Capacity of cytochrome and alternative path in coupled and uncoupled root respiration of Pisum and Plantago

A critical duration of darkness must be exceeded for the photoperiodic induction of flowering in short-day plants. This requires detection of the light/dark transition at dusk and the coupling of this information to a time-measuring system.
Lowering the P_fr/P_tot, ratio photochemically at the end of the day did not accelerate the onset of dark timing in Pharbitis nil Choisy cv. Violet. Time-measurement was initiated when, with no change in spectral quality, the irradiance fell below a threshold value. Thus, if the light/dark transition at dusk is sensed by a reduction in P_fr, this reduction can be achieved as rapidly through thermal reactions as through photochemical ones. When given at hourly intervals during a 6-h extension of a 24-h main light period in white light, pulses of red light were as effective as continuous red light in delaying the onset of timing; pulses every 2 or 3 h were less effective. The effectiveness of intermittent red light indicates that phytochrome is the photoreceptor and the requirement for frequent exposures suggests that P_fr is lost rapidly in the dark. However, the red light pulses could not be reversed by far-red light, which argues against this hypothesis. An alternative explanation is that the perception of light as being continuous occurs only when "new" P_fr is regenerated sufficiently frequently.
The nature of the coupling of the dusk signal to the time-measuring system is discussed and it is suggested that the effect of each red light pulse is to delay the phase of the photoperiodic rhythm by 1–3 h.     

Oxygen dependence of respiration in rat spinotrapezius muscle in situ

The oxygen dependence of respiration in striated muscle in situ was studied by measuring the rate of decrease of interstitial Po(2) [oxygen disappearance curve (ODC)] following rapid arrest of blood flow by pneumatic tissue compression, which ejected red blood cells from the muscle vessels and made the ODC independent from oxygen bound to hemoglobin. After the contribution of photo-consumption of oxygen by the method was evaluated and accounted for, the corrected ODCs were converted into the Po(2) dependence of oxygen consumption, Vo(2), proportional to the rate of Po(2) decrease. Fitting equations obtained from a model of heterogeneous intracellular Po(2) were applied to recover the parameters describing respiration in muscle fibers, with a predicted sigmoidal shape for the dependence of Vo(2) on Po(2). This curve consists of two regions connected by the point for critical Po(2) of the cell (i.e., Po(2) at the sarcolemma when the center of the cell becomes anoxic). The critical Po(2) was below the Po(2) for half-maximal respiratory rate (P(50)) for the cells. In six muscles at rest, the rate of oxygen consumption was 139 ± 6 nl O(2)/cm(3)·s and mitochondrial P(50) was k = 10.5 ± 0.8 mmHg. The range of Po(2) values inside the muscle fibers was found to be 4-5 mmHg at the critical Po(2). The oxygen dependence of respiration can be studied in thin muscles under different experimental conditions. In resting muscle, the critical Po(2) was substantially lower than the interstitial Po(2) of 53 ± 2 mmHg, a finding that indicates that Vo(2) under this circumstance is independent of oxygen supply and is discordant with the conventional hypothesis of metabolic regulation of the oxygen supply to tissue.     

Structure of coupled and uncoupled cell junctions

Cells of Chironomus salivary glands and Malpighian tubules have junctions of the "septate" kind. This is the only kind of junction discerned which is large enough to effect the existing degree of intercellular communication. The electron microscopic observations of the "septate" junction conform to a honeycomb structure, with 80-A-thick electron-opaque walls and 90-A-wide transparent cores, connecting the cellular surface membranes. A projection pattern of light and dark bands (the "septa") with a 150-A periodicity results when the electron beam is directed normal to any set of honeycomb walls. Treatment of the salivary gland cells with media, which interrupt cellular communication (without noticeable alteration of cellular adhesion) by reducing junctional membrane permeability or perijunctional insulation, produces no alterations in the junctional structure discernible in electron micrographs of glutaraldehyde-fixed cell material.     

Intrinsic uncoupling in proton-pumping cytochrome c oxidase: pH dependence of cytochrome c oxidation in coupled and uncoupled phospholipid vesicles

The pH dependence of the transient aerobic kinetics of cytochromes c and a has been investigated with cytochrome oxidase reconstituted in phospholipid vesicles in the absence and presence of an uncoupler and an ionophore. The cytochrome a reduction level immediately after the burst phase was 60-80% and was not significantly changed by the addition of uncoupler and/or ionophore. The coupled rate of ferro-cytochrome c oxidation increases linearly with decreasing pH in the range 8.4-5.4. The increase in rate on uncoupling becomes less with decreasing pH and low cytochrome c concentration, being almost zero at pH 5.4. The coupled rate is increased by a lowering of the outside pH when the inside pH is constant. Varying the inside pH with a constant outside pH of 7.4 has little effect on the rate. It is suggested that the electrochemical potential has two separate effects on the coupled rate: the pH gradient mainly slows down the intramolecular electron transfer, but the membrane potential also lowers the second-order rate constant for the reaction with cytochrome c. The results are interpreted in terms of a model in which protonation of an acid-base group with a pKa of 6.4 from the inside increases the catalytic constant. Protonation from the outside, on the other hand, leads to an intrinsic uncoupling, because the protonated enzyme in the output state can return to the input state. This has no adverse physiological effect, since it becomes significant only at pH values well below 7.     

UCP4C mediates uncoupled respiration in larvae of Drosophila melanogaster

Larvae of Drosophila melanogaster reared at 23°C and switched to 14°C for 1 h are 0.5°C warmer than the surrounding medium. In keeping with dissipation of energy, respiration of Drosophila melanogaster larvae cannot be decreased by the F‐ATPase inhibitor oligomycin or stimulated by protonophore. Silencing of Ucp4C conferred sensitivity of respiration to oligomycin and uncoupler, and prevented larva‐to‐adult progression at 15°C but not 23°C. Uncoupled respiration of larval mitochondria required palmitate, was dependent on Ucp4C and was inhibited by guanosine diphosphate. UCP4C is required for development through the prepupal stages at low temperatures and may be an uncoupling protein.     

Oxygen dependence of nuclear DNA replication in Ehrlich ascites cells

Oxygen was excluded from cultured Ehrlich ascites cells for 5-7 h and then readmitted. During the anaerobic period and for about 1 h following reoxygenation the DNA synthesis of the cells was studied by determining the DNA synthesis rate from [3H] thymidine incorporation data, by evaluation of the thymidine (pulse labelling) index, by DNA fibre autoradiography, and by alkaline sucrose gradients in order to follow the maturation of the daughter chains. The DNA synthesis rate was found to decay to a few percent of the initial value within 5-7 h after deoxygenation. Immediately after reoxygenation it increased to exceed the control level within 0.5-1 h. The only partial process of the genome replication definitely responding to deoxygenation/reoxygenation was the initiation of new replicon units, while progress of the replication forks and maturation of the new daughter chains were not significantly affected. The coordination of replicon initiation within groups or clusters was maintained throughout. The interruption of replication at the level of initiation of clusters upon deoxygenation was interpreted as a regulatory response of the cells to ensure basic viability under unfavourable conditions.     

Phosphorylation and uncoupled respiration in the tissue of rats during the development of homoiothermy

A marked increase in the rate of mitochondrial respiration, not coupled with ADP phosphorylation, was noted during the transformation of newborn poikilothermic animals into homoeothermic ones in the experiment on the rat tissue homogenates. Uncoupled respiration, as well as coupled one, is realized by the mitochondrial respiration chain, is observed upon oxidation of NADH, succinate, ascorbate and is expressed by a high rate of O2 consumption in the absence of added ADP. During ontogenesis, uncoupled respiration is activated to a greater extent in the heart and skeletal muscle and to a lesser extent in the liver and brown fat. The rates of phosphorylating oxidation of different substrates in tissue homogenates of animals from various age groups differ insignificantly. It is supposed that the postnatal development of homoeothermism in rats is ensured by the formation in many tissues of a system of uncoupled respiration, which takes part in heat production without preliminary ATP synthesis.     

Spatial and temporal dependence of the cerebral endothelial cells elasticity

The reliable determination of the mechanical properties of a living cell is one of the most important challenges of the atomic force microscopic measurements. In the present study the spatial and temporal dependency of the force measurements on cerebral endothelial cells was investigated. Besides imaging the cells, two different sequences of force measurements were applied: Acquisition of force curves in short time at several points across the cell surface investigating spatial dependence of the elasticity. Acquisition of force curves for long time at a previously determined place, over the cell nucleus, which provides the temporal stability/variation of the measured forces/values. Three different stages of endothelial cell cultures of the hCMEC/D3 cells were used: sub-confluent living, confluent living, and confluent fixed cells. The Young's modulus was calculated from the force curves using the Hertz model and the results were plotted against time or location correspondingly. The rational of using the three stage of culture was to clarify whether the observed effect belongs to the individual cell, to the ensemble of cells or just to some, not living cell component. In case of sub-confluent cells the results revealed a softer nuclear region compared to the periphery, while an attenuated oscillation like fluctuation in time, with a period of about 10-30 min, was observed. Confluent living cells showed similar tendencies to the sub-confluent cells, but the changes were larger and the temporal oscillations had longer period. The spatial dependency of the elasticity on confluent cells was confirmed by force-volume measurement too. In case of fixed cells neither spatial nor temporal differences were observed between the nuclear and peripheral region, however the Young's modulus and the error of the measurement was larger, compared to the sub-confluent living cells.     

The respiration of cells and mitochondria of porin deficient yeast mutants is coupled

Several mutants of yeast lacking the porin gene have been found stable and viable on glucose or glycerol media. Ethanol-supported respiration of porin-free mutant and wild cells appeared equally coupled in vivo being similarly depressed by inhibitors of ADP/ATP translocase or of ATP synthase and stimulated by the uncoupler FCCP. The absence of porin in isolated mutant mitochondria hardly impaired the electron flux but increased the requirement for Mg2+ (or Ca2+) and for ADP and carboxyatractylate concentrations necessary to drive effectively state 3 - state 4 and state 4 - state 3 transitions, respectively. The existence of another porin species, possibly controlled by bivalent cations, is postulated.     

The oxygen dependence of the mitochondrial respiration rate in ascites tumor cells.

The effect of the oxygen concentration on the rate of oxygen consumption by 786 and TA3 ascites tumor cell lines has been determined under steady-flow conditions with a membraneless fast-responding O2 electrode and using ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine as electron donors. The reaction was initiated by rapid injection of O2 into anaerobically incubated test system. The time-dependence of the intact cell respiration showed three distinct phases; an early very fast but short duration phase, a subsequent slow phase that prevailed for most of the reaction period and a third phase which preceded the reestablishment of anaerobiosis. Kinetic analysis of the reaction indicated a linkage between the catalytic efficiency and the transmembrane electrochemical potential. The rates of O2 uptake, obtained in the presence of both protonophores and ionophores, were monotonic and pseudo-first order over 90% of the course of O2 consumption. Extrapolation of the observed rates to zero time, at which zero delta mu H+ and thus constant flow prevails, was used to calculate the oxygen concentration for the half-maximal respiratory rate, which was found to be in the range 1.55-2.10 microM O2. No noticeable variation in the value of this kinetic parameter was found between the two cell lines used. Possible reasons for discrepancies in published reports on the oxygen dependence of the cytochrome c oxidase activity in various mitochondrial and reconstituted systems are discussed.     

Some effects of hydrolytic enzymes on coupled and uncoupled electron flow in chloroplasts

Digestion of spinach chloroplasts with pancreatic lipase or trypsin effectively uncoupled electron transport. Continued digestion led to inhibition of saturated rates of Hill reaction activity and a decrease in quantum yield. Irradiation with ultraviolet light decreased the quantum yield and inhibited Hill activity, but did not uncouple. Ascorbate-dichlorophenol-indophenol-mediated reduction of nicotinamide adenine dinucleotide phosphate was not appreciably inhibited by treatment with either of the enzymes or by ultraviolet irradiation.     

The effect of respiration substrates on the ATPase activity of uncoupled mitochondria

Mg-dependent ATPase activity in aging uncoupled mitochondria is 30% reduced by 2 mM of succinate. The results show that redox state of mitochondria electron-transport chain affects the activity and apparently modifies the structure of the enzyme performing ATP synthesis and hydrolysis.     

Oxygen diffusion and dark respiration in aquatic macrophytes

Abstract. The supply of oxygen to respiring shoot tissue was investigated for three submerged macrophytes (Potamogeton crispus L., Egeria densa Planch, and Myriophyllum triphyllum Orchard). For all species, the response of oxygen uptake rates to the external O₂ concentration was a rectangular hyperbola over the range 0–5.0 × 10^?3m³ m^?3. However, the response pattern for material with water-infiltrated lacunar airspaces was non-hyperbolic over this range. The change in response was interpreted as an increased substrate (O₂) limitation, resulting from lower radial diffusion rates within the infiltrated material. Neither the uninfiltrated nor the infiltrated responses obeyed the linear and logarithmic formulae of the type observed for submerged macrophytes by earlier authors. These results suggest that the responses observed are affected by factors such as water velocity, internal restrictions to diffusion and the range of oxygen tensions investigated. Therefore, it is unlikely that one response formula can adequately account for the effects of oxygen concentration on submerged macrophyte oxygen uptake. The lacunar airspaces also represent a possible oxygen source for dark respiration. The consumption of oxygen from the airspaces was investigated by displacing the gas from the lacunae and measuring the subsequent increase in the rate of oxygen assimilation from the external liquid. Approximately 30% of the oxygen consumed by E. densa and P. crispus, and more than 40% of that consumed by M. triphyllum, was derived from the lacunar system. This O₂ supply is a consequence of the higher oxygen concentration in the lacunae than in the external medium, due to the low solubility of oxygen in water. Storage of photosynthetically-produced oxygen in the lacunae could not be identified during a light/dark transient, due to rate changes caused by the effects of light on the respiratory metabolism. However, O₂ partial pressure gradients artificially set up between the lacunae and water equilibrated within an hour, suggesting that excess oxygen would be lost to the water within this time.