Self-aggregation of free base porphyrins in aqueous solution and in DMPC vesicles

Free base porphyrin (PPhe), derivatized with aminosulfonyl groups linked to the aromatic amino acid phenylalanine at the meso-positions, was mixed with DMPC vesicles. The resulting interaction was studied by absorption, steady-state and transient state fluorescence, at different pHs. At pH=2 to pH=9, the aforementioned porphyrin predominates as an aggregated species, with a co-facial arrangement of the molecules taking into account the blue shift of the Soret band (414 nm for the monomer and 401 nm for the aggregate). Upon interaction with DMPC vesicles, the competing hydrophobic interactions with the bilayer destabilize the aggregated species in favor of monomer incorporation. Fluorescence lifetimes also show that the long component assigned to the monomer contributes only 30% to the overall decay in solution (e.g. pH=7.0) whereas in DMPC vesicles this contribution increases up to 85% independent of the solution pH, which confirms a location of the probe in an environment "protected" from free water. The picture changes completely in the case of TSPP, an anionic porphyrin which does not incorporate in DMPC vesicles. Remarkably, at pH=2.5 all the experimental findings point to the self-assembling of the porphyrin units in J-aggregates induced at the surface of the DMPC vesicle. In fact, upon removal of the aqueous solvent, we could define by fluorescence lifetime imaging microscopy (FLIM) regions where the fluorescence lifetime is that characteristic of the J-aggregate ( 0.11 ns).     

Effect of ubiquinone on phospholipid metabolism in radiation injury

The synthesis and phospholipid content in the liver, intestine and spleen in normal and irradiated rats administered ubiquinone-9 were studied with the use of 3H-serine. Ubiquinone markedly activated decarboxylation of phosphatidylserine and suppressed transformation of phosphatidylethanolamine to phosphatidylcholine in rat liver and spleen. The effect was also observed in the organs of irradiated animals. In rat intestine, administration of ubiquinone normalized a sharp gamma-irradiation-induced inhibition of transformation of phosphatidylcholine from phosphatidylethanolamine. The catabolism of phospholipids under the action of ubiquinone and radiation was inhibited in the liver and, on the contrary, was activated in radiosensitive organs.     

Effect of ethanol in vivo on enzymes which detoxify oxygen free radicals

The effects of ethanol administered as a 15% solution in drinking fluid on weight gain, soluble liver protein and the activity of the three enzymes of oxygen radical metabolism (i.e., superoxide dismutase, catalase, and glutathione peroxidase) were studied in five inbred strains of mice (129/ReJ, BALB/c, C3H/HeSnJ, C57BL/6J, Csb) and Sprague Dawley rats, relative to age, sex, and genotype matched controls. Animals maintained on ethanol exhibited lower weight gains and elevation of soluble liver protein than controls. Total superoxide dismutase, catalase and glutathione peroxidase activity in ethanol-treated animals were in general reduced in comparison to that of their matched controls, with each strain showing genotype specific enzyme activity. Such ethanol feeding results are attributed to the direct and indirect effects of this treatment protocol and raise the possibility that ethanol-fed animals may be susceptible to free radical damage and at least some of the cellular damages observed following ethanol challenges could be attributed to the reduced level of these protective enzymes.     

Generation of free oxygen radicals in heart mitochondria: Effect of hypoxia-reoxygenation

The relationship between the rate of generation of superoxide radicals and the duration of hypoxia has been studied in isolated heart mitochondria with the use of the spin trap sodium 4,5dihydroxybenzene-1,3-disulfonate. The EPR spectra were recorded from a mitochondrial suspension placed in a gas-permeable capillary under conditions of regulated partial oxygen pressure. Earlier we have shown that the mitochondria isolated from perfused hearts after 30-min ischemia display a higher rate of superoxide generation than those from controls. However, in isolated mitochondria the EPR signal from 4,5-dihydroxybenzene-1,3-disulfonate increased already after 10-min hypoxia, but its intensity remained the same in the mitochondria subjected to 30-, 45-, and 60-min hypoxia. Thus, the isolated mitochondria in the incubation medium are less sensitive to hypoxia than the mitochondria from cardiomyocytes of an ischemic heart.     

High-frequency ultrasonic absorption spectroscopy on aqueous suspensions of phospholipid bilayer vesicles

Between 1 MHz and 3 GHz the ultrasonic absorption coefficient has been precisely measured as a function of frequency for some aqueous suspensions of single-walled phospholipid bilayer vesicles. All solutions of the specially purified phospholipids clearly show excess absorption, reflecting three molecular relaxation processes with discrete relaxation times. Typical values for these times are 50, 3 and 0.5 ns. The attempt is made to relate these relaxation processes to mechanisms of rotational isomerization in the hydrocarbon chains. Some other molecular mechanisms which could also contribute to the ultrasonic excess absorption spectra are also briefly discussed.     

Cell damage by oxygen free radicals

The exposure of isolated and cultured cells to oxygen free radicals generated extracellularly or intracellularly during the metabolism of foreing compounds results in the development of damage that eventually lead to cell death. Multiple mechanisms are involved in these cytopathological processes, including direct attack of free radicals to macromolecules essential for cell life, as well as indirect activation of catabolic processes such as proteases, endonucleases and phospholipases. A key role in triggering these indirect events is played by Ca²⁺ whose cytosolic concentration during oxidative stress raises well above the physiological limits.     

Sulfur oxidation of free methionine by oxygen free radicals

The oxidation of free methionine to methionine sulfoxide by chemically or enzymatically generated oxygen free radicals is presented. The physiological significance of this process in living cells is suggested.     

Permeability of bilayer phospholipid membranes to superoxide oxygen radicals

Lecithin monolayer liposomes (1000 A in diameter) loaded with cytochrome c were placed into the external solution, in which O2 superoxide radicals were regenerated by the xanthine-xanthine oxidase system. The penetration of superoxide radicals across the liposomal membranes was followed by cytochrome c reduction in the interval volume of the liposomes. The effects of lipid membrane modifiers and temperature on this process were investigated. The results obtained were used for calculation of the permeability coefficients of bilayer lipid membranes for O(2) (P'O(2) = (7.6 +/- 0.3) . 10(-8) cm . s-1) or HO . 2(P'HO(2) = 4.9 x 10(-4) cm . s-1). The effect of the transmembrane electric potential (concentration gradient of H+, valinomycin) on the permeability of liposomal membranes for the superoxide radical was studied. The superoxide radical was down to penetrate across the bilayer lipid membranes in an unloaded state. Using an intramolecular cholesterol-amphotericin B-complex, the superoxide radicals were shown to penetrate across the bilayer lipid membranes, predominantly via the anionic channels.     

活性氧、自由基与植物的衰老

介绍近 1 0年来有关活性氧、自由基的产生 ,对植物的伤害及植物对活性氧、自由基清除的研究进展。     

Proton dissociation dynamics in the aqueous layer of multilamellar phospholipid vesicles

Summary The water layers interspacing between the phospholipid membranes of a multilamellar vesicle are 3–10 water layers across and their width is adjusted by osmotic pressure (Parsegian, V.A., et al., 1986.Methods Enzymol. 127:400–416).In these thin water layers we dissolved pyranine (8 hydroxypyrene 1,3,6 trisulfonate), a compound which, upon photo excitation, ejects it hydroxy proton with time constant of 100 psec. (Gutman, M. 1986.Methods Enzymol. 127:522–538).In the present study we investigated how the width of the aqueous layer, the density of phosphomoieties on the membrane's surface and the activity of water in the layer affect the capacity of protons to diffuse out from the electrostatic cage of the excited anion before it decays to the ground state.Using a combination of steady-state and subnanosecond time-resolved fluorescence measurements we determined the average number of proton excited-anion recombinations before the proton escapes from the Coulomb cage.The probability of recombination in thin water layer is significantly higher than in bulk. The factor contributing most to enhancement of recombination is the diminished water activity of the thin aqueous layer.The time frame for proton escape from an electrostatic trap as big as a membrane-bound protein is 3 orders of magnitude shorter than turnover time of membrane-bound enzymes. Thus the effects of local forces on proton diffusion, at the time scale of physiological processes, is negligible.     

Viscosity of the internal aqueous phase of unilamellar phospholipid vesicles

The viscosity of the internal aqueous phase of unilamellar vesicles comprised of purified soybean phospholipids (asolectin) was measured using the fluorescence polarization of the entrapped hydrophilic probe 8-hydroxy-1,3,6-pyrenetrisulfonate (pyranine). At 20 °C the rotational relaxation time (?) for pyranine in bulk solution was 0.55 ns as compared to a rotational time of 1.8 ns for pyranine within the internal aqueous compartment. Similar large increases in ? for internal pyranine were noted over the temperature range 5–35 °C suggesting that in these small vesicles the internal water is more viscous than bulk water.     

Serendipitous findings while researching oxygen free radicals

This review is based on the honor of receiving the Discovery Award from the Society of Free Radical Biology and Medicine. The review is reflective and presents our thinking that led to experiments that yielded novel observations. Critical questioning of our understanding of oxygen free radicals in biomedical problems led us to use and develop more direct and extremely sensitive methods. This included nitrone free radical spin trapping and HPLC–electrochemical detection. This technology led to the pioneering use of salicylate to trap hydroxyl free radicals and show increased flux in ischemia/reperfused brain regions and also to first sensitively detect 8-hydroxyl-2-deoxyguanosine in oxidatively damaged DNA and help assess its role in cancer development. We demonstrated that methylene blue (MB) photoinduces formation of 8-hydroxyguanine in DNA and RNA and discovered that MB sensitively photoinactivates RNA viruses, including HIV and the West Nile virus. Studies in experimental stroke led us serendipitously to discover that α-phenyl-tert-butylnitrone (PBN) was neuroprotective if given after the stroke. This led to extensive commercial development of NXY-059, a PBN derivative, for the treatment of stroke. More recently we discovered that PBN nitrones have potent anti-cancer activity and are active in preventing hearing loss caused by acute acoustical trauma.     

Myocardial injuries mediated by free oxygen radicals

Dog experiments were performed to establish the lipid peroxidation of heart tissue (measured by formation of malone-dialdehyde--MDA) and the natural scavenger action (measured by determination of superoxide dismutase--SOD and of reduced glutathione--GSH). Experimental groups were: control dogs having intact heart, dogs ventilated with hypoxic gas (N2O and O2 at a ratio of 10:1) for 1, 2 and 3 hours and dogs having acute coronary ligature for 1, 2, 3 and 24 hours. Acute hypoxia caused a gradual increase of MDA concentration, a moderate increase of the GSH level and a sharp decrease in SOD activity. In ischaemic heart tissue, these changes were very distinctive. High MDA values were found after 3 hours. GSH level and SOD activity decreased continually. Increased MDA formation indicates breakdown of polyunsaturated fatty acids (PUFA) in the membranes, decreased GSH and SOD levels indicate impairment of the natural scavenging, clearly outlining the extent of disintegration of the membrane structure and function due to the effect of toxic free oxygen radicals.     

Role of oxygen free radicals in carcinogenesis and brain ischemia

Even though oxygen is necessary for aerobic life, it can also participate in potentially toxic reactions involving oxygen free radicals and transition metals such as Fe that damage membranes, proteins, and nucleic acids. Oxygen free radical reactions and oxidative damage are in most cases held in check by antioxidant defense mechanisms, but where an excessive amount of oxygen free radicals are produced or defense mechanisms are impaired, oxidative damage may occur and this appears to be important in contributing to several pathological conditions including aging, carcinogenesis, and stroke. Several newer methods, such as in vivo spin-trapping, have become available to monitor oxygen free radical flux and quantitate oxidative damage. Using a combination of these newer methods collectively focused on one model, recent results show that oxidative damage plays a key role in brain injury that occurs in stroke. Subtle changes, such as oxidative damage-induced loss of glutamine synthetase activity, may be a key event in stroke-induced brain injury. Oxygen free radicals may play a key role in carcinogenesis by mediating formation of base adducts, such as 8-hydroxyguanine, which can now be quantitated to very low levels. Evidence is presented that a new class of free radical blocking agents, nitrone spin-traps, may help not only to clarify if free radical events are involved, but may help prevent the development of injury in certain pathological conditions.     

Mechanoformation of neutral giant phospholipid vesicles in high ionic strength solution

We present new and simple method for formation of giant unilamellar vesicles (GUVs) in high ionic strength solutions, such as phosphate buffered saline (PBS). Mechanoformation method is an alternative method to electroformation method. The advantage of the mechanoformation procedure is that there are no limitations with respect to the ionic strength of the aqueous solutions, because there is no applied electric potential thus no current flow through the formation cell and no electrolysis is induced.     

Retention of aqueous contents during divalent cation-induced fusion of phospholipid vesicles

The relative kinetics of intermixing and release of liposome aqueous contents during Ca²⁺-induced membrane fusion has been investigated. Fusion was monitored by the Tb-dipicolinic acid (DPA) fluorescence assay. Release was followed by the relief of self-quenching of carboxyfluorescein or by Tb fluorescence, with essentially identical results. Fusion of large unilamellar vesicles (LUV) made of phosphatidylserine (PS) in 100 mM NaCl (pH 7.4) at 25°C was initially non-leaky, whereas the fusion of small unilamellar vesicles (SUV) was accompanied by partial release of contents. After several rounds of fusion, the internal aqueous space of the vesicles collapsed. The rate of intermixing of lipids, measured by a resonance energy transfer assay, and the rate of coalescence of aqueous contents during fusion were similar over a range of Ca²⁺ concentrations. Most of the aqueous contents were retained after the fusion of SUV (PS) in 5 mM NaCl and 1 mM Ca²⁺. LUV made of a 1:1 mixture of Bacillus subtilis cardiolipin and dioleoylphosphatidylcholine went through about two rounds of fusion in the presence of Ca²⁺ at 10°C, with complete retention of contents. Similar results were obtained with vesicles composed of phosphatidate/PS/phosphatidylethanolamine/cholesterol (1:2:3:2) in the presence of Ca²⁺ and synexin at 25°C. These results emphasize the diversity of the relative kinetics of fusion and release in different phospholipid vesicle systems under various ionic conditions, and indicate that the initial events in the fusion of LUV are in general, non-leaky.     

The effects of oxygen free radicals on the preserved kidney

This study evaluated the effect of specific scavengers of oxygen derived free radicals on the results of kidney preservation. The immediate function of rabbit kidneys preserved for 24 hr by hypothermic perfusion was studied on an ex vivo shunt. A significant improvement in creatinine clearance was seen when the perfusate was treated with superoxide dismutase (SOD) and catalase (CAT), with values of 261 ± 82 ml/hr vs control values of 203 ± 72 ml/hr, P < 0.05. This effect was enhanced if a long-persistent polyethylene glycol-linked form of SOD, namely PEG-SOD, was used (330 ± 58 ml/hr, P < 0.01). Recipient treatment and other modifications designed to protect against free radicals resulted in similar improvement in function. In contrast, no effect of free radical scavengers could be demonstrated in kidneys which were preserved by flush cooling, whether the agents were added to the flushing solution, given to the recipient, or both.