The changes in the contents of adenosine 5’-triphosphate (ATP) and its related breakdown compounds were investigated in the adductor muscle, mantle, gill and body trunk of oyster (Crassostrea gigas) during frozen sto...The changes in the contents of adenosine 5’-triphosphate (ATP) and its related breakdown compounds were investigated in the adductor muscle, mantle, gill and body trunk of oyster (Crassostrea gigas) during frozen storage at -20℃ and -30℃ and compared with that of the fresh oyster. The investigation was performed using an HPLC system. Different extents of ATP decomposi- tion were found in various tissues frozen at the two temperatures. The K, K’ and A.E.C values were calculated as the chemical fresh- ness indices. Considering the results of sensory evaluation, the A.E.C. value in body trunk at -20℃ and -30℃ could be used as the best freshness index for frozen oyster.展开更多
Applying a stimulating current to acupoints through acupuncture needles–known as electroacupuncture–has the potential to produce analgesic effects in human subjects and experimental animals. When acupuncture was app...Applying a stimulating current to acupoints through acupuncture needles–known as electroacupuncture–has the potential to produce analgesic effects in human subjects and experimental animals. When acupuncture was applied in a rat model, adenosine 5-triphosphate disodium in the extracellular space was broken down into adenosine, which in turn inhibited pain transmission by means of an adenosine A1 receptor-dependent process. Direct injection of an adenosine A1 receptor agonist enhanced the analgesic effect of acupuncture. The analgesic effect of acupuncture appears to be mediated by activation of A1 receptors located on ascending nerves. In neuropathic pain, there is upregulation of P2X purinoceptor 3 （P2X3） receptor expression in dorsal root ganglion neurons. Conversely, the onset of mechanical hyperalgesia was diminished and established hyperalgesia was significantly reversed when P2X3 receptor expression was downregulated. The pathways upon which electroacupuncture appear to act are interwoven with pain pathways, and electroacupuncture stimuli converge with impulses originating from painful areas. Electroacupuncture may act via purinergic A1 and P2X3 receptors simultaneously to induce an analgesic effect on neuropathic pain.展开更多
BACKGROUND： Most of the currently available information on purinergic receptors （P2Rs） involved in pain transmission is based on results obtained in dorsal root ganglion or the spinal cord. However, the mechanism o...BACKGROUND： Most of the currently available information on purinergic receptors （P2Rs） involved in pain transmission is based on results obtained in dorsal root ganglion or the spinal cord. However, the mechanism of P2Rs in trigeminal neuralgia remains unclear. OBJECTIVE： To investigate changes in the P2R-mediated calcium signaling pathway in nociceptive trigemJnal ganglion neurons. DESIGN, TIME AND SETTING： In vitro experiments were conducted at the Patch-Clamp Laboratory of Comprehensive Experiment Center of Anhui Medical University, China from September 2008 to June 2009. MATERIALS： Thapsigargin, caffeine, suramin, and adenosine 5＇-triphosphate were purchased from Sigma, USA. METHODS： Using Fura-2-based microfluorimetry, intracellular calcium concentration （[Ca^2＋]i） was measured in freshly isolated adult rat small trigeminal ganglion neurons before and after drug application. MAIN OUTCOME MEASURES： Fluorescent intensities were expressed as the ratio F340/F380 to observe [Ca^2＋]i changes. RESULTS： In normal extracellular solution and Ca^2＋-free solution, application of thapsigargin （1 μmol/L）, a sarcoplasmic reticulum Ca^2＋ pump adenosine 5＇-triphosphate inhibitor, as well as caffeine （20 mmol/L）, a ryanodine receptor agonist, triggered [Ca^2＋]i increase in small trigeminal ganglion neurons. A similar response was induced by application of adenosine 5＇-triphosphate （100 μmol/L）. In Ca^2＋-free conditions, adenosine 5＇-triphosphate-induced [Ca^2＋]i transients in small trigeminal ganglion neurons were inhibited in cells pre-treated with thapsigargin （P 〈 0.01）, but not by caffeine （P 〉 0.05）. In normal, extracellular solution, adenosine 5＇-triphosphate-induced [Ca^2＋]i transients in small trigeminal ganglion neurons were partly inhibited in cells pre-treated with thapsigargin （P 〈 0.05）. CONCLUSION： Inositol-1,4, 5-triphosphate （IP3）- and ryanodine-sensitive Ca^2＋ stores exist in rat nociceptive trigeminal ganglion neurons. Tw展开更多
Phosphatidylinositol(3,4,5)-triphosphate(PIP3),acting as a fundamental second messenger,is emerging as a promising biomarker for disease diagnosis and prognosis.However,the real time analysis of phosphoinositide in li...Phosphatidylinositol(3,4,5)-triphosphate(PIP3),acting as a fundamental second messenger,is emerging as a promising biomarker for disease diagnosis and prognosis.However,the real time analysis of phosphoinositide in living cells remains a key challenge owing to the low basal abundance and its fast metabolic rate.Herein,we design an optogenetic system that uses light sensitive protein-protein interaction between Arabidopsis cryptochrome 2(CRY2)and CIB1 to spatiotemporally visualize the PIP3 production with sub-second timescale.In this system,a CIBN is anchored on the plasma membrane,whereas a CRY2 fused with a constitutively active PI3-kinase(acPI3K)would be driven from the cytosol to the membrane by the blue-light-activated CRY2-CIB1 interaction upon light irradiation.The PIP3 production is visualized via a fused fluorescent protein by the translocation of a Pleckstrin Homology(PH)domain(GRP1)from the cytosol to the plasma membrane with high specificity.We demonstrated the fast dynamics and reversibility of the optogenetic system initiated PIP3 synthesis on the plasma membrane.Notably,the real-time cell movements were also observed upon localized light stimulation.The established optogenetic method provides a novel spatiotemporal strategy for specific PIP3 visualization,which is beneficial to improve the understanding of PIP3 functions.展开更多
An adenine nucleotide derivative 2-aminoadenosine 5＇-triphosphate was chemically synthesized through four steps and was characterized with 1H NMR, 31p NMR, 13C NMR, EA and FT-IR. Its ultraviolet and fluorescence prop...An adenine nucleotide derivative 2-aminoadenosine 5＇-triphosphate was chemically synthesized through four steps and was characterized with 1H NMR, 31p NMR, 13C NMR, EA and FT-IR. Its ultraviolet and fluorescence properties at various pH values were studied. Two pKa values for the compound were determined by the curves of UV absorption dependency on pH, Which were 0.68 and 4.83, respectively. The values were consistent with those calculated from ACD/Labs software. In addition, hydrolysis of the adenine nucleotide derivative in the catalysis of potato apyrase was studied. The competition of the ATP analogue with ATP for potato apyrase＇ active site was proved to be a sequential reaction mechanism.展开更多
Selective recognition of adenosine 5＇-triphosphate （ATP） is of great significance owing to its indispensable functions to organisms. Also, it is a challenging task because other nucleosides triphosphate hold the sa...Selective recognition of adenosine 5＇-triphosphate （ATP） is of great significance owing to its indispensable functions to organisms. Also, it is a challenging task because other nucleosides triphosphate hold the same triphosphate group and structurally planar bases as ATP. It is known that metal-organic frameworks （MOFs） are a new type of sensing material. In this work, highly selective recognition of ATP against other nucleosides triphosphate is successfully achieved with a luminescent MOF of [Zn（BDC）（H2O）2]n （BDC2- = 1,4-benzenedicarboxylate）. [Zn（BDC）（H2O）2]n dispersed in water shows a remarkable redshift of the emission wavelength upon addition of ATP, while cytidine 5＇-triphosphate （CTP）, uridine 5＇-triphosphate （UTP） and guanosine 5＇-triphosphate （GTP）, as well as some inorganic anions such as P2074- or PO43- can＇t induce such spectral change as ATP. 1H NMR, 31p NMR and Raman spectra indicate that both π-π stacking interactions and the coordination of Zn（II） with adenine and the phosphate group are involved in the interaction of [Zn（BDC）（H2O）2],, with ATP. In addition, the experimental results showed that the redshift extent of the emission wavelength of [Zn（BDC）（HzO）2]n has the linear relation- ship with the concentration of ATP in the range of 0.3-1.8 mmol/L. Based on this, the detection of ATP content in the sample of ATP injection was made with satisfactory results. This system pioneers the application of MOFs in the recognition of nucle- otides, and testifies that the participation of base in the recognition process can improve the selectivity against the other nucleotides.展开更多
Background: Molnupiravir, N4-hydroxycytidine-5’-isopropyl ester, is an oral prodrug of N4-deoxycytidine (NHC), a nucleoside analog, which has in vitro activity against SARS-CoV-2. NHC is phosphorylated in cells to NH...Background: Molnupiravir, N4-hydroxycytidine-5’-isopropyl ester, is an oral prodrug of N4-deoxycytidine (NHC), a nucleoside analog, which has in vitro activity against SARS-CoV-2. NHC is phosphorylated in cells to NHC triphosphate (NHC-TP), which is incorporated into viral RNA, leading to epigenetic catastrophe of the viral genome and inhibition of viral replication. The antiviral activity against SARS-CoV-2 is dependent on the number of molecules of NHC-TP incorporated into viral RNA. Clinical studies in patients with COVID-19 showed that treatment with molnupiravir for 5 days decreases the risk of hospitalization and death as compared with placebo. Objective: It should be possible to enhance the antiviral activity of NHC-TP against SARS-CoV-2 by the use of the biochemical modulator, 3-deazauridine (3DU). 3DU is an inhibitor of CTP synthetase. Inhibition of this enzyme results in a reduction in the intracellular pool size of CTP. Since NHC-TP competes with CTP for incorporation into viral RNA in the reaction catalyzed by the SARS-CoV-2 viral RNA-dependent RNA polymerase, the reduction in the level of CTP should result in a significant enhancement of the incorporation of NHC-TP into viral RNA and an enhancement of its antiviral activity. Methods: Analysis of the publications of 3DU and cytosine nucleoside analogues support the hypothesis that 3DU enhances the pharmacological action of the analogues. Results: 3-DU increased the incorporation of 5-azacytidine into RNA and 5-aza-deoxycytidine into DNA of leukemic cells with an enhancement of their antineoplastic action. 3-DU potentiated the antiviral activity against HIV-1 activity by the cytosine nucleoside analogues: 2’-deoxy-3’-thiacytidine (3TC;lamivudine) and 2’,3’-dideoxycytidine (ddC). This anti-HIV-1 activity of 3DU was associated with a reduction in the intracellular pool size of dCTP and increased incorporation of triphosphates of 3TC and ddC into DNA by the HIV-1 reverse transcriptase. The reduction of CTP levels in cells by 3-DU also leads to a展开更多
In this paper, some new results on the selective weak interaction between Na-4-tosyl-L-arginine methyl ester hydrochloride （TAME） and adenosine-5＇-triphosphate （ATP） have been reported. Fluorescence spectrophotom...In this paper, some new results on the selective weak interaction between Na-4-tosyl-L-arginine methyl ester hydrochloride （TAME） and adenosine-5＇-triphosphate （ATP） have been reported. Fluorescence spectrophotometry and Fourier transform infrared （FT-IR） spectroscopy were used to investigate this kind of weak interaction. In fluorescence experiments, obvious fluorescence quenching phenomena were observed when TAME was added, which indicated the weak interactions between TAME and ATP. It has been identified by fluorescence titration experiments that TAME exhibited high selectivity to ATP over ADP and AMP. FT-IR spectral results showed that an ATP-TAME adduct was formed. The experimental results indicated that the interaction sites were the guanidinium group of TAME main-chain and the γ-phosphate group of ATP, and the interaction took place through hydrogen bonding and electrostatic force. In addition, the effects of metal ions on the weak interaction between ATP and TAME, or between ATP and analogues of L-arginine were studied.展开更多
文摘The changes in the contents of adenosine 5’-triphosphate (ATP) and its related breakdown compounds were investigated in the adductor muscle, mantle, gill and body trunk of oyster (Crassostrea gigas) during frozen storage at -20℃ and -30℃ and compared with that of the fresh oyster. The investigation was performed using an HPLC system. Different extents of ATP decomposi- tion were found in various tissues frozen at the two temperatures. The K, K’ and A.E.C values were calculated as the chemical fresh- ness indices. Considering the results of sensory evaluation, the A.E.C. value in body trunk at -20℃ and -30℃ could be used as the best freshness index for frozen oyster.
文摘Applying a stimulating current to acupoints through acupuncture needles–known as electroacupuncture–has the potential to produce analgesic effects in human subjects and experimental animals. When acupuncture was applied in a rat model, adenosine 5-triphosphate disodium in the extracellular space was broken down into adenosine, which in turn inhibited pain transmission by means of an adenosine A1 receptor-dependent process. Direct injection of an adenosine A1 receptor agonist enhanced the analgesic effect of acupuncture. The analgesic effect of acupuncture appears to be mediated by activation of A1 receptors located on ascending nerves. In neuropathic pain, there is upregulation of P2X purinoceptor 3 （P2X3） receptor expression in dorsal root ganglion neurons. Conversely, the onset of mechanical hyperalgesia was diminished and established hyperalgesia was significantly reversed when P2X3 receptor expression was downregulated. The pathways upon which electroacupuncture appear to act are interwoven with pain pathways, and electroacupuncture stimuli converge with impulses originating from painful areas. Electroacupuncture may act via purinergic A1 and P2X3 receptors simultaneously to induce an analgesic effect on neuropathic pain.
基金the National Natural Science Foundation of China, No.30670694 the Natural Science Foundation of Anhui Province Department of Education in China, No.2006KJ361B+2 种基金 the National Science Fund for Distinguished Young Scholars of Anhui Medical University, No.GJJQ-0801 the Scientific Research Foundation for Doctor of Anhui Medical University, No. XJ2005006the Special Foundation for Young Scientists in Higher Education Institutions of Anhui Province, No.2010SQRL078
文摘BACKGROUND： Most of the currently available information on purinergic receptors （P2Rs） involved in pain transmission is based on results obtained in dorsal root ganglion or the spinal cord. However, the mechanism of P2Rs in trigeminal neuralgia remains unclear. OBJECTIVE： To investigate changes in the P2R-mediated calcium signaling pathway in nociceptive trigemJnal ganglion neurons. DESIGN, TIME AND SETTING： In vitro experiments were conducted at the Patch-Clamp Laboratory of Comprehensive Experiment Center of Anhui Medical University, China from September 2008 to June 2009. MATERIALS： Thapsigargin, caffeine, suramin, and adenosine 5＇-triphosphate were purchased from Sigma, USA. METHODS： Using Fura-2-based microfluorimetry, intracellular calcium concentration （[Ca^2＋]i） was measured in freshly isolated adult rat small trigeminal ganglion neurons before and after drug application. MAIN OUTCOME MEASURES： Fluorescent intensities were expressed as the ratio F340/F380 to observe [Ca^2＋]i changes. RESULTS： In normal extracellular solution and Ca^2＋-free solution, application of thapsigargin （1 μmol/L）, a sarcoplasmic reticulum Ca^2＋ pump adenosine 5＇-triphosphate inhibitor, as well as caffeine （20 mmol/L）, a ryanodine receptor agonist, triggered [Ca^2＋]i increase in small trigeminal ganglion neurons. A similar response was induced by application of adenosine 5＇-triphosphate （100 μmol/L）. In Ca^2＋-free conditions, adenosine 5＇-triphosphate-induced [Ca^2＋]i transients in small trigeminal ganglion neurons were inhibited in cells pre-treated with thapsigargin （P 〈 0.01）, but not by caffeine （P 〉 0.05）. In normal, extracellular solution, adenosine 5＇-triphosphate-induced [Ca^2＋]i transients in small trigeminal ganglion neurons were partly inhibited in cells pre-treated with thapsigargin （P 〈 0.05）. CONCLUSION： Inositol-1,4, 5-triphosphate （IP3）- and ryanodine-sensitive Ca^2＋ stores exist in rat nociceptive trigeminal ganglion neurons. Tw
基金the National Natural Science Foundation of Guangdong Province project(No.2018A030310579)the Medical Research Foundation of Guangdong Province project(No.A2019024)CREST(No.JPMJCR1752 to T.O.)from Japan Science and Technology(JST),and the Japan Society for the Promotion of Science(JSPS)KAKENHI(Grants-in-Aid for Scientific Research(A)19H00900 and Innovative Areas JP 18K19092 to T.0.).
文摘Phosphatidylinositol(3,4,5)-triphosphate(PIP3),acting as a fundamental second messenger,is emerging as a promising biomarker for disease diagnosis and prognosis.However,the real time analysis of phosphoinositide in living cells remains a key challenge owing to the low basal abundance and its fast metabolic rate.Herein,we design an optogenetic system that uses light sensitive protein-protein interaction between Arabidopsis cryptochrome 2(CRY2)and CIB1 to spatiotemporally visualize the PIP3 production with sub-second timescale.In this system,a CIBN is anchored on the plasma membrane,whereas a CRY2 fused with a constitutively active PI3-kinase(acPI3K)would be driven from the cytosol to the membrane by the blue-light-activated CRY2-CIB1 interaction upon light irradiation.The PIP3 production is visualized via a fused fluorescent protein by the translocation of a Pleckstrin Homology(PH)domain(GRP1)from the cytosol to the plasma membrane with high specificity.We demonstrated the fast dynamics and reversibility of the optogenetic system initiated PIP3 synthesis on the plasma membrane.Notably,the real-time cell movements were also observed upon localized light stimulation.The established optogenetic method provides a novel spatiotemporal strategy for specific PIP3 visualization,which is beneficial to improve the understanding of PIP3 functions.
文摘An adenine nucleotide derivative 2-aminoadenosine 5＇-triphosphate was chemically synthesized through four steps and was characterized with 1H NMR, 31p NMR, 13C NMR, EA and FT-IR. Its ultraviolet and fluorescence properties at various pH values were studied. Two pKa values for the compound were determined by the curves of UV absorption dependency on pH, Which were 0.68 and 4.83, respectively. The values were consistent with those calculated from ACD/Labs software. In addition, hydrolysis of the adenine nucleotide derivative in the catalysis of potato apyrase was studied. The competition of the ATP analogue with ATP for potato apyrase＇ active site was proved to be a sequential reaction mechanism.
基金the National Natural Science Foundation of China(21175109)for the financial support
文摘Selective recognition of adenosine 5＇-triphosphate （ATP） is of great significance owing to its indispensable functions to organisms. Also, it is a challenging task because other nucleosides triphosphate hold the same triphosphate group and structurally planar bases as ATP. It is known that metal-organic frameworks （MOFs） are a new type of sensing material. In this work, highly selective recognition of ATP against other nucleosides triphosphate is successfully achieved with a luminescent MOF of [Zn（BDC）（H2O）2]n （BDC2- = 1,4-benzenedicarboxylate）. [Zn（BDC）（H2O）2]n dispersed in water shows a remarkable redshift of the emission wavelength upon addition of ATP, while cytidine 5＇-triphosphate （CTP）, uridine 5＇-triphosphate （UTP） and guanosine 5＇-triphosphate （GTP）, as well as some inorganic anions such as P2074- or PO43- can＇t induce such spectral change as ATP. 1H NMR, 31p NMR and Raman spectra indicate that both π-π stacking interactions and the coordination of Zn（II） with adenine and the phosphate group are involved in the interaction of [Zn（BDC）（H2O）2],, with ATP. In addition, the experimental results showed that the redshift extent of the emission wavelength of [Zn（BDC）（HzO）2]n has the linear relation- ship with the concentration of ATP in the range of 0.3-1.8 mmol/L. Based on this, the detection of ATP content in the sample of ATP injection was made with satisfactory results. This system pioneers the application of MOFs in the recognition of nucle- otides, and testifies that the participation of base in the recognition process can improve the selectivity against the other nucleotides.
文摘Background: Molnupiravir, N4-hydroxycytidine-5’-isopropyl ester, is an oral prodrug of N4-deoxycytidine (NHC), a nucleoside analog, which has in vitro activity against SARS-CoV-2. NHC is phosphorylated in cells to NHC triphosphate (NHC-TP), which is incorporated into viral RNA, leading to epigenetic catastrophe of the viral genome and inhibition of viral replication. The antiviral activity against SARS-CoV-2 is dependent on the number of molecules of NHC-TP incorporated into viral RNA. Clinical studies in patients with COVID-19 showed that treatment with molnupiravir for 5 days decreases the risk of hospitalization and death as compared with placebo. Objective: It should be possible to enhance the antiviral activity of NHC-TP against SARS-CoV-2 by the use of the biochemical modulator, 3-deazauridine (3DU). 3DU is an inhibitor of CTP synthetase. Inhibition of this enzyme results in a reduction in the intracellular pool size of CTP. Since NHC-TP competes with CTP for incorporation into viral RNA in the reaction catalyzed by the SARS-CoV-2 viral RNA-dependent RNA polymerase, the reduction in the level of CTP should result in a significant enhancement of the incorporation of NHC-TP into viral RNA and an enhancement of its antiviral activity. Methods: Analysis of the publications of 3DU and cytosine nucleoside analogues support the hypothesis that 3DU enhances the pharmacological action of the analogues. Results: 3-DU increased the incorporation of 5-azacytidine into RNA and 5-aza-deoxycytidine into DNA of leukemic cells with an enhancement of their antineoplastic action. 3-DU potentiated the antiviral activity against HIV-1 activity by the cytosine nucleoside analogues: 2’-deoxy-3’-thiacytidine (3TC;lamivudine) and 2’,3’-dideoxycytidine (ddC). This anti-HIV-1 activity of 3DU was associated with a reduction in the intracellular pool size of dCTP and increased incorporation of triphosphates of 3TC and ddC into DNA by the HIV-1 reverse transcriptase. The reduction of CTP levels in cells by 3-DU also leads to a
基金Project supported by the National Natural Science Foundation of China （No. 90210027）.Acknowledgments The authors especially thank Zhide Hu in Lanzhou University for kindly allowing the use of the RF-5301PC spectrofluorophotometer （Shimadzu） in this work and Wenying He in Lanzhou University for active discussion as well as zealous help during the fluorescence experiments.
文摘In this paper, some new results on the selective weak interaction between Na-4-tosyl-L-arginine methyl ester hydrochloride （TAME） and adenosine-5＇-triphosphate （ATP） have been reported. Fluorescence spectrophotometry and Fourier transform infrared （FT-IR） spectroscopy were used to investigate this kind of weak interaction. In fluorescence experiments, obvious fluorescence quenching phenomena were observed when TAME was added, which indicated the weak interactions between TAME and ATP. It has been identified by fluorescence titration experiments that TAME exhibited high selectivity to ATP over ADP and AMP. FT-IR spectral results showed that an ATP-TAME adduct was formed. The experimental results indicated that the interaction sites were the guanidinium group of TAME main-chain and the γ-phosphate group of ATP, and the interaction took place through hydrogen bonding and electrostatic force. In addition, the effects of metal ions on the weak interaction between ATP and TAME, or between ATP and analogues of L-arginine were studied.
