Potential-sensitive Fluorescent Probe Research Articles

Water-soluble sulfur quantum dots as a potential sensitive fluorescent probe for quercetin detection and cell imaging

A simple fluorescent quenching probe based on polyethylene glycol-400 capped sulfur quantum dots (PEG-SQDs) was fabricated to determine quercetin (QT) quantitatively. As anticipated, the PEG-SQDs exhibited favourable luminescent properties, stability and low cytotoxicity. QT effectively quenched the fluorescence of the PEG-SQDs through static quenching and the inner filter effect. Moreover, the PEG-SQDs showed rapid QT detection within a linear range of 0.100–45.0 μM, with a limit of detection of 0.014 μM (3σ/k). This fluorescent probe successfully detected QT in human serum, quercetin supplement capsules and red wine, achieving a standard recovery of 92.6 %–105 %.

Conjugates of Chloramphenicol Amine and Berberine as Antimicrobial Agents.

In order to obtain antimicrobial compounds with improved properties, new conjugates comprising two different biologically active agents within a single chimeric molecule based on chloramphenicol (CHL) and a hydrophobic cation were synthesized and studied. Chloramphenicol amine (CAM), derived from the ribosome-targeting antibiotic CHL, and the plant isoquinoline alkaloid berberine (BER) are connected by alkyl linkers of different lengths in structures of these conjugates. Using competition binding, double reporter system, and toeprinting assays, we showed that synthesized CAM-Cn-BER compounds bound to the bacterial ribosome and inhibited protein synthesis like the parent CHL. The mechanism of action of CAM-C5-BER and CAM-C8-BER on the process of bacterial translations was similar to CHL. Experiments with bacteria demonstrated that CAM-Cn-BERs suppressed the growth of laboratory strains of CHL and macrolides-resistant bacteria. CAM-C8-BER acted against mycobacteria and more selectively inhibited the growth of Gram-positive bacteria than the parent CHL and the berberine derivative lacking the CAM moiety (CH3-C8-BER). Using a potential-sensitive fluorescent probe, we found that CAM-C8-BER significantly reduced the membrane potential in B. subtilis cells. Crystal violet assays were used to demonstrate the absence of induction of biofilm formation under the action of CAM-C8-BER on E. coli bacteria. Thus, we showed that CAM-C8-BER could act both on the ribosome and on the cell membrane of bacteria, with the alkylated berberine fragment of the compound making a significant contribution to the inhibitory effect on bacterial growth. Moreover, we showed that CAM-Cn-BERs did not inhibit eukaryotic translation in vitro and were non-toxic for eukaryotic cells.

Metal Substrate Selectivity and Mechanism of Transport in a Transmembrane Zn‐pump Revealed by in‐vitro Real‐time Transport

P1B-type ATPases are primary active transmembrane metal pumps conserved in all kingdoms of life that play a pivotal role in controlling cellular metal ion levels and homeostasis. In bacteria, they are responsible for selectively exporting essential and toxic metal ions across the cell membrane to maintain concentrations below toxic levels. They are critical virulence factors in pathogenic bacteria and allow selected microorganisms to survive in extreme metal-rich environments. The P1B-2-type Zn(II) ATPase pump subfamily possesses a wide promiscuity towards metal substrate translocation by plastic selection of Zn(II)/Cd(II)/Hg(II)/Pb(II) at their high-affinity transmembrane metal binding site. Despite these substrates stimulate ATP-hydrolysis, direct translocation of all these metals and their mechanism of transport remain to be demonstrated. In this work, we established a platform for purification and in-vitro functional reconstitution in artificial phospholipid bilayer vesicles (proteoliposomes) of ZntA from Cupriavidus metallidurans to dissect the molecular mechanism of divalent metal ion translocation and substrate promiscuity towards first-, second- and the third-row transition metals. Metal-dependent ATPase activity assays of CmΔZntA166–794 in detergent micelles and proteoliposomes revealed stimulation of the ATPase activity by Pb(II), Zn(II), Cd(II) and Hg(II) substrates with the maximal velocities (Vmax) following the order Pb(II) > Zn(II) > Cd(II) > Hg(II), confirming the successful functional reconstitution in lipid vesicles. By selecting and encapsulating divalent metal sensitive fluorescent probes in the proteoliposome lumen, we performed real-time metal transport assays to reveal the ATP-dependent translocation of Pb(II), Zn(II), Cd(II) and Hg(II) across the lipid bilayer, establishing that the translocation rates correlate to the order observed following ATP hydrolysis. Furthermore, to dissect the substrate transport mechanism, we encapsulated the pH sensitive fluorescent probe pyranine to demonstrated that, differently than other P-type ATPases, translocation is not coupled to H+ counter-flux for all the M(II) substrates. By encapsulating the membrane potential sensitive fluorescent probe Oxonol-VI we also revealed that, with all substrates, Zn-pumps act as primary active electrogenic uniporters capable of generating a transmembrane potential. This work establishes a platform to dissect the mechanisms of translocation of various metal substrates in P-type ATPases which is applicable to study other uncharacterized transporter families and to reveal the bioinorganic chemistry of metal ion transport at molecular level.

Effect of Glycyrrhizic Acid and Arabinogalactan on the Membrane Potential of Rat Thymocytes Studied by Potential-Sensitive Fluorescent Probe.

The effect of the natural saponin glycyrrhizic acid (GA) and polysaccharide arabinogalactan (AG) on the transmembrane potential of rat thymocytes was investigated using the potential-sensitive fluorescent probe 4-(p-dimethylaminostyryl)-1-methylpyridinium (DSM). Incubation of cells with GA in micellar form resulted in a decrease of the amplitude of observed fluorescence kinetics that points out to a decrease of the transmembrane potential. The proposed mechanism is an increase of membrane ion permeability (passive ion transport) of the plasma cell membrane due to GA incorporation. The incorporation of GA molecules into the cell membrane is extremely sensitive to the degree of GA dissociation. The neutral form of glycyrrhizic acid enters the lipid bilayer in contrast to the deprotonated anionic form. The incubation of rat thymocytes with anionic form of GA, namely with its disodium salt, has no effect on the fluorescence kinetics. The possible reasons of this phenomenon are discussed in the light of the nuclear magnetic resonance (NMR) and molecular dynamics (MD) data. The treatment of thymocytes with AG affects only the initial rate of the probe incorporation. The proposed mechanism is that AG covers the surface of the cell membrane and forms a barrier for the probe. Additionally, our experiments demonstrated that both polysaccharide AG and GA in the neutral form (but not Na2GA) effectively capture the cationic probe in an aqueous solution and then deliver it to the cell membrane.

Sulfur doped graphene quantum dots as a potential sensitive fluorescent probe for the detection of quercetin

In this work, a novel, selective and sensitive fluorescent probe (sulfur doped graphene quantum dots, SGQDs) was designed for real-time detection of quercetin in red wine samples. SGQDs were synthesized by pyrolyzing citric acid (CA) and 3-Mercaptopropionic acid (MPA) and characterized through advanced techniques. It was observed that fluorescence intensity of SGQDs could be substantially quenched by the addition of quercetin through inner filter effect (IFE) mechanism. Additionally, a visual color change (colorless to light yellow) was also noticed after addition of quercetin into a solution of SGQDs. The change in SGQDs fluorescence intensity with varying quercetin content revealed good linearity in the 0–50.0 μM range with regression coefficient of 0.9943 and a lowest detection limit of 0.006 μg/mL. To authenticate the real-time application of SGQDs as a potential fluorescent probe, red wine samples having different quercetin concentrations were used for quantitative analysis, after the optimization of several analytical parameters.

Acetylenic fatty acids from Porcelia macrocarpa (Annonaceae) against trypomastigotes of Trypanosoma cruzi: Effect of octadec-9-ynoic acid in plasma membrane electric potential

Porcelia macrocarpa (Warm.) R. E. Fries (Annonaceae) is an endemic plant in Brazil where its tasty pulp has been eaten fresh. The hexane extract from its flowers was subjected to chromatographic procedures to afford four acetylene derivatives identified as octadec-9-ynoic (stearolic acid – 1), (11E)-octadec-11-en-9-ynoic (santalbic acid – 2), 8-hydroxyoctadec-9,11-diynoic (3) and 8-hydroxyoctadec-17-en-9,11-diynoic (isanolic acid – 4) acids by NMR and HRESIMS. Among tested compounds against trypomastigote forms of T. cruzi, octadec-9-ynoic acid (1) displayed higher potential with IC50 = 27.6 µM and a selectivity index (SI) higher than 7. Compounds 2 and 3 showed IC50 of approximately 60 µM while compound 4 was inactive. The lethal action of the compound 1 was investigated using spectrofluorometric techniques to detect ROS content, plasma membrane permeability and plasma membrane potential by flow cytometry. Compound 1 showed no alteration in the production of ROS of treated trypomastigotes and no alteration of the plasma membrane permeability was observed as detected by the fluorescent probe SYTOX-green after 120 min of incubation. However, by using the potential-sensitive fluorescent probe DiSBAC2(3), compound 1 caused depolarization of the plasma membrane potential when compared to untreated parasites. Our results demonstrated the anti-T. cruzi effects of compounds 1–3 isolated from flowers of P. macrocarpa and indicated that the lethal effect of compound 1 in T. cruzi could be associated to the plasma membrane disturbance of the parasite.

Electrochemical potential of the inner mitochondrial membrane and Ca(2+) homeostasis of myometrium cells

We demonstrated using Ca(2+)-sensitive fluorescent probe, mitochondria binding dyes, and confocal laser scanning microscopy, that elimination of electrochemical potential of uterus myocytes' inner mitochondrial membrane by aprotonophore carbonyl cyanide m-chlorophenyl hydrazone (10 μM), and by a respiratory chain complex IV inhibitor sodium azide (1 mM) is associated with substantial increase of Ca2+ concentration in myoplasm in the case of the protonophore effect only, but not in the case of the azide effect. In particular, with the use of nonyl acridine orange, a mitochondria-specific dye, and 9-aminoacridine, an agent that binds to membrane compartments in the presence of proton gradient, we showed that both the protonophore and the respiratory chain inhibitor cause the proton gradient on mitochondrial inner membrane to dissipate when introduced into incubation medium. We also proved with the help of 3,3'-dihexyloxacarbocyanine, a potential-sensitive carbocyanine-derived fluorescent probe, that the application of these substances results in dissipation of the membrane's electrical potential. The elimination of mitochondrial electrochemical potential by carbonyl cyanide m-chlorophenyl hydrazone causes substantial increase in fluorescence of Ca(2+)-sensitive Fluo-4 AM dye in myoplasm of smooth muscle cells. The results obtained were qualitatively confirmed with flow cytometry of mitochondria isolated through differential centrifugation and loaded with Fluo-4 AM. Particularly, Ca2+ matrix influx induced by addition of the exogenous cation is totally inhibited by carbonyl cyanide m-chlorophenyl hydrazone. Therefore, using two independent fluorometric methods, namely confocal laser scanning microscopy and flow cytometry, with Ca(2+)-sensitive Fluo-4 AM fluorescent probe, we proved on the models of freshly isolated myocytes and uterus smooth muscle mitochondria isolated by differential centrifugation sedimentation that the electrochemical gradient of inner membrane is an important component of mechanisms that regulate Ca2+ homeostasis in myometrium cells.

Dipolar rearrangement during micellization explored using a potential-sensitive fluorescent probe

Dipole potential is the potential difference within the membrane bilayer, which originates due to the nonrandom arrangement of lipid dipoles and water molecules at the membrane interface. Although dipole potential is generally used in the context of bilayer membranes, the nonrandom arrangement of amphiphiles and water dipoles would also contribute to dipole potential in organized molecular assemblies such as micelles. In this work, we show that the process of micelle formation from monomers for a representative variety of detergents is associated with dipolar rearrangement. We monitor the dipolar reorganization upon micellization as a change in dipole potential, measured by the dual wavelength ratiometric approach utilizing the potential-sensitive membrane probe di-8-ANEPPS. We further utilized this phenomenon to estimate the critical micelle concentration (CMC) of a variety of detergents. CMC determined by this method are in overall agreement with the literature values of CMC for these detergents. To the best of our knowledge, these results constitute the first report showing dipolar reorientation during micellization. We conclude that dipole potential measurements could provide a novel approach to explore micellar organization.

The Lantibiotic NAI-107 Binds to Bactoprenol-bound Cell Wall Precursors and Impairs Membrane Functions

The lantibiotic NAI-107 is active against Gram-positive bacteria including vancomycin-resistant enterococci and methicillin-resistant Staphylococcus aureus. To identify the molecular basis of its potency, we studied the mode of action in a series of whole cell and in vitro assays and analyzed structural features by nuclear magnetic resonance (NMR). The lantibiotic efficiently interfered with late stages of cell wall biosynthesis and induced accumulation of the soluble peptidoglycan precursor UDP-N-acetylmuramic acid-pentapeptide (UDP-MurNAc-pentapeptide) in the cytoplasm. Using membrane preparations and a complete cascade of purified, recombinant late stage peptidoglycan biosynthetic enzymes (MraY, MurG, FemX, PBP2) and their respective purified substrates, we showed that NAI-107 forms complexes with bactoprenol-pyrophosphate-coupled precursors of the bacterial cell wall. Titration experiments indicate that first a 1:1 stoichiometric complex occurs, which then transforms into a 2:1 (peptide: lipid II) complex, when excess peptide is added. Furthermore, lipid II and related molecules obviously could not serve as anchor molecules for the formation of defined and stable nisin-like pores, however, slow membrane depolarization was observed after NAI-107 treatment, which could contribute to killing of the bacterial cell.

Modulation of myometrium mitochondrial membrane potential by calmodulin antagonists

Influence of calmodulin antagonists on mitochondrial membrane potential was investigated using a flow cytometry method, confocal microscopy and fluorescent potential-sensitive probes TMRM and MTG. Influence of different concentrations of calmodulin antagonists on mitochondrial membrane potential was studied using flow cytometry method and a fraction of myometrium mitochondria of unpregnant rats. It was shown that 1-10 microM calmidazolium gradually reduced mitochondria membrane potential. At the same time 10-100 microM trifluoperazine influenced as follows: 10 microM--increased polarization, while 100 microM--caused almost complete depolarization of mitochondrial membranes. In experiments which were conducted with the use of confocal microscopy method and myometrium cells it was shown, that MTG addition to the incubation medium led to the appearance of fluorescence signal in a green range. Addition of the second probe (TMRM) resulted in the appearance of fluorescent signal in a red range. Mitochondrial membrane depolarization by 1 microM CCCP or 10 mM NaN3 was accompanied by the decline of "red" fluorescence intensity, "green" fluorescence was kept. The 10-15 minute incubation of myometrium cells in the presence 10 microM calmidazolium or 100 microM trifluoperazine was accompanied by almost complete decrease of the TMRM fluorescent signal. Thus, with the use of potential-sensitive fluorescent probes TMRM and MTG it was shown, that calmodulin antagonists modulate mitochondrial membrane potential of myometrium cells.

Calix[4]arenes C-136 and C-137 hyperpolarize myometrium mitochondria membranes

Calixarenes--supramolecular compounds interacting with bioactive molecules and ions that causes the changes in biochemical and biophysical processes. The aim of this work was to study the effects of calix[4]arenes C-136, C-137 and C-138 on the level of polarization of rat myometrium mitochondria membrane. Structure of synthesized calix[4]arene molecules was confirmed by the methods of 1H NMR and infra-red spectroscopy. Calix[4]arenes C-136 and C-137 possess two chalcone amide moieties at the lower rim, while the calix[4]arene C-138--only one. In case of calix[4]arenes C-136 and C-137 take place, accordingly, absence or presence of phenolic hydroxyl groups at the lower rim on the calix[4]arene skeleton. It was shown that calix[4]arenes C-136, C-137 and C-138 form micelles in a water medium and in the dimethylformamide (DMF). The irradiation of micelles with argon laser on flow cytometer results in appearance of autofluorescence. In the water medium calix[4]arene micelles interact with positively charged potential-sensitive fluorescent probe TMRM, that can testify to the presence of negative charge in these structures. However calix[4]arene micelles in DMF solution do not interact with TMRM. Mitochondrial membrane potential was measured using fluorescent dyes MTG and TMRM with confocal microscopy and fluorescent dye TMRM with flow cytometry. Experiments were conducted on myometrium cells in culture and on suspension of digitonin-permeabilized uterus myocytes. It was shown that a fluorescent signal was stable during time of experiment. Calix[4]arenes C-136 and C-137 (10 μM) hyperpolarize mitochondria membranes. A maximal effect was 173%. At the same time calix[4]arene C-138 did not influence on mitochondria membrane potential. Connection comes into question between structural organization of investigated calix[4]arene molecules and their influence on polarization of mitochondria membrane.

Functional Characterization and Determination of the Physiological Role of a Calcium-Dependent Potassium Channel from Cyanobacteria

Despite the important achievement of the high-resolution structures of several prokaryotic channels, current understanding of their physiological roles in bacteria themselves is still far from complete. We have identified a putative two transmembrane domain-containing channel, SynCaK, in the genome of the freshwater cyanobacterium Synechocystis sp. PCC 6803, a model photosynthetic organism. SynCaK displays significant sequence homology to MthK, a calcium-dependent potassium channel isolated from Methanobacterium thermoautotrophicum. Expression of SynCaK in fusion with enhanced GFP in mammalian Chinese hamster ovary cells' plasma membrane gave rise to a calcium-activated, potassium-selective activity in patch clamp experiments. In cyanobacteria, Western blotting of isolated membrane fractions located SynCaK mainly to the plasma membrane. To understand its physiological function, a SynCaK-deficient mutant of Synechocystis sp. PCC 6803, ΔSynCaK, has been obtained. Although the potassium content in the mutant organisms was comparable to that observed in the wild type, ΔSynCaK was characterized by a depolarized resting membrane potential, as determined by a potential-sensitive fluorescent probe. Growth of the mutant under various conditions revealed that lack of SynCaK does not impair growth under osmotic or salt stress and that SynCaK is not involved in the regulation of photosynthesis. Instead, its lack conferred an increased resistance to the heavy metal zinc, an environmental pollutant. A similar result was obtained using barium, a general potassium channel inhibitor that also caused depolarization. Our findings thus indicate that SynCaK is a functional channel and identify the physiological consequences of its deletion in cyanobacteria.

Human Erythrocytes And Mononuclear Leukocytes Are Capable Of Concentrating HIV-1 Fusion Inhibitor Peptides In Their Membranes

Following the successful approval of the first HIV-1 fusion inhibitor, enfuvirtide (formerly T-20), T-1249 was developed to be one of the next generation drug of this type. Previous studies, based on tryptophan intrinsic fluorescence, showed that these peptides interact with membrane model systems (large unilamellar vesicles) of different lipid compositions. Studies with human blood cell membranes were necessary to further establish the role of membranes on these peptides mode of action. An experimental strategy was applied taking into account the membrane dipole potential as measured by the potential sensitive fluorescent probe di-8-ANEPPS. Successful labelling was performed with erythrocytes and peripheral blood mononuclear cells (PBMC) isolated from human blood samples.For erythrocytes, membrane bound di-8-ANEPPS excitation spectra were blue shifted, indicating a decrease in the dipole potential due to peptide-membrane interactions. Accordingly, a decrease in the probe fluorescence excitation ratio (a measure of the spectral shift) dependent of peptide concentration was observed. The quantitative analysis of these variations indicated that T-1249 had the higher affinity towards erythrocyte membrane. This is in agreement with the previously known adsorption of this peptide on cholesterol-rich membrane domains.Preliminary results show that the behaviour is similar in the case of PBMC, with a decrease in dipole potential that is more pronounced for T-1249. As there is strong suggestion that HIV also associates with erythrocytes in vivo, the peptide concentration effect of the erythrocytes and lymphocyte membranes can correlate with the stronger efficacy of T-1249.

Cytotoxicity and cell membrane depolarization induced by aluminum oxide nanoparticles in human lung epithelial cells A549

The cytotoxicity of 13 and 22 nm aluminum oxide (Al2O3) nanoparticles was investigated in cultured human bronchoalveolar carcinoma-derived cells (A549) and compared with 20 nm CeO2 and 40 nm TiO2 nanoparticles as positive and negative control, respectively. Exposure to both Al2O3 nanoparticles for 24 h at 10 and 25 µg mL−1 doses significantly decreased cell viability compared with control. However, the cytotoxicity of 13 and 22 nm Al2O3 nanoparticles had no difference at 5–25 µg mL−1 dose range. The cytotoxicity of both Al2O3 nanoparticles were higher than negative control TiO2 nanoparticles but lower than positive control CeO2 nanoparticles (TiO2 < Al2O3 < CeO2). A real-time single cell imaging system was employed to study the cell membrane potential change caused by Al2O3 and CeO2 nanoparticles using a membrane potential sensitive fluorescent probe DiBAC4(3). Exposure to the 13 nm Al2O3 nanoparticles resulted in more significant depolarization than the 30 nm Al2O3 particles. On the other hand, the 20 nm CeO2 particles, the most toxic, caused less significant depolarization than both the 13 and 22 nm Al2O3. Factors such as exposure duration, surface chemistry, and other mechanisms may contribute differently between cytotoxicity and membrane depolarization.

Interaction of peptides with biomembranes assessed by potential‐sensitive fluorescent probes

Peptide-membrane interaction is an important step to be evaluated in a study of the activity and mode of action of several bioactive peptides. A variety of methods are available; however, few of them satisfy the criteria of being sensitive, biocompatible, versatile, easy to perform, and allowing real-time monitoring as the use of potential-sensitive fluorescent probes. Here we review methods for detecting the effects of membrane-active peptides, even those that are not intrinsically fluorescent, on the different types of membrane potentials, with a special emphasis on studies conducted with living cells. FPE is a probe sensitive to surface potential and detects electrostatic interactions at the water-lipid interface. Di-8-ANEPPS is sensitive to dipole potential and detects membrane incorporations. Transmembrane potential changes reveal major membrane destabilizations, such as in pore formation. The combination of the information obtained from the three potential variations can lead to a more elucidative picture of the mechanisms of the interaction of relevant peptides with biomembranes.

Stimulating effect of erythropoietin on thymocyte energetics established in vitro with a potential-sensitive fluorescent probe

Effects of a peptide hormone—human recombinant erythropoietin (EPO)—on transmembrane potential (TMP) and a number of active mitochondria in rat thymocytes were studied in vitro using the fluorescent cationic probe 4-(p-dimethylaminostyryl)-1-methylpyridinium (DSM). It was established that EPO changes electric potentials on the surface of cellular membranes of rat thymocytes. Changes in fluorescent signals of DSM were found to depend on the EPO concentration and physiological status of thymus cells. EPO concentrations sufficient to increase average fluorescence intensity of DSM ( $$\tilde F$$ ) in thymus cell mitochondria were established in vitro. These effects were stimulated by increasing the average number of energized mitochondria (N m ) able to accumulate the fluorescent cationic probe. These changes can also be due to elevation of proton potential on the mitochondrial membrane and/or of electric potential of the plasma membrane. Experimental values of positive correlation coefficients between mean values of $$\tilde F$$ and N m in experimental (EPO) thymus cell samples differed from those in control. In the presence of EPO, $$\tilde F$$ increased nonlinearly with N m due to different responsiveness of thymocytes to EPO and change in polarization of the outer mitochondrial membrane in some EPO-stimulated cells. In the majority of cases, the peak in the distribution histogram of $$\tilde F$$ was shifted towards augmentation of the DSM signal. The EPO response of cells isolated from different thymuses depended on the initial level of the average fluorescent signal of DSM in mitochondria, which testifies to differences in physiological statuses of animal thymuses and experimental animals at large.

Phenylarsine oxide is able to dissipate synaptic vesicle acidic pool

Phenylarsine oxide (PAO) has a number of targets in the neurons, one of them is exocytotic process. In this study, we have focused on the mechanisms of phenylarsine oxide action on Ca 2+-dependent and Ca 2+-independent neurotransmitter release from rat brain synaptosomes. We investigated the influence of phenylarsine oxide on: (i) l-[ 14C]glutamate and [ 3H]GABA release and uptake; (ii) plasma membrane potential using a potential-sensitive fluorescent probe rhodamine 6G; (iii) exo/endocytotic process using a pH-sensitive fluorescent probe acridine orange (AO). It has been found that phenylarsine oxide induced deacidification of synaptic vesicles. This effect was completely abolished by preliminary treatment of synaptosomes with a protonophore FCCP indicating that both reagents injured a proton electrochemical gradient. Dissipation of the proton gradient by low concentrations of phenylarsine oxide (not exceed 1 μM) did not prevent KCl-triggered exocytotic response, but essentially modified endocytotic one. At higher concentrations of phenylarsine oxide (up to 10 μM), the proton gradient dissipation was intensified and the exocytotic response was fully abolished. The reagent did not change plasma membrane potential, but depolarized mitochondria. It also caused potent inhibition of the Ca 2+-stimulated l-[ 14C]glutamate and [ 3H]GABA release and increase the Ca 2+-independent release of l-[ 14C]glutamate, but not of [ 3H]GABA. Disulfide-reducing reagents (dithiothreitol and β-mercaptoethanol) completely prevented phenylarsine oxide-evoked injuries. They could also restore the initial levels of the mitochondrial potential, the exocytotic response to KCl and the release and uptake of neurotransmitters. Our data provide the evidence that phenylarsine oxide causes dissipation of synaptic vesicle acidic pool resulting in the reduction of vesicle filling and as consequence in attenuation of Ca 2+-stimulated neurotransmitter release.

Ratiometric fluorescence measurements of membrane potential generated by yeast plasma membrane H +-ATPase reconstituted into vesicles

Potential-sensitive fluorescent probes oxonol V and oxonol VI were employed for monitoring membrane potential (Δ ψ) generated by the Schizosaccharomyces pombe plasma membrane H +-ATPase reconstituted into vesicles. Oxonol VI was used for quantitative measurements of the Δ ψ because its response to membrane potential changes can be easily calibrated, which is not possible with oxonol V. However, oxonol V has a superior sensitivity to Δ ψ at very low concentration of reconstituted vesicles, and thus it is useful for testing quality of the reconstitution. Oxonol VI was found to be a good emission-ratiometric probe. We have shown that the reconstituted H +-ATPase generates Δ ψ of about 160 mV on the vesicle membrane. The generated Δ ψ was stable at least over tens of minutes. An influence of the H + membrane permeability on the Δ ψ buildup was demonstrated by manipulating the H + permeability with the protonophore CCCP. Ratiometric measurements with oxonol VI thus offer a promising tool for studying processes accompanying the yeast plasma membrane H +-ATPase-mediated Δ ψ buildup.

Characteristics of the cell membrane fluidity, actin fibers, and mitochondrial dysfunctions of frozen-thawed two-cell mouse embryos.

Physical and chemical alterations caused by the freezing and thawing and their effects on survivals/developments in vitro were investigated. Of a total of 452 two-cell mouse embryos, the overall survival rate of the frozen-thawed embryos was 76.1% (344/452). The blastocyst formation of the frozen-thawed embryos was 32.6% (44/136) compared to 74.5% (117/157) in the fresh embryos (P<0.05). The total number of cells in a blastocyst also decreased from 96.0 +/- 19.0 (n=26) in the fresh embryos to 42.0 +/- 11 .34 (n=30) in the frozen-thawed embryos (P<0.05). Fluorescence recovery after photobleaching (FRAP) measurement revealed about 5-fold decrease in the cell membrane fluidity with a characteristic time constant (tau) of 1.46 +/- 0.13 sec (n=5) in the frozen-thawed embryos as opposed to 0.28 +/- 0.04 sec (n=5) in the fresh embryos (P<0.05). The relative amount of H(2)O(2) in an embryo as quantified by the fluorescence intensity of 2',7'-dichlorofluorescein (DCF) showed 62.8 +/- 23.5 (n=24) and 34.2 +/- 14.5 (n=20) in the frozen-thawed embryos and in the fresh embryos, respectively (P<0.05). The distribution of actin filaments in the frozen-thawed embryos revealed an uneven distribution, particularly discontinuities at the "actin band," which contrasted to an even distribution shown in the fresh embryos. Mitochondrial staining by Rhodamine 123 showed that there was no significant difference between the two treatments in the number and in the distribution of viable mitochondria, but a marked aggregation was seen in the arrested embryos. No Annexin V binding was detected in two-cell or four-cell embryos while the binding was positive in the arrested embryos. The mitochondrial membrane potential measured by a membrane potential-sensitive fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazol- carbocyanine iodide (JC-1) revealed a marked depolarization in the frozen-thawed embryos. Finally, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) was employed to quantify the DNA fragmentation. In 75.0% cells of blastocysts (n=24) in the frozen-thawed embryos, the DNA fragmentation was detected as opposed to 37.0% in the fresh embryos (n=20) (P<0.05). Taken together, it is proposed that during the cryopreservation, two-cell mouse embryos are subjected to physical and chemical alterations, including destruction of the cell membrane integrity, redistribution of actin fibers, mitochondrial depolarizations, and increased reactive oxygen species (ROS) productions, which then may trigger the apoptotic cascade leading to a decrease in the survival rate and in the developmental rate of the embryos.

A peptide analogue to a fusion domain within photoreceptor peripherin/rds promotes membrane adhesion and depolarization

Photoreceptor peripherin/rds promotes membrane fusion, through a putative fusion domain located within the C-terminus (Boesze-Battaglia et al., Biochemistry 37 (1998) 9477–9487). A peptide analogue to this region, PP-5, competitively inhibits peripherin/rds mediated fusion in a cell free assay system. To characterize how this region is involved in the fusion process we investigated two of the individual steps in membrane fusion, membrane adhesion and membrane destabilization inferred from depolarization studies. Membrane depolarization was measured as the collapse of a valinomycin induced K + diffusion potential in model membranes, using a potential sensitive fluorescent probe, diS-C 2-5. PP-5 induced membrane depolarization in a concentration dependent manner. PP-5 has been shown by Fourier transform infrared spectroscopy to be an amphiphilic α-helix. Therefore, the requirement for an amphiphilic α-helix to promote depolarization was tested using two mutant peptides designed to disrupt either the amphiphilic nature of PP-5 (PP-5AB) or the α-helical structure (PP-5HB). PP-5AB inhibited PP-5 induced depolarization when added in an equimolar ratio to PP-5. Neither mutant peptide alone or in combination with PP-5 had any effect on calcium dependent vesicle aggregation. Using non-denaturing gel electrophoresis and size exclusion chromatography techniques PP-5 was shown to form a tetrameric complex. Equimolar mixtures of PP-5 and PP-5AB formed a heterotetramer which was unable to promote membrane depolarization. The hypothesis that PP-5 tetramers promote membrane depolarization is consistent with the calculated Hill coefficient of 3.725, determined from a Hill analysis of the depolarization data.