SOME RELATIONSHIPS BETWEEN ULTRAVIOLET LIGHT AND HEME-PROTEIN-INDUCED PEROXIDATIVE LIPID BREAKDOWN IN LIPOSOMES, AS REFLECTED BY FLUORESCENCE CHANGES: THE EFFECT OF NEGATIVE SURFACE CHARGE

Abstract— The water soluble, photolabile nitrene precursor,azidonaphthalene–2,7-disulfonic acid (ANDS) was encapsulated in small unilamellar, isoelectric (egg PC) or negatively charged (egg PC + dihexadecylphosphate) liposomes. The individual and combined effects of heme-proteins and UV irradiation on the fluorescence of these vesicles under aerobic conditions were studied. Consistent with the catalytic action of heme-proteins on lipid peroxidation and peroxide decomposition, addition of cytochrome c (positively charged) or catalase (negatively charged) to the vesicles elicited immediate formation of a fluorescence band at 470 nm, characteristic of Schiff bases that form from aldehyde byproducts of decomposing hydroperoxides. Ultraviolet irradiation of liposomes for 5 min caused no significant changes in the fluorescence spectrum, in spite of the radiolysis of ANDS inside the vesicles with consequent formation of nitrene radicals. When isoelectric vesicles were irradiated with UV light in the presence of cytochrome c or catalase, Schiff base formation was further increased by2–3 fold, which effect was not observed in the absence of internal ANDS, or in the presence of negative surface charge on the vesicles. These findings suggest that (a) UV irradiation, by itself, cannot trigger lipid decomposition even when it is assisted by photoproduced nitrene radicals, (b) there is a ternary synergism between UV light, heme-proteins and nitrene radicals in promoting peroxidative lipid breakdown, and (c) negative surface charge inhibits the above synergism, which effect is unlikely to be due to electrostatic interaction between the vesicles and the protein or the ANDS.     

EFFECT OF TRANSMEMBRANE Ca~(2+) GRADIENT ON CONFORMATION AND ENZYME ACTIVITY OF RECONSTITUTED ADENYLATE CYCLASE

Adenylate cyclase from bovine brain cortex was reconstituted into asolectin liposomes with (500-fold) or without transmembrane Ca~(2+) gradient. The enzyme activity of four types of proteoliposomes (the active center of enzyme exposing outside) was compared. The highest adenylate cyclase activity was observed in the vesicles with outside lower Ca~(2+)concentration (≈10~(-6) mol/L, similar to thephysiological condition). If the transmembrane Ca~(2+) gradient was in the inverse direction (i.e. outside higher Ca~(2+) concentration, 0.5 mmol/L), a lowest enzymatic activity would appear. The difference in enzymatic activity between the two types of proteoliposomes could be diminished following the addition of Ca~(2+) ionophore A23187. Proteoliposomes without transmembrane Ca~(2+) gradient exhibited intermediate activities.The conformation difference of adenylatecyclases in the above-mentioned proteoliposomes was also detected by measuring intrinsic fluorescence and fluorescence quenching with KI.     

双电层相互作用时胶体表面电位的分布及电荷密度的计算

本文以实验为基础,运用计算机数值解解出双电层相互作用时平面胶体表面电位ψ随距离X的分布,依据作者导出的双电层相互作用时胶体表面阴离子负吸附方程,计算出胶体表面外Hclmhotz面(OHP)处的表面电荷密度σ_δ;讨论了双电层相互作用程度(ψ_d/ψ_δ)对胶体表面电位分布及其表面电荷密度的影响。     

EFFECT OF FAR-UV AND NEAR-UV RADIATION ON THE CELL SURFACE CHARGE OF THE PROTOZOAN

Abstract— Cell electrophoresis was used to detect the effect of far-UV or near-UV radiation on the cell surface charge of the pathogenic protozoan Tritrichomonas foerus . Either far-UV or near-UV radiation interfered with the surface charge of T. foetus at fluences which inhibited cell growth by 50%. Both UV-radiations induced a significant decrease on surface charge of T. foetus , as evaluated by measurement of its electrophoretic mobility (EPM). Determinations of EPM of protozoa in solution of low ionic strength indicated that the decrease in the EPM induced by far-UV is much less pronounced than that observed for near-UV or control cells.     

脂质体在分析化学研究中的应用

脂质体由脂质双分子组成,内部为水相的闭合囊泡,在生命科学、膜工程学、药学、临床医学等领域有着广泛的应用.对脂质体分类和制备以及在分析化学研究中的应用进行综述,以期促进脂质体在分析化学研究中的发展.     

STUDY OF THE EFFECTS OF ULTRAVIOLET LIGHT ON BIOMEMBRANES—IV. THE EFFECT OF OXYGEN ON UV-INDUCED HEMOLYSIS AND LIPID PHOTOPEROXIDATION IN RAT ERYTHROCYTES AND LIPOSOMES

Abstract— Hemolysis induced by irradiation with ultraviolet (UV) light at 254 nm showed a pronounced oxygen effect: under irradiation in vacuum, the rate of hemolysis was decreased by an order of magnitude. Irradiation at 254 nm in air but not under vacuum caused the peroxidation of erythrocyte membrane lipids. These results suggest that membrane lipid photoperoxidation is one of the causative factors of UV hemolysis. Irradiation at different wavelengths showed that UV-induced lipid photoperoxidation in erythrocyte membranes developed while the antioxidant α-tocopherol was directly photooxidized. It is shown that the process of lipid photolysis in erythrocyte membranes involves sensitization, possibly by protoporphyrin, whose presence in liposomes accelerates the photoperoxidation at 254 and 365 nm of unsaturated fatty acid residues in lecithin. Possible mechanisms of photochemical damage to erythrocyte membranes are discussed.     

SINGLET OXYGEN GENERATION BY FUROCOUMARINS: EFFECT OF DNA AND LIPOSOMES

The quantum yield of singlet oxygen generation by aqueous furocoumarins was measured at 365 nm using the photosensitized inactivation of subtilisin Carlsberg as the probe with the following results: psoralen (0.18), 5-methoxypsoralen (0.013), and 8-methoxypsoralen (0.035). Singlet oxygen formation was significant for dark complexes of 8-MOP with calf thymus DNA and the covalent DNA photoadducts. Incorporation of 8-MOP in sonicated egg phosphatidylcholine liposomes did not inhibit photosensitization of subtilisin Carlsberg and also led to lipid peroxidation, with positive tests for the involvement of singlet oxygen. Peroxidation of the liposomes was inhibited by the presence of α-tocopherol and promoted by the presence of cholesterol in the membranes.     

EFFECT OF SINGLET OXYGEN QUENCHERS ON OXIDATIVE DAMAGE TO LIPOSOMES INITIATED BY PHOTOSENSITIZATION OR BY RADIOFREQUENCY DISCHARGE

Abstract —Lipid model membrane systems, liposomes, may be oxidized by both toluidine blue Osensitized photo-oxidation and radiofrequency-discharge-generated singiet oxygen. Oxidation can be followed by the appearance of lipid peroxides, malondialdehyde formation, and ultimately by the lysis of the liposomes. Inhibition of oxidation is observed in both systems using either β-carotene or DABCO. These observations are consistent with the view that type II (singlet oxygen) reactions are involved in this photodynamic system.     

乳化剂OP对饮料中铅铜溶出增敏作用的研究及应用

探讨了乳化剂ＯＰ对饮料中铅,铜溶出的增敏作用,不同类型饮料中基质成分的影响与检出限的关系。研究了活性剂的种类,反应介质,溶出条件及共存物质的影响,拟定出同位镀汞标准加入法同时测定饮料中铅,铜的最佳分析条件。方法较已往的电位溶出分析相比,具有分辨率高,选择性强,高灵敏度,简便,快速,准确等特点,有很好的实用推广价值。     

EFFECT OF INTRACELLULAR INJECTION OF EGTA AND TETRAETHYLAMMONIUM CHLORIDE ON THE RECEPTOR POTENTIAL OF LOCUST PHOTORECEPTORS

Abstract— The depolarizing photoreceptor potential of locust, Valanga irregularis , as that of other arthropods, has an initial transient phase that declines to a stable sustained phase. In this work, the effect of intracellular injection of EGTA and TEA on these phases was studied.
(1) Reversal potential of the transient phase was higher than that of the sustained phase.
(2) EGTA and TEA enhanced both phases and the enhancement was more prominent in the sustained phase.
(3) Resting potential was shifted slightly upward by the injection. The amplitude of the maximum response was also increased.
(4) Current injected through an electrode filled with 2 M KCl had a significant effect on the shape of the response depending on the direction of the current.
(5) It is suggested that the activation of K⁺ conductance induced by an increase of intracellular free Ca²⁺ concentration causes a decrease in amplitude of the sustained phase.     

BEHAVIOR OF HETEROEXCIMER SYSTEMS IN SINGLE BILAYER LIPOSOMES

Abstract— Behavior of heteroexcimers formed between pyrene moieties incorporated at varying depths from the charged surface of vesicles and exogeneous N, N-dimethylaniline during the lifetime of excited pyrene moiety have been investigated by means of fluorescence measurements and laser photolysis. Fluorescence studies show that the fluorescence quenching kinetics in liposomes is rather similar to that in homogeneous bulk solutions, in contrast to the case of ordinary micellar solutions. Weak heteroexcimer fluorescence as well as transient absorption bands of ion radicals produced as a consequence of electron transfer from the amine to the excited pyrenyl group were observed. Comparison of the present results with those in homogeneous solutions indicates that the polar environment where dielectric constant is as large as 10 is attained in the hydrocarbon core of the liposomes. The polar environment of the fatty acid chains suggests the presence of water molecules penetrated into the hydrocarbon core.     

STUDIES ON THE MECHANISM OF THE HEMATOPORPHYRIN-SENSITIZED PHOTOOXIDATION OF 1,3-DIPHENYLISOBENZOFURAN IN ETHANOL and UNILAMELLAR LIPOSOMES

The 5 microM hematoporphyrin-sensitized photooxidation of 1,3-diphenylisobenzofuran (DPBF) was studied in homogeneous ethanolic solutions and in aqueous dispersions of unilamellar liposomes of dipalmitoylphosphatidylcholine; both the porphyrin and DPBF are embedded in the phospholipid bilayer. The rate and quantum yield of DPBF photooxidation were found to increase upon increasing the substrate concentration and were higher in the liposome system, while they were unaffected by the fluidity of the phospholipid bilayer. Time-resolved spectroscopic measurements showed that the photooxidation of DPBF in ethanol solution proceeds by a type II O2(1 delta g)-involving mechanism. In the liposomal vesicles the high local concentration of hematoporphyrin (Hp) and DPBF in the phospholipid bilayer (ca 2000-fold higher than the stoichiometric concentration) enhances the probability of energy transfer from triplet Hp to DPBF with generation of triplet DPBF; hence O2 (1 delta g) formation can be promoted by both triplet Hp and triplet DPBF. A minor fraction of triplet DPBF quenchings appears to generate radical species which propagate DPBF damage by chain reaction.     

ON THE POSSIBILITY OF PROTEIN–CHROMOPHORE CHARGE TRANSFER IN VISUAL PIGMENTS

Abstract— Quantum chemical calculations have been performed on the retinylidene Schiff base assumed to be protonated or hydrogen bonded at the nitrogen and perturbed by a carboxylate ion at its β-ionone end. The results indicate that weak charge transfer type bands might exist due to electron transfer from the carboxylate ion to the Schiff base and that the intense (π, π*) band in the visible spectrum might acquire a significant degree of charge transfer character.     

HYDRODYNAMIC EFFECTS IN THE PHOTOSENSITIZED LYSIS OF LIPOSOMES*

Abstract— The photosensitized lysis of phosphatidylcholine liposomes incorporating methylene blue in the membrane or in the presence of external methylene blue is promoted by hydrodynamic agitation concurrent with or subsequent to irradiation with red light. The results implicate the attack of singlet oxygen on an unsaturated lipid component as the key photochemical step, which is followed by additional membrane damage induced by hydrodynamic action.     

DIFFERENTIAL EFFECT OF CALCIUM ON CHLOROPLAST MOVEMENT IN MOUGEOTIA

The flat, ribbon-shaped chloroplast in the filamentous green alga Mougeotia sp. undergoes light-induced orientational movement controlled by an intracellular tetrapolar gradient of the active form of phytochrome, P_fr. Some substructural and physiological aspects of this reaction were studied. An intracellular pattern of microfilaments (diameter: 5–10nm), presumably related to chloroplast movement, was identified in situ. In addition, it could be shown that chloroplast movement decreases parallel to a nitric acid soluble fraction of intracellular calcium. These results might indicate that phytochrome governs chloroplast movement in Mougeotia via control of the binding state of calcium.     

EFFECT OF HEMATOPORPHYRIN DERIVATIVE ON HEMATOLOGICAL PARAMETERS IN RATS

Abstract— Wistar rats were injected with hematoporphyrin derivative (Hpd) intraperitoneally and kept in the dark. Rats were sacrificed 2, 24, 48 and 72 h after injection. It was observed that Hpd in the dark did not affect the hemoglobin content and number of erythrocytes, while the leukocyte count was increased and blood pH decreased. Blood levels of glucose and lactate were increased significantly. Because the food intake was similar in all the groups, glycogenolysis was suspected to be the source of increased glucose levels in blood. However, a significant increase in the glycogen content of the livers of Hpd-treated rats was observed, which rules out glycogenolysis. Hyperglycemia may result due to a number of reasons such as stimulation of the central nervous pathways innervating the liver and adrenal medulla, excessive glucogenesis in liver from glycogen and noncarbohydrate sources, emotional stress, anesthesia and hormonal effects. The present study rules out hyperglycemia due to anesthesia and glucogenesis in the liver. Maintenance of blood glucose levels is a highly complex mechanism. Further investigations to understand these mechanisms are in progress.     

EFFECT OF CALCIUM ON DARK ADAPTATION IN Phycornyces PHOTOTROPISM

Adaptation processes enable phototropism of Phycomyces to operate over a 10-decade range of blue-light intensity (1 nW m-2-10 W m-2). To investigate the influence of calcium on dark adaptation, the phototropic latency method was employed with the modification that sporangiophores were temporarily immersed in solutions containing CaCl2 or LaCl3. Following such treatment, the time course of bending was found to have two components with distinct latencies and bending rates. After immersion in darkness for 30 min in LaCl3 solution or 1 h in a solution of CaCl2, MgCl2, or the calcium chelator EGTA, each sporangiophore was adapted to a blue light beam (1 W m-2) for 45 min by rotation around its vertical axis. Cessation of rotation defined the onset of the phototropic stimulus, at which time the intensity was reduced by as much as 10(3)-fold. For a 10(2)-fold reduction (to 10(-2) W m-2), immersion in CaCl2 (10-100 microM) reduces the latency 13 min for the early bending component and 18 min for the late component, whereas treatment with the calcium-channel blocker lanthanum (0.1-11 microM LaCl3) increases the latency 12 min for the early component and 13 min for the late component. EGTA (10 microM) also had an inhibitory effect, increasing the latency of the first and the second components by 7 and 10 min, respectively. In experiments performed similarly, but without the light adaptation treatment after immersion, no differences between calcium-treated and control sporangiophores were found. The bending rates of both components show only a weak dependence on calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

SPECTROSCOPIC PROPERTIES OF THE POTENTIOMETRIC PROBE MEROCYANINE-540 IN SOLUTIONS AND IN LIPOSOMES

Absorption, fluorescence and resonance Raman spectra of the membrane dye merocyanine-540 (MC540) were measured. The aggregation of the dye, its binding to lipid membranes and its response to crossmembrane electric potential differences were studied. The dye was found to aggregate even at micromolar concentrations in water, but not in organic solvents. The dimerization constant was evaluated by spectroscopic techniques. The binding constant to liposomes was estimated by a spectroscopic titration method. Resonance Raman spectra of MC540 were measured for the first time. Distinct changes were observed in the vibrational spectrum upon the generation of a valinomycin-induced K⁺ diffusion potential (Nernst potential) on liposomes. The ratio of Raman band intensities, which was found to be related to the membrane potential, can be used to evaluate the absolute value of the electric potential.