Measuring Ca^(2+) influxes of TRPC1-dependent Ca^(2+) channels in HL-7702 cells with Non-invasive Micro-test Technique

AIM： To explore the possibility of using the Noninvasive Micro-test Technique （NMT） to investigate the role of Transient Receptor Potential Canonical 1 （TRPC1） in regulating Ca^2＋ influxes in HL-7702 cells, a normal human liver cell line.METHODS： Net Ca^2＋ fluxes were measured with NMT, a technology that can obtain dynamic information of specific/selective ionic/molecular activities on material surfaces, non-invasively. The expression levels of TRPCl were increased by liposomal transfection, whose effectiveness was evaluated by Western-blotting and single cell reverse transcription-polymerase chain reaction.RESULTS： Ca^2＋ influxes could be elicited by adding 1 mmol/L CaCl2 to the test solution of HL-7702 cells. They were enhanced by addition of 20 μmol/L noradrenalin and inhibited by 100 μmol/L LaCl3 （a non-selective Ca^2＋ channel blocker）; 5 μmol/L nifedipine did not induce any change. Overexpression of TRPCl caused increased Ca^2＋ influx. Five micromoles per liter nifedipine did not inhibit this elevation, whereas 100 μmol/L LaCI3 did.CONCLUSION： In HL-7702 cells, there is a type of TRPCl-dependent Ca^2＋ channel, which could be detected v/a NMT and inhibited by La^3＋.

Measurement of Ca^(2+) Flow in Cochlear Cells Using Non-Invasive Micro-Test Technique

Objective To test Calcium ion(Ca2+) flow at the head and end of outer hair cells(OHCs) in resting state and in response to Nimodipine treatment.Methods Non-invasive micro-test techniques were used to study Ca2+ in isolated OHCs in adult guinea pigs.Results Four types of Ca2+ transport were identified in OHCs on basilar membrane tissue fragments:influx at the head of with efflux at the bottom(type 1):efflux at the head of OHCs with influx at the bottom(type 2);influx at the both head and bottom(type 3);and efflux at the both head and bottom(type 4).However,only type 1 and type 3 of Ca2+ ion transport were detected in the cochlea.We propose that Ca2+ ion transport exists in adult guinea pig cochlear OHCs in resting state and is variable.Ca2 + flow in OHC can be inhibited by Nimodipine in resting state.

基于非损伤微测技术监测贮期鸡蛋氧呼吸规律

鸡蛋作为一个生命体,呼吸作用是其产后重要的生理活动,也是影响鸡蛋贮运效果的重要因素,研究鸡蛋氧呼吸规律对其贮藏保鲜意义重大。该文应用非损伤微测技术,搭建特有的微测试验平台,实时监测鸡蛋与外界微环境的O2交换速率以及交换强度。通过预试验选定探针与鸡蛋表面的最佳距离为50μm,选定鸡蛋最佳测试位置为钝端。试验选取一天中的4个时间点(6:00,12:00,18:00,24:00)来测试鸡蛋O2呼吸,借以研究鸡蛋在一天不同时间点的氧呼吸强弱;另外在每天同一时间点测试鸡蛋O2流速,连续测一个月,来研究鸡蛋贮藏期间的氧呼吸变化规律。试验发现:一天中鸡蛋的氧呼吸存在凌晨高下午低的规律性,无论是受精蛋还是非受精蛋,在每天的不同时间点呼吸强度有差异,受精蛋比非受精蛋的生命活动及新陈代谢变化更强烈,昼夜呼吸差异显著;一个月中,鸡蛋在贮期会产生2次呼吸峰值,分别出现在质变的转折点,并且第2次的峰值明显高于第1次的峰值。试验结果表明鸡蛋存在自身特有呼吸规律,生命代谢活动强时其对应氧呼吸活动也强,反之则弱;刚产下的鸡蛋的呼吸较为活跃。该研究可为如何优化贮藏条件以及提高种蛋的孵化率提供参考。

Effects and mechanisms of store-operated calcium channel blockade on hepatic ischemia-reperfusion injury in rats

AIM:To further investigate the important role of store-operated calcium channels (SOCs) in rat hepatocytes and to explore the effects of SOC blockers on hepatic ischemia-reperfusion injury (HIRI).METHODS:Using freshly isolated hepatocytes from a rat model of HIRI (and controls),we measured cyto-solic free Ca 2+ concentration (by calcium imaging),net Ca 2+ fluxes (by a non-invasive micro-test technique),the SOC current (I SOC ;by whole-cell patch-clamp record-ing),and taurocholate secretion [by high-performance liquid chromatography and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays].RESULTS:Ca 2+ oscillations and net Ca 2+ fluxes medi-ated by Ca 2+ entry via SOCs were observed in rat he-patocytes.I SOC was significantly higher in HIRI groups than in controls (57.0 ± 7.5 pA vs 31.6 ± 2.7 pA,P <0.05) and was inhibited by La 3+.Taurocholate secretion by hepatocytes into culture supernatant was distinctly lower in HIRI hepatocytes than in controls,an effect reversed by SOC blockers.CONCLUSION:SOCs are pivotal in HIRI.SOC blockers protected against HIRI and assisted the recovery of se-cretory function in hepatocytes.Thus,they are likely to become a novel class of effective drugs for prevention or therapy of HIRI patients in the future.

Cellular Cd^(2+)fluxes in roots confirm increased Cd availability to rice(Oryza sativa L.)induced by soil acidifications

Soil acidifications become one of the main causes restricting the sustainable development of agriculture and causing issues of agricultural product safety.In order to explore the effect of different acidification on soil cadmium(Cd)availability,soil pot culture and hydroponic(soil potting solution extraction)were applied,and non-invasive micro-test technique(NMT)was combined.Here three different soil acidification processes were simulated,including direct acidification by adding sulfuric acid(AP1),acid rain acidification(AP2)by adding artificial simulated acid rain and excessive fertilization acidification by adding(NH_(4))_(2)SO_(4)(AP3).The results showed that for direct acidification(AP1),DTPA-Cd concentration in field soils in Liaoning(S1)and Zhejiang(S2)increased by 0.167-0.217 mg/kg and 0.181-0.346 mg/kg,respectively,compared with control group.When soil pH decreased by 0.45 units in S1,the Cd content of rice stems,leaves and roots increased by 0.48 to 6.04 mg/kg and 2.58 to 12.84mg/kg,respectively,When the pH value of soil S1 and S2 decreased by 0.20 units,the average velocity of Cd^(2+)at 200μm increased by 10.03-33.11 pmol/cm~2/sec and 21.33-52.86pmol/cm^(2)/sec,respectively,and followed the order of AP3>AP2>AP1.In summary,different acidification measures would improve the effectiveness of Cd,under the same pH reduction condition,fertilization acidification increased Cd availability most significantly.

Phosphorus removal from sediments by Potamogeton crispus:New high-resolution in-situ evidence for rhizosphere assimilation and oxidization-induced retention

Macrophytes are usually chosen for phytoremediation tools to remove P in eutrophic aquatic ecosystems,but the lack of test methods hinders the understanding of removal mechanism and application.In this study,we used the novel technologies combined of Diffusive gradients in thin films(DGT),Planar optode(PO),and Non-invasive micro-test technology(NMT)to explore P dynamics in water-sediment continuum and rhizosphere of Potamogeton crispus over time.Results of the high-resolution in situ measurement showed that labile P(LPDGT)fluxes at the surficial sediment significantly decreased from approximate 120,140,and 200 pg/(cm^(2)·sec)via 30 days incubation period to 17,40,and 56 pg/(cm2•sec)via that of 15 days.Obvious synchronous increase of LPDGT was not detected in overlying water,suggesting the intense assimilation of dissolve reactive P via root over time.PO measurement indicated that O_(2)concentration around the rhizosphere remarkably increased and radially diffused into deeper sediment until 100%saturation along with the root stretch downwards.NMT detection of roots showed the obvious O_(2)inflow into root tissue with the uppermost flux of 30 pmol/(cm2•sec)from surroundings via aerenchyma on different treatment conditions.Different from previous reports,gradually saturating O_(2)concentrations around the rhizosphere was principally driven by O_(2)penetration through interspace attributing to root stretch downward rather than root O_(2)leakage.Increased O_(2)concentrations in deep sediment over time finally induced the oxidization of labile Fe(II)into Fe(III)bound P and local P immobilization.

Micro-nondestructive detection of the moisture and ion of rice seeds during germination under salt stress

Salt stress is one of the major stress factors limiting rice productivity.Its damaging effects include water deficit due to osmotic stress,and ionic toxicity caused by ionic stress.It is very important to study the salt-tolerance mechanism of rice under salt stress,in order to improve the salt-tolerance capacity of rice and thereby increase the yield.In this experiment,the low field nuclear magnetic resonance(LF NMR)technique and the traditional dry-weight weighing method,the non-invasive micro-test technique(NMT)and the inductively coupled plasma emission spectrometry(ICP-AES)were applied to analyze the distribution of water and the flow of K^(+)and Na^(+)of rice seeds during germination under NaCl stress.The results suggested that for all different NaCl concentrations,as germination hours grew,the amplitude of NMR signals of the bound water that of the free water and the total amplitude all increased gradually.And the higher the NaCl concentration is,the weaker the increase trend is.In addition,the moisture content of the seeds and the total amplitude of NMR signals were positively correlated.The regression equation was y=191.53x+1463.6,the correlation coefficient was R=0.9823,and the determination coefficient was R2=0.9650.By this regression equation,the moisture content of each state of water during seed germination can be calculated.When without NaCl stress,the rice seeds absorbed K^(+)in the germination process.However,when under NaCl stress at different concentrations,K^(+)efflux was detected.The contents of K^(+)and K^(+)/Na^(+)were lower than that under the control condition.The higher NaCl concentration is,the lower the K^(+)and K^(+)/Na^(+)contents are.These results are in consistence with the K^(+)and Na^(+)contents detected by the inductively coupled technique.These empirical data offer a reference for the study of rice-seeds’response mechanism under salt stress during germination and the screening of germplasm resources,and also put forward a new method of biopsy micro-nondestructive test for plants under stress.

Differentiating between fertilized and unfertilized eggs prior to incubation based on oxygen flux measurement

One unresolved challenge in the egg industry is how to efficiently and non-invasively detect unfertilized eggs prior to incubation.This detection ability would not only significantly improve hatching rates and reduce costs but also conserve incubator space and prevent poor-quality embryos from contributing to the spread of infections.This study demonstrates a procedure for distinguishing between fertilized and unfertilized eggs prior to incubation by studying the respiratory differences between fertilized and unfertilized eggs using the Non-invasive Micro-test Technique(NMT).A customized micro-testing examination platform,NMT Egg Testing System(NMT-ETS)was constructed for the real-time monitoring of the intensity and rate of oxygen exchange between the egg and its external environment.The results from this study revealed that at room temperature,there is a significant difference in gas exchange rates between fertilized and unfertilized eggs.The results indicate that the oxygen flux of fertilized eggs exceed 20 pmol/(cm^(2)·s),whereas unfertilized eggs show a much lower oxygen flux.Based on the results,the NMT method can be used to effectively distinguish between fertilized and unfertilized chicken eggs.

K^(+)and Na^(+)fluxes in roots of two Chinese Iris populations

Maintenance of ion homeostasis,particularly the regulation of K^(+)and Na^(+)uptake,is important for all plants to adapt to salinity.Observations on ionic response to salinity and net fluxes of K^(+),Na^(+)in the root exhibited by plants during salt stress have highlighted the need for further investigation.The objectives of this study were to compare salt adaptation of two Chinese Iris(Iris lactea Pall.var.chinensis(Fisch.)Koidz.)populations,and to improve understanding of adaptation to salinity exhibited by plants.Plants used in this study were grown from seeds collected in the Xinjiang Uygur Autonomous Region(Xj)and Beijing Municipality(Bj),China.Hydroponicallygrown seedlings of the two populations were supplied with nutrient solutions containing 0.1(control)and 140 mmol·L^(–1) NaCl.After 12 days,plants were harvested for determination of relative growth rate and K^(+),Na^(+)concentrations.Net fluxes of K^(+),Na^(+)from the apex and along the root axis to 10.8 mm were measured using noninvasive micro-test technique.With 140 mmol·L^(–1) NaCl treatment,shoots for population Xj had larger relative growth rate and higher K^(+)concentration than shoots for population Bj.However,the Na^(+)concentrations in both shoots and roots were lower for Xj than those for Bj.There was a lower net efflux of K^(+)found in population Xj than by Bj in the mature zone(approximately 2.4^(–1)0.8 mm from root tip).However,no difference in the efflux of Na^(+)between the populations was obtained.Population Xj of I.lactea continued to grow normally under NaCl stress,and maintained a higher K^(+)/Na^(+)ratio in the shoots.These traits,which were associated with lower K^(+)leakage,help population Xj adapt to saline environments.