Magnetic resonance imaging of isolated single liposome by magnetic resonance force microscopy.

Magnetic resonance imaging (MRI) is very useful spectroscopy to visualize a three-dimensional (3D) real structure inside the sample without physical destruction. The spatial resolution of the readily available MRI spectrometer is, however, limited by a few ten to hundreds of microns due to a technological boundary of generating larger magnetic field gradient and to the insensitivity inherent to the inductive signal detection. Magnetic resonance force microscopy (MRFM) is new alternative MRI spectroscopy which is anticipated to significantly surpass the conventional MRI in both resolution and sensitivity. We report two imaging experiments on our MRFM spectrometer operated at room temperature and in vacuum approximately 10(-3)Pa. One is for approximately 20 microm liposome membrane labeled entirely by a nitroxide imaging agent and the other for approximately 15 microm DPPH particles, both are nearly the same size as that of human cell. The reconstructed images at spatial resolution approximately 1 microm were in satisfactory agreement with the scanning electron microscope images. The potential capability of visualizing intrinsic radicals in the cell is suggested to investigate redox process from a microscopic point of view.

An electron spin resonance study on alkylperoxyl radical in thin-sliced renal tissues from ferric nitrilotriacetate-treated rats: the effect of alpha-tocopherol feeding.

Formation of excess free radical causes cellular oxidative stress, which has been shown to be associated with a variety of pathologic conditions. While electron spin resonance (ESR) spectroscopy has been the only method to demonstrate the presence of free radicals, its application to tissue samples has been challenging. We report here the successful ESR detection in thin-sliced fresh tissues or frozen sections in a rat model. Ferric nitrilotriacetate (Fe-NTA) induces oxidative renal tubular damage that ultimately leads to high incidence of renal carcinoma in rodents. Twenty minutes after administration of 5 mg iron/kg Fe-NTA to rats, a thin-slice of the kidney was mounted on a tissue-type cell and analyzed by ESR spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). An ESR signal from alkylperoxyl radical adduct was obtained, and the signal was inversely proportional to renal alpha-tocopherol content which was modulated through diet. Furthermore, we undertook ex vivo study using frozen sections. Fe-NTA (1 mM) was added to a rat kidney frozen section for 10 min. After washing the specimen was mounted on a tissue-type cell and analyzed with ESR spin trapping using DMPO. Alkylperoxyl radical signal was dependent on thickness, incubation time and renal tissue levels of alpha-tocopherol, and was reduced by preincubation with catalase or dimethyl sulfoxide but not with alpha-tocopherol outside tissue. This versatile method facilitates identification of free radicals in pathologic conditions, and may be useful for selection of antioxidants.

Electron spin resonance studies of dipalmitoylphosphatidylcholine liposomes containing soybean-derived sterylglucoside.

The effects of soybean-derived sterylglucoside (SG) on the fluidity of liposomal membrane composed of dipalmitoylphosphatidylcholine (DPPC) were investigated compared with those of soybean-derived sterol (SS) and cholesterol (Ch) using an electron spin resonance spectrometer. Three kinds of liposomes were prepared in the molar ratio of DPPC/X=7/4, where X is SS, Ch or SG. The fluidity close to the polar head groups increased with an increase of temperature in the DPPC membrane containing SS, Ch and SG in the range 35 to 45 degrees C. Those near the hydrophobic end changed with an increase in temperature in liposomes containing SS, Ch and SG, which had a fluidizing effect on the DPPC membrane below the transition temperature (Tm, 41.9 degrees C) and a condensing effect over the Tm. The fluidizing effects of these compounds around 37 degrees C near the polar head group and the hydrophobic end increased in the following order: Ch < SG < or = SS and SS < Ch < SG, respectively. SG increased the fluidity of liposomal membrane dramatically above the Tm (35.4 degrees C). These results suggest that the high fluidity close to the hydrophobic end of the liposomal membranes around 37 degrees C, the decrease of Tm, and the sigmoidal nature of fluidity vs. temperature are important factors in the effectiveness of liposomes containing SG as a carrier of drugs.

Protective role of nitric oxide synthase against ischemia-reperfusion injury in guinea pig myocardial mitochondria.

In guinea-pig myocardial mitochondria preparation, lowering the Ca2+ concentration or pH level in the perfusate rapidly elevated the fura-2 Ca2+ signal ([Ca2+]m). Pretreatment with 10(-4) M L-Arg inhibited the rapid [Ca2+]m influx, whereas administration of 10(-4) M L-NAME did not, suggesting some association between nitric oxide (NO*) synthase (NOS) activation and Ca2+ kinetics in mitochondria. Immunoblotting analysis showed that endothelial (e)-NOS was present in mitochondria, but not inducible (i)-NOS or brain (b)-NOS. Electron microscopy observations revealed that the e-NOS antibody-reactive site in the mitochondria was the inner cristae. The production of reactive oxygen species and NO* in isolated mitochondria was detected by the spin trapping technique with electron paramagnetic resonance (EPR) spectrometry. Pretreatment with 10(-5) M S-nitroso-N-acetyl-DL-penicillamine (SNAP) and 10(-5) M 3-[2-Hydroxy-1-(1-methylethyl)-2-nitrosohydrazino]-1-propananin e (NOC 5), which spontaneously generate NO*, completely inhibited the [Ca2+]m uptake. In addition, N-morpholino sydnonimine hydrochloride (SIN-1) (10(-5) M), which simultaneously generates NO* as well as *O2- and peroxynitrite anion (ONOO-), inhibited the increase in [Ca2+]m. ONOO- (3 x 10(-4) M) itself also inhibited this increase. Pretreatment with the *O2(-)-scavenger manganese superoxide dismutase or catalase (200 units/ml) completely inhibited the increase in [Ca2+]m caused by lowering of either the Ca2+ concentration or the pH in the perfusate. These results suggested that the formation of reactive oxygen species promoted the [Ca2+]m influx. The agents that inhibited the [Ca2+]m influx improved contractility even in Langendorff preparations after ischemia. Based on these findings, we concluded that e-NOS exists in mitochondria and that NO* may play an important protective role in reperfusion cardiac injury after ischemia, by inhibiting the Ca2+ influx into mitochondria which are otherwise damaged by *O2-.

Excimer laser-induced hydroxyl radical formation and keratocyte death in vitro.

PURPOSE: To characterize the type of reactive oxygen species (ROS) produced by excimer photoablation of aqueous solutions and to show the effects of ROS and antioxidants on corneal stromal cells in vitro. METHODS: Electron spin-resonance spectroscopy was performed using the spin-trapping agent 5,5-dimethyl-1-pyrroline N-oxide (DMPO) for the detection of the superoxide anion and the hydroxyl radical in an acellular DMPO solution irradiated with the excimer laser. Hydroxyl radicals were produced by the Fenton reaction in vitro by the mixture of hydrogen peroxide and ferrous iron (Fe2+), and the effects on cultured corneal fibroblasts were observed by fluorescent microscopy using the cell death marker, propidium iodide (PI) and TdT-mediated dUTP nick-end labeling (TUNEL). RESULTS: Excimer photoablation of a 1% DMPO solution produced a species-specific spin-trapping adduct for the hydroxyl radical ('OH), but not for the superoxide anion or other unidentified free radical. The signals were inhibited dose dependently by the hydroxyl radical scavenger dimethylsulfoxide (DMSO) and an L-ascorbic acid analogue, EPCK-1. The production of *OH in the supernatant of cultured rabbit corneal fibroblasts by the Fenton reaction caused an increase in PI (+) and TUNEL (+) cells by 90 minutes, which was significantly inhibited by the addition of DMSO. CONCLUSIONS: Hydroxyl radicals may be partly responsible for stromal fibroblast cell apoptosis after excimer photoablation.

[Distribution of nitroxide radical compounds and its reducing activity in liver, spleen and kidney of rat by electron spin resonance (ESR) spectroscopy].

The time courses of intensity changes of X-band electron spin resonance (ESR) spectra in the blood, liver, spleen, kidney and porta hepatis of rats were examined after 3-carbamoyl-2,2,5,5-tetramethylpyrrolidin-1-yloxyl (carbamoyl-PROXYL) was perorally administered. The quenching activities of nitroxide radicals were determined using homogenate of the organs of individual rats. It was found that administrated nitroxide radicals were delivered to the liver, spleen, and kidney after peroral administration, where ESR signal intensities in the blood decreased gradually. The concentration of the elivered nitroxide radical varied with the individual organs. The quenching activity of the nitroxide radical for the homogenate of the porta hepatis was the highest before administration, while the concentration due to the reduced deoxidized-nitroxide radical compounds after 4 h of peroral administration was shown to be high in the kidney. It is concluded that the nitroxide radical compound is quenched in the porta hepatis of the liver, while the reduced nitroxide radical compound is delivered to the kidney.

Three dimensional electron spin resonance (ESR) imaging of internal organs in living mice.

In vivo electron spin resonance imaging (ESR imaging) was applied to living mice after peroral administration of a nitroxide radical spin probe. A 3D ESR imaging procedure was applied in vivo in order to obtain the exact distribution of the spin probe in a living animal. The imaging pictures demonstrated that the administered spin probe was firstly located in the stomach, then delivered to the liver, kidney and heart of the animal.

Spin trapping for nitric oxide produced in LPS-treated mouse using various new dithiocarbamate iron complexes having substituted proline and serine moiety.

Four dithiocarbamate derivatives of 4-substituted L-proline and N-methyl-L-serine were synthesized, and their iron complexes were prepared in Tris-HCl buffer solution. These complexes were used as spin trapping reagents for nitric oxide in ESR spectrometry, and compared with each other in regard to their spin trapping properties in vivo. When the synthesized complexes were injected to lipopolysaccharide-treated mice intravenously, the nitric oxide adducts were detected both in the liver and in the blood except N-dithiocarboxy-4-(methoxymethyl)oxy-L-proline iron complex, whose nitric oxide adduct was detected mostly in the blood. When the exogenous nitric oxide adduct of this complex was injected, it was not detected in the liver, too. It is considered that this complex can trap nitric oxide in the blood by excluding the accumulation of the nitric oxide adduct in the liver.

[Effect of shikonin and alkannin on hydroxyl radical generation system concerned with iron ion].

The effects of shikonnin (SK) and its optical isomer alkannin (AK) on the hydroxyl radical (HO.) generation system including iron ions were evaluated using the spin trap method by ESR spectroscopy. 5,5-Dimethyl-1-pyrroline-1-oxide (DMPO) was used as a spin trap agent and HO. was generated by a reaction between an iron ion and hydrogen peroxide, which is called Fenton reaction system. SK inhibited the HO. spin adduct (DMPO-OH) yielded in a dose-dependent manner. In this effect no difference was observed between SK and AK. When different concentrations of DMPO were used for the confirmation of its competitive reaction, no difference was also observed in the concentration of SK required to reduce the amount of the DMPO-OH by 50% (ID50). These findings suggested that the inhibitory effect of SK against the thus yielded DMPO-OH was not generated by the scavenging for HO., but by the inhibition on the Fenton reaction system. The mechanism of the inhibition on this system may be based on the formation of a complex between SK and the iron ion. The molar ratio of SK to the iron ion in the complex was considered 2 to 1 (2:1), because the concentrations of the observed ID50 and the used iron ion exhibited the same value. In addition, the same result was also obtained from the study using spectroscopic analysis.

Antioxidative Activity of Water Extracts of Lagerstroemia speciosa Leaves.

In order to develop naturally occurring antioxidants from edible plants, the antioxidative effect of hot water extracts of Lagerstroemia speciosa leaves, known by the Tagalog name of banaba in the Phillipines, was studied. The content of tannin in banaba extract was 36.8% in dry weight. Banaba extract showed strong antioxidative activity in a linoleic acid autoxidation system. Banaba extract was found to have a potent radical scavenging action on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and superoxide radicals (O(2-) ) generated by a hypoxanthine (HPX)/xanthine oxidase (XOD) system. In vitro lipid peroxidation of rat liver homogenate induced by tert-butyl hydroperoxide (BHP) was inhibited by the addition of banaba extract in a dose-dependent manner. From these results, banaba extract was demonstrated to be useful as an antioxidant or free radical scavenger to protect biological systems against oxidative stress.

Kinetic analysis of the Fenton reaction by ESR-spin trapping.

A quantitative analysis of hydroxyl radical (.OH) generated in the Fe(2+)-hydrogen peroxide reaction system was explored by a spin-trapping method using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) combined with electron spin resonance spectroscopy. Based on the numerical analysis of Fenton-related reactions, the reduction of DMPO-OH adduct from 1:1 stoichiometry prominent at high concentrations of Fe2+ was consistent with a reaction model in which a molar amount of hydrogen peroxide was reduced by two molar amounts of Fe2+. Furthermore, time-dependent decrease in DMPO-OH quantity, apparent at much higher concentration of Fe2+, was proved due to the reaction not with Fe2+ but with Fe3+.

Guanidino compounds generate reactive oxygen species.

Methylguanidine, guanidinoacetic acid and guanidinosuccinic acid are endogenous substances in body tissues. Extremely high levels of these substances are known to be related to the pathogenesis of epilepsy and renal failure such as uremia. In this study it was demonstrated that methylguanidine, guanidinoacetic acid and guanidinosuccinic acid, and arginine generate hydroxyl radicals in aqueous solution. These findings suggest that a high level of guanidino compounds accumulating near or within cells such as neurons (in an epileptogenic focus) or nephrons (in uremic patients) may cause free radical damage leading to these clinical disorders. Arginine may have a similar role in the pathogenesis of hyperarginemia.

alpha-Guanidinoglutaric acid as a free radical generator.

alpha-Guanidinoglutaric acid (alpha-GGA) was first isolated from the cobalt-induced epileptic focus of cat cerebral cortex by us in 1980. alpha-GGA could induce behavioral convulsion as well as electroencephalography-documented epileptic seizures, when it was administered into the brain. alpha-GGA was also found to be a potent nitric oxide synthase inhibitor, suggesting that suppression of this activity may result in epileptic seizures. It is now observed that alpha-GGA generates reactive oxygen species as superoxide and hydroxyl radicals in aqueous solution. These findings suggest that reactive oxygen species may damage cell membranes, thus leading to neuronal depolarization, which is closely related to epileptogenesity.

ESR demonstration of nitric oxide production from nitroglycerin and sodium nitrite in the blood of rats.

Electron spin resonance (ESR) spectra of iron-metal complexes formed by the reaction between nitric oxide (NO) and hemoglobin (Hb), referred to as nitrosylhemoglobin (HB-NO), were observed in rat blood treated in vitro and in vivo with nitroglycerin (GTN) at 77K. The same types of spectra were also detected in rats treated with sodium nitrite (NaNO2). Two types of Hb-NO, which were identified by ESR parameters of g values and superhyperfine coupling constants (shfcc), were the 6- and 5-coordinated complexes. These two types of Hb-NO were generated in a dose-dependent manner in the blood after intraperitoneal administration of 1.5-6 mg of GTN. At the higher dose of GTN (6 mg), the 6-coordinated complex was the major species generated initially, but within 10 min, the 5-coordinated complex increased time-dependently. Quantitative analysis of Hb-NO revealed that when GTN 0.3 mg and 0.6 mg was administered sublingually in rats, the concentration of Hb-NO observed in rat blood was 30% higher than the estimated concentration of GTN. The methemoglobin and peroxide complex of hemoglobin were observed in the blood incubated with GTN at 37 degrees C. These results suggest that the function of GTN was related to oxidative stress with the generation of Hb-NO. Therefore, monitoring of Hb-NO levels may be useful as an indicator of the function of various vasodilators.

Bromocriptine protects mice against 6-hydroxydopamine and scavenges hydroxyl free radicals in vitro.

Pretreatment with bromocriptine (5 mg/kg, i.p., 7 days) completely protected against the decrease in mouse striatal dopamine and its metabolites induced by intraventricular injection of 6-hydroxydopamine after intraperitoneal administration of desipramine, but similar pretreatment with L-DOPA/carbidopa (75/7.5 mg/kg, i.p., 7 days) showed only partial protective effect. Furthermore, in an in vitro system that generated.OH from FeSO4-H2O2, bromocriptine dose-dependently reduced the number of .OH radicals. These findings indicate that bromocriptine has a neuroprotective effect against neurotoxins such as 6-hydroxydopamine, probably due, in part, to its hydroxyl radical scavenging activity and inhibiting effect on dopamine turnover rate. This suggests that early introduction of bromocriptine in the therapy of Parkinson's disease may be superior to treatment with L-DOPA alone.

Age-related changes in the content of non-reducible crosslinks in rat mandibular bone.

Age-related changes in the content of non-reducible crosslink amino acids, pyridinoline and histidinoalanine, in rat mandibular bone were studied. The pyridinoline content markedly increased up to 12 months of age and thereafter slightly increased. The histidinoalanine content was low until 3 months of age, but thereafter increased significantly up to 24 months. Total collagen content remained constant throughout the experiment. From these results, pyridinoline and histidinoalanine may serve as markers for the maturation and senescence, respectively, of mandibular bone.