The present work was undertaken to characterize kinetics, including activation, desensitization and deactivation, of responses mediated by GABAA and GABAc receptors on carp retinal bipolar cells, using the whole-cell ...The present work was undertaken to characterize kinetics, including activation, desensitization and deactivation, of responses mediated by GABAA and GABAc receptors on carp retinal bipolar cells, using the whole-cell patch-clamp technique. It was revealed that the GABAC response was generally slower in kinetics than the GABAA response. Activation kinetics of both the receptors could be well fit by monoexponential functions with time constants r, being 44.57 ms (GABAC) and 10.86 ms (GABAA) respectively. Desensitization of the GABAA response was characterized by a fast and a slow exponential component with time constants of τfast = 2.16 s and τslow = 19.78 s respectively, whereas desensitization of the GABAc response was fit by a monoexponential function of the time constant T = 6.98 s. Deactivation at both the receptors was adequately described by biexponential functions with time constants being much higher for the GABAC response (τfast = 674.8 ms; τslow = 2 090 ms) than those for the GABAA response (τfast = 42.07 ms; τslow = 275.1 ms). These differences in kinetics suggest that GABAC and GABAA receptors may be involved in processing signals in different frequency domains.展开更多
Receptor-ligand interactions in blood flow are crucial to initiate the biological processes as inflammatory cascade, platelet thrombosis, as well as tumor metastasis. To mediate cell adhesions, the interacting recepto...Receptor-ligand interactions in blood flow are crucial to initiate the biological processes as inflammatory cascade, platelet thrombosis, as well as tumor metastasis. To mediate cell adhesions, the interacting receptors and ligands must be anchored onto two apposing surfaces of two cells or a cell and a substratum, i.e., the two-dimensional （2D） binding, which is different from the binding of a soluble ligand in fluid phase to a receptor, i.e., （3D） binding. While numerous works have been focused on 3D kinetics of receptor-ligand interactions in immune systems, 2D kinetics and its regulations have less been understood, since no theoretical framework and experimental assays have been established until 1993. Not only does the molecular structure dominate 2D binding kinetics, but the shear force in blood flow also regulates cell adhesions mediated by interacting receptors and iigands. Here we provided the overview of current progresses in 2D bindings and regulations. Relevant issues of theoretical frameworks, experimental measurements, kinetic rates and binding affinities, and force regulations, were discussed.展开更多
Nicotinic acetylcholine receptors(N2-ChRs)were synthesized in Xenopus oocytes after injection of mRNAs extracted from denervated rat muscle and mRNAs transcribed from Torpedo N2-ChR subunit cDNAs in vitro.Membrane inw...Nicotinic acetylcholine receptors(N2-ChRs)were synthesized in Xenopus oocytes after injection of mRNAs extracted from denervated rat muscle and mRNAs transcribed from Torpedo N2-ChR subunit cDNAs in vitro.Membrane inward current in the injected oocytes wa展开更多
As a general mechanism for governing the bioactivity of membrane receptors,allosteric modulation is critical in cell signaling and cell communication but remains difficult to measure in situ.Herein,we introduce a data...As a general mechanism for governing the bioactivity of membrane receptors,allosteric modulation is critical in cell signaling and cell communication but remains difficult to measure in situ.Herein,we introduce a data mining-integrated tracking microscopy(DMITM)to investigate allosteric modulation of membrane receptors in the native state in live cells.Using Kmeans clustering-based hidden Markov modeling to uncover the ligand binding and unbinding events with diffusivity variations of ligand-conjugated nanoprobes as observations.展开更多
In this work,a calculation method of chemical kinetics was established for labeling reaction of 99mTc-N-ethyl-N2S2-memantine,a potential NMDA receptor imaging agent prepared in our laboratory.Four groups of vials (3 v...In this work,a calculation method of chemical kinetics was established for labeling reaction of 99mTc-N-ethyl-N2S2-memantine,a potential NMDA receptor imaging agent prepared in our laboratory.Four groups of vials (3 vials per group) were added with 0.02 mL (1 mg/mL) N-ethyl-N2S2Memantine,0.08 mL (40 mg/mL) GH,0.05 mL (10 mg/mL) EDTA-2Na,0.035 mL (2 mg/mL) SnF2,0.8 mL phosphate buffer(1mol/L,pH 6.5) and 37 MBq Na99mTcO4.The vials were incubated at 70℃,80℃,90℃ or 100℃.Samples were taken with capillary from the vials at 2,5,10,20,30,40 and 60min.Labeling yields were determined by TLC.Order of reaction n,rate constant k,activation energy Ea and half life t1/2 of labeling reaction were calculated with the kinetics software we compiled.Mean labeling yields of 99m Tc-N-ethyl-N2S2-memantine at 2,5,10,20,30,40 and 60min were (1) 13.5,15.7,34.0,64.8,81.9,91.4 and 95.4 at 70℃;(2) 13.2,20.5,40.1,70.0,88.2,94.5 and 95.6 at 80℃;(3) 15.6,22.9,43.7,74.3,87.2,93.4 and 96.1 at 90℃;and (4) 20.5,25.8,45.3,81.1,92.2,95.6 and 96.0 at 100℃.The other parameters were;n =1;k=0.053,0.061,0.063 and 0.076 L/min at 70℃,80℃,90℃ and 100℃,respectively;Ea=12.38 kJ/L;t1/2=13.11,11.45,11.05 and 9.07min at 70℃,80℃,90℃ and 100℃,respectively.The mean labeling yield increased with temperature and time,optimized at 100℃ and 40-60min.The concentration of 99mTc-N-ethyl-N2S2-Memantine was larger than that of Na99mTcO4,so n=1.The k increased with reaction,hence the accelerated reaction rate at higher temperatures.The labeling reaction was not so difficult because of the low Ea.The t1/2 decreased with increasing reaction temperature,hence the acceleration of labeling reaction.展开更多
The activation of Statl by the interferon-gamma （IFN-γ） receptor complex is responsible for the transcription of a significant portion of IFN-γ induced genes. Many of these genes are responsible for the induction ...The activation of Statl by the interferon-gamma （IFN-γ） receptor complex is responsible for the transcription of a significant portion of IFN-γ induced genes. Many of these genes are responsible for the induction of an apoptotic state in response to IFN-γ. In the absence of Stat 1 activation, IFN-γ instead induces a proliferative response. Modifying Stat 1 activation by IFN-γ may have pharmacological benefits. We report that the rate of activation of Statl can be altered in HeLa cells by overexpressing either the IFN-γ R1 chain or the IFN-γ R2 chain. These alterations occur in hematopoietic cell lines： Raji cells and monocytic cell lines, which have average and above-average IFN-γ R2 surface expression, activate Statl similarly to HeLa cells and HeLa cells overexpressing IFNγR2, respectively. The rapid Statl activation seen in HeLa cells can be inhibited by overexpressing a chimeric IFN-γR2 chain that does not bind Jak2 or （when high concentrations of IFN-γ are used） by overexpressing IFN-γR1. These data are consistent with a model in which the recruitment of additional Jak2 activity to a signaling complex accelerates the rate of Statl activation. We conclude that the rate of activation of Statl in cells by IFN-γ can be modified by regulating either receptor chain and speculate that pharmacological agents which modify receptor chain expression may alter IFN-γ receptor signal transduction.展开更多
Receptor-ligand interactions in blood flow are crucial to initiate such biological processes as inflammatory cascade,platelet thrombosis,as well as tumor metastasis.To mediate cell adhesion,the interacting receptors a...Receptor-ligand interactions in blood flow are crucial to initiate such biological processes as inflammatory cascade,platelet thrombosis,as well as tumor metastasis.To mediate cell adhesion,the interacting receptors and ligands must be anchored onto two apposing surfaces of two cells or a cell and a substratum,i.e.,two-dimensional（2D）binding,which is different from the binding of a soluble ligand in fluid phase to a receptor,i.e.,three-dimensional（3D） binding.While numerous works have been focused on3 D kinetics of receptor-ligand interactions in the immune system,2D kinetics and its regulations have been less understood,since no theoretical framework or experimental assays were established until 1993.Not only does the molecular structure dominate 2D binding kinetics,but the shear force in blood flow also regulates cell adhesion mediated by interacting receptors and ligands.Here,we provide an overview of current progress in 2D binding and regulations,mainly from our group.Relevant issues of theoretical frameworks,experimental measurements,kinetic rates and binding affinities,and force regulations are discussed.展开更多
A technique for studying in vivo the production rate and turnover rate constant of mouse brain M-receptors was established. A single injection of 25 mg / kg of Benzilylcholine Mustard to living mice resulted in 90 % i...A technique for studying in vivo the production rate and turnover rate constant of mouse brain M-receptors was established. A single injection of 25 mg / kg of Benzilylcholine Mustard to living mice resulted in 90 % irreversible block of brain M-receptors. The time course of the receptor density was then monitored by 3H-QNB binding assay and the production rate and turnover rate constant were calculated from the time course curve with a computer program. It was found that in normal mice the turnover rate constant was about 0.035 h-1 (half-life was about 20 h) and the production rate was 30-42 fmol / (h ·mg protein). Parallel experiments revealed a significant slow down of the turnover of brain M-receptors in hypothyroid mice (turnover rate constant was 0.0257±0.0012 h-1 in hypothyroid vs. 0.0356±0.0021 h-1 in normal) while the production rate was not changed significantly. The results suggest that thyroid hormones have a regulatory action on the turnover of brain M-receptors and the elevation of brain M-receptor density together with slow down of the turnover of brain M- receptors is probably one of the important mechanisms relevant to the brain dysfunction in hypothyroidism.展开更多
文摘The present work was undertaken to characterize kinetics, including activation, desensitization and deactivation, of responses mediated by GABAA and GABAc receptors on carp retinal bipolar cells, using the whole-cell patch-clamp technique. It was revealed that the GABAC response was generally slower in kinetics than the GABAA response. Activation kinetics of both the receptors could be well fit by monoexponential functions with time constants r, being 44.57 ms (GABAC) and 10.86 ms (GABAA) respectively. Desensitization of the GABAA response was characterized by a fast and a slow exponential component with time constants of τfast = 2.16 s and τslow = 19.78 s respectively, whereas desensitization of the GABAc response was fit by a monoexponential function of the time constant T = 6.98 s. Deactivation at both the receptors was adequately described by biexponential functions with time constants being much higher for the GABAC response (τfast = 674.8 ms; τslow = 2 090 ms) than those for the GABAA response (τfast = 42.07 ms; τslow = 275.1 ms). These differences in kinetics suggest that GABAC and GABAA receptors may be involved in processing signals in different frequency domains.
文摘Receptor-ligand interactions in blood flow are crucial to initiate the biological processes as inflammatory cascade, platelet thrombosis, as well as tumor metastasis. To mediate cell adhesions, the interacting receptors and ligands must be anchored onto two apposing surfaces of two cells or a cell and a substratum, i.e., the two-dimensional （2D） binding, which is different from the binding of a soluble ligand in fluid phase to a receptor, i.e., （3D） binding. While numerous works have been focused on 3D kinetics of receptor-ligand interactions in immune systems, 2D kinetics and its regulations have less been understood, since no theoretical framework and experimental assays have been established until 1993. Not only does the molecular structure dominate 2D binding kinetics, but the shear force in blood flow also regulates cell adhesions mediated by interacting receptors and iigands. Here we provided the overview of current progresses in 2D bindings and regulations. Relevant issues of theoretical frameworks, experimental measurements, kinetic rates and binding affinities, and force regulations, were discussed.
文摘Nicotinic acetylcholine receptors(N2-ChRs)were synthesized in Xenopus oocytes after injection of mRNAs extracted from denervated rat muscle and mRNAs transcribed from Torpedo N2-ChR subunit cDNAs in vitro.Membrane inward current in the injected oocytes wa
基金supported by theNationalNatural Science Foundation of China(grant nos.21874039,21605045,21890744,and 21521063)Fundamental Research Funds for Central Universities at Hunan University。
文摘As a general mechanism for governing the bioactivity of membrane receptors,allosteric modulation is critical in cell signaling and cell communication but remains difficult to measure in situ.Herein,we introduce a data mining-integrated tracking microscopy(DMITM)to investigate allosteric modulation of membrane receptors in the native state in live cells.Using Kmeans clustering-based hidden Markov modeling to uncover the ligand binding and unbinding events with diffusivity variations of ligand-conjugated nanoprobes as observations.
基金Supported by the Jiangsu Natural Science Foundation (BK2008111,BK2010157)the National Natural Science Foundation of China (30770602)
文摘In this work,a calculation method of chemical kinetics was established for labeling reaction of 99mTc-N-ethyl-N2S2-memantine,a potential NMDA receptor imaging agent prepared in our laboratory.Four groups of vials (3 vials per group) were added with 0.02 mL (1 mg/mL) N-ethyl-N2S2Memantine,0.08 mL (40 mg/mL) GH,0.05 mL (10 mg/mL) EDTA-2Na,0.035 mL (2 mg/mL) SnF2,0.8 mL phosphate buffer(1mol/L,pH 6.5) and 37 MBq Na99mTcO4.The vials were incubated at 70℃,80℃,90℃ or 100℃.Samples were taken with capillary from the vials at 2,5,10,20,30,40 and 60min.Labeling yields were determined by TLC.Order of reaction n,rate constant k,activation energy Ea and half life t1/2 of labeling reaction were calculated with the kinetics software we compiled.Mean labeling yields of 99m Tc-N-ethyl-N2S2-memantine at 2,5,10,20,30,40 and 60min were (1) 13.5,15.7,34.0,64.8,81.9,91.4 and 95.4 at 70℃;(2) 13.2,20.5,40.1,70.0,88.2,94.5 and 95.6 at 80℃;(3) 15.6,22.9,43.7,74.3,87.2,93.4 and 96.1 at 90℃;and (4) 20.5,25.8,45.3,81.1,92.2,95.6 and 96.0 at 100℃.The other parameters were;n =1;k=0.053,0.061,0.063 and 0.076 L/min at 70℃,80℃,90℃ and 100℃,respectively;Ea=12.38 kJ/L;t1/2=13.11,11.45,11.05 and 9.07min at 70℃,80℃,90℃ and 100℃,respectively.The mean labeling yield increased with temperature and time,optimized at 100℃ and 40-60min.The concentration of 99mTc-N-ethyl-N2S2-Memantine was larger than that of Na99mTcO4,so n=1.The k increased with reaction,hence the accelerated reaction rate at higher temperatures.The labeling reaction was not so difficult because of the low Ea.The t1/2 decreased with increasing reaction temperature,hence the acceleration of labeling reaction.
文摘The activation of Statl by the interferon-gamma （IFN-γ） receptor complex is responsible for the transcription of a significant portion of IFN-γ induced genes. Many of these genes are responsible for the induction of an apoptotic state in response to IFN-γ. In the absence of Stat 1 activation, IFN-γ instead induces a proliferative response. Modifying Stat 1 activation by IFN-γ may have pharmacological benefits. We report that the rate of activation of Statl can be altered in HeLa cells by overexpressing either the IFN-γ R1 chain or the IFN-γ R2 chain. These alterations occur in hematopoietic cell lines： Raji cells and monocytic cell lines, which have average and above-average IFN-γ R2 surface expression, activate Statl similarly to HeLa cells and HeLa cells overexpressing IFNγR2, respectively. The rapid Statl activation seen in HeLa cells can be inhibited by overexpressing a chimeric IFN-γR2 chain that does not bind Jak2 or （when high concentrations of IFN-γ are used） by overexpressing IFN-γR1. These data are consistent with a model in which the recruitment of additional Jak2 activity to a signaling complex accelerates the rate of Statl activation. We conclude that the rate of activation of Statl in cells by IFN-γ can be modified by regulating either receptor chain and speculate that pharmacological agents which modify receptor chain expression may alter IFN-γ receptor signal transduction.
基金supported by Natural Science Foundation of China(grants 10042001,10072071,10128205,30225027, 10332060,30730032,11072251,and 31110103918)National Key Basic Research Foundation of China(grants 2006CB910303 and 2011CB710904)+2 种基金National High Technology Research and Development Program of China(grants 2007AA02Z306 and 2011AA020109)Chinese Academy of Sciences(grants KJCX2-L02,KJCX2-SW-L06, 2005-1-16,KJCX2-YW-L08,Y2010030,XDA01030102,XDA04073 801)NIH Fogarty International Research Collaboration Award TW 05774-01
文摘Receptor-ligand interactions in blood flow are crucial to initiate such biological processes as inflammatory cascade,platelet thrombosis,as well as tumor metastasis.To mediate cell adhesion,the interacting receptors and ligands must be anchored onto two apposing surfaces of two cells or a cell and a substratum,i.e.,two-dimensional（2D）binding,which is different from the binding of a soluble ligand in fluid phase to a receptor,i.e.,three-dimensional（3D） binding.While numerous works have been focused on3 D kinetics of receptor-ligand interactions in the immune system,2D kinetics and its regulations have been less understood,since no theoretical framework or experimental assays were established until 1993.Not only does the molecular structure dominate 2D binding kinetics,but the shear force in blood flow also regulates cell adhesion mediated by interacting receptors and ligands.Here,we provide an overview of current progress in 2D binding and regulations,mainly from our group.Relevant issues of theoretical frameworks,experimental measurements,kinetic rates and binding affinities,and force regulations are discussed.
文摘A technique for studying in vivo the production rate and turnover rate constant of mouse brain M-receptors was established. A single injection of 25 mg / kg of Benzilylcholine Mustard to living mice resulted in 90 % irreversible block of brain M-receptors. The time course of the receptor density was then monitored by 3H-QNB binding assay and the production rate and turnover rate constant were calculated from the time course curve with a computer program. It was found that in normal mice the turnover rate constant was about 0.035 h-1 (half-life was about 20 h) and the production rate was 30-42 fmol / (h ·mg protein). Parallel experiments revealed a significant slow down of the turnover of brain M-receptors in hypothyroid mice (turnover rate constant was 0.0257±0.0012 h-1 in hypothyroid vs. 0.0356±0.0021 h-1 in normal) while the production rate was not changed significantly. The results suggest that thyroid hormones have a regulatory action on the turnover of brain M-receptors and the elevation of brain M-receptor density together with slow down of the turnover of brain M- receptors is probably one of the important mechanisms relevant to the brain dysfunction in hypothyroidism.
