Mg~(2+)加强嵌有H~+-ATP酶脂酶体脂质分子的堆积(packing)

用亲脂性的灵敏的荧光MC 540标记在有Mg~(2+)(1mM)与无Mg~(2+)条件下重建的线粒体H~+-ATP酶脂酶体,后者的荧光强度较前者增加30%左右.这提示,含Mg~(2+)的脂酶体的脂质分子间的堆积紧密度增加.在N-AF系列(n=27和16)探剂与MC 540之间的能量转移效率,又以反应靠近脂双层表面变化的2-AP与MC 54O之间最高.这进一步表明,含Mg~(2+)的脂酶体具有较适合流动性是与Mg~(2+)通过调节靠近脂双层表面的脂质分子具有适度的堆积相关的.这对阐明我们已提出的Mg~(2+)促进线粒体H~+-ATP酶重建作用模型进一步提供了较直接的证据.     

Synthesis of a new vanadyl(IV) complex with trehalose (TreVO): insulin-mimetic activities in osteoblast-like cells in culture

Vanadium compounds show interesting biological and pharmacological properties. Some of them display insulin-mimetic effects and others produce anti-tumor actions. The bioactivity of vanadium is present in inorganic species like the vanadyl(IV) cation or vanadate(V) anion. Nevertheless, the development of new vanadium derivatives with organic ligands which improve the beneficial actions and decrease the toxic effects is of great interest. On the other hand, the mechanisms involved in vanadium bioactivity are still poorly understood. A new vanadium complex of the vanadyl(IV) cation with the disaccharide trehalose (TreVO), Na(6)[VO(Tre)(2)].4H(2)O, here reported, shows interesting insulin-mimetic properties in two osteoblast cell lines, a normal one (MC3T3E1) and a tumoral one (UMR106). The complex affected the proliferation of both cell lines in a different manner. On tumoral cells, TreVO caused a weak stimulation of growth at 5 microM but it inhibited cell proliferation in a dose-response manner between 50 and 100 microM. TreVO significantly inhibited UMR106 differentiation (15-25% of basal) in the range 5-100 microM. On normal osteoblasts, TreVO behaved as a mitogen at 5-25 microM. Different inhibitors of the MAPK pathway blocked this effect. At higher concentrations (75-100 microM), the complex was a weak inhibitor of the MC3T3E1 proliferation. Besides, TreVO enhanced glucose consumption by a mechanism independent of the PI3-kinase activation. In both cell lines, TreVO stimulated the ERK phosphorylation in a dose- and time-dependent manner. Different inhibitors (PD98059, wortmannin, vitamins C and E) partially decreased this effect, which was totally inhibited by their combination. These results suggest that TreVO could be a potential candidate for therapeutic treatments.     

Staining of Viable and Nonviable Myotubes and of Myofibrils by the Fluorescent Dye Merocyanine 540

Merocyanine 540 (MC 540) has been reported to interact specifically with excitable plasma membranes in live cells [3]. Here we show that the MC 540 fluorescence staining pattern previously believed to be characteristic of viable myotubes [3] is observed in formaldehyde-fixed cells. In contrast, viable myotubes show an MC 540 fluorescence staining pattern that is characteristic of cell surface staining (no internal structures fluoresce). The specific I-band and H-zone fluorescence of isolated myofibrils is also consistent with the interpretation that the fluorescence patterns previously reported for viable myotubes are in fact characteristic of cells with disrupted plasma membranes. Time-course observations of MC 540 and trypan blue staining of myotubes suggest that when plasma membrane integrity is lost, MC 540 fluorescence can be visualized inside the cell 5–10 min before trypan blue absorbance. Thus the trypan blue viability assay can be misleading when applied to myotubes.     

Staining of viable and nonviable myotubes and of myofibrils by the fluorescent dye merocyanine 540

Merocyanine 540 (MC 540) has been reported to interact specifically with excitable plasma membranes in live cells [3]. Here we show that the MC 540 fluorescence staining pattern previously believed to be characteristic of viable myotubes [3] is observed in formaldehyde-fixed cells. In contrast, viable myotubes show an MC 540 fluorescence staining pattern that is characteristic of cell surface staining (no internal structures fluoresce). The specific I-band and H-zone fluorescence of isolated myofibrils is also consistent with the interpretation that the fluorescence patterns previously reported for viable myotubes are in fact characteristic of cells with disrupted plasma membranes. Time-course observations of MC 540 and trypan blue staining of myotubes suggest that when plasma membrane integrity is lost, MC 540 fluorescence can be visualized inside the cell 5-10 min before trypan blue absorbance. Thus the trypan blue viability assay can be misleading when applied to myotubes.     

Temperature dependence of the lipid packing in thylakoid membranes studied by time- and spectrally resolved fluorescence of Merocyanine 540

The lipid packing of thylakoid membranes is an important factor for photosynthetic performance. However, surprisingly little is known about it and it is generally accepted that the bulk thylakoid lipids adopt the liquid-crystalline phase above -30 degrees C and that a phase transition occurs only above 45 degrees C. In order to obtain information on the nature of the lipid microenvironment and its temperature dependence, steady-state and time-resolved fluorescence measurements were performed on the fluorescence probe Merocyanine 540 (MC540) incorporated in isolated spinach thylakoids and in model lipid systems (dipalmitoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine) adopting different phases. It is demonstrated that the degree and way of incorporation differs for most lipid phases--upon selective excitation at 570 nm, the amplitude of the fluorescence component that corresponds to membrane-incorporated MC540 is about 20% in gel-, 60% in rippled gel-, and 90% in liquid-crystalline and inverted hexagonal phase, respectively. For thylakoids, the data reveal hindered incorporation of MC540 (amplitude about 30% at 7 degrees C) and marked spectral heterogeneity at all temperatures. The incorporation of MC540 in thylakoids strongly depends on temperature. Remarkably, above 25 degrees C MC540 becomes almost completely extruded from the lipid environment, indicating major rearrangements in the membrane.     

Synthesis of vanadium(IV,V) hydroxamic acid complexes and in vivo assessment of their insulin-like activity

We synthesized vanadyl (oxidation state +IV) and vanadate (oxidation state +V) complexes with the same hydroxamic acid derivative ligand, and assessed their glucose-lowering activities in relation to the vanadium biodistribution behavior in streptozotocin-induced diabetic mice. When the mice received an intraperitoneal injection of the complexes, the vanadate complex more effectively lowered the elevated glucose levels compared with the vanadyl one. The glucose-lowering effect of the vanadate complex was linearly related to its dose within the range from 2.5 to 7.5 mg V/kg. In addition, pretreatment of the vanadate complex induced a larger insulin-enhancing effect than the vanadyl complex. Both complexes were more effective than the corresponding inorganic vanadium compounds. The vanadyl and vanadate complexes, but not the inorganic vanadium compounds, resulted in almost the same organ vanadium distribution. Consequently, the observed differences in the insulin-like activity between the complexes would reflect the potency of the two compounds in the +IV and +V oxidation states in the subcellular region.     

Tetravalent Vanadium Releases Ferritin Iron which Stimulates Vanadium-Dependent Lipid Peroxidation

The iron storage protein, ferritin, represents a possible source of iron for oxidative reactions in biological systems. It has been shown that superoxide and several xenobiotic free radicals can release iron from ferritin by a reductive mechanism. Tetravalent vanadium (vanadyl) reacts with oxygen to generate superoxide and pentavalent vanadium (vanadate). This led to the hypothesis that vanadyl causes the release of iron from ferritin. Therefore, the ability of vanadyl and vanadate to release iron from ferritin was investigated. Iron release was measured by monitoring the generation of the Fe²⁺-fcrrozine complex. It was found that vanadyl but not vanadate was able to mobilize ferritin iron in a concentration dependent fashion. Initial rates. and iron release over 30 minutes. were unaffected by the addition of superoxide dismutase. Glutathione or vanadate added in relative excess to the concentration of vanadyl, inhibited iron release up to 45%. Addition of ferritin at the concentration used for measuring iron release prevented vanddyl-induced NADH oxidation. Vanadyl promoted lipid peroxidation in phospholipid liposomes. Addition of ferritin to the system stimulated lipid peroxidation up to 50% above that with vanadyl alone. Fcrritin alone did not promote significant levels of lipid peroxidation.     

Tetravalent Vanadium Releases Ferritin Iron which Stimulates Vanadium-Dependent Lipid Peroxidation

The iron storage protein, ferritin, represents a possible source of iron for oxidative reactions in biological systems. It has been shown that superoxide and several xenobiotic free radicals can release iron from ferritin by a reductive mechanism. Tetravalent vanadium (vanadyl) reacts with oxygen to generate superoxide and pentavalent vanadium (vanadate). This led to the hypothesis that vanadyl causes the release of iron from ferritin. Therefore, the ability of vanadyl and vanadate to release iron from ferritin was investigated. Iron release was measured by monitoring the generation of the Fe²⁺-fcrrozine complex. It was found that vanadyl but not vanadate was able to mobilize ferritin iron in a concentration dependent fashion. Initial rates. and iron release over 30 minutes. were unaffected by the addition of superoxide dismutase. Glutathione or vanadate added in relative excess to the concentration of vanadyl, inhibited iron release up to 45%. Addition of ferritin at the concentration used for measuring iron release prevented vanddyl-induced NADH oxidation. Vanadyl promoted lipid peroxidation in phospholipid liposomes. Addition of ferritin to the system stimulated lipid peroxidation up to 50% above that with vanadyl alone. Fcrritin alone did not promote significant levels of lipid peroxidation.     

Alteration of membrane lipid biophysical properties and resistance of human lung adenocarcinoma A549 cells to cisplatin

Alterations of membrane lipid biophysical properties of sensitive A₅₄₉ and resistant A₅₄₉/DDP cells to the Cis-dichlorodiammine platinum (Cisplatin) were performed by measurements of fluorescence and flow cytometry approaches using fluorescence dyes of DPH, N-AS and Merocyanine 540 (MC 540) respectively. Fatty acids of membrane lipid of the two cell lines were analyzed by gas chromatography. The results indicated clearly that fluorescence polarization (P) of the DPH probe is 0.169 for the sensitive A₅₄₉ cell and 0.194 for the resistant A₅₄₉/DDP cells. Statistical analysis showed significant difference between the two cell lines. The polarizations of 2-AS and 7-AS which reflect the fluidity of surface and middle of lipid bilayer are 0.134 and 0.144 for the sensitive A549 cells as well as 0.171 and 0.178 for the resistant A₅₄₉/DDP cells respectively, but there is no significant difference of the polarization of 12-AS between the two cell lines. This shows that alterations of the membrane fluidity of both cells were mainly located on the surface and middle of the lipid bilayer. In addition, the packing density of phospholipid molecules in the membrane of the two cell lines detected by MC540 probe indicated that lipid packing of A₅₄₉ cell membranes was looser than that of the A₅₄₉/DDP cells. And unsaturation degree of plasma membrane fatty acids of the A₅₄₉/DDP cells was also lower than that of A₅₄₉ cells. Taken together, it was proposed that the alteration of membrane lipid biophysical state may be involved in the resistance of A₅₄₉/DDP cells to cisplatin.     

Vanadyl-and Vanadate-Induced Lipid Peroxidation in Mitochondria and in Phosphatidylcholine Suspensions

Vanadyl (V_(IV)) was found to induce rapidly developing lipid peroxidation in intact and sonicated mitochondria as well as in phosphatidylcholine suspension. The ability of vanadate (V_(V)) to induce lipid peroxidation was much less pronounced compared to that of vanadyl. The peroxidative action of vanadate on phosphatidylcholine much increased in the presence of NADH and ascorbate. Preincubation of vanadate with glucose had the same effect.

Vanadyl-induced lipid peroxidation was not essentially influenced by SOD, catalase and ethanol but was completely inhibited by butylated hydroxytoluene.

All these effects of vanadyl and vanadate are thought to participate in the insulin-like and other biological actions of vanadium.     

Importance of hydroxyl radical in the vanadium-stimulated oxidation of NADH

Vanadium compounds are known to stimulate the oxidation of NAD(P)H, but the mechanism remains unclear. This reaction was studied spectrophotometrically and by electron spin resonance spectroscopy (ESR) using vanadium in the reduced state (+4, vanadyl) and the oxidized state (+5, vanadate). In 25 mM sodium phosphate buffer at pH 7.4, vanadyl was slightly more effective in stimulating NADH oxidation than was vanadate. Addition of a superoxide generating system, xanthine/xanthine oxidase, resulted in a marked increase in NADH oxidation by vanadyl, and to a lesser extent, by vanadate. Decreasing the pH with superoxide present increased NADH oxidation for both vanadate and vanadyl. Addition of hydrogen peroxide to the reaction mixture did not change the NADH oxidation by vanadate, regardless of concentration or pH. With vanadyl however, addition of hydrogen peroxide greatly enhanced NADH oxidation which further increased with lower pH. Use of the spin trap DMPO in reaction mixtures containing vanadyl and hydrogen peroxide or a superoxide generating system resulted in the detection by ESR of hydroxyl. In each case, the hydroxyl radical signal intensity increased with vanadium concentration. Catalase was able to inhibit the formation of the DMPO--OH adduct formed by vanadate plus superoxide. These results show that the ability of vanadium to act in a Fenton-type reaction is an important process in the vanadium-stimulated oxidation of NADH.     

Alteration of membrane lipid biophysical properties and resistance of human lung adenocarcinoma A549 cells to cisplatin

Alterations of membrane lipid biophysical properties of sensitive A₅₄₉ and resistant A₅₄₉/DDP cells to the Cis-dichlorodiammine platinum (Cisplatin) were performed by measurements of fluorescence and flow cytometry approaches using fluorescence dyes of DPH, N-AS and Merocyanine 540 (MC 540) respectively. Fatty acids of membrane lipid of the two cell lines were analyzed by gas chromatography. The results indicated clearly that fluorescence polarization (P) of the DPH probe is 0.169 for the sensitive A₅₄₉ cell and 0.194 for the resistant A₅₄₉/DDP cells. Statistical analysis showed significant difference between the two cell lines. The polarizations of 2-AS and 7-AS which reflect the fluidity of surface and middle of lipid bilayer are 0.134 and 0.144 for the sensitive A549 cells as well as 0.171 and 0.178 for the resistant A₅₄₉/DDP cells respectively, but there is no significant difference of the polarization of 12-AS between the two cell lines. This shows that alterations of the membrane fluidity of both cells were mainly located on the surface and middle of the lipid bilayer. In addition, the packing density of phospholipid molecules in the membrane of the two cell lines detected by MC540 probe indicated that lipid packing of A₅₄₉ cell membranes was looser than that of the A₅₄₉/DDP cells. And unsaturation degree of plasma membrane fatty acids of the A₅₄₉/DDP cells was also lower than that of A₅₄₉ cells. Taken together, it was proposed that the alteration of membrane lipid biophysical state may be involved in the resistance of A₅₄₉/DDP cells to cisplatin.     

Vanadyl-and Vanadate-Induced Lipid Peroxidation in Mitochondria and in Phosphatidylcholine Suspensions

Vanadyl (V_(IV)) was found to induce rapidly developing lipid peroxidation in intact and sonicated mitochondria as well as in phosphatidylcholine suspension. The ability of vanadate (V_(V)) to induce lipid peroxidation was much less pronounced compared to that of vanadyl. The peroxidative action of vanadate on phosphatidylcholine much increased in the presence of NADH and ascorbate. Preincubation of vanadate with glucose had the same effect.

Vanadyl-induced lipid peroxidation was not essentially influenced by SOD, catalase and ethanol but was completely inhibited by butylated hydroxytoluene.

All these effects of vanadyl and vanadate are thought to participate in the insulin-like and other biological actions of vanadium.     

Metabolism of added orthovanadate to vanadyl and high-molecular-weight vanadates by Saccharomyces cerevisiae

The effect of vanadium oxides on living systems may involve the in vivo conversion of vanadate and vanadyl ions. The addition of 5 mM orthovanadate (VO4(3-), V(V)), a known inhibitor of the (Na,K)-ATPase, to yeast cells stopped growth. In contrast, the addition of 5 mM vanadyl (VO2+, V(IV) stimulated growth. Orthovanadate addition to whole cells is known to stimulate various cellular processes. In yeast, both ions inhibited the plasma membrane Mg2+ ATPase and were transported into the cell as demonstrated with [48V]VO4(3-) and VO2+. ESR spectroscopy has been used to measure the cell-associated paramagnetic vandyl ion, while 51V NMR has detected cell-associated diamagnetic vanadium (e.g. V(V)). Cells were exposed to both toxic (5 mM) and nontoxic (1 mM) concentrations of vanadate in the culture medium. ESR showed that under both conditions, vanadate became cell associated and was converted to vanadyl which then accumulated in the cell culture medium. 51V NMR studies showed the accumulation of new cell-associated vanadium resonances identified as dimeric vanadate and decavanadate in cells exposed to toxic amounts of medium vanadate (5 mM). These vanadate compounds did not accumulate in cells exposed to 1 mM vanadate. These studies confirm that the inhibitory form of vanadium usually observed in in vitro experiments is vanadate, in one or more of its hydrated forms. These data also support the hypothesis that the stimulatory form of vanadium usually observed in whole cell experiments is the vanadyl ion or one or more of its liganded derivatives.     

Digalactosyl-diacylglycerol-deficiency lowers the thermal stability of thylakoid membranes

We investigated the effects of digalactosyl-diacylglycerol (DGDG) on the organization and thermal stability of thylakoid membranes, using wild-type Arabidopsis thaliana and the DGDG-deficient mutant, dgd1. Circular-dichroism measurements reveal that DGDG-deficiency hampers the formation of the chirally organized macrodomains containing the main chlorophyll a/b light-harvesting complexes. The mutation also brings about changes in the overall chlorophyll fluorescence lifetimes, measured in whole leaves as well as in isolated thylakoids. As shown by time-resolved measurements, using the lipophylic fluorescence probe Merocyanine 540 (MC540), the altered lipid composition affects the packing of lipids in the thylakoid membranes but, as revealed by flash-induced electrochromic absorbance changes, the membranes retain their ability for energization. Thermal stability measurements revealed more significant differences. The disassembly of the chiral macrodomains around 55°C, the thermal destabilization of photosystem I complex at 61°C as detected by green gel electrophoresis, as well as the sharp drop in the overall chlorophyll fluorescence lifetime above 45°C (values for the wild type—WT) occur at 4–7°C lower temperatures in dgd1. Similar differences are revealed in the temperature dependence of the lipid packing and the membrane permeability: at elevated temperatures MC540 appears to be extruded from the dgd1 membrane bilayer around 35°C, whereas in WT, it remains lipid-bound up to 45°C and dgd1 and WT membranes become leaky around 35 and 45°C, respectively. It is concluded that DGDG plays important roles in the overall organization of thylakoid membranes especially at elevated temperatures.     

Spectral imaging of MC540 during murine and human colon carcinoma cell differentiation.

We studied the staining pattern of merocyanine 540 (MC540) by spectral imaging of murine CT26 and human HT29 colon carcinoma cells incubated with the dye MC540. This dye, usually considered a potential membrane probe, localized mainly in the cytoplasmic vesicles of the colon carcinoma cells. However, in cells incubated in an environment similar to that of a tumor (pH 6.7), high fluorescence was detected in the nuclear membrane and nucleoli. Under these acidic conditions, resembling the Krebs effect, a population of CT26 cells displayed fluorescent plasma membranes. In differentiating cells, exhibiting cell cycle arrest at G(1)-phase and an elevated level of alkaline phosphatase, MC540 fluorescence was confined to cytoplasmic vesicles and was not detected in the plasma membrane or in the nucleoli. Cell sorting analysis of both cell types at pH 5.0 revealed higher fluorescence intensity in proliferating cells compared to differentiating cells. The fluorescence intensity of MC540-stained cells reached a maximum at pH 5.0, although the fluorescence of MC540 dye was maximal at pH 7.2. This phenomenon may result from increased binding of MC540 monomers to the cells because disaggregation of the dye with Triton X-100 produced similar results. We conclude that nucleolar localization of MC540 and an elevated fluorescence intensity can be used as indicators for proliferating cells in the characteristically acidic tumor environment. (J Histochem Cytochem 49:147-153, 2001)     

钒对人红细胞膜蛋白和膜脂质过氧化的影响

对钒酸根V（V）与红细胞膜相互作用研究表明V（V）使膜蛋白内源荧光淬灭（KD,37＝2．23,KD,20＝4．17）和膜巯基含量降低,但对膜脂质过氧化影响较小,提示V（V）主要与膜蛋白作用．与V（V）不同,V（V）与红细胞膜的作用虽使膜蛋白就基含量下降,但不显著,其主要作用是引起膜脂质过氧化．     

Perturbations of membrane structure by optical probes: I. Location and structural sensitivity of merocyanine 540 bound to phospholipid membranes

Summary The maximum monomer absorption wavelength of a frequently used external membrane probe, Merocyanine 540, can be related to the location of the binding site for the dye within lipid membranes. Solvent studies indicate the occurrence of very specific and mutual perturbances between the probe and its microenvironment, that are of relevance, when investigating structural and functional events in biomembranes with the aid of this dye. Merocyanine 540 (MC 540) is an excellent probe for structural altions in the lipids including phase transitions. The extinction coefficient and _max place the location of the dye-chromophore slightly above the domain of the glycerol of backbone of neutral and charged phospholipids. This explains the sensitivity of MC 540 to structural variations in the head-group region of several synthetic dipalmitoyl-lecithin analogues. The major physical parameters involved in variations of the optical signals associated with changes in the membrane structure are the dye/lipid partition coefficient and the monomer-dimer dissociation constant of the dye bound to the lipids. A temperature dependent transition from the liquid-crystalline to the crystalline state leads mainly to an exclusion of the dye from the lipid phase with a concomitant dimerization of the dye molecules still in contact with the polarhead group region of the lipid. The relevance of this finding for the mechanism of transient optical signals in connection with the occurrence of action potentials in excitable membranes is discussed. Our findings underline the necessary caution when applying external optical probes and analyzing membrane features from the spectral data, because of inevitable perturbances in the microenvironment of every probe molecule.     

Selective staining of immature hemopoietic cells with merocyanine 540 in flow cytometry

Merocyanine 540 (MC540) has been reported to bind hemopoietic cells specifically. In this study, MC540 was used as a probe for the cytofluorometric discrimination of hemopoietic cells. In PHA-stimulated lymphocytes or HL-60 cells induced to differentiate with DMSO, MC540 binding was increased in actively proliferating cells and undifferentiated cells as compared with the more differentiated cells of the same lineage. Mononuclear bone marrow cells exhibited a discrete distribution of MC fluorescence. Sorting a population with high MC540 fluorescence (MC+ population) produced a 9-14-fold enrichment of granulocyte-macrophage progenitors (CFU-GM), and a recovery of all S-G2M phase cells (BrdUrd/DNA analysis). Cytological examination of the sorted MC+ population confirmed the enrichment in immature cells from all lineages. Double-labeling experiments using MC540 and Hoechst 33342 on total bone marrow or peripheral blood cells confirmed that the MC+ population included all the cycling cells. The proportion of S-G2M phase cells in this MC+ population was 29.3 +/- 7.8 for 15 bone marrow samples and 16.3 +/- 6.8 for 10 blood samples. MC540 could therefore be used as a marker for human hemopoietic cells, and it represents a useful tool for investigation of hemopoiesis in normal or leukemic bone marrow.     

Proliferative and morphological changes induced by vanadium compounds on Swiss 3T3 fibroblasts

Vanadium compounds are shown to have a mitogenic effect on fibroblast cells. The effects of vanadate, vanadyl and pervanadate on the proliferation and morphological changes of Swiss 3T3 cells in culture are compared. Vanadium derivatives induced cell proliferation in a biphasic manner, with a toxic-like effect at doses over 50mM, after 24h of incubation. Vanadyl and vanadate were equally potent at 2.5–10mM. At 50mM vanadate inhibited cell proliferation, whereas slight inhibition was observed at 100mM of vanadyl. At 10mM pervanadate was as potent as vanadate and vanadyl in stimulating fibroblast proliferation, but no effect was observed at lower concentrations. A pronounced cytotoxic-like effect was induced by pervanadate at 50mM. All of these effects were accompanied by morphological changes: transformation of fibroblast shape from polygonal to fusiform; retraction with cytoplasm condensation; and loss of lamellar processes. The magnitude of these transformations correlates with the potency of vanadium derivatives to induce a cytotoxic-like effect: pervanadate>vanadate>vanadyl. These data suggest that the oxidation state and coordination geometry of vanadium determine the degree of the cytotoxicity.