Regulating Brownian fluctuations with tunable microscopic magnetic traps.

A major challenge to achieving positional control of fluid borne submicron sized objects is regulating their Brownian fluctuations. We present a magnetic-field-based trap that regulates the thermal fluctuations of superparamagnetic beads in suspension. Local domain-wall fields originating from patterned magnetic wires, whose strength and profile are tuned by weak external fields, enable the bead trajectories within the trap to be managed and easily varied between strong confinements and delocalized spatial excursions that are described remarkably well by simulations.

Stokes vector imaging of the polarized sky-dome.

A practical method has been developed for obtaining partial Stokes vector (IQU_) and derivative (IPT_) images of the polarized sky-dome. This method takes advantage of a four-lens stereoscopic camera, a dome mirror, photo CD processing, and commercially available digital image-processing software.

Measuring the oxidation of heme compounds in heart homogenates from rats supplemented with dietary antioxidants.

Protection by antioxidant nutrients against oxidative damage in rat heart homogenates was studied. Following spontaneous oxidation of heart homogenates from rats fed vitamin E, selenium, or beta-carotene, oxidized heme proteins (OHP) and thiobarbituric acid reactive substances (TBARS) were measured. The absorbance spectra of oxidized and reduced heme proteins were analyzed with a heme spectral analysis program (HSAP) developed in this laboratory. HSAP is a multicomponent analysis program that uses successive approximations and computer spread-sheet solver functions to deconvolute a complex absorbance spectrum into individual heme protein spectra. Vitamin E markedly decreased formation of OHP, and vitamin E, selenium, or beta-carotene significantly lowered the production of TBARS during spontaneous oxidation of heart homogenates compared with homogenates from rats fed antioxidant-deficient diets. Pyridine hemochrome analysis showed that the total amounts of heme proteins present in the homogenates decreased during the oxidative incubation period. The formation of OHP correlated significantly with the amount of TBARS produced and could be simulated as a function of the oxidative and protective reactions involved in the oxidation of rat heart homogenates.

Multicomponent analysis of heme protein spectra in biological materials.

Absorption spectra of heme proteins in a tissue homogenate contain information about the oxidation status of the biological sample. Deconvolution of absorption spectra can be used to quantitate the amount of individual heme proteins present in the mixture. The heme spectral analysis program (HSAP), a computer spreadsheet program, was used to quantitatively calculate values for heme proteins measured spectrally (390 to 450 and 500 to 640 nm) in tissue homogenates undergoing oxidation. The amount of oxidized heme proteins obtained by HSAP can be compared to other measurements of tissue oxidation. Precise quantitation of the amount of heme proteins present in a homogenate sample provided accurate assessment of the oxidized heme proteins calculated by HSAP. This quantitation was achieved through modification of existing pyridine hemochrome methods. Input into HSAP of the total heme protein content via the pyridine hemochrome value generated reproducible values for oxidized heme proteins. The program has broad potential as a multicomponent analysis tool. Modification of HSAP led to the development of a difference spectra analysis program (DSAP) which was used to quantitate the type and amount of heme proteins observed in mitochondrial difference spectra. In the present application, HSAP and DSAP provide methods for interpreting complex spectral information of multicomponent biological samples that undergo oxidation.

Multiple antioxidants protect against heme protein and lipid oxidation in kidney tissue.

The effect of antioxidant nutrients in rat kidney homogenates was studied by measuring the formation of oxidized heme proteins (OHP) and thiobarbituric acid reactive substances (TBARS) during spontaneous oxidation at 37 degrees. OHP were analyzed using a modified spreadsheet protocol; the Heme Protein Spectra Analysis Program (HPSAP). Male Sprague-Dawley (SD) rats were fed a basal diet fortified with vitamin E, selenium, or beta-carotene, or a combination of all three antioxidants. A second group of male SD rats received a basal diet fortified with Trolox, ascorbic acid palmitate, acetylcysteine, beta-carotene, coenzyme Q10, coenzyme Q0, and (+)-catechin. A control group of rats was given a vitamin E- and selenium-deficient basal diet. The amount of TBARS production during a 1 h reaction decreased as the relative antioxidant effectiveness of the dietary treatments increased. Dietary treatments providing nine antioxidants significantly reduced the formation of OHP and methemoglobin during the 1 h reaction compared to the dietary treatment providing only two antioxidant nutrients. These data suggest that increasing the diversity and quantity of antioxidants in the diet provides significantly more protection for heme proteins and lipids in kidney tissue than individual antioxidants or a combination of vitamin E and selenium.

Polyunsaturated fatty acids increase lipid radical formation induced by oxidant stress in endothelial cells.

Lipid-derived free radicals were detected by electron paramagnetic resonance (EPR) spectrometry when cultured endothelial cells attached to Cytodex beads were exposed to iron-induced oxidant stress in the presence of the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN). Radical adduct formation was enhanced greatly when the cells were supplemented during growth with polyunsaturated fatty acids. The largest EPR signal intensity was observed in cells enriched with docosahexaenoic acid (DHA) or eicosapentaenoic acid, but enhanced radical adduct production also occurred after exposure to arachidonic, alpha-linolenic, gamma-linolenic, or linoleic acids. Radical adduct formation increased as the DHA content of the cells increased and approached a maximum after only 6 h of exposure to DHA. Ascorbic acid, acting as a pro-oxidant, enhanced radical adduct formation in cells enriched with DHA. The EPR signal intensity was reduced when the cells were tested 6 h after replacement of the DHA-enriched medium with a medium containing 5-20 microM oleic acid, indicating that the increased endothelial responsiveness to oxidant stress is reversible. Likewise, when U937 monocytes enriched with DHA were exposed subsequently to 20 microM oleic acid, a 35-45% decrease in radical adduct formation also occurred. These findings suggest that the endothelium may become more susceptible to oxidative injury when it is exposed to elevated amounts of polyunsaturated fatty acids. However, the effect appears to be temporary. The protective action of oleic acid against oxidant stress is not confined to the endothelium; it applies to monocytes as well.

Cell fatty acid composition affects free radical formation during lipid peroxidation.

Lipid-derived free radicals generated from intact human U937 monocytes exposed to iron-induced oxidative stress were detected by electron paramagnetic resonance (EPR) with the spin trap alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (POBN). Lipid radical formation was enhanced when the cells were enriched with n-3 or n-6 polyunsaturated fatty acids. Computer simulation indicated that at least two POBN spin adducts were formed, having spectral characteristics consistent with carbon-centered radicals (aN = 15.9 G and aH = 2.6 G; aN = 15.1 G and aH = 2.8 G). These alkyl radicals are probably formed by beta-scission of alkoxyl radicals. POBN spin adduct formation correlated with ethane generation. Addition of ascorbate to the assay medium greatly increased the radical signal intensity. Although radical generation was cell dependent and POBN spin adducts were observed in cell homogenates, the adducts formed by the intact cells were detected only in the extracellular medium. These findings indicate that the extent of lipid radical formation in response to oxidative stress can be influenced by changes in the polyunsaturated fatty acid composition of the cell lipids and suggest the possibility that carbon-centered lipi radicals may interact with extracellular structures.

In vitro virucidal effects of Allium sativum (garlic) extract and compounds.

Garlic (Allium sativum) has been shown to have antiviral activity, but the compounds responsible have not been identified. Using direct pre-infection incubation assays, we determined the in vitro virucidal effects of fresh garlic extract, its polar fraction, and the following garlic associated compounds: diallyl thiosulfinate (allicin), allyl methyl thiosulfinate, methyl allyl thiosulfinate, ajoene, alliin, deoxyalliin, diallyl disulfide, and diallyl trisulfide. Activity was determined against selected viruses including, herpes simplex virus type 1, herpes simplex virus type 2, parainfluenza virus type 3, vaccinia virus, vesicular stomatitis virus, and human rhinovirus type 2. The order for virucidal activity generally was: ajoene > allicin > allyl methyl thiosulfinate > methyl allyl thiosulfinate. Ajoene was found in oil-macerates of garlic but not in fresh garlic extracts. No activity was found for the garlic polar fraction, alliin, deoxyalliin, diallyl disulfide, or diallyl trisulfide. Fresh garlic extract, in which thiosulfinates appeared to be the active components, was virucidal to each virus tested. The predominant thiosulfinate in fresh garlic extract was allicin. Lack of reduction in yields of infectious virus indicated undetectable levels of intracellular antiviral activity for either allicin or fresh garlic extract. Furthermore, concentrations that were virucidal were also toxic to HeLa and Vero cells. Virucidal assay results were not influenced by cytotoxicity since the compounds were diluted below toxic levels prior to assaying for infectious virus. These results indicate that virucidal activity and cytotoxicity may have depended upon the viral envelope and cell membrane, respectively. However, activity against non-enveloped virus may have been due to inhibition of viral adsorption or penetration.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of lipid radicals by electron paramagnetic resonance spin trapping using intact cells enriched with polyunsaturated fatty acid.

Electron paramagnetic resonance (EPR) spin trapping was used to detect lipid-derived free radicals generated by iron-induced oxidative stress in intact cells. Using the spin trap alpha-(4-pyridyl 1-oxide)-N-tert-butylnitrone (POBN), carbon-centered radical adducts were detected. These lipid-derived free radicals were formed during incubation of ferrous iron with U937 cells that were enriched with docosahexaenoic acid (22:6n-3). The EPR spectra exhibited apparent hyperfine splittings characteristic of a POBN/alkyl radical, aN = 15.63 +/- 0.06 G and aH = 2.66 +/- 0.03 G, generated as a result of beta-scission of alkoxyl radicals. Spin adduct formation depended on the FeSO4 content of the incubation medium and the number of 22:6-enriched cells present; when the cells were enriched with oleic acid (18:1n-9), spin adducts were not detected. This is the first direct demonstration, using EPR, of a lipid-derived radical formed in intact cells in response to oxidant stress.