Role of nitric oxide and mitochondria in control of firefly flash

In light-producing cells (photocytes) of the firefly light organ,mitochondria are clustered in the cell periphery, positionedbetween the tracheolar air supply and the oxygen-requiring bioluminescentreactants which are sequestered in more centrally-localizedperoxisomes. This relative positioning suggests that mitochondriacould control oxygen availability for the light reaction. Wehypothesized that active cellular respiration would make theinterior regions of the photocytes relatively hypoxic, and thatthe "on" signal for production of bioluminescence might dependon inhibition of mitochondrial oxygen consumption, which wouldallow delivered oxygen to pass through the peripheral mitochondrialzone to reach peroxisomes deep in the cell interior. We publishedrecently that exogenous NO induces bioluminescence in the intactfirefly; that NO mediates octopamine-induced bioluminescencein the dissected lantern, and that nitric oxide synthase isabundant in cells of the tracheolar system of the light organ.Additional experiments showed that nitric oxide gas (NO) inhibitsrespiration in isolated lantern mitochondria. Inhibition isreversed by bright light, and this inhibition is relieved whenthe light is turned off. Altogether, the results support theidea that NO triggers light production by reversible inhibitionof mitochondrial respiration in lantern cells, and probablyin tracheolar cells as well. The data also suggest that thelight of bioluminescence itself relieves NO inhibition thuscontributing to rapid on/off switching. While other mechanismsmay be in play, NO production that is directly related to neuralinput appears to have a key role in the oxygen gating that controlsflash communication signals.     

Ventilatory Mechanism and Control in Grasshoppers

SYNOPSIS. Grasshoppers exhibit a diversity of ventilatory patternsdepending on activity status. For each pattern, the mechanismand control of gas exchange is analyzed in terms of a two-stepmodel, consisting of tracheolar and trans-spiracular steps inseries. During the intermittent gas exchange that characterizesthe most quiescent grasshoppers, spiracles open and close inresponse to changing carbon dioxide, and trans-spiracular resistancecontrols gas exchange. In resting but alert grasshoppers, abdominalpumping occurs, and gas exchange is controlled equally by tracheolarand trans-spiracular resistances; tracheal oxygen and carbondioxide are regulated by variation in abdominal pumping andspiracular opening. During hopping, abdominal pumping does notoccur, and bulk gas flow is driven by cuticular deformationsassociated with locomotion. Increased cellular oxygen consumptiondepends on use of internal oxygen stores and increased partialpressure gradients. After hopping ceases, abdominal pumpingincreases dramatically and restores tracheal gas composition;however, the rise in abdominal pumping after hopping is notaffected by tracheal gas levels. During flight, bulk flow tothe flight muscles is driven by tidal thoracic auto-ventilation,while the remainder of the body is ventilated by abdominal pumping.During both hopping and flight, the greatest resistances togas transport exist in the tracheolar rather than the trans-spiracularstep.     

Mosquito (Aedes aegypti ) aquaporin, present in tracheolar cells, transports water, not glycerol, and forms orthogonal arrays in Xenopus oocyte membranes.

Previous results showed that mRNA encoding a putative aquaporin (AQP) (GenBank accession number AF218314) is present in the tracheolar cells associated with female Aedes aegypti Malpighian tubules. In this study, immunohistochemistry detected the protein, AeaAQP, also in tracheolar cells, suggesting its involvement in water movement in the respiratory system. When expressed in Xenopus oocytes, AeaAQP increased the osmotic water permeability from 15 x 10(-6) to 150 x 10(-6) m x s-1, which was inhibited by mercury ions. No permeability to glycerol or other solute was observed. AeaAQP expressed in oocytes was solubilized as a homotetramer in nondenaturing detergent as deduced from velocity centrifugation on density gradients. Phylogenetic analysis of MIP (major intrinsic protein) family sequences shows that AeaAQP clusters with other native orthogonal array forming proteins. Specific orthogonal arrays were detected by freeze-fracture analysis of AeaAQP oocyte membranes. We conclude that, in tracheolar cells of A. aegypti, AeaAQP is probably a highly water-permeable homotetrameric MIP which natively can form 2D crystals.     

Structure and function of the respiratory epithelium in the tracheal gills of stonefly larvae

The respiratory epithelium in the tracheal gills of Perla, Protonemura, and Taeniopteryx has numerous tracheoles beneath the cuticle, which are extracellularly located in deep indentations of the plasma membrane and run parallel to the longitudinal axis of the gill filaments. In the latter two species, the tracheoles are extremely densely packed at interspaces normally not exceeding the tracheolar diameters, which vary between 0·2 and 1 μ. This type of tracheation, when compared with the highly ordered tracheation in the tracheal gills of caddis fly larvae, requires a surplus of tracheolar material for the full utilization of the respiratory surface area. The functional significance of the tracheation types involving a minimum or surplus of tracheolar material is interpreted in terms of the diffusion theory of respiration.     

Zur Morphogenese des respiratorischen Epithels der Tracheenkiemen bei Larven der Limnephilini KOL. (Insecta,Trichoptera)

Zusammenfassung Das respiratorische Epithel der Tracheenkiemen ist durch ein hochgeordnetes Tracheolengerüst charakterisiert. Die Tracheolen liegen parallel zur Längsachse der fadenförmigen Tracheenkiemen dicht unter der Cuticula in statistisch gleichmäßigem Abstand zueinander. Die Regelmäßigkeit dieses subcuticularen Tracheolengerüsts weist auf ein physiologisch optimal arbeitendes System hin. Der Abstand zwischen zwei Tracheolen ist sehr wahrscheinlich gleich dem doppelten Radius der tracheolaren Einzugsgebiete. Auf diese Weise wird bei einem Minimum an Tracheolenmaterial der gesamte diffundierende Sauerstoff der respiratorischen Oberfläche von den Tracheolen erfaßt. Die Morphogenese dieser Strukturregelmäßigkeit wird während der larvalen Entwicklung verfolgt. Dabei zeigt sich, daß mit jeder Häutung zahlreiche neue Tracheolen in das respiratorische Epithel geordnet eingebaut werden und die Abstände zwischen den Tracheolen in Korrelation zum Radius der Tracheolen von Larvenstadium zu Larvenstadium geregelt abnehmen.

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Morphogenesis of the respiratory epithelium in the tracheal gills of larval Limnephilini KOL. (Insecta, Trichoptera)

Summary The respiratory epithelium of the tracheal gills of the larval Limnephilini KOL. (Insecta, Trichoptera) is characterized by a highly organized tracheolar framework. The tracheoles are found parallel to the longitudinal axis of the thread-like tracheal gills and lie closely underneath the cuticle at statistically uniform distances. The regular distribution of the subcuticular tracheoles represents an optimum physiological system with the tracheolar interspace probably corresponding to twice the radius of the tracheolar catchment area. This arrangement ensures that all oxygen diffusing across the respiratory gill surface is taken up by the tracheoles with a minimum of tracheolar material. The morphogenesis of this regular distribution was studied during the larval development. With each moult numerous new tracheoles are added to the regular distribution. The distances between the tracheoles decrease regularly in correlation to the decreasing radius of the tracheoles from one larval stage to the next.

     

Permeable sites in the firefly lantern tracheal system: Use of osmium tetroxide vapor as a tracer

Flashing fireflies were permitted to breathe osmium tetroxide vapor, after which the lanterns were removed and the sites of absorption of the osmium into the tissues were detected in two ways: (1) by sonication to remove soft tissues, that is, those that had not been fixed by the osmium gas, and (2) by intensification with thiocarbohydrazide and silver nitrate, in a modification of the osmium–thiocarbohydrazide–osmium (OTO) stain technique. The results of both procedures indicate that the gas first enters into the tissues at the level of the tracheoles. These findings may be interpreted as underscoring the importance of the tracheolar cell and the tracheal end organ in the control of oxygen entry into the lantern tissues, and the implications of the results in the oxygen regulation theory of flash control are discussed.     

The effect of model approximations on single-collision distributions of low-energy electrons in liquid water

The development of cross sections for the inelastic interaction of low-energy electrons with condensed tissue-like media is best accomplished within the framework of the dielectric theory. In this work we investigate the degree to which various model approximations, used in the above methodology, influence electron single-collision distributions. These distributions are of major importance to Monte Carlo track structure codes, namely, the energy-loss spectrum, the inelastic inverse mean free path, and the ionization efficiency. In particular, we make quantitative assessment of the influence of (1) the optical data set, (2) the dispersion algorithm, and (3) the perturbation and exchange Born corrections. It is shown that, although the shape and position of the energy-loss spectrum remains almost fixed, its peak height may vary by up to a factor of 1.5. Discrepancies in the calculated inelastic inverse mean free path are largely within 20-30% above 100 eV; they increase drastically, though, at lower energies. Exchange and perturbation Born corrections increase gradually below 1 keV leading to a approximately 30 to 40% reduction of the inverse mean free path at 100 eV. The perturbation effect contributes more than the exchange effect to this reduction. Similar to the dispersion situation, the effect of Born corrections at lower energies is also unclear since the models examined disagree strongly below 100 eV. In comparison, the vapor data are higher than the liquid calculations by 20 to 50% as the energy decreases from 1 to 0.1 keV, respectively. The excitation contribution is the main cause of this difference, since the ionization efficiency in the liquid levels off at approximately 90%, whereas the plateau value for the vapor is approximately 70%. It is concluded that electron inelastic distributions for liquid water, although in some respects distinctively different from the vapor phase, have associated uncertainties that are comparable in magnitude to the phase differences. The situation below 100 eV is uncertain.     

A difference equation method for the calculation of the probability that two molecules will collide

The probability that one molecule will collide with another is calculated for the case in which the molecule is confined to equally spaced rings about the second larger molecule except when it jumps from one ring to the next. The result is practically the same as that obtained for an infinitesimal mean free path, not having first-order terms in the ratio of the mean free path to the particle radius as does the result of the calculations of R. Wijsman (1952).     

Mechanism(s) for the metabolism of mitoxantrone: electron spin resonance and electrochemical studies

Mitoxantrone has been reported to lack certain properties that characterize quinone containing antitumor agents that undergo enzymatic reduction. These properties are the stimulation of NADPH oxidation, the stimulation of oxygen consumption by microsomes and reductases and, the absence of oxygen free radicals during these reactions. Having these properties implies the presence of a futile redox cycle that requires the generation and the oxidation of a semiquinone free radical. It would follow that if mitoxantrone does not redox cycle in the presence of reductases, then the semiquinone free radical is not produced or, if it is formed, it reacts quickly to form diamagnetic products. However, using liver microsomes, there are reports of the formation of the mitoxantrone free radial anion. In this paper we investigated the mitoxantrone free radical anion generated electrochemically and found that in the presence of oxygen it behaved like other semiquinones. That is, it is oxidized to the parent compound (presumably generating oxygen free radicals), indicating the ability to redox cycle. The reduction potential to generate such free radical in aqueous medium is very high (-0.79 V) when compared to diaziquone (-0.36 V) and Adriamycin (-0.6 V). This suggests that mitoxantrone may not be a substrate for reductases. Under reductive conditions with purified NADPH cytochrome P-450 reductase which very easily reduces diaziquone and Adriamycin, mitoxantrone was not reduced. However, under the same conditions, mitoxantrone was oxidized by the prototype oxidase horseradish peroxidase with the production of a mitoxantrone free radical. This oxidation was accompanied by a drastic change in color and the formation of a dark precipitate. Because microsomes contain a variety of enzymes, we suggest that the previously observed free radical in microsomes is probably due to the oxidation of mitoxantrone. In this theory, this product is probably a polymer which would not require oxygen to be formed. Thus, under oxidative conditions, the mitoxantrone free radical cation will also display impaired redox activity.     

The transition metal-mediated formation of the hydroxyl free radical during the reduction of molecular oxygen by ferredoxin-ferredoxin:NADP+ oxidoreductase

The NADPH-supported enzymatic reduction of molecular oxygen by ferredoxin-ferredoxin:NADP+ oxidoreductase was investigated. The ESR spin trapping technique was employed to identify the free radical metabolites of oxygen. The spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to trap and identify the oxygen-derived free radicals. [17O]Oxygen was employed to demonstrate that the oxygen-centered radicals arose from molecular oxygen. From the data, the following scheme is proposed: (Formula:see text). The formation of the free hydroxyl radical during the reduction of oxygen was demonstrated with quantitative competition experiments. The hydroxyl radical abstracted hydrogen from ethanol or formate, and the resulting scavenger-derived free radical was trapped with known rate constants. If H2O2 was added to the enzymatic reaction, a stimulation of the production of the hydroxyl radical was obtained. This stimulation was manifested in both the concentration and the rate of formation of the DMPO/hydroxyl radical adduct. Catalase was shown to inhibit formation of the hydroxyl radical adduct, further supporting the formation of hydrogen peroxide as an intermediate during the reduction of oxygen. All three components, ferredoxin, ferredoxin:NADP+ oxidoreductase, and NADPH, were required for reduction. Ferredoxin:NADP+ oxidoreductase reduces ferredoxin, which in turn is responsible for the reduction of oxygen to hydrogen peroxide and ultimately the hydroxyl radical. The effect of transition metal chelators on the DMPO/hydroxyl radical adduct concentration suggests that the reduction of chelated iron by ferredoxin is responsible for the reduction of hydrogen peroxide to the hydroxyl radical via Fenton-type chemistry.     

Effect of Design Parameters on Oxygen Mass Transfer Coefficient in a Free Jet Bioreactor

The rate of oxygen delivery is a very important factor in aerobic processes. In many cases, this determines the efficiency of processes such as biological wastewater treatment. The availability of oxygen is essential for sustaining the growth of microorganisms necessary for degrading the organic matter. In this work, a continuous, re‐circulation, constant hold‐up plunging free jet‐loop system was designed to study the effects of design parameters on the oxygen mass transfer. This was investigated using three different nozzle diameters 6, 10 and 14 mm. For a fixed value of diameter, the distance between the nozzle mouth and the free liquid surface was varied to cover the range 14–26 cm. The experimental results showed significant effects of these two parameters. The oxygen mass transfer coefficient was found to increase with either increasing the nozzle position or decreasing its diameter.     

Oximetry in cells and tissues using a nitroxide-liposome system

In order to avoid the complication of reduction of nitroxides in biological media during oxygen measurements, liposomes containing a water-soluble nitroxide, 2,2,6,6-tetramethyl-piperidine-N-oxyl-4-trimethylammonium (Cat1), were used in studies of oxygen consumption by thymus-bone-marrow cells. The superhyperfine structure of Cat1 contained in liposomes was found to be sensitive to oxygen concentration in a fashion similar to that of free Cat1. Measurements of cellular respiration using Cat1 contained in liposomes agreed well with the results obtained using free Cat1. Using this nitroxide-liposome system, the respiration of liver slices was measured successfully, whereas such measurements using free cat1 were complicated by rapid reduction of the nitroxide. This nitroxide-liposome system also could be used in conjunction with a membrane permeable nitroxide and an extracellular broadening agent to measure intracellular and extracellular oxygen concentrations simultaneously.     

In situ oxygen consumption rates of cells in V-79 multicellular spheroids during growth

The rate of consumption of oxygen by V-79 cells in multicellular spheroids was measured as a function of the spheroid diameter. In situ consumption was equal to that of exponentially growing cells for spheroids less than 200 micron in diameter. The rate of oxygen consumption decreased for cells in spheroids between 200 and 400 micron diameter to a value one-fourth the initial, then remained constant with further spheroid growth. Comparison of consumption rates for spheroid-derived cells before and after dissociation from the spheroid structure indicated that the spheroid microenvironment accounted for only 20% of the change in oxygen consumption rate. Cell-cell contact, cell packing, and cell volume were not critical parameters. Plateau-phase cells had a fivefold lower rate of oxygen consumption than exponential cells, and it is postulated that the spheroid quiescent cell population accounts for a large part of the intrinsic alteration in oxygen consumption of cells in spheroids. Some other mechanism must be involved in the regulation of cellular oxygen consumption in V-79 spheroids to account for the remainder of the reduction observed in this system.     

Use of a microbial sensor: a new approach to the measurement of inhibitory effects on the microbial activity of activated sludge

This paper presents a new method for the measurement of inhibitory effects in wastewater treatment plants on the basis of a continuous measurement of the microbial respiration product (CO(2)). The microbial sensor developed for this purpose consists of a small conical fluidized bed reactor connected to a cylindrical chamber that comprises part of the sample recirculation system. Activated sludge microbes are immobilized on spherical (diameter=1-2 mm) reticulated sinter glass carriers. Pure oxygen is supplied via the cylindrical chamber in order to sustain a highly dense population of microbial mass. The mean hydraulic retention time in the microbial sensor ranges between 30 and 40 min, while temperature is maintained at 30 degrees C, and pH 6.4. Carbon dioxide in the off-gas, which reflects the microbial activity, is continuously analyzed by means of an infrared analyzer. Inhibition of microbial activity (toxicity) can be determined as the mean percent reduction in carbon dioxide concentration. Several substances were tested and proved toxic to the microbes. With this microbial sensor, early detection of toxic substances becomes feasible, preventing them from entering an activated sludge unit operation.     

AN INVESTIGATION INTO THE CAUSE OF THE SPONTANEOUS AGGREGATION OF FLAGELLATES AND INTO THE REACTIONS OF FLAGELLATES TO DISSOLVED OXYGEN : PART II.

Spontaneous aggregations of flagellates are formed under the cover-glass because the organisms are attracted to and remain in regions where the concentration of dissolved oxygen is less than the saturation concentration under atmospheric partial pressure. These regions of lessened oxygen content arise towards the center of the liquid beneath the cover-glass, owing to the oxygen consumed by the flagellates in respiration not being replaced here by the solution of atmospheric oxygen, as it is along the edges of the liquid. The flagellates, however, are insensitive to the attraction of regions of lessened oxygen concentration when the oxygen concentration throughout the liquid is above a certain value. Therefore, for the aggregations to form, either the initial concentration of dissolved oxygen must be below this limiting value, or an interval of time must first elapse after the making of the preparation until the respiration of the organisms has reduced the oxygen concentration throughout the liquid down to this limiting value. The aggregations will then form because the flagellates have become positively chemotropic to the lower concentration of oxygen at the center of the liquid. Once established, such an aggregation of flagellates does not remain long in the same form. An area free from flagellates appears at the center of the aggregation so that the organisms lie in a circular band surrounding the clear area. The latter increases in size and its bordering band of flagellates in diameter, the band gradually becoming less circular and more square in shape, if the cover-glass is a square one. The clear central area is a region where the oxygen consumption of the flagellates has reduced the oxygen content to such a low value that the organisms are forced to leave the region. They collect in a band where the concentration of dissolved oxygen is an optimum for them. It is the equilibrium position between the oxygen consumed at the center and that diffusing in from the edges of the liquid. As the consumption at the center is more rapid than the replacement from the edge, the flagellate band moves outwards until it becomes stationary at a position where the rates of consumption and replacement of oxygen are equal. Although the flagellates collect in this manner in regions of optimum oxygen concentration, yet greater concentrations of dissolved oxygen have no injurious effect on them. Concentrations of dissolved oxygen lower than the optimum have the effect of inhibiting the movement of the flagellates. They recover their activity, however, immediately they are given access to dissolved oxygen again. Work done in the past on chemotropism of flagellates will have to be revised in the light of the above facts, since the oxygen content of solutions used has never been taken into account.     

Spatial energy transfer with observation of bimolecular singlet oxygen emission using quantum dots as donors and zinc‐phthalocyanine as acceptors

This study reports the influence of CdSe–ZnS core–shell quantum dots (QDs) for formation of singlet oxygen using zinc‐phthalocyanine (ZnPc) dyes in colloidal solutions. Using a microluminescence surface scan technique it was possible to measure accurately the photon diffusion length, or photon mean free path, inside the medium. Analyses were performed for a range of QD concentrations. Photon diffusion length was assigned to the bimolecular singlet oxygen emission at 707 nm. Related singlet oxygen emission was predicted by observing quenching of the photon diffusion length measured at the specific oxygen emission as a function of QD concentration, being a nontrivial phenomenon related to the QD donors. Diffusion length measured at 707 nm increased with QD concentration; in the absence of QDs, as in pure ZnPc samples, the emission peak at 707 nm was not observed.     

Physiological aspects of free and immobilized Aspergillus niger cultures producing citric acid under various glucose concentrations

Similarities and differences between cultures of free and immobilized Aspergillus niger were identified under various glucose concentrations. Growth and citric acid production rates were compared, and the macro-morphology and fine structure of the mycelia examined to determine which parameters were significant in the production of citric acid. With free cultures the diameter of pellets was inversely related to glucose concentration, while biomass levels were lower for immobilized cultures than the equivalent free cultures. Rates of citric acid production were higher with immobilized mycelium, especially at higher glucose levels. The morphology that characterized high citric acid productivity was that of swollen hyphal tips which were seen at concentrations over 100 kg/m³ glucose in both free and immobilized mycelium. Although there is a characteristic morphology associated with high productivity it does not account for the difference observed between free and immobilized mycelia. The increased glucose uptake and productivity was not due to an increased surface area either, since the immobilized system was slightly lower in total surface area than the equivalent free cultures. The major difference was in the mean diffusion path in the two systems.     

Transport calculations for light scattering in blood.

In vivo measurement of the oxygen saturation levels in blood may be obtained from relative amounts of backscattered monochromatic light at two different wavelengths, as measured with a fiber-optic catheter oximeter. Because of the short mean free path length of light in blood, the backscattering can be well approximated by a previously-derived, one-wavelength transport theory solution for the half-space searchlight problem. This solution, unlike simple diffusion approximations has the advantage that the boundary condition describing illumination of a localized area of blood by a monodirectional light beam can be rigorously satisfied. Sample calculations using the solution are compared with experimental values of the reflectance of blood.     

Localization of the Site of Oxygen Radical Generation inside the Complex I of Heart and Nonsynaptic Brain Mammalian Mitochondria

Mitochondrial production of oxygen radicals seems to be involved in many diseases and aging. Recent studies clearly showed that a substantial part of the free radical generation of rodent mitochondria comes from complex I. It is thus important to further localize the free radical generator site within this respiratory complex. In this study, superoxide production by heart and nonsynaptic brain submitochondrial particles from up to seven mammalian species, showing different longevities, were studied under different conditions. The results, taking together, show that rotenone stimulates NADH-supported superoxide generation, confirming that complex I is a source of oxygen radicals in mammals, in general. The rotenone-stimulated NADH-supported superoxide production of the heart and nonsynaptic brain mammalian submitochondrial particles was inhibited both by p-chloromercuribenzoate and by ethoxyformic anhydride. These results localize the complex I oxygen radical generator between the ferricyanide and the ubiquinone reduction site, making iron—sulfur centers possible candidates, although unstable semiquinones can not be discarded. The results also indicate that the previously described inverse correlation between rates of mitochondrial oxygen radical generation and mammalian longevity operates through mechanisms dependent on the presence of intact functional mitochondria.     

Niridazole metabolism by rat embryos in vitro

We report the results of studies on the reductive activation of the schistosomicidal agent, niridazole (NDZ). Intact rat embryos in vitro reduced this compound, generating a stable metabolite in the presence of 5% O2. By contrast, embryo and yolk sac homogenates or liver microsomes appeared to require anaerobiasis. Malformation incidence--specifically, axial asymmetry--showed a strong correlation with nitroreductase activity rates when the latter were modulated by oxygen tension. Data presented here suggest that when embryos are exposed to NDZ under conditions of low oxygen in vitro, redox cycling ensues with molecular oxygen serving to oxidize early reduction products. This process continues, regenerating the parent compound until oxygen is depleted locally. The basis of this localized depletion is unknown, but inability of the immature supply system to replete oxygen or demand by precociously aerobic tissues may be involved. Once local anaerobiasis is attained, further reduction could generate toxic metabolites capable of covalently binding cellular macromolecules. Localized hypoxia represents another potential mechanism of dysmorphogenesis.