Pressure-adaptive differences in the binding and catalytic properties of muscle-type (M4) lactate dehydrogenases of shallow- and deep-living marine fishes

Summary The pressure sensitivities of substrate (pyruvate) and cofactor (NADH) binding and catalytic rate of purified muscle-type (M₄) lactate dehydrogenases (LDH, EC 1.1.1.27; NAD⁺: lactate oxidoreductase) from shallow- and deep-living teleost fishes were compared. The LDH's of the shallow species are significantly more pressure-sensitive than the LDH's of the deep-living fishes. The apparent Michaelis constant (K _m)¹ of pyruvate of the deep-living species' LDH's is pressure-insensitive over the entire pressure range used in these studies, 1 to 476 atmospheres (Fig. 1). For the LDH's of the shallow species, theK _m of pyruvate increases significantly between 1 and 68 atmospheres, and then remains stable up to 476 atmospheres. TheK _m of NADH displays a much higher pressure sensitivity. For the LDH's of the deep species, theK _m of NADH increases slightly (approximately 32%) between 1 and 68 atmospheres, and then remains stable up to 476 atmospheres (Fig. 1). TheK _m of the shallow species' LDH's rises sharply (approximately 113%) between 1 and 68 atmospheres, and then continues to increase at a slower rate up to 476 atmospheres. This marked inhibition of cofactor binding by pressure for the shallow species' LDH's may be of sufficient magnitude to seriously impair the function of these LDH's at pressures typical of those encountered by the deeper-living species.Pressure effects on optimal velocity, measured under high (optimal) concentrations of pyruvate and NADH, were generally lower for the LDH's of the deep species (Table 1).These results indicate that M₄-LDH's of shallow water fishes are not pre-adapted for function at deepsea pressures, and that the reduction of pressure sensitivities ofK _m's and catalysis may be a ubiquitous feature of adaptation to life at depth. The virtually identical pressure responses of M₄-LDH's from deepliving teleosts belonging to four different families represents a striking example of convergent evolution at the molecular level.     

Effect of High Oxygen Tension on Potassium Retentivity and Colony Formation of Bakers' Yeast

The ability of yeast cells to retain potassium and to form colonies was studied after exposure to pressures ranging from 2 to 143 atmospheres of oxygen. The investigations allow comparison of these responses with those found after x-ray exposure. Exposure to 2 to 8 atmospheres of oxygen for 2, 20, and 40 hours showed decreased potassium leakage as measured by an elution technique. Further experiments using 0.5 to 22 hour exposures to 10 to 143 atmospheres of oxygen showed decreased potassium leakage when glucose was present in the test media, but increased leakage (as did x-ray effects) in the absence of substrate. There was increased potassium leakage into the suspending media (distilled water) during oxygen exposure but this usually did not affect the leakage rates measured subsequently. Marked inability to form colonies was observed after 20 hour exposures to 100 atmospheres of oxygen, with a much smaller response at lower pressures. Increased oxygen concentrations, not pressure, evidently caused these effects, since comparable pressures of nitrogen produced almost no change. The ratio of potassium leakage to survival sensitivity was found to be approximately unity when comparing exposures causing 50 per cent damage. This is quite different from that seen with x-ray or ultraviolet irradiation.     

Oxygen-dependent aging of seeds

When seeds of soybeans (Glycine max Amsoy var.) or safflower were stored under high O₂ concentrations, their per cent germination declined rapidly. For example, soybean seeds stored under 7.7 atmospheres O₂ pressure at 25°C and 17% moisture lost all viability within 22 days, whereas under 7.7 atmospheres N₂, the per cent germination remained greater than 80%. Germination decreased continually in O₂ pressures ranging from 0 to 7.7 atmospheres. High levels of O₂, moisture, or temperature each acted independently to cause losses of germination, but when applied simultaneously, these factors acted synergistically. Soybean seeds were also aged under conditions of high temperature (44°C) and humidity (100% RH), which have been routinely used to accelerate aging. Under these conditions, no O₂ dependence of seed death was observable.

Increased lipid oxidation was not detected in seeds that had lost germination ability due to high O₂ treatment. Seeds of two safflower varieties that contained either high oleic or high linoleic fatty acid compositions were subjected to high O₂ treatment. Although the lipid of the high oleic variety is markedly more stable to oxidative degradation, we detected no significant difference in the O₂ tolerance of these seeds.

     

The opposing physiological effects of high pressures and inert gases.

The physiological effects on mammals of elevated pressures (approximately 100 atmospheres) must be considered in the context of the inert gases breathed. The most striking effect of pressure per se is a central hyperexcitability manifest at first by trembling of the entremities and finally by convulsions. Paralysis and death occur at higher pressures. The primary effects of the inert gases breathed are inert gas narcosis and general anesthesia. The exciting effects of pressure per se and the depressive effects of the inert gases tend to oppose each other. Thus consciousness may be restored to anesthetized mice by raising the pressure, and conversely the threshold pressure that causes convulsions is elevated in the presence of anesthetics. These mutually antagonistic effects can be rationalized in terms of model which proposes that both anesthetics and pressure non-specifically perturb thelipid bilayer regions of neutral membranes. This model is termed the critical volume hypothesis. Anthesthetics dissolve in and expand these lipid bilayer regions, while pressure causes mechanical compression. Expansion leads to anesthesia and compression to convulsions if a critical degree of change is achieved. At elevated partial pressures of inert gas the gas-induced expansion is opposed by the compression of pressure per se. With very insoluble gases, such as helium, this expansion is so small that net compression results and the effects of helium differ little from those of pressure per se. With more soluble gases, such as nitrogen, net expansion results in inert gas narcosis and anesthesia. The critical volume hypothesis enables "safe" mixtures of "expanding" and "compressing" gases to be defined. These enable higher pressures to be better tolerated by mammals.     

Thermal polymerization of amino acids under various atmospheres or at low pressures

The kinds and proportions of amino acids formed in two simulated prebiotic experiments or detected in hydrolyzed extracts of three extraterrestrial samples were found to polymerize thermally under various atmospheres or at low pressures. Yields, tested properties, and amino acid compositions of the polymers were not influenced by the type of enveloping atmosphere, including two simulated prebiotic atmospheres and five pure gases. However, polyamino acids prepared at low pressure (0.02, 10^?4 atm) were obtained in appreciably greater yield than those synthesized at 1 atm; amino acid composition was somewhat influenced by low pressure. The results indicate that polyamino acids could have been formed thermally under a variety of possible prebiotic atmospheres and on planetary bodies of low atmospheric pressure.     

Carbon dioxide effects on ethanol production, pyruvate decarboxylase, and alcohol dehydrogenase activities in anaerobic sweet potato roots

The effect of varied anaerobic atmospheres on the metabolism of sweet potato (Ipomoea batatas [L.] Lam.) roots was studied. The internal gas atmospheres of storage roots changed rapidly when the roots were submerged under water. O₂ and N₂ gases disappeared quickly and were replaced by CO₂. There were no appreciable differences in gas composition among the four cultivars that were studied. Under different anaerobic conditions, ethanol concentration in the roots was highest in a CO₂ environment, followed by submergence and a N₂ environment in all the cultivars except one. A positive relationship was found between ethanol production and pyruvate decarboxylase activity from both 100% CO₂-treated and 100% N₂-treated roots. CO₂ atmospheres also resulted in higher pyruvate decarboxylase activity than did N₂ atmospheres. Concentrations of CO₂ were higher within anaerobic roots than those in the ambient anaerobic atmosphere. The level of pyruvate decarboxylase and ethanol in anaerobic roots was proportional to the ambient CO₂ concentration. The measurable activity of pyruvate decarboxylase that was present in the roots was about 100 times less than that of alcohol dehydrogenase. Considering these observations, it is suggested that the rate-limiting enzyme for ethanol biosynthesis in sweet potato storage roots under anoxia is likely to be pyruvate decarboxylase rather than alcohol dehydrogenase.     

Tissue culture in synthetic atmospheres: diffusion rate effects on cytokinin-induced callus growth and isoflavonoid production in soybean [Glycine max (L.) Merr. cv. Acme]

Concentration is one factor that is known to determine how metabolic gases influence the growth and secondary metabolism of plant tissues in culture. How actual gas bioavailability influences these processes has not been studied despite its potential importance in specialized applications. A simple model system, soybean [Glycine max (L.) Merr. cv. Acme] callus culture, was selected for experiments because exogenous cytokinin (6-benzylaminopurine; BAP) elicits two types of responses: (1) enhanced callus proliferation, and (2) rapid induction of the isoflavonoid daidzein (7,4′-dihydroxyisoflavone). Synthetic atmospheres supplying metabolic gases with higher or lower bioavailability than in air were created by replacing the nitrogen moiety in standard air with either helium or argon, respectively. Callus was cultured on agar or in liquid shake cultures according to standard procedures. At an optimal cytokinin concentration for stimulation of callus proliferation, 4.4 × 10⁻⁷ M BAP, increased diffusion rates for the metabolic gases resulted in greater weight gain in agar cultures. Weight gain was 11% higher for He-treated and 39% lower for Ar-treated cultures than for the nitrogen control. In contrast, there was no significant effect of metabolic gas diffusion rate on daidzein production in either agar or liquid cultures. Apart from the potential application of these synthetic atmospheres for enhancing plant tissue culture growth, they may have unique value for the space program as an effective way of replicating the gas exchange limitations posed for plants by microgravity (Ar atmosphere), and as a countermeasure for this limitation (He atmosphere).     

Thermodynamic Constrains for Life Based on Non-Aqueous Polar Solvents on Free-Floating Planets

Free-floating planets (FFPs) might originate either around a star or in solitary fashion. These bodies can retain molecular gases atmospheres which, upon cooling, have basal pressures of tens of bars or more. Pressure-induced opacity of these gases prevents such a body from eliminating its internal radioactive heat and its surface temperature can exceed for a long term the melting temperature of a life-supporting solvent. In this paper two non-aqueous but still polar solvents are considered: hydrogen sulfide and ammonia. Thermodynamic requirements to be fulfilled by a hypothetic gas constituent of a life-supporting FFP’s atmosphere are studied. The three gases analyzed here (nitrogen, methane and ethane) are candidates. We show that bodies with ammonia oceans are possible in interstellar space. This may happen on FFPs of (significantly) smaller or larger mass than the Earth. Generally, in case of FFP smaller in size than the Earth, the atmosphere exhibits a convective layer near the surface and a radiative layer at higher altitudes while the atmosphere of FFPs larger in size than Earth does not exhibit a convective layer. The atmosphere mass of a life-hosting FFP of Earth size is two or three orders of magnitude larger than the mass of Earth atmosphere. For FFPs larger than the Earth and specific values of surface pressure and temperature, there are conditions for condensation (in the ethane atmosphere). Some arguments induce the conclusion than the associated surface pressures and temperatures should be treated with caution as appropriate life conditions.     

Experimental biology of ammonia-rich environments. Germination of allium seed, a novel capability among angiosperms

Seed populations from a majority of species and cultivars of Allium yield up to 15% germination under atmospheres containing NH₃ together with equal volumes of N₂ or air. Selected onion and leek cultivars tested in a range of NH₃ or O₂ compositions show little or no dependency upon quantitative variations in these gases.     

Biomass fast pyrolysis in a fluidized bed reactor under N2, CO2, CO, CH4 and H2 atmospheres

Biomass fast pyrolysis is one of the most promising technologies for biomass utilization. In order to increase its economic potential, pyrolysis gas is usually recycled to serve as carrier gas. In this study, biomass fast pyrolysis was carried out in a fluidized bed reactor using various main pyrolysis gas components, namely N₂, CO₂, CO, CH₄ and H₂, as carrier gases. The atmosphere effects on product yields and oil fraction compositions were investigated. Results show that CO atmosphere gave the lowest liquid yield (49.6%) compared to highest 58.7% obtained with CH₄. CO and H₂ atmospheres converted more oxygen into CO₂ and H₂O, respectively. GC/MS analysis of the liquid products shows that CO and CO₂ atmospheres produced less methoxy-containing compounds and more monofunctional phenols. The higher heating value of the obtained bio-oil under N₂ atmosphere is only 17.8 MJ/kg, while that under CO and H₂ atmospheres increased to 23.7 and 24.4 MJ/kg, respectively.     

Comparisons of turkey embryos incubated in tenuous or dense gas environments--II. Organ growth

1. Eggs from large white turkeys were incubated in tenuous and dense gas atmospheres. 2. Tenuous gases resulted in heavier embryos until the onset of the plateau stage in oxygen consumption when dense gas environments caused heavier embryos. 3. Tenuous gases decreased heart and lung weight but increased liver weight. 4. Oxygen supplementation in tenuous gases increased liver weights but had no effect on lung or heart weights. 5. The data suggest an interaction of gas density and partial pressures of individual gases which affects breathing and the physiology of developing poult embryos.     

Applicability of Henry's law to hydrogen, helium, and nitrogen solubilities in water and olive oil at 37 degrees C and pressures up to 300 atmospheres

The solubilities of pure hydrogen, helium, and nitrogen in water and olive oil were measured at 37 degrees C at gas-saturation pressures from 25 to 300 atmospheres. Rigorous thermodynamic criteria were used to assess the applicability of Henry's law to the pressure dependence of the gas solubility in each system. The solubilities of the three gases in water and helium in olive oil followed Henry's law as given by the Krichevsky-Kasarnovsky equation. In contrast, hydrogen and nitrogen in olive oil each attained concentrations high enough to cause significant concentration-dependent variations of the dissolved gas activity coefficient and/or partial molal volume. The consequent deviations from Henry's law were greatest in the nitrogen-oil system, where mole fraction nitrogen solubilities calculated from the Krichevsky-Kasarnovsky equation exceeded measured values by 8, 14, and 23% at 50, 100, and 250 atm, respectively. Incorporation of results into the critical volume model of nitrogen anesthesia, using olive oil as a model of the physiological anesthetic site and literature data for the anesthetic potency of nitrogen in mice breathing high-pressure He-N2-O2 atmospheres, shows that nonideal solution behavior may become important for gases dissolved in physiological hydrophobic regions at biologically active concentrations, even if dissolved gas binding to proteins or other macromolecules is not involved.     

Prediction of barometric pressures at high altitudes with the use of model atmospheres

West, John B. Prediction of barometric pressures athigh altitudes with the use of model atmospheres. J. Appl. Physiol. 81(4): 1850-1854, 1996.It wouldbe valuable to have model atmospheres that allow barometric pressures(PB) to bepredicted at high altitudes. Attempts to do this in the past using theInternational Civil Aviation Organization or United StatesStandard Atmosphere model have brought such models into disreputebecause the predicted pressures at high altitudes are usually much toolow. However, other model atmospheres have been developed bygeophysicists. The critical variable is the change of air temperaturewith altitude, and, therefore, model atmospheres have been constructedfor different latitudes and seasons of the year. These different modelsgive a large range of pressures at a given altitude. For example, the maximum difference of pressure at an altitude of 9 km is from 206 to248 Torr, i.e., ~20%. However, the mean of the model atmospheres forlatitude of 15° (in all seasons) and 30° (in thesummer) predicts PB at manylocations of interest at high altitude very well, with predictionswithin 1%. The equation is PB(Torr) = exp (6.63268  0.1112 h  0.00149 h²), where h is the altitude inkilometers. The predictions are good because many high mountain sitesare within 30° of the equator and also many studies are made duringthe summer. Other models should be used for latitudes of 45° andabove. Model atmospheres have considerable value in predictingPB at high altitude if proper account is taken of latitude and season of the year.

     

Effects of helium group gases and nitrous oxide on HeLa cells

The helium group gases and nitrous oxide at superatomospheric pressures depress multiplication of HeLa cells in monolayer cultures. The effectiveness of these gases in eliciting the pressure-dependent response follows the order N₂O, Xe > Kr > Ar > > Ne and He. The response correlates with lipid solubility of the gases. Depression of growth by 4.2 atm Xe is reversible after exposure for one and two days. Cultures exposed to 7.2 atm Xe show irreversible damage including cytoplasmic vacuolization. Cell attachment is strongly inhibited by Xe; 36% of the cell inoculum were not attached after 24 hours. Affinity for hydrophobic sites in the cell is suggested as determining the order of effectiveness of the gases in evoking the response.     

GERMINATION OF HELEOCHLOA ALOPECUROIDES (POACEAE) IN VARIOUS CONCENTRATIONS OF HELIUM,NITROGEN, AND CARBON DIOXIDE

The percentages of germination of Heleochloa alopecuroides (Pill and Mitterp) Host seeds were tested in various controlled atmospheres of He, N₂, and CO₂. Atmospheres of 16.7%, 33.3 %, and 88.9 % of each of these gases (thus reducing O₂ concentrations to 17.5 %, 14.0 %, and 2.3% respectively) were introduced into 1000-ml stoppered vacuum flasks, each containing 100 seeds placed on substrates of either sterilized soil or filter paper. The flasks were placed in growth chambers with an 8-hr light period at 20 C and a 16-hr dark period at 5 C. The germination rate was accelerated directly with increased concentrations of He and N₂ but was retarded by increased percentages of CO₂. Control flasks eventually reached nearly the same percentages of germination as those of He and N₂ but at a later point in time.     

Prevention of oxygen toxicity with superoxide dismutase and catalase in premature lambs

The use of high oxygen concentrations and high mean airway pressures during mechanical ventilation of premature newborn infants with respiratory distress syndrome leads in 20%–30% of the survivors to chronic lung disease. This study explores if exogenous polyethylene glycol conjugated superoxide dismutase (PEG-SOD) and catalase (PEG-CAT) mitigate oxygen toxicity in premature lambs with respiratory distress syndrome. Six pairs of premature lambs were delivered by cesarean section and treated by tracheal instillation of 60 mg natural sheep surfactant/kg/body weight. After birth, all lambs were ventilated with 100% oxygen, and one of each pair received a single intravenous injection of 1 million U/kg PEG-CAT and 50,000 U/kg PEG-SOD. At 8 h of age or after respiratory failure was established, the lambs were killed and the lungs were removed intact. Lung damage was assessed by microscopy. The arterial blood gases, pH, and mean airway pressures of the lambs treated with PEG-SOD/PEG-CAT did not differ from those of the controls. Mean PaO₂ was > 140 mmHg during the first 4 h of the experiments. In the lambs treated with PEG-SOD/PEG-CAT, SOD and CAT levels were very high during the study period and less bronchiolar epithelial damage and lung hemorrhages were found at microscopy.     

Reversal of P-glycoprotein-mediated multidrug resistance by doxorubicin and quinine co-loaded liposomes in tumor cells

Multidrug resistance (MDR) is a major obstacle to successful clinical cancer chemotherapy. Currently, there is still unsatisfactory demand for innovative strategies as well as effective and safe reversing agent to overcome MDR. In this study, we developed a novel nanoformulation, in which doxorubicin hydrochloride (DOX) and quinine hydrochloride (QN) were simultaneously loaded into liposomes by a pH-gradient method for overcoming MDR and enhancing cytotoxicity in a doxorubicin-resistant human breast cancer cell line (MCF-7/ADR). The various factors were investigated to optimize the formulation and manufacturing conditions of DOX and QN co-loaded liposomes (DQLs). The DQL showed uniform size distribution and high encapsulation efficiency (over 90%) for both the drugs. Furthermore, DQLs significantly displayed high intracellular accumulation and potential of MDR reversal capability in MCF-7/ADR cells through the cooperation of DOX with QN, in which QN played the role as a MDR reversing agent. The IC₅₀ of DQL0.5:1 with the DOX/QN/SPC weight ratio of 0.5:1:50 was 1.80?±?0.03?μg/mL, which was 14.23 times lower than that of free DOX in MCF-7/ADR cells. And the apoptotic percentage induced by DQL0.5:1 was also increased to 62.2%. These findings suggest that DQLs have great potential for effective treatment of MDR cancer.     

The inhibition of mitochondrial cytochrome oxidase by the gases carbon monoxide, nitric oxide, hydrogen cyanide and hydrogen sulfide: chemical mechanism and physiological significance

The four gases, nitric oxide (NO), carbon monoxide (CO), hydrogen sulfide (H₂S) and hydrogen cyanide (HCN) all readily inhibit oxygen consumption by mitochondrial cytochrome oxidase. This inhibition is responsible for much of their toxicity when they are applied externally to the body. However, recently these gases have all been implicated, to greater or lesser extents, in normal cellular signalling events. In this review we analyse the chemistry of this inhibition, comparing and contrasting mechanism and discussing physiological consequences. The inhibition by NO and CO is dependent on oxygen concentration, but that of HCN and H₂S is not. NO and H₂S are readily metabolised by oxidative processes within cytochrome oxidase. In these cases the enzyme may act as a physiological detoxifier of these gases. CO oxidation is much slower and unlikely to be as physiologically important. The evidence for normal physiological levels of these gases interacting with cytochrome oxidase is equivocal, in part because there is little robust data about their steady state concentrations. A reasonable case can be made for NO, and perhaps CO and H₂S, inhibiting cytochrome oxidase in vivo, but endogenous levels of HCN seem unlikely to be high enough.     

Microbial growth modification by compressed gases and hydrostatic pressure

Studies of the growth-modifying actions for Escherichia coli, Saccharomyces cerevisiae, and Tetrahymena thermophila of helium, nitrogen, argon, krypton, xenon, and nitrous oxide led to the conclusion that there are two definable classes of gases. Class 1 gases, including He, N(2), and Ar, are not growth inhibitors; in fact, they can reverse the growth inhibitory action of hydrostatic pressures. Class 2 gases, including Kr, Xe, and N(2)O, are potent growth inhibitors at low pressures. For example, at 24 degrees C, 50% growth-inhibitory pressures of N(2)O were found to be ca. 1.7 MPa for E. coli, 1.0 MPa for S. cerevisiae, and 0.5 MPa for T. thermophila. Class 1 gases could act as potentiators for growth inhibition by N(2)O, O(2), Kr, or Xe. Hydrostatic pressure alone is known to reverse N(2)O inhibition of growth, but we found that it did not greatly alter oxygen toxicity. Therefore, potentiation by class 1 gases appeared to be a gas effect rather than a pressure effect. The temperature profile for growth inhibition of S. cerevisiae by N(2)O revealed an optimal temperature for cell resistance of ca. 24 degrees C, with lower resistance at higher and lower temperatures. Overall, it appeared that microbial growth modification by hyperbaric gases could not be related to their narcotic actions but reflected definably different physiological actions.     

Influence of Modified Atmosphere Storage on Aflatoxin Production in High Moisture Corn

Samples of freshly harvested corn and remoistened corn were inoculated with Aspergillus flavus and stored for 4 weeks at about 27 C in air and three modified atmospheres. Aflatoxins and fat acidity were determined weekly. Corn stored in the modified atmospheres did not accumulate over 15 μg of aflatoxin B₁ per kg and 20 μg of total aflatoxins per kg. Corn from the high CO₂ treatment (61.7% CO₂, 8.7% O₂, and 29.6% N₂) was visibly molded at 4 weeks and had a higher fat acidity than the other treatments. In the N₂ (99.7% N₂ and 0.3% O₂) and controlled atmosphere (13.5% CO₂, 0.5% O₂, 84.8% N₂) treatments, a fermentation-like odor was detected. When the corn was removed from the modified atmospheres it deteriorated rapidly and was soon contaminated with aflatoxins.