Effect of Quercetin on the Proliferation and Mitochondrial Transmembrane Potential of CBRH-7919 Cells

[Objective] To investigate the effect of quercetin on the proliferation and mitochondrial transmembrane potential of CBRH-7919 cells. [Method] The CBRH-7919 cells of hepatocarcinoma were cultured in vitro. After treated with different concentrations of quercetin, the OD405 nm of CBRH-7919 cells was detected by using the acid phosphatase assy （APA）; morphologic changes of the cells were observed under inverted microscope; the mitochondrial transmembrane potential （△ψm） intensity changes of CBRH-7919 cells were analyzed by flow cytometry after stained with Rhodamine 123. [Result] Quercetin inhibited the proliferation of CBRH-7919 cells significantly, and the growth inhibitory effect presented time- and dose-dependent relationship. Typical decrease of cell density was observed by optical microscopy on the quercetin-treated cells. With the effect of 10 μg/ml quercetin on CBRH-7919 cells for 12, 24 and 48 h, the percentage of Rhodamine 123 stained hypofluorescence cells increased, while the mitochondrial transmembrane potential（△ψm） intensity of CBRH-7919 cells decreased. [Conclusion] Quercetin could inhibit the proliferation of CBRH-7919 cells in vitro, causing the decrease in mitochondrial transmembrane potential.

Berbamine induces apoptosis in human hepatoma cell line SMMC7721 by loss in mitochondrial transmembrane potential and caspase activation

Objective： To investigate the effect ofberbamine on human hepatoma cell line SMMC7721. Methods： The effects of 24 h and 48 h incubation with different concentrations （0-64 μg/ml） of the berbamine on SMMC7721 cells were evaluated using 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide （MTT） assay. Hoechst 33258 staining was conducted to distinguish the apoptotic cell, and the appearance of sub-G1 stage was determined by PI （propidium iodide） staining, the percentage of apoptotic cell was determined by flow cytometry following annexin V/PI staining. Flow cytometry was performed to analyze the cell cycle distribution and the mitochondrial membrane potential （△ψm）, the expression of activated caspase3 and caspase9 was analyzed by Western-blot. Results： The proliferation of SMMC7721 was decreased after treatment with berbamine in a dose- and time-dependent manner. Berbamine could induce apoptosis in SMMC7721 cells and could cause cell cycle arrest in G0/G1 phase, to induce loss of mitochondrial membrane potential （AVm） and activate caspase3 and caspase9. Berbamine-induced apoptosis could be blocked by the broad caspase inhibitor z-VAD-fmk. Conclusion： Berbamine exerts antiproliferative effects on human hepatocellular carcinoma SMMC7721 cells. The anticancer activity of berbamine could be attributed partly to its inhibition of cell proliferation and induction of apoptosis in cancer cells through loss in mitochondrial transmembrane potential and caspase activation.

Interventional effect of phycocyanin on mitochondrial membrane potential and activity of PC12 cells after hypoxia/reoxygenation

BACKGROUND： Phycocyanin can relieve decrease of mitochondrial membrane potential through reducing production of active oxygen so as to protect neurons after hypoxia/reoxygenation. OBJECTIVE： To observe the effect of phycocyanin on activity of PC12 cells and mitochondrial membrane potential after hypoxia/reoxygenation. DESIGN： Randomized controlled study SETTING ： Cerebrovascular Disease Institute of Affiliated Hospital, Medical College of Qingdao University MATERIALS： The experiment was carried out at the Key Laboratory of Prevention and Cure for cerebropathia in Shandong Province from October to December 2005. PC12 cells, rat chromaffin tumor cells, were provided by Storage Center of Wuhan University; phycocyanin was provided by Ocean Institute of Academia Sinica; Thiazoyl blue tetrazolium bromide （MTT） and rhodamine 123 were purchased from Sigma Company, USA; RPMI-1640 medium, fetal bovine serum and equine serum were purchased from Gibco Company, USA. METHODS： ① Culture of PC12 cells： PC12 cells were put into RPMI-1640 medium which contained 100 g/L heat inactivation equine serum and 0.05 volume fraction of fetal bovine serum and incubated in CO2 incubator at 37℃. Number of cells was regulated to 4 × 10^5 L 1, and cells were inoculated at 96-well culture plate. The final volume was 100μL. ② Model establishing and grouping： Cultured PC12 cells were randomly divided into three groups： phycocyanin group, model control group and non-hypoxia group. At 24 hours before hypoxia, culture solution in phycocyanin group was added with phycocyanin so as to make sure the final concentration of 3 g/L , but cells in model control group did not add with phycocyanin. Cells in non-hypoxia group were also randomly divided into adding phycocyanin group （the final concentration of 3 g/L） and non-adding phycocyanin group. Cells in model control group and phycocyanin group were cultured with hypoxia for 1 hour and reoxygenation for 1, 2 and 3 hours; meanwhile, cells in non-hypoxia group were cultured with oxygen and were measured at 1 hour after hypoxia/reoxygenation. ③ Detecting items： At 1, 2 and 3 hours after reoxygenation, absorbance （A value） of PC12 cells was measured with MTT technique so as to observe activity and quantity of cells. Fluorescence intensity of PC12 cells marked by rhodamine 123 was measured with confocal microscope in order to observe changes of mitochondrial membrane potential. MAEN OUTCOME MEASURES： Comparisons between quantity and activity of PC12 cells and mitochondria membrane potential at 1, 2 and 3 hours after reoxygenation. RESULTS： ① Effect of phycocyanin on quantity and activity of PC12 cells： A value was 0.924±0.027 in adding phycocyanin group and 0.924±0.033 in non-adding phycocyanin group. A value was lower in model control group and phycocyanin group than that in non-hypoxia group at 1, 2 and 3 hours after reoxygenation （0.817±0.053, 0.838±0.037, 0.875±0.029; 0.842±0.029, 0.872±0.025, 0.906±0.023, P 〈 0.05）. A value was higher in phycocyanin group than that in model control group at 1, 2 and 3 after culture （P 〈 0.05）. With culture time being longer, A value was increased gradually in phycocyanin group and model control group after reoxygenation （P 〈 0.05）. ~ Effect of phycocyanin on mitochondrial membrane potential of PC12 cells： Fluorescence intensity was 2.967±0.253 in adding phycocyanin group and 2.962±0.294 in non-adding phycocyanin group. Fluorescence intensity was lower in model control group and phycocyanin group than that in non-hypoxia group at 1, 2 and 3 hours after hypoxia/reoxygenation （1.899±0.397, 2.119±0.414, 2.287±0.402; 2.191±0.377, 2.264±0.359, 2.436±0.471, P 〈 0.05）; but it was higher in phycocyanin group than that in model control group at 1, 2 and 3 after reoxygenation （P 〈 0.05）. With culture time being longer, fluorescence intensity was increased gradually in phycocyanin group and model control group after reoxygenation （P 〈 0.05）. CONCLUSION： Phycocyanin and reoxygenation can protect PC12 cells after hypoxia injury through increasing mitochondrial membrane potential and cellular activity, and the effect is improved gradually with prolonging time of reoxygenation.

Effect of rhTNF-α on Mitochondrial Transmembrane Potential and Motility of Human Sperm in vitro by Flow Cytometry and Computer Aided of Semen Analysis

To evaluate effect of recombined human tumor necrosis factor （rhTNF- α） on mitochondrial transmembrane potential and motility of human sperm in vitro Methods Semen samples for study were obtained from 40 health men （average age 26 ± 1.2 years） with normal semen analysis. Sperm suspension with computer aided of semen analysis （CASA） technique; 2） were stained in the presence of 10 μg/ml Rh123 and PI, mitochondrial transmembrane potential of those was analyzed by flow cytometry （FCM）. Results Significant differences were found between experimental groups and control groups on viability, straight line velocity, curvilinear velocity, average path velocity, progressive motility of human sperm and number of sperm with normal mitochondrial transmembrane potential （P〈0.01） expect final concentration 30 pg/ml group （P〉0. 05）. Sperm motility lowed with increasing rhTNF-α concentration and incubating time （P〈0. 01）. Number of sperm with normal mitochondrial transmembrane potential decreased with increasing rhTNF-α concentration and incubating time （P〈0.01）. Conclusion rh TNF-α can decrease human sperm motility function in vitro, which can interfere the function of human sperm mitochondrial transmembrane potential and may inhibit sperm mitochondrial enzymatic activities.

Inhibition of phosphodiesterase 4 by FCPR16 protects SH-SY5Y cells against MPP^+ -induced decline of mitochondrial membrane potential and oxidative stress

Parkinson disease(PD) is a chronic neurodegenerative disorder caused by progressive dopaminergic neuronal death in the substantia nigra pars compacta within the midbrain.There still is no cure,effective treatments for PD,available therapies are only capable of offering temporary and symptomatic relief to the patients.There are certain patents that claim phosphodiesterase(PDE) inhibitors as possible anti-PD drugs,PDE4 is a promising target for the treatment of PD and the underlying mechanism has not yet been well elucidated.PDE4 is an enzyme that specifically hydrolyzes intracellular cyclic adenosine monophosphate(cAMP)throughout the body,including the brain.Most of the available PDE4 inhibitors exert unpleasant and serious side effects,such as emesis and nausea,which hinder its clinical application.Therefore,more efforts are needed before PDE4 inhibitors with high therapeutic indices are available for treatment of PD.FCPR16 is a novel PDE4 inhibitor with little emetic potential,which exhibits excellent enzyme inhibition activity(IC50=90 nmol·L^(-1)).METHODS SH-SY5 Y cell was induced with 1-methyl-4-phenylpyridinium(MPP+)to mimic PD cell injury in vitro,and CCK-8 assay was used to investigate the viability effects of different concentration of FCPR16(3.1-50 μmol·L^(-1)) on MPP+-injured SH-SY5 Y cells.Detection of apoptosis was performed by flow cytometry.The level of ntracellular reactive oxygen species was detected with the fluorescent probe DCFH-DA,and the mitochondrial membrane potential of cells in different experimental groups was detected with the JC-1 fluorescent probe.AO staining and Lysotracker Red staining were used to detect the intracellular antophagy changes.The expression of apoptosis related proteins,autophagy and other related signal molecules were demonstrated by Western blotting.Different cellular signaling pathway inhibitors were used to invesitigate the specific cellular mechanisms of FCPR16 protecting MPP+-induced cell injury.RESULTS FCPR16(12.5-50 μmol·L^(-1)) dose-dependently reduced MPP+-induced decline of cell viability,accompanied by reductions in nuclear condensation and lactate dehydrogenase release.The level of cleaved caspase 3 and the ratio of Bax/Bcl-2 were also decreased after treatment with FCPR16 in MPP+-treated cells.Furthermore,FCPR16(25 μmol·L^(-1)) significantly suppressed the accumulation of reactive oxygen species(ROS),prevented the decline of mitochondrial membrane potential(Δψm) and attenuated the expression of malonaldehyde level.Further studies disclosed that FCPR16 enhanced the levels of cA MP and the exchange protein directly activated by cA MP(Epac) in SHSY5 Y cel s.Western blotting analysis revealed that FCPR16 increased the phosphorylation of c AMP response element-binding protein(CREB) and protein kinase B(Akt)down-regulated by MPP+in SHSY5 Y cells.Moreover,the inhibitory effects of FCPR16 on the production of ROS and Δψm loss could be blocked by PKA inhibitor H-89 and Akt inhibitor KRX-0401.CONCLUSION The novel PDE4 inhibitor FCPR16 can protect against damaging pathways including oxidative stress,mitochondrial dysfunction and apoptosis in SH-SY5 Y cells.FCPR16 preventes MPP+-induced neurotoxicity through activation of cAMP/PKA/CREB and Epac/Akt signaling pathways.These may lead to develop mechanism based therapeutics and improved pharmacotherapy for PD.It is reasonable to assume that FCPR16 is a potential candidate for the prevention and treatment of PD.

JTE-522-induced apoptosis in human gastric adenocarinoma cell line AGS cells by caspase activation accompanying cytochrome C release,membrane translocation of Bax and loss of mitochondrial membrane potential

AIM: To investigate the role of the mitochondrial pathway in JTE-522-induced apoptosis and to investigate the relationship between cytochrome C release, caspase activity and loss of mitochondrial membrane potential (Deltapsim). METHODS: Cell culture, cell counting, ELISA assay, TUNEL, flow cytometry, Western blot and fluorometric assay were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanism. RESULTS: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Caspases 8 and 9 were activated during apoptosis as judged by the appearance of cleavage products from procaspase and the caspase activities to cleave specific fluorogenic substrates. To elucidate whether the activation of caspases 8 and 9 was required for the apoptosis induction, we examined the effect of caspase-specific inhibitors on apoptosis. The results showed that caspase inhibitors significantly inhibited the apoptosis induced by JTE-522. In addition, the membrane translocation of Bax and cytosolic release of cytochrome C accompanying with the decrease of the uptake of Rhodamin 123, were detected at an early stage of apoptosis. Furthermore, Bax translocation, cytochrome C release, and caspase 9 activation were blocked by Z-VAD.fmk and Z-IETD-CHO. CONCLUSION: The present data indicate a crucial association between activation of caspases 8, 9, cytochrome C release, membrane translocation of Bax, loss of Deltapsim and JTE-522-induced apoptosis in AGS cells.

YB-1 downregulation attenuates UQCRC1 protein expression level in H9C2 cells and decreases the mitochondrial membrane potential

UQCRC1 is one of the 10 mitochondrial complex III subunits,this protein has a role in energy metabolism,myocardial protection,and neurological diseases.The upstream mechanism of the UQCRC1 protective effect on cardiomyocytes is currently unavailable.In order to explore the upstream molecules of UQCRC1 and elucidate the protective mechanism of UQCRC1 on cardiomyocytes in more detail,we focused on the nuclease-sensitive elementbinding protein 1(YB-1).We hypothesized YB-1 acts as an upstream regulatory molecule of UQCRC1.This study found that YB-1 RNAi significantly reduces the expression of the UQCRC1 protein level(p<0.05)and obviously decreases the mitochondrial membrane potential(p<0.05),and that YB-1 interacts with UQCRC1 protein in vivo,but YB-1 RNAi has little effect on the UQCRC1 gene transcription.

Cannabidiol (CBD) Prevents Palmitic Acid-Induced Drop in Mitochondrial Membrane Potential

Exposure of macrophages and microglia cells to the saturated palmitic acid (PA) leads to reduction in the mitochondrial membrane potential (), shrinkage of the cells and apoptosis. Here we show that the Cannabis component Cannabidiol (CBD) rescues both macrophages and microglia cells from the detrimental effects of PA. CBD prevents the shrinkage in cell size and the reduction incaused by PA. The protective effect of CBD on the macrophage mitochondria is important for sustaining the macrophage population even under the immunosuppressed conditions caused by this drug. To a similar extent, the antagonistic effect of CBD on PA-mediated microglia cytotoxicity is important for its role in neuroprotection.

Effect on apoptosis、mitochondrial membrane potential and Ca^(2+) concentration in HepG-2 by CSEO

Objective To study on the mechanism of growth inhibiting and apoptosis inducing effect of total alkaloid in the CSEO(Capparis spinosa L.essential oil,CSEO)on human hepatocarcinoma cell Line HepG-2.Methods The growth inhibiting effect of the CSEO on human hepatocarcinoma cell Line HepG-2 was measured by MTT method.Morphological observation of the HepG-2 cells was completed by fluorescence microscope.The changing of mitochondrion membrane potential induced by CSEO was observed by staining with Rhodamine123.Effect of the CSEO on intracellular Ca2+ level of the HepG-2 cells was measured by laser confocal microscope.Results The CESO has obvious growth inhibiting effect on the HepG-2 and seems to be dose-dependent,and its IC50 is 127.5 μg·mL-1.The characteristic apoptosis morpha of HepG-2 cells has been observed,and the apoptosis percentage increase to 44.447% in the 300 μg·mL-1 dosage group.In addition,the progress of cells cycle of G1 period has been blocked,and the cellular proportion in S and G2 period is decreased in the 75 μg·mL-1 and 150 μg·mL-1 dosage groups by the function of CSEO for 48 h.The mitochondria membrane potential(Δψm)effected by CESO is decreased,while the curve moves toward left.In addition,the intracellular Ca2+ level is increased by the function of CESO in the middle and high dose groups.Conclusions The CESO has obviously growth inhibiting and apoptosis inducing effect on human hepatocarcinoma cell Line HepG-2 by the mechanism of decreasing the mitochondria membrane potential and increasing the intracellular Ca2+ level.

Immp2l Mutation Induces Mitochondrial Membrane Depolarization and Complex Ⅲ Activity Suppression after Middle Cerebral Artery Occlusion in Mice

Objective We previously reported that mutations in inner mitochondrial membrane peptidase 2-like(Immp2l)increase infarct volume,enhance superoxide production,and suppress mitochondrial respiration after transient cerebral focal ischemia and reperfusion injury.The present study investigated the impact of heterozygous Immp2l mutation on mitochondria function after ischemia and reperfusion injury in mice.Methods Mice were subjected to middle cerebral artery occlusion for 1 h followed by 0,1,5,and 24 h of reperfusion.The effects of Immp2l^(+/−)on mitochondrial membrane potential,mitochondrial respiratory complex III activity,caspase-3,and apoptosis-inducing factor(AIF)translocation were examined.Results Immp2l^(+/−)increased ischemic brain damage and the number of TUNEL-positive cells compared with wild-type mice.Immp2l^(+/−)led to mitochondrial damage,mitochondrial membrane potential depolarization,mitochondrial respiratory complex III activity suppression,caspase-3 activation,and AIF nuclear translocation.Conclusion The adverse impact of Immp2l^(+/−)on the brain after ischemia and reperfusion might be related to mitochondrial damage that involves depolarization of the mitochondrial membrane potential,inhibition of the mitochondrial respiratory complex III,and activation of mitochondria-mediated cell death pathways.These results suggest that patients with stroke carrying Immp2l^(+/−)might have worse and more severe infarcts,followed by a worse prognosis than those without Immp2l mutations.

Restoration of Mitochondrial Structure and Function within Helicobacter pylori VacA Intoxicated Cells

The Helicobacter pylori vacuolating cytotoxin (VacA) is an intracellular, mitochondrial-targeting exotoxin that rapidly causes mitochondrial dysfunction and fragmentation. Although VacA targeting of mitochondria has been reported to alter overall cellular metabolism, there is little known about the consequences of extended exposure to the toxin. Here, we describe studies to address this gap in knowledge, which have revealed that mitochondrial dysfunction and fragmentation are followed by a time-dependent recovery of mitochondrial structure, mitochondrial transmembrane potential, and cellular ATP levels. Cells exposed to VacA also initially demonstrated a reduction in oxidative phosphorylation, as well as increase in compensatory aerobic glycolysis. These metabolic alterations were reversed in cells with limited toxin exposure, congruent with the recovery of mitochondrial transmembrane potential and the absence of cytochrome c release from the mitochondria. Taken together, these results are consistent with a model that mitochondrial structure and function are restored in VacA-intoxicated cells.

Treatment of a patient with severe lactic acidosis and multiple organ failure due to mitochondrial myopathy:A case report

BACKGROUND Mitochondrial myopathy is a rare genetic disease with maternal inheritance that may involve multiple organ systems.Due to the lack of typical characteristics,its clinical diagnosis is difficult,and it is often misdiagnosed or even missed.CASE SUMMARY The patient was a young college student.When he presented at the hospital,he had severe lactic acidosis,respiratory failure,and shock with multiple organ dysfunction syndrome(MODS).He was treated by mechanical ventilation,venoarterial extracorporeal membrane oxygenation,and other organ support.However,his condition continued to worsen.After a thorough and detailed medical and family history was taken,a mitochondrial crisis was suspected.A muscle biopsy was taken.Further genetic testing confirmed a mitochondrial gene mutation(TRNL13243A>G).The final diagnosis of mitochondrial myopathy was made.Although there is no known specific treatment,intravenous methylprednisone and intravenous immunoglobulin were started.The patient’s shock eventually improved.The further course was complicated by severe infection in multiple sites,severe muscle weakness,and recurrent MODS.After 2 mo of multidisciplinary management and intensive rehabilitation,the patient could walk with assistance 4 mo after admission and walk independently 6 mo after admission.CONCLUSION More attention should be paid to mitochondrial myopathy to avoid missed diagnosis and misdiagnosis.

Changes of plasma membrane ATPase activity, membrane potential and transmembrane proton gradient in Kandelia candel and Avicennia marina seedlings with various salinities

The salt-secreting mangrove, Avicennia marina, and non-salt-secreting mangrove, Kandelia candel were cultivated in sand with various salinities(0‰, 10‰, 20‰, 30‰, 40‰) for 60 d. Plasma membrane vesicles of high-purity in leaves and roots of A.marina and K. candel seedlings were obtained by two-phase partitioning. The function of the plasma membranes, the activity of ATPase, membrane potential and transmembrane proton gradient, at various salinities were investigated. The results showed that within a certain range of salinity(A. marina and roots of K. candel: 0—30‰; leaves of K.candel: 0—20‰), the activity of ATPase increased with increasing salinity, while high salinity(above 30‰ or 20‰) inhibited ATPase activity. In comparison with A. marina, K. candel appeared to be more sensitive to salinity. The dynamics of membrane potential and transmembrane proton gradient in leaves and roots of A. marina and K. candel seedlings were similar to that of ATPase. When treated directly by NaCl all the indexes were inhibited markedly: there was a little increase within 0—10‰(K. candel) or 0—20‰(A. marina) followed by sharp declining. It indicated that the structure and function of plasma membrane was damaged severely.

Parkinson’s disease-associated VPS35 mutant reduces mitochondrial membrane potential and impairs PINK1/Parkinmediated mitophagy

Background:Mitochondrial dysfunction plays a prominent role in the pathogenesis of Parkinson’s disease(PD),and several genes linked to familial PD,including PINK1(encoding PTEN-induced putative kinase 1[PINK1])and PARK2(encoding the E3 ubiquitin ligase Parkin),are directly involved in processes such as mitophagy that maintain mitochondrial health.The dominant p.D620N variant of vacuolar protein sorting 35 ortholog(VPS35)gene is also associated with familial PD but has not been functionally connected to PINK1 and PARK2.Methods:To better mimic and study the patient situation,we used CRISPR-Cas9 to generate heterozygous human SH-SY5Y cells carrying the PD-associated D620N variant of VPS35.These cells were treated with a protonophore carbonyl cyanide m-chlorophenylhydrazone(CCCP)to induce the PINK1/Parkin-mediated mitophagy,which was assessed using biochemical and microscopy approaches.Results:Mitochondria in the VPS35-D620N cells exhibited reduced mitochondrial membrane potential and appeared to already be damaged at steady state.As a result,the mitochondria of these cells were desensitized to the CCCPinduced collapse in mitochondrial potential,as they displayed altered fragmentation and were unable to accumulate PINK1 at their surface upon this insult.Consequently,Parkin recruitment to the cell surface was inhibited and initiation of the PINK1/Parkin-dependent mitophagy was impaired.Conclusion:Our findings extend the pool of evidence that the p.D620N mutation of VPS35 causes mitochondrial dysfunction and suggest a converging pathogenic mechanism among VPS35,PINK1 and Parkin in PD.

Effect of solanine on the membrane potential of mitochondria in HepG_2 cells and [Ca^(2+)]i in the cells

AIM： To observe the effect of solanine on the membrane potential of mitochondria in HepG2 cells and [Ca^2＋]i in the cells, and to uncover the mechanism by which solanine induces apoptosis.METHODS： HepG2 cells were double stained with AO/EB, and morphological changes of the cells were observed using laser confocal scanning microscopy （LCSM）. HepG2 cells were stained with TMRE, and change in the membrane potential of mitochondria in the cells were observed using LCSM. HepG2 cells were double stained with Fluo-3/AM, and change of [Ca^2＋]i in the cells were observed using LCSM. HepG2 cells were double stained with TMRE and Fluo-3/AM, and both the change in membrane potential of mitochondria and that of [Ca^2＋]i in the cells were observed using LCSM.RESULTS： Cells in treated groups showed typical signs of apoptosis. Staining with TMRE showed that solanine could lower membrane potential; staining with Fluo-3/AM showed that solanine could increase the concentration of Ca^2＋ in tumor cells; and those of double staining with TMRE and Fluo-3/AM showed that solanine could increase the concentration of Ca^2＋ in the cells at the same time as it lowered the membrane potential of mitochondria.CONCLUSION： Solanine opens up the PT channels in the membrane by lowering the membrane potential, leading to Ca^2＋ being transported down its concentration gradient, which in turn leads to the rise of the concentration of Ca^2＋ in the cell, turning on the mechanism for apoptosis.

Effects of NaCI and Ca^(2+) on Membrane Potential of Epidermal Cells of Maize Roots

The effects of salt-stress on plants involve not only the water stress caused by low osmotic pressure, but also the toxicity of excess Na^＋. A large amount of Na^＋ entering cells would reduce K^＋ uptake, which leads to an imbalance of K：Na ratio in cells. One of the reasons for the reduced K^＋-uptake is the closure of K^＋-channel which is controlled by membrane potential. Calcium is usually applied to improve the growth of plants on saline soils and shows positive influence in the integrality of cell membrane. This study applied glass microelectrode technique to monitoring the NaCl-induced changes of membrane potential of root epidermal cells of maize （Zea mays L., Denghai 11） seedlings at NaCl concentrations of 0, 8, 20, 50, 100, 200 mmol L^-1, respectively. The effect of Ca^2＋ on the changes of membrane potential caused by NaCl was also studied. The results showed that： NaCl caused cell membrane depolarization. The depolarization became greater and faster with increasing of NaCl concentration. Moreover, the extent of depolarization was positively correlated with NaCl concentration. The addition of calcium postponed the depolarization, and decreased the degree of depolarization caused by NaCl. High NaCl concentration leads to depolarization of maize root cell membrane, which can partly be counteracted by calcium.

Phosphatidylglycerol-containing ER-transport vesicles built and restore outer mitochondrial membrane and deliver nuclear DNA translation products to generate cardiolipin in the inner mitochondrial membrane

Phosphatidylglycerol (PG) an important membrane phospholipid required for the synthesis of diphos-phatidylglycerol (DPG) commonly known as cardiolipin (CL) was identified in the fraction of endo-plasmic reticulum (ER)-derived transport vesicles which had no affinity for Golgi. The vesicles were produced in the presence of Brefeldin A (BFA), the agent known to inhibit ER-Golgi transport, and found to display affinity to mitochondria. The analysis revealed that their cargo was not containing proteins that are transported to Golgi, and that their membrane was free of phosphatidylinositol (PI) and ceramides (Cer). The incubation of PG-containing transport vesicles with mitochondria afforded incorporation of their membrane into the Outer Mito-chondrial Membrane (OMM) and formation of lyso-phosphatidylglycerol (LPG). In turn, upon further incubation with fresh transport active cytosol, the mitochondrial LPG was converted to PG. The results of analysis of the OMM, Inner Mitochondrial Mem-brane (IMM) and Inner Mitochondrial Space Components (IMSC) strongly suggest that PG-containing transport vesicles deliver nuclear DNA translation products to the IMSC and thus facilitate CL synthesis in the IMM. In summary, our studies provide evidence that ER-generated PG-enriched transport vesicles represent the general pathway for restitution of mitochondrial membranes and the delivery of nuclear DNA translation products that generate CL, and thus sustain the mitochondrial matrix CL-dependent metabolic reactions.

The Measurement of Membrane Potential and NO3 Activity in Root Cells Using Ion-Selective Microelectrodes

Remobilisation of nitrate in plants, especially in vacuole of plant, is mostly related to the qua- lity of agricultural products and the high nitrogen use efficiency in plants. Ion-selective microelectrodes offer a non-destructive and non-interruptive method to measure NO 3 gradients and electric potential differences across both the plasma membrane and tonoplast. Thus, a double-barrelled microelectrode backfilled with a membrane sensor for NO 3 embedded in poly vinyl chloride (PVC) can record the NO 3 activity in cytoplasm and vacuole of a cell. This paper presented how to make this kind of microelectrode and how to do the intracellular measurements on intact plants. Our result showed that nitrate activity was about 2.7 mmol L 1 in cytoplasm while 70 mmol L 1 in vacuole, which implicated that vacuole was a pool of nitrate in plants.

Mitochondrial membrane protein Bcl-xL, a regulator of adult neuronal growth and synaptic plasticity: multiple functions beyond apoptosis

The B-cell lymphoma 2 （Bcl2） family of proteins participates in cell death or survival through a mitochondrial pathway. The pro-apoptotic members of the Bcl2 family such as Bim, Bid, Bax and Bak trigger cell death by contributing to the enhancement of mitochondrial outer membrane permeabil- ity to pro-apoptotic factors such as cytochrome c, with the subsequent activation of caspases. The anti-apoptotic mem- bers, such as B-cell lymphoma-extra large （Bd-xL）, block the pro-apoptotic Bcl2 members and prevent cell death. Bcl-xL is abundantly expressed during development and in mature neurons, suggesting that it plays a role in protection from death from untoward events occurring in adult life such as ischemia, inflammation or trauma. When these neurotoxic in- sults occur, Bcl-xL translocates to mitochondria and prevents activation and homo-oligomerization of pro-apoptotic family members such Bax and Bak. Numerous studies have shown pro-survival roles for Bcl-xL in adult neurons using various models; nevertheless, the role of Bcl-xL outside of the field of neuronal death, i.e., in adult neuronal growth, excitability or synaptic plasticity, has not been studied in depth.

Effects of La^(3+)on H^+ Transmembrane Gradient and Membrane Potential in Rice Seedling Roots

The effects of LaCl 3 on membrane potential and transmembrane proton gradient for rice ( Oryza sativa ) seedling roots were studied. Highly purified plasma membrane was isolated by aqueous two phase partitioning method. Both the gradient of transmembrane proton and membrane potential were stimulated by certain low concentration of LaCl 3 and depressed by high concentration of LaCl 3. The optimal concentration of La 3+ is around 40～60 μmol·L -1 for transmembrane proton gradient and membrane potential. It shows that La 3+ can influence the generations and maintenances of membrane potential and transmembrane proton gradient in rice seedling roots.