Effect of heavy water on the viability of bacteria

Influence of heavy water (D2O) on the membrane energization, the efflux of hydrogen ions and the respiration of bacteria E. coli M-17 was studied. As has been shown, heavy water of a low concentration (0.05-0.20% v/v) activates and of a high concentration (above 10%) inhibits the absorption of lipophilic cation tetraphenylphosphonium (TPP+) and of oxygen by cells. The return of these characteristics to the initial levels after the removal of D2O points to a reversible action of D2O. A protective effect of D2O towards membrane energization and rate of respiration on dried cells was observed. This fact is in agreement with the data on viability of bacteria. The indicated protective action increases at the stage of rehydration in the presence of D2O.     

A simple and easy method for the monitoring of environmental pollutants using oligotrophic bacteria

Sphingomonas paucimobilis KPS01, an oligotrophic bacterium isolated from soil, may be a useful tool for monitoring heavy metals. Previous methods relying on counting of viable cells require a relatively long time and some skill; we have developed a method based on optical density (O.D.) measurements which is significantly faster and does not require skilled personnel. The results of the O.D. and viable count methods were consistent; both methods detected heavy metals at concentrations ranging from 10-3 to 10-5 mmol l-1 and identified heavy metal contamination in 13 of 18 river water samples. Our results demonstrate that biological detection using this O.D. method and S. paucimobilis KPS01 may be useful for routine environmental monitoring of heavy metals, particularly in water sources.     

D2O as a modifier of ionic specificity of Na, K-ATPase

Effect of heavy water D2O on the rate of hydrolysis of ATP and pNPP by Na,K-ATPase was studied. Heavy water of high concentration inhibits the rate of ATPase reaction in all the studied ratios of the ions Na/K at constant ionic strength 150 mM. Activation of the enzyme was observed in the solution with low concentration of heavy water (less than 5%). The value of isotope effects depended on the ratio between sodium and potassium ion concentrations in the medium. At low temperature no activation of the enzyme with heavy water in low concentration was observed. Substitution of usual water for the heavy one was accompanied by a decrease of apparent constants of enzyme activation with sodium and potassium ions. During pNPP hydrolysis with Na,K-ATPase an increase of reaction rate in the medium with heavy water was observed. Substitution of potassium ions by cesium resulted in an increase of isotope effects during ATP and pNPP hydrolysis. Analysis of isotope effects in terms of the molecular model of sodium pump proposed permits a conclusion that the isotope effects of heavy water are explained by its influence as a solvent, the binding centres of potassium and sodium ions are localized in different regions of the enzyme differing in physico-chemical properties. The structure of sodium centres is controlled by hydrogen bonds, and of potassium ones--by hydrophobic interactions; the transport of ions by the enzyme is accompanied by dehydration of ions.     

Visualizing ocular lens fluid dynamics using MRI: manipulation of steady state water content and water fluxes

Studies using various MRI techniques have shown that a water-protein concentration gradient exists in the ocular lens. Because this concentration is higher in the core relative to the lens periphery, a gradient in refractive index is established in the lens. To investigate how the water-protein concentration profile is maintained, bovine lenses were incubated in different solutions, and changes in water-protein concentration ratio monitored using proton density weighted (PD-weighted) imaging in the absence and presence of heavy water (D(2)O). Lenses incubated in artificial aqueous humor (AAH) maintained the steady state water-protein concentration gradient, but incubating lenses in high extracellular potassium (KCl-AAH) or low temperature (Low T-AAH) caused a collapse of the gradient due to a rise in water content in the core of the lens. To visualize water fluxes, lenses were incubated in D(2)O, which acts as a contrast agent. Incubation in KCl-AAH and low T-AAH dramatically slowed the movement of D(2)O into the core but did not affect the movement of D(2)O into the outer cortex. D(2)O seemed to preferentially enter the lens cortex at the anterior and posterior poles before moving circumferentially toward the equatorial regions. This directionality of D(2)O influx into the lens cortex was abolished by incubating lenses in high KCl-AAH or low T-AAH, and resulted in homogenous influx of D(2)O into the outer cortex. Taken together, our results show that the water-protein concentration ratio is actively maintained in the core of the lens and that water fluxes preferentially enter the lens at the poles.     

Examination of microgravity effects on spontaneous Ca2+ oscillations in AtT20 pituitary cells using heavy water

Effects of heavy water (D2O) on various organisms have been extensively studied and a majority of D2O actions were generally ascribed to the viscosity (1.23 times of H2O) and a larger inter-molecule force of D2O that may eventually alternate molecular structure of various enzymes and ion channels. It is reported that chronic application of D2O induces toxic effects and the 35% substitution of whole body water with D2O induced fatal effects in the mouse. Mitosis of a fertile egg of sea urchin was completely inhibited by 75% D2O but the paused segmentation was recovered after rinse of D2O. In addition, we also observed that neuronal development of the Lymnaea stagnalis was reversibly inhibited by D2O (M. Sakakibara, unpublished data). However, mechanism of the toxicity of D2O and the effects of D2O on cellular events have not been fully understood. The spontaneous oscillation in cytosolic free Ca2+ concentration is one of the typical physiological events in living secretory cells. We previously demonstrated that the Ca2+ oscillations are regulated by voltage-sensitive Ca2+ channels (VSCC), Ca2+ ATPases, and Ca(2+)-induced Ca2+ release from intracellular stores. To analyze the site(s) of action of D2O in the living cellular systems, the present study examined effects of D2O on the Ca2+ mobilization and resting membrane potentials in AtT20 mouse pituitary cells.     

Small angle neutron scattering, total cross-sections and mass density measurements of concentrated NaCl and KCl solutions in H2O or D2O

The mass densities, total cold neutron cross sections and small angle scattering of concentrated NaCl and KCl solutions in H2O or D2O (2H2O) were measured at 20 degrees C. The partial specific volumes of both salts increase with salt concentration and are significantly smaller in D2O than in H2O, showing that these salt solutions cannot be considered as isomorphous in H2O and D2O. As salt concentration increases for both salts, the total coherent cross sections for neutrons of the solutions also increase while the coherent small angle scattering decreases-observations that are consistent, respectively, with increasing correlations involving the ion and water components and a decrease in the particle number density and/or concentration fluctuations, in the solutions. Changes in incoherent scattering with salt concentration are essentially those expected from the solution compositions and densities.     

Effect of heavy water on protein flexibility

The effects of heavy water (D(2)O) on internal dynamics of proteins were assessed by both the intrinsic phosphorescence lifetime of deeply buried Trp residues, which reports on the local structure about the triplet probe, and the bimolecular acrylamide phosphorescence quenching rate constant that is a measure of the average acrylamide diffusion coefficient through the macromolecule. The results obtained with several protein systems (ribonuclease T1, superoxide dismutase, beta-lactoglobulin, liver alcohol dehydrogenase, alkaline phosphatase, and apo- and Cd-azurin) demonstrate that in most cases D(2)O does significantly increase the rigidity the native structure. With the exception of alkaline phosphatase, the kinetics of the structure tightening effect of deuteration are rapid compared with the rate of H/D exchange of internal protons, which would then assign the dampening of structural fluctuations in D(2)O to a solvent effect, rather than to stronger intramolecular D bonding. Structure tightening by heavy water is generally amplified at higher temperatures, supporting a mostly hydrophobic nature of the underlying interaction, and under conditions that destabilize the globular fold.     

Modifications of sodium channel gating in Myxicola giant axons by deuterium oxide, temperature, and internal cations.

In dialyzed Myxicola axons substitution of heavy water (D2O) externally and internally slows both sodium and potassium kinetics and decreases the maximum conductances. Furthermore, this effect is strongly temperature dependent, the magnitude of the slowing produced by D2O substitution decreasing with increasing temperature over the range 3-14 degrees C with a Q10 of approximately 0.71. The relatively small magnitude of the D2O effect, combined with its strong temperature dependence, suggests that the rate limiting process producing a conducting channel involves appreciable local changes in solvent structure. Maximum conductances in the presence of D2O were decreased by approximately 30%, while the voltage dependences of both gNa and gK were not appreciably changed. In contrast to the effects of heavy water substitution on the ionic currents, membrane asymmetry currents were not altered by D2O, suggesting that gating charge movement may preceed by several steps the final transformation of the Na+ channel to a conducting state. In Myxicola axons the effect of temperature alone on asymmetry current kinetics can be well described via a simple temporal expansion equivalent to a Q10 of 2.2, which is somewhat less than the Q10 of GNa activation. The integral of membrane asymmetry current, representing maximum charge movement, is however not appreciably altered by temperature.     

Accumulation of deuterium oxide in body fluids after ingestion of D2O-labeled beverages

A simple low-cost procedure was developed to compare the temporal profiles of deuterium oxide (D2O) accumulation in body fluids after ingestion of D2O-labeled solutions. D2O concentration was measured in plasma and saliva samples taken at various intervals after ingestion of 20 ml of D2O mixed with five solutions differing in carbohydrate and electrolyte concentrations. An infrared spectrometer was used to measure D2O in purified samples obtained after a 48-h incubation period during which the water (D2O and H2O) in the sample was equilibrated with an equal volume of distilled water in a sealed diffusion dish. The procedure yields 100% recoveries of 60-500 ppm D2O with an average precision of 5%. When compared with values for distilled water, D2O accumulation in serial samples of plasma and saliva was slower for ingested solutions containing 40 and 15% glucose and faster for hypotonic saline and a 6% carbohydrate-electrolyte solution. These differences appear to reflect known differences in gastric emptying and intestinal absorption of these beverages. Therefore this technique may provide a useful index of the rate of water uptake from ingested beverages into the body fluids.     

底物种类和浓度对污泥重金属生物淋滤效果的影响

以生污泥为材料,研究单质硫(浓度分别为5、10、20 g/L)、FeSO4?7H2O (浓度分别为10、20 g/L）和硫代硫酸钠（浓度分别为10、20 g/L）三种底物的不同浓度对污泥中Zn、Cu、Pb、Cd、Cr和Ni六种重金属生物淋滤效果的影响。结果表明：单质硫的致酸性最好,第4天pH值就达到2.0左右,Zn、Cu、Pb、Cr和Ni的滤出量最大（原污泥Cd未检出）,并以底物投放浓度在10～20 g/L为佳。FeSO4?7H2O的致酸性和重金属滤出量较单质硫弱。硫代硫酸钠在浓度10 g/L时,致酸性和重金属的滤出量介于单质硫和FeSO4?7H2O之间,但浓度20 g/L时滤液呈碱性,重金属难于滤出甚至不滤出。     

Effect of D2O on maize plasmalemma ATPase and electron transport coupled proton pumping.

Heavy water (D2O) has been used as a putative inhibitor of the plasma membrane H(+)-ATPase and the plasma membrane redox system. Concentrations above 50% D2O inhibited H+ secretion and the plasma membrane redox system of Zea mays L. roots. Inhibition of H+ secretion by vanadate was reduced in presence of D2O. The plasma membrane of roots was transiently depolarized after the addition of heavy water in concentrations above 5%. The repolarization of the plasma membrane that takes place while the H+ secretion is still reduced by heavy water indicates that, despite the overall inhibiting effect of D2O, the plant is still able to regulate the membrane potential.     

Kinetic Studies on the Initial Crystallization Process of Lysozyme in the Presence of D2O and H2O

In the initial stages of the crystallization of egg-white lysozyme, monomeric lysozyme aggregates rapidly and forms a nucleus in the presence of high salt concentrations. The formation process of the aggregates was examined to make clear the difference between the situations in heavy water and in water at the same sodium ion concentration. The aggregation in both cases was observed at unsaturated and/or saturated lysozyme concentrations. The turbidity at 350 nm of lysozyme increased remarkably within 60 min under each experimental condition and showed no appreciable changes over 60 min. The increase of turbidity in H₂O was much slower than in D₂O at the same salt concentration (3%). Lysozyme showed a critical concentration for nucleus formation whose value in H₂O was lower than in D₂O at 3% salt concentration. There are two different aggregation models, depending on the concentration of lysozyme. However, similar results were not obtained at 3% sodium ions in H₂O. The initial aggregation rate was also dependent on the concentrations of both lysozyme and NaCI. Therefore, the effect of lysozyme concentration on the aggregation process in H₂O may be smaller than in D₂O.     

Kinetic Studies on the Initial Crystallization Process of Lysozyme in the Presence of D2O and H2O

In the initial stages of the crystallization of egg-white lysozyme, monomeric lysozyme aggregates rapidly and forms a nucleus in the presence of high salt concentrations. The formation process of the aggregates was examined to make clear the difference between the situations in heavy water and in water at the same sodium ion concentration. The aggregation in both cases was observed at unsaturated and/or saturated lysozyme concentrations. The turbidity at 350 nm of lysozyme increased remarkably within 60 min under each experimental condition and showed no appreciable changes over 60 min. The increase of turbidity in H₂O was much slower than in D₂O at the same salt concentration (3%). Lysozyme showed a critical concentration for nucleus formation whose value in H₂O was lower than in D₂O at 3% salt concentration. There are two different aggregation models, depending on the concentration of lysozyme. However, similar results were not obtained at 3% sodium ions in H₂O. The initial aggregation rate was also dependent on the concentrations of both lysozyme and NaCI. Therefore, the effect of lysozyme concentration on the aggregation process in H₂O may be smaller than in D₂O.     

Aggregation and adsorption at the air-water interface of bacteriophage phiX174 single-stranded DNA

We study the phase behavior of phage phiX174 single-stranded DNA in very dilute solutions in the presence of monovalent and multivalent salts, in both water (H(2)O) and heavy water (D(2)O). DNA solubility depends on the nature of the salts, their concentrations, and the nature of the solvent. The appearance of attractive interactions between the monomers of the DNA chains in the bulk of the solution is correlated with an adsorption of the chains at the air-water interface. We characterize this correlation in two types of aggregation processes: the condensation of DNA induced by the trivalent cation spermidine and its salting out in the presence of high concentrations (molar and above) of monovalent (sodium) cations, both in water and in heavy water. The overall solubility of single-stranded DNA is decreased in D(2)O compared to H(2)O, pointing to a role of DNA hydration in addition to electrostatic factors in the observed phase separations. DNA adsorption involves attractive van der Waals forces, and these forces are also operating in the bulk aggregation process.     

Appearance of D2O in sweat after oral and oral-intravenous rehydration in men

Intravenous (IV) rehydration is common in athletics, but its thermoregulatory benefits and ergogenicity have not been elucidated. Availability of orally ingested fluid is dependent on gastric emptying and intestinal absorption rate. Deuterium oxide (D2O) has been used to demonstrate that fluid ingested during exercise appears in sweat within 10 minutes. The purpose of this study was to determine the effect of concurrent IV rehydration on D2O appearance in sweat samples after per ora rehydration with D2O labeled fluid. We hypothesized that the combination method would not be superior to the oral method. Ten fit men (age 23 ± 4, VO2max 59.49 ± 4.09 L·min(-1)) underwent 20 hours of fluid restriction resulting in 1.95 ± 0.25% body weight loss before beginning treadmill exercise and cycling. Exercise was performed in an environmental chamber (35.6 ± 0.2° C, 35.0 ± 1.8% relative humidity) for 2 hours at 55% VO2max, and the participants exhibited a mean body weight deficit of 4.50 ± 0.04%. Thermoregulatory measures were recorded while subjects were rehydrated with oral (OR) or oral combined with intravenous (IVO) fluid traced with D2O. After 30 minutes of rehydration and 30 minutes of seated recovery, the subjects began treadmill exercise at 55-60% VO2max. Forehead sweat samples were collected 0, 5, 10, 20, and 75 minutes from the start of rehydration. The samples were analyzed for D2O via isotope ratio mass spectrometry. D2O did not appear in the sweat within 20 minutes of rehydration; however, it did appear during the subsequent exercise bout. There was no significant difference between rehydration modes. Plasma volume increases and decreased volume of orally ingested fluid did not significantly alter transit time from ingestion to appearance in excreted sweat. The IVO method does not appear to be superior to the traditional OR method of rehydration.     

Stabilizing action of heavy water (D20) on the cell

Literature data and our own findings show convincingly that heavy water (D2O) stabilizing proteins against denaturants and proteinases increases cell thermostability in plants and animals and that of microorganisms. In Drosophila, along with cell and organism thermostabilities, it enhances thermophilia. Evidence is given in favour of the suggestion that the protein stabilization is responsible for the above biological effects of D2O, although heavy water may increase the melting temperature of nucleic acids and, but slightly, of lipids.     

Medium Isotope Effect in [3H]Diazepam Binding to Benzodiazepine Receptors of Synaptic Membranes

Replacement of H2O by D2O resulted in significantly higher amount of [3H]diazepam specifically bound to synaptic membranes. The isotope effect arises from increased number of binding sites in D2O and is associated with a stronger solvation of membrane receptors by heavy water.     

The Effects of Temperature and Heavy Water on Cell Division in Heat-Synchronized Cells of Tetrahymena*

SYNOPSIS. A study was undertaken of the rates of cell division of heat-synchronized cells of Tetrahymena pyriformis GL at various temperatures in water and 20, 30 and 40% heavy water. The results suggest that division rate is limited by a protein which undergoes both high and low temperature denaturation and that this protein is partially stabilized against heat-denaturation while becoming more susceptible to cold denaturation in the presence of heavy water. Thus, the optimum temperature for division shifts upward as the heavy water concentration is increased, with a maximum shift of 1 C occurring in 40% heavy water. In addition, division activity occurs in heavy water at 34 C, a temperature at which cells kept in water are blocked. Furthermore, the sharp increase in slope seen in the low temperature portion of Arrhenius plots of the data, occurs at higher temperatures when heavy water is present. Finally, at virtually all temperatures, except the highest, heavy water has a depressive effect on division rate indicating a general inhibitory influence of deuterium-substituted water on rate processes within the cell.     

H/D isotope effects on protein hydration and interaction in solution

An approach has been suggested to study the H/D isotope effect on protein-water and protein-protein intermolecular interactions by determining the content of non-freezing water using low-temperature (1)H NMR in mixed (H2O/D2O) water solutions. Direct data are obtained on the amount of H2O adsorbed (absolute hydration) in presence of the heavy isotope (deuterium D), and isothermals of H2O/D2O fractionation at protein surface groups are presented for temperatures between -10 degrees C and -35 degrees C and solutions of varying composition. The fractionation factor, phi = [x/(1 - x)]/[x(0)/(1 - x(0))], where x and x(0) are the fractions of deuterons in hydration and bulk water, respectively, appeared to be extremely high: phi > 1 at 0.03 < x(0) < 0.10. The high values of phi indicate a decrease in apparent hydration of protein molecules. A probable reason of the effect can be an inter-protein molecular solvent-mediated interaction induced by D2O. The excess of phi over 1 appears to provide a quantitative estimate of the fraction of hydration water affected by such interaction.     

Effect of deuterium oxide on actomyosin motility in vitro.

Actin filament velocities in an in vitro motility assay system were measured both in heavy water (deuterium oxide, D(2)O) and water (H(2)O) to examine the effect of D(2)O on the actomyosin interaction. The dependence of the sliding velocity on pD of the D(2)O assay solution showed a broad pD optimum of around pD 8.5 which resembled the broad pH optimum (pH 8.5) of the H(2)O assay solution, but the maximum velocity (4.1+/-0.5 microm/s, n=11) at pD 8.5 in D(2)O was about 60% of that (7.1+/-1.1 microm/s, n=11) at pH 8.5 in H(2)O. The K(m) values of 95 and 80 microM and V(max) values of 3.2 and 5.1 microm/s for the D(2)O and H(2)O assay were obtained by fitting the ATP concentration dependence of the velocity (at pD and pH 7.5) to the Michaelis-Menten equation. The K(m) value of actin-activated Mg-ATPase activity of myosin subfragment 1 (S1) was decreased from 50 microM [actin] in H(2)O to 33 microM [actin] in D(2)O without any significant changes in V(max) (9.4 s(-1) in D(2)O and 9.3 s(-1) in H(2)O). The rate constants of ADP release from the acto-S1-ADP complex measured by the stopped flow method were 361+/-26 s(-1) (n=27) in D(2)O and 512+/-39 s(-1) (n=27) in H(2)O at 6 degrees C. These results suggest that the decrease in the in vitro actin-myosin sliding velocity in D(2)O results from a slowing of the release of ADP from the actomyosin-ADP complex and the increase in the affinity of actin for myosin in the presence of ATP in D(2)O.