Electron spin resonance oximetry.
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Cyanobacteria↗
Biomedical subjects
L Packer
Publications and source records attributed to L Packer.
Determination of pH gradients in intact cyanobacteria by electron spin resonance spectroscopy.
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Cyanobacteria↗
Cytoplasmic membrane changes during adaptation of the fresh water cyanobacterium Synechococcus 6311 to salinity.
In this investigation, changes were characterized in cell structure and cytoplasmic membrane organization that occur when the freshwater cyanobacterium Synechococcus 6311 is transferred from 'low salt' (0.03 molar NaCl) to 'high salt' (0.5 molar NaCl) media (i.e. sea water concentration). Cells were examined at several time points after the imposition of the salt stress and compared to control cells, in thin sections and freeze fracture electron microscopy, and by flow cytometry. One minute after exposure to high salt, i.e. 'salt shock', virtually all intracellular granules disappeared, the density of the cytoplasm decreased, and the appearance of DNA material was changed. Glycogen and other granules, however, reappeared by 4 hours after salt exposure. The organization of the cytoplasmic membrane undergoes major reorganization following salt shock. Freeze-fracture electron microscopy showed that small intramembrane particles (diameter 7.5 and 8.5 nanometers) are reduced in number by two- to fivefold, whereas large particles, (diameters 14.5 and 17.5 nanometers) increase two- to fourfold in frequency, compared to control cells grown in low salt medium. The changes in particle size distribution suggest synthesis of new membrane proteins, in agreement with the known increases in respiration, cytochrome oxidase, and sodium proton exchange activity of the cytoplasmic membrane.
Cell Membrane↗
Blood glutathione oxidation during human exercise.
To examine the effects of increased O2 utilization on the glutathione antioxidant system in blood, eight moderately trained male volunteers were exercised to peak O2 consumption (VO2peak) and for 90 min at 65% of VO2peak on a cycle ergometer. Blood samples were taken during exercise, and for up to 4 days of recovery from submaximal exercise. During exercise to VO2peak, blood reduced glutathione (GSH) and total glutathione [GSH + oxidized glutathione (GSSG)] did not change significantly. Lactate (L), pyruvate (P), and L/P increased significantly from rest values (P less than 0.01). During prolonged submaximal exercise, GSH decreased 60% from control, and GSSG increased 100%. Total glutathione, glucose, pyruvate, and lactate concentrations and L/P did not change significantly during sustained exercise. During recovery, GSH and GSH/GSSG increased from exercise levels and significantly overshot preexercise levels, reaching maximum values after 3 days. Oxidation of GSH during submaximal exercise and its reduction in recovery suggest increased formation of active O2-. species in blood during physical exercise in moderately trained males.
Adult↗
Effect of cross linkers on the bacteriorhodopsin photocycle.
A general behavior of bacteriorhodopsin in purple membranes from Halobacterium halobium has been observed upon modification resulting in cross-linking of carboxyl and lysine groups. The rise of the M-intermediate contained two components with approximately 50-50% intensity; its decay showed three components with approximately 25-50-25% intensity respectively in a pH range of 5-9. The significance of these remarkably similar data with respect to the proton translocation mechanism in bacteriorhodopsin is that chemical modification allows us to conclude that disturbing parts of the hypothetical "proton conducting chain" does not inhibit proton translocation.
Bacteriorhodopsins↗
Effects of endurance training on a mitochondrial reticulum in limb skeletal muscle.
High voltage electron microscopy at 1500 kV, was used to examine the effects of endurance training on mitochondrial morphology in rat skeletal muscle. The soleus, deep portions of the vastus lateralis, and superficial portions of the vastus lateralis muscles were examined to represent slow-twitch-oxidative, fast-twitch-oxidative-glycolytic, and fast-twitch-glycolytic skeletal muscle fiber types, respectively. Muscle samples were removed from endurance trained and untrained control female Wistar rats (n = 6, each group). Tissues were fixed using standard electron microscopic techniques and sectioned transversely with respect to muscle fiber orientation to approximately, 0.5 micron thickness. The sections were stained on grids with uranyl acetate and Reynolds' lead citrate. Results confirmed the presence of a mitochondrial reticulum in all three skeletal muscle fiber types of both groups. Stereologic analyses indicated volume densities of intermyofibrillar mitochondria increased significantly (P less than 0.05) with endurance training in the three skeletal muscle fiber types. Surface-to-volume ratio of mitochondria was significantly decreased (P less than 0.05) after training only in the deep portion of the vastus lateralis muscle. It was concluded that the mitochondria in mammalian limb skeletal muscle are a reticulum which adapts to endurance training by proliferating.
Animals↗
Determination of dissolved oxygen in photosynthetic systems by nitroxide spin-probe broadening.
Concentrations of dissolved oxygen were monitored by following the width of the midfield line of the electron spin resonance spectrum of a nitroxide spin-probe. Measurements of peak-to-trough widths of first derivative spectra yielded accurate data over a high range of O2 concentrations (up to 5mM). Continuous traces of second harmonic line heights yielded similar results and proved to be advantageous for kinetic measurements, but were nonlinear and less sensitive at O2 levels above 2 mM. Photosynthetic oxygen evolution and respiratory oxygen uptake in the cyanobacterium Agmenellum quadruplicatum were thus examined over a broad range of oxygen concentrations. Upon prolonged (greater than 1 min) illumination, effects of photooxidative damage to both photosynthesis and respiration were demonstrated in the same experimental system. With the addition of an impermeable paramagnetic broadening agent, rapid transients in intracellular concentrations of dissolved O2 also could be measured.
Cyanobacteria↗
Quantitative determination of vitamin E and oxidized and reduced coenzyme Q by high-performance liquid chromatography with in-line ultraviolet and electrochemical detection.
We describe a sensitive quantitative high-performance liquid chromatographic (HPLC) assay for the simultaneous determination of vitamin E isomers (alpha-, gamma- and delta-tocopherol), oxidized coenzyme Q species (ubiquinone 9, ubiquinone 10) and reduced coenzyme Q homologues (ubiquinol 9, ubiquinol 10) in various tissues, including blood and plasma. The compounds of interest are quantitatively extracted with a fast one-step lipid extraction procedure and subjected to HPLC without further purification. The extract is separated on a reversed-phase column and the eluted compounds are monitored by sequential UV and electrochemical detection. Ubiquinones are detected at their 275 nm absorbance maximum, by the UV detector, whereas tocopherols and ubiquinols are monitored by the electrochemical detector with high sensitivity and selectivity. The method can detect as little as 1 pmol of the individual ubiquinones. Detection limits for tocopherols and ubiquinols are at least two orders of magnitude lower.
Animals↗
Reduction and destruction rates of nitroxide spin probes.
A series of nitroxides was tested for rates of one-electron reduction in a chemical, a photochemical, and two biological systems by ESR assays. In all cases, piperidine and hydropyridine nitroxides were reduced consistently more rapidly than pyrroline and pyrrolidine nitroxides. Substituents on the nitroxides also affected reduction rates, although not as greatly as ring structure. One of the reduction systems, consisting of the photosensitizer FMN and the photoreductant EDTA, was used to study both anaerobic reduction and O2-dependent reoxidation of some of the nitroxides. Reduced piperidine and hydropyridine nitroxides were also oxidized more rapidly than the reduced pyrroline and pyrrolidine nitroxides. Reoxidation subsequent to reduction was partially inhibited by superoxide dismutase, indicating that superoxide radicals are involved in the process. Even after prolonged reoxidation, not all of the probe molecules were returned to their oxidized form, implying an irreversible "destruction" of the spin probe concomitant with its chemical reduction. Probe destruction was studied more specifically with a photochemical system for generating methyl radicals, which showed that these carbon-centered radicals destroyed different nitroxides at rates which were much less influenced by the nitroxide structures than one-electron reduction was.
Animals↗
23Na and 31P NMR studies of the effects of salt stress on the freshwater cyanobacterium Synechococcus 6311.
We have used 23Na and 31P nuclear magnetic resonance (NMR) spectroscopy to elucidate some of the bioenergetic changes that occur in the freshwater cyanobacterium Synechococcus 6311 after a transition from growth medium (Na concentration 0.01 M) to medium containing 0.5 M NaCl. 23Na NMR analysis showed Na rapidly penetrates the cells under dark aerobic conditions; cells grown for several days in high salt medium, however, reestablish a low internal sodium content, comparable to control cells. For 31P NMR analysis, a system was devised to aerate and illuminate cell suspensions during spectral acquisition. The NMR spectra showed that when cells are presented with 0.5 M NaCl (final concentration), nucleotide triphosphate peaks decrease, the inorganic phosphate peak increases, and the cytoplasmic pH transiently increases from 7.4 to 7.9. Pyrophosphate added to cell suspensions is hydrolyzed to inorganic phosphate apparently by an extracellular phosphatase, allowing external and internal pools of inorganic phosphate to be distinguished. Nucleotide triphosphate levels fall almost as much when cells are incubated in darkness as under anoxia, indicating that both respiration and photosynthesis contribute to the maintenance of intracellular ATP levels. Cells grown in high salt medium for several generations exhibited a pattern of 31P metabolites similar to control cells, except that they produced more (and more intense) peaks in the monoester phosphate region, presumably signals from sugar phosphates.
Anaerobiosis↗
Application of photosynthetic N2-fixing cyanobacteria to the CELSS program.
The feasibility of using photosynthetic microalgae (cyanobacteria) as a subsystem component for the CELSS program, with particular emphasis on the manipulation of the biomass (protein/carbohydrate) has been addressed. Using factors which retard growth rates, but not photosynthetic electron flux, the partitioning of photosynthetically derived reductant may be dictated towards CO2 fixation (carbohydrate formation) and away from N2 fixation (protein formation). Cold shock treatment of fairly dense cultures markedly increases the glycogen content from 1% to 35% (dry weight), and presents a useful technique to change the protein/carbohydrate ratio of these organisms to a more nutritionally acceptable form.
Biomass↗
Proton gradients in intact cyanobacteria.
The internal pH values of two unicellular cyanobacterial strains were determined with electron spin resonance probes, over an external pH range of 6 to 9, in the light and in the dark. The slow growing, thylakoid-lacking Gloeobacter violaceus was found to have a low capacity for maintaining a constant internal pH. The distribution pattern of weak acid and amine nitroxide spin probes across the cell membranes of this organism, in the light and in the dark, was consistent with the assumption that it contains a single intracellular compartment. At an external pH of 7.0, intracellular pH was 6.8 in the dark and 7.2 in the light. The cells of Agmenellum quadruplicatum, a marine species, were found to contain two separate compartments; in the dark, the pH of the cytoplasmic and the intrathylakoid spaces were calculated to be 7.2 and 5.5, respectively. Upon illumination, the former increased and the latter decreased by about 0.5 pH units.
Biological Transport↗
Effect of exercise training on tissue vitamin E and ubiquinone content.
Endurance exercise training led to an adaptive increase in the ubiquinone content and cytochrome c reductase activity of red quadriceps and soleus muscles and adipose tissues, but not of cardiac or white quadriceps muscle. These findings are consistent with the well-known positive adaptation of skeletal muscle mitochondria to endurance training. However, there was no concomitant increase in the vitamin E content of tissues, which showed an increase in mitochondrial content. Since ubiquinone is located in the mitochondrial inner membrane and the major pool of vitamin E is also associated with mitochondrial membranes, the results suggest that training causes a substantial decrease in vitamin E concentration in the proliferating muscle mitochondrial membranes, thus depleting muscle mitochondria of their major lipid antioxidant. Since vitamin E is the major cellular, lipid-soluble, chain-breaking antioxidant, these findings indicate increased free radical reactions in the tissues of exercising animals.
Adipose Tissue, Brown↗
Selenium deficiency, endurance exercise capacity, and antioxidant status in rats.
Increased O2 metabolism imposed by physical exercise is likely to augment the production of active O2 species that have been shown to react with lipids, proteins, and DNA. Antioxidants and antioxidant enzymes, such as the selenium enzyme glutathione peroxidase, minimize or prevent such potentially toxic reactions. This study shows that selenium deficiency decreases glutathione peroxidase activity in liver and muscle (less than 80%, P less than 0.001), increases total glutathione in liver, muscle, and plasma (P less than 0.05) and increases muscle cytochrome oxidase activity, and ubiquinone content (P less than 0.05) but has no effect on endurance capacity. Exercise to exhaustion resulted in a significant (P less than 0.001) elevation of total and oxidized glutathione (GSSG) and a significant (P less than 0.05) decrease of vitamin E in plasma of control and selenium-deficient rats. Acute exercise also increased tissue GSSG levels in both control and selenium-deficient groups of rats. Hence, despite a large depletion of selenium-deficient glutathione peroxidase, pronounced oxidation of glutathione to GSSG can be produced by the increased oxidative metabolism during physical exercise. The results suggest that the residual glutathione peroxidase activity is sufficient to detoxify hydroperoxides in exercising selenium-deficient animals and to prevent the impairment of endurance capacity.
Animals↗
Simultaneous determination of tocopherols, ubiquinols, and ubiquinones in blood, plasma, tissue homogenates, and subcellular fractions.
A fast single-step lipid extraction procedure and high-performance liquid chromatography with in-line uv and electrochemical detection are used for the simultaneous quantitative determination of tocopherols, ubiquinols, and ubiquinones in blood, plasma, tissue homogenates, and subcellular fractions. The compounds of interest can be quantitatively extracted into hexane from a sodium dodecyl sulfate-treated aqueous homogenate after precipitation of protein by addition of an equal volume of ethanol. alpha-, gamma-, and delta-Tocopherol, ubiquinol 9, ubiquinol 10, and ubiquinones 9 and 10 can be well separated on a reversed phase column. Ubiquinones are detected at 275 nm by the uv detector, and ubiquinols and tocopherols by the electrochemical detector in the oxidative mode. Quantitation is done by comparing chromatographic peak heights to those of a standard solution containing known amounts of tocopherols, ubiquinols 9 and 10, and ubiquinones 9 and 10, analyzed under identical conditions. The high sensitivity of the electrochemical detection allows operation at low potentials (+0.5 V) with low detector response, but high selectivity for the easily oxidizable tocopherols and ubiquinols and decreased baseline noise. The uv detection limits the overall sensitivity of the procedure to 2 pmol ubiquinone, corresponding to 0.1 microM ubiquinone in the lipid extract. The ranges of values obtained for rat and guinea pig tissues, for rat liver mitochondria, and for blood and plasma from rats and humans are given.
Adipose Tissue, Brown↗
Sulfate-dependent iron oxidation by Thiobacillus ferrooxidans: characterization of a new EPR detectable electron transport component on the reducing side of rusticyanin.
Iron(II) oxidation by pH 2.5 HCl-washed cells of Thiobacillus ferrooxidans is known to be sulfate dependent. Sulfate dependence of the autooxidation of a novel component in the electron transport pathway is demonstrated. This component exhibits an electron paramagnetic resonance (EPR) signal in the oxidized state at g = 2.005 distinguishable from the g = 2.08 signal attributed to rusticyanin. The novel component is proposed to be a three-iron-sulfur cluster based upon the g value, lineshape, and temperature dependence. Oxyanion specificity for the EPR signal has the same dependence on sulfate as does iron(II) oxidation. By using azide to inhibit electron transfer to oxygen, sulfate was shown to be involved in electron transfer from the g = 2.005 component to the copper of rusticyanin.
Anions↗
The role of respiration during adaptation of the freshwater cyanobacterium Synechococcus 6311 to salinity.
Growth of the freshwater cyanobacterium Synechococcus 6311 under saline conditions stimulated respiration tenfold during the first 24 h, while growth and photosynthesis were inhibited. The elevated respiration rate was seen under both light and dark conditions, was uncoupler and cyanide sensitive, and did not decrease upon salt removal. Membrane preparations from salt-grown cells exhibited a tenfold increase in cytochrome oxidase activity, while electron transfer rates from NADPH to cytochrome c only increased threefold. Cytochrome oxidase activities were correlated with levels of EPR detectable Cu2+ in the salt and control membranes. Sodium-driven proton (antiproter) gradients in salt-grown cells were sensitive to cyanide but not dicyclohexylcarbodiimide, indicating the direct role of respiratory electron transport in maintaining low intracellular sodium levels.
Acridine Orange↗
Aggregation and proton release of purple and white membranes following cleavage of the carboxyl-terminal tail of bacteriorhodopsin.
Our results indicate that the previously reported decrease in proton release by proteolyzed purple membrane sheets was due merely to the aggregation state of these preparations and not to the loss of the carboxyl-terminal tail. Changes in H+/M412 ratios obtained for purple and white membrane preparations correlate with the measured aggregation. White membrane preparations consistently exhibit H+/M412 ratios more than twice those measured for native purple membranes under the same conditions. Quasi-elastic light scattering was used to characterize the size of isolated purple and white membrane sheets before and after proteolysis. The results clearly show that native purple membrane preparations are larger in size than would be expected and that, following trypsin treatment, they are on average more than an order of magnitude larger. Negative staining electron microscopy showed that the purple membrane became aggregated in stacked arrays. Bleaching and reconstitution with retinal also affect aggregation, but iodination or nitration of purple membrane does not affect the measured size. The average size of white membranes is smaller; this is consistent with results of electron microscopy and the size increase is much less than that of purple membranes following trypsin treatment. No size change occurs with retinal reconstitution. In aggregated purple membrane preparations, protons and other cations are unable to exchange freely with the aqueous medium, explaining why proteolysis lowers the proton release from purple membrane sheets in suspension.
Bacteriorhodopsins↗