AIM： To evaluate the safety and efficacy of cellular photoablation using BCECF-AM [2, T-bis-（2-carboxyethyl） -5-（and-6）-carboxyfluorescein, acetoxymethyl ester mixed isomersl as a method to control postoperative ...AIM： To evaluate the safety and efficacy of cellular photoablation using BCECF-AM [2, T-bis-（2-carboxyethyl） -5-（and-6）-carboxyfluorescein, acetoxymethyl ester mixed isomersl as a method to control postoperative fibrosis in subscleral trabeculectomy （SST） compared to mitomycin C （MMC） in a rabbit model. METHODS： A comparative prospective case-control animal study was conducted. Fourteen rabbits were subjected to SST with intraoperaUve use of wound modulating agents （MMC or BCECF-AM） of the right eye （study groups I and II respectively） and SST without use of intraoperative wound modulating agents for the left eye （control group II）. Two rabbits 4 eyes were considered as control group I with no surgical intervention. BCECF-AM was injected subconjunctivally 30min before surgery followed by intraoperative illumination with diffuse blue light for 10min. Antifibrotic efficacy was established by clinical response and histological examination. Clinical response was assessed by measuring intraocular pressure （lOP） at day 1, 3, 5, 7, 14, 21 postoperatively, Success was defined by 〉20.0% reduction in lOP from the preoperative values without anti-glaucoma medications. RESULTS： The mean percentage of reduction was 35.0% in the study group I with only one eye （14.3%） had 12.5% reduction. The mean percentage of reduction was 28.0% in the study group U with two eyes （28.6%） in study group II had 14.2% reduction each. Regarding the control group II, the mean percentage of reduction was 14.3% with 64.3% eyes had 〈20.0% reduction. There was a highly statistically significant difference between each of the study groups （right eyes） and the corresponding control group II （left eyes） as regards the mean postoperative lOP values started from day 5 in both study groups and this highly significant difference remained so till the end of the follow up period. Histologically, MMC treated blebs showed thinning of conjunctival epithelium with marked reduction of the goblet cells relative to control. Marked sub-epithelial edema was seen along with variable collagen dispersion. Mild cellularity was noted in sub-epithelial tissue. BCECF-AM treated blebs showed normal conjunctival epithelial thickness with abundant goblet cells. Mild sub- epithelial edema was noted along with moderate collagen dispersion. No histological abnormality was noted in the ciliary body or the cornea in any of the studied groups. CONCLUSION： Cellular photoablation using BCECF-AM is a safe and effective wound modulating agent to control postoperative fibrosis in trabeculectomy. However MMC considered as a more potent adjuvant to trabeculectomy than BCECF-AM in promoting IOP reduction.展开更多
AIM To establish a functional and molecular model of the intracellular pH(pH_i) regulatory mechanism in human induced pluripotent stem cells(hiPSCs).METHODS hiP SCs(HPS0077) were kindly provided by Dr. Dai from the Tr...AIM To establish a functional and molecular model of the intracellular pH(pH_i) regulatory mechanism in human induced pluripotent stem cells(hiPSCs).METHODS hiP SCs(HPS0077) were kindly provided by Dr. Dai from the Tri-Service General Hospital(IRB No. B-106-09). Changes in the pH_i were detected either by microspectrofluorimetry or by a multimode reader with a pH-sensitive fluorescent probe, BCECF, and the fluorescent ratio was calibrated by the high K^+/nigericin method. NH_4Cl and Na-acetate prepulse techniques were used to induce rapid intracellular acidosis and alkalization, respectively. The buffering power(β) was calculated from the ΔpH_i induced by perfusing different concentrations of(NH_4)_2SO_4. Western blot techniques and immunocytochemistry staining were used to detect the protein expression of pH_i regulators and pluripotency markers.RESULTS In this study, our results indicated that(1) the steadystate pH_i value was found to be 7.5 ± 0.01(n = 20) and 7.68 ± 0.01(n =20) in HEPES and 5% CO_2/HCO_3^- buffered systems, respectively, which were much greater than that in normal adult cells(7.2);(2) in a CO_2/HCO_3^--buffered system, the values of total intracellular buffering power(β) can be described by the following equation: β_(tot) = 107.79(pH_i)~2-1522.2(pH_i) + 5396.9(correlation coefficient R^2 = 0.85), in the estimated pH_i range of 7.1- 8.0;(3) the Na^+/H^+ exchanger(NHE) and the Na^+/HCO_3^- cotransporter(NBC) were found to be functionally activated for acid extrusion for pHi values less than 7.5 and 7.68, respectively;(4) V-ATPase and some other unknown Na^+-independent acid extruder(s) could only be functionally detected for pHi values less than 7.1;(5) the Cl^-/OH^- exchanger(CHE) and the Cl^- /HCO_3 anion exchanger(AE) were found to be responsible for the weakening of intracellular proton loading;(6) besides the CHE and the AE, a Cl^--independent acid loading mechanism was functionally identified; and(7) in hiPSCs, a strong positive correlation was observed between the loss of pluripotency and the weakening of the intracellular acid extrusion mechanism, which included a decrease in the steady-state pH i value and diminished the functional activity and protein expression of the NHE and the NBC.CONCLUSION For the first time, we established a functional and molecular model of a pHi regulatory mechanism and demonstrated its strong positive correlation with hiPSC pluripotency.展开更多
The plant vacuole plays a fundamental role in cell homeostasis.The successful application of patch-clamp tech-nique on isolated vacuoles allows the determination of the functional characteristics of tonoplast ion chan...The plant vacuole plays a fundamental role in cell homeostasis.The successful application of patch-clamp tech-nique on isolated vacuoles allows the determination of the functional characteristics of tonoplast ion channels and transporters.The parallel use of a sensor-based fluorescence approach capable of detecting changes in calcium and proton concentrations opens up new possibilities for investigation.In excised patch,the presence of fura-2 in the vacuolar solution reveals the direct permeation of calcium in plant TPC channels.In whole-vacuole,the activity of non-electrogenic NHX potassium proton antiporters can be measured by using the proton sensitive dye BCECF loaded in the vacuolar lumen by the patch pipette.Both vacuolar NHXs and CLCa(chloride/nitrate antiporter)are inhibited by the phosphoinositide PI(3,5)P2,suggesting a coordinated role of these proteins in salt accumulation.Increased knowledge in the molecular mechanisms of vacuolar ion channels and transporters has the potential to improve our understanding on how plants cope with a rapidly changing environment.展开更多
文摘AIM： To evaluate the safety and efficacy of cellular photoablation using BCECF-AM [2, T-bis-（2-carboxyethyl） -5-（and-6）-carboxyfluorescein, acetoxymethyl ester mixed isomersl as a method to control postoperative fibrosis in subscleral trabeculectomy （SST） compared to mitomycin C （MMC） in a rabbit model. METHODS： A comparative prospective case-control animal study was conducted. Fourteen rabbits were subjected to SST with intraoperaUve use of wound modulating agents （MMC or BCECF-AM） of the right eye （study groups I and II respectively） and SST without use of intraoperative wound modulating agents for the left eye （control group II）. Two rabbits 4 eyes were considered as control group I with no surgical intervention. BCECF-AM was injected subconjunctivally 30min before surgery followed by intraoperative illumination with diffuse blue light for 10min. Antifibrotic efficacy was established by clinical response and histological examination. Clinical response was assessed by measuring intraocular pressure （lOP） at day 1, 3, 5, 7, 14, 21 postoperatively, Success was defined by 〉20.0% reduction in lOP from the preoperative values without anti-glaucoma medications. RESULTS： The mean percentage of reduction was 35.0% in the study group I with only one eye （14.3%） had 12.5% reduction. The mean percentage of reduction was 28.0% in the study group U with two eyes （28.6%） in study group II had 14.2% reduction each. Regarding the control group II, the mean percentage of reduction was 14.3% with 64.3% eyes had 〈20.0% reduction. There was a highly statistically significant difference between each of the study groups （right eyes） and the corresponding control group II （left eyes） as regards the mean postoperative lOP values started from day 5 in both study groups and this highly significant difference remained so till the end of the follow up period. Histologically, MMC treated blebs showed thinning of conjunctival epithelium with marked reduction of the goblet cells relative to control. Marked sub-epithelial edema was seen along with variable collagen dispersion. Mild cellularity was noted in sub-epithelial tissue. BCECF-AM treated blebs showed normal conjunctival epithelial thickness with abundant goblet cells. Mild sub- epithelial edema was noted along with moderate collagen dispersion. No histological abnormality was noted in the ciliary body or the cornea in any of the studied groups. CONCLUSION： Cellular photoablation using BCECF-AM is a safe and effective wound modulating agent to control postoperative fibrosis in trabeculectomy. However MMC considered as a more potent adjuvant to trabeculectomy than BCECF-AM in promoting IOP reduction.
基金Supported by Ministry of Science and Technology Grants of Taiwan,No.MOST 106-2320-B-016-003-MY2(to Loh SH)and No.MOST 106-2314-B-016-037-MY3(to Tsai YT)National Defense Medical Center Grants of Taiwan,No.MAB-106-033(to Loh SH),No.MAB-105-043 and No.MAB-106-034(to Dai NZ)Teh-Tzer Study Group for Human Medical Research Foundation of Taiwan,No.A1061037 and No.A1061054(to Loh SH)
文摘AIM To establish a functional and molecular model of the intracellular pH(pH_i) regulatory mechanism in human induced pluripotent stem cells(hiPSCs).METHODS hiP SCs(HPS0077) were kindly provided by Dr. Dai from the Tri-Service General Hospital(IRB No. B-106-09). Changes in the pH_i were detected either by microspectrofluorimetry or by a multimode reader with a pH-sensitive fluorescent probe, BCECF, and the fluorescent ratio was calibrated by the high K^+/nigericin method. NH_4Cl and Na-acetate prepulse techniques were used to induce rapid intracellular acidosis and alkalization, respectively. The buffering power(β) was calculated from the ΔpH_i induced by perfusing different concentrations of(NH_4)_2SO_4. Western blot techniques and immunocytochemistry staining were used to detect the protein expression of pH_i regulators and pluripotency markers.RESULTS In this study, our results indicated that(1) the steadystate pH_i value was found to be 7.5 ± 0.01(n = 20) and 7.68 ± 0.01(n =20) in HEPES and 5% CO_2/HCO_3^- buffered systems, respectively, which were much greater than that in normal adult cells(7.2);(2) in a CO_2/HCO_3^--buffered system, the values of total intracellular buffering power(β) can be described by the following equation: β_(tot) = 107.79(pH_i)~2-1522.2(pH_i) + 5396.9(correlation coefficient R^2 = 0.85), in the estimated pH_i range of 7.1- 8.0;(3) the Na^+/H^+ exchanger(NHE) and the Na^+/HCO_3^- cotransporter(NBC) were found to be functionally activated for acid extrusion for pHi values less than 7.5 and 7.68, respectively;(4) V-ATPase and some other unknown Na^+-independent acid extruder(s) could only be functionally detected for pHi values less than 7.1;(5) the Cl^-/OH^- exchanger(CHE) and the Cl^- /HCO_3 anion exchanger(AE) were found to be responsible for the weakening of intracellular proton loading;(6) besides the CHE and the AE, a Cl^--independent acid loading mechanism was functionally identified; and(7) in hiPSCs, a strong positive correlation was observed between the loss of pluripotency and the weakening of the intracellular acid extrusion mechanism, which included a decrease in the steady-state pH i value and diminished the functional activity and protein expression of the NHE and the NBC.CONCLUSION For the first time, we established a functional and molecular model of a pHi regulatory mechanism and demonstrated its strong positive correlation with hiPSC pluripotency.
文摘The plant vacuole plays a fundamental role in cell homeostasis.The successful application of patch-clamp tech-nique on isolated vacuoles allows the determination of the functional characteristics of tonoplast ion channels and transporters.The parallel use of a sensor-based fluorescence approach capable of detecting changes in calcium and proton concentrations opens up new possibilities for investigation.In excised patch,the presence of fura-2 in the vacuolar solution reveals the direct permeation of calcium in plant TPC channels.In whole-vacuole,the activity of non-electrogenic NHX potassium proton antiporters can be measured by using the proton sensitive dye BCECF loaded in the vacuolar lumen by the patch pipette.Both vacuolar NHXs and CLCa(chloride/nitrate antiporter)are inhibited by the phosphoinositide PI(3,5)P2,suggesting a coordinated role of these proteins in salt accumulation.Increased knowledge in the molecular mechanisms of vacuolar ion channels and transporters has the potential to improve our understanding on how plants cope with a rapidly changing environment.
