Systemic deuteration of SCID mice using the water-isotopologue deuterium oxide (D2 O) inhibits tumor growth in an orthotopic bioluminescent model of human pancreatic ductal adenocarcinoma.

Since its initial discovery as a natural isotopologue of dihydrogen oxide (1 H2 O), extensive research has focused on the biophysical, biochemical, and pharmacological effects of deuterated water (2 H2 O [D2 O, also referred to as "heavy water"]). Using a panel of cultured human pancreatic ductal adenocarcinoma (PDAC) cells we have profiled (i) D2 O-induced phenotypic antiproliferative and apoptogenic effects, (ii) redox- and proteotoxicity-directed stress response gene expression, and (iii) phosphoprotein-signaling related to endoplasmic reticulum (ER) and MAP-kinase stress response pathways. Differential array analysis revealed early modulation of stress response gene expression in both BxPC-3 and PANC-1 PDAC cells elicited by D2 O (90%; ≤6 h; upregulated: HMOX1, NOS2, CYP2E1, CRYAB, DDIT3, NFKBIA, PTGS1, SOD2, PTGS2; downregulated: RUNX1, MYC, HSPA8, HSPA1A) confirmed by independent RT-qPCR analysis. Immunoblot-analysis revealed rapid (≤6 h) onset of D2 O-induced MAP-kinase signaling (p-JNK, p-p38) together with ER stress response upregulation (p-eIF2α, ATF4, XBP1s, DDIT3/CHOP). Next, we tested the chemotherapeutic efficacy of D2 O-based drinking water supplementation in an orthotopic PDAC model employing firefly luciferase-expressing BxPC-3-FLuc cells in SCID mice. First, feasibility and time course of systemic deuteration (30% D2 O in drinking water; 21 days) were established using time-resolved whole-body proton magnetic resonance imaging and isotope-ratio mass spectrometry-based plasma (D/H)-analysis. D2 O-supplementation suppressed tumor growth by almost 80% with downregulated expression of PCNA, MYC, RUNX1, and HSP70 while increasing tumor levels of DDIT3/CHOP, HO-1, and p-eIF2α. Taken together, these data demonstrate for the first time that pharmacological induction of systemic deuteration significantly reduces orthotopic tumor burden in a murine PDAC xenograft model.

Monitoring bacterial spore metabolic activity using heavy water-induced Raman peak evolution.

Endospore-forming bacteria are associated with food spoilage, food poisoning, and infection in hospitals. Therefore, methods to monitor spore metabolic activity and verify sterilization are of great interest. However, current methods for tracking metabolic activity are time-consuming and resource intensive. This work investigates isotope labeling and Raman microscopy as a low-cost rapid alternative. Specifically, we monitor the Raman spectrum of enterotoxic B. cereus spores undergoing germination and cell division in D2O-infused broth. During germination and cell division, water is metabolized and deuterium from the broth is incorporated into proteins and lipids, resulting in the appearance of a Raman peak related to C-D bonds at 2190 cm-1. We find that a significant C-D peak appears after 2 h of incubation at 37 °C. Further, we found that the peak appearance coincides with the observed first cell division indicating little metabolic activity during germination. Lastly, the germination and cell growth rate of spores were not affected by adding 30% heavy water to the broth. This shows the potential for real-time monitoring of metabolic activity from a bacterial spore to a dividing cell. In conclusion, our work proposes tracking the evolution of the C-D Raman peak in spores incubated with D2O-infused broth as an effective and time-, and cost-efficient method to monitor the outgrowth of a spore population, simultaneously allowing us to track for how long the bacteria have grown and divided.

Prenatal Molecular Hydrogen Administration Ameliorates Several Findings in Nitrofen-Induced Congenital Diaphragmatic Hernia.

Oxidative stress plays a pathological role in pulmonary hypoplasia and pulmonary hypertension in congenital diaphragmatic hernia (CDH). This study investigated the effect of molecular hydrogen (H2), an antioxidant, on CDH pathology induced by nitrofen. Sprague-Dawley rats were divided into three groups: control, CDH, and CDH + hydrogen-rich water (HW). Pregnant dams of CDH + HW pups were orally administered HW from embryonic day 10 until parturition. Gasometric evaluation and histological, immunohistochemical, and real-time polymerase chain reaction analyses were performed. Gasometric results (pH, pO2, and pCO2 levels) were better in the CDH + HW group than in the CDH group. The CDH + HW group showed amelioration of alveolarization and pulmonary artery remodeling compared with the CDH group. Oxidative stress (8-hydroxy-2'-deoxyguanosine-positive-cell score) in the pulmonary arteries and mRNA levels of protein-containing pulmonary surfactant that protects against pulmonary collapse (surfactant protein A) were significantly attenuated in the CDH + HW group compared with the CDH group. Overall, prenatal H2 administration improved respiratory function by attenuating lung morphology and pulmonary artery thickening in CDH rat models. Thus, H2 administration in pregnant women with diagnosed fetal CDH might be a novel antenatal intervention strategy to reduce newborn mortality due to CDH.

Deuterium oxide protects against myocardial injury induced by ischemia and reperfusion in rats.

Objectives. Although deuterium oxide (D2O) has preservative property on the extracted organ, whether D2O also protects the in situ myocardial injury remains unknown. Using cardiac microdialysis, local administration of D2O through dialysis probe was applied in situ rat heart. We examined the effect of the D2O on the myocardial injury induced ischemia, reperfusion, and chemical hypoxia. Methodology. We measured dialysate myoglobin levels during 30 min of coronary occlusion and reperfusion in the absence and presence of D2O. Furthermore, to confirm the effect of D2O on NaCN induced myocardial injury, we measured the dialysate myoglobin levels with local perfusion of NaCN in the absence and presence of D2O. Results. The dialysate myoglobin levels increased from 177 ± 45 ng/mL at baseline to 3030 ± 1523 ng/mL during 15-30 min of coronary occlusion and further increased to 8588 ± 1684ng/mL at 0-15 min of reperfusion. The dialysate myoglobin levels with 60 min local perfusion of NaCN increased to 1214 ± 279 ng/mL. D2O attenuated myocardial myoglobin release during 15-30 min of coronary occlusion and 0-30 min of reperfusion and 15-60 min of local perfusion of NaCN. Conclusions. D2O might have a beneficial effect of myocardium against ischemia, reperfusion and chemical hypoxia.

Novel confocal Raman microscopy method to investigate hydration mechanisms in human skin.

BACKGROUND: Skin hydration is essential for maintaining stratum corneum (SC) flexibility and facilitating maturation events. Moisturizers contain multiple ingredients to maintain and improve skin hydration although a complete understanding of hydration mechanisms is lacking. The ability to differentiate the source of the hydration (water from the environment or deeper skin regions) upon application of product will aid in designing more efficacious formulations. MATERIALS AND METHODS: Novel confocal Raman microscopy (CRM) experiments allow us to investigate mechanisms and levels of hydration in the SC. Using deuterium oxide (D2 O) as a probe permits the differentiation of endogenous water (H2 O) from exogenous D2 O. Following topical application of D2 O, we first compare in vivo skin depth profiles with those obtained using ex vivo skin. Additional ex vivo experiments are conducted to quantify the kinetics of D2 O diffusion in the epidermis by introducing D2 O under the dermis. RESULTS: Relative D2 O depth profiles from in vivo and ex vivo measurements compare well considering procedural and instrumental differences. Additional in vivo experiments where D2 O was applied following topical glycerin application increased the longevity of D2 O in the SC. Reproducible rates of D2 O diffusion as a function of depth have been established for experiments where D2 O is introduced under ex vivo skin. CONCLUSION: Unique information regarding hydration mechanisms are obtained from CRM experiments using D2 O as a probe. The source and relative rates of hydration can be delineated using ex vivo skin with D2 O underneath. One can envision comparing these depth-dependent rates in the presence and absence of topically applied hydrating agents to obtain mechanistic information.

Using [2H]water to quantify the contribution of de novo palmitate synthesis in plasma: enabling back-to-back studies.

An increased contribution of de novo lipogenesis (DNL) may play a role in cases of dyslipidemia and adipose accretion; this suggests that inhibition of fatty acid synthesis may affect clinical phenotypes. Since it is not clear whether modulation of one step in the lipogenic pathway is more important than another, the use of tracer methods can provide a deeper level of insight regarding the control of metabolic activity. Although [2H]water is generally considered a reliable tracer for quantifying DNL in vivo (it yields a homogenous and quantifiable precursor labeling), the relatively long half-life of body water is thought to limit the ability of performing repeat studies in the same subjects; this can create a bottleneck in the development and evaluation of novel therapeutics for inhibiting DNL. Herein, we demonstrate the ability to perform back-to-back studies of DNL using [2H]water. However, this work uncovered special circumstances that affect the data interpretation, i.e., it is possible to obtain seemingly negative values for DNL. Using a rodent model, we have identified a physiological mechanism that explains the data. We show that one can use [2H]water to test inhibitors of DNL by performing back-to-back studies in higher species [i.e., treat nonhuman primates with platensimycin, an inhibitor of fatty acid synthase]; studies also demonstrate the unsuitability of [13C]acetate.

Aquaporin 11-Dependent Inhibition of Proliferation by Deuterium Oxide in Activated Hepatic Stellate Cells.

Deuterium oxide (D2O) has been reported to be active toward various in vitro cell lines in combination with phytochemicals. Our objective was to describe, for the first time, the effect of D2O on the proliferation of hepatic stellate cells (HSCs). After D2O treatment, the p53-cyclin-dependent kinase (CDK) pathway was stimulated, leading to inhibition of the proliferation of HSCs and an increase in the [ATP]/[ADP] ratio. We also evaluated the role of aquaporin (AQP) 11 in activated HSCs. We found that D2O treatment decreased AQP11 expression levels. Of note, AQP11 levels elevated by a genetic approach counteracted the D2O-mediated inhibition of proliferation. In addition, the expression levels of AQP11 negatively correlated with those of p53. On the other hand, cells transfected with an AQP11-targeted small interfering RNA (siRNA) showed enhanced inhibition of proliferation. These findings suggest that the inhibition of cell proliferation by D2O in activated HSCs could be AQP11 dependent. Our previous studies have documented that bisdemethoxycurcumin (BDMC) induces apoptosis by regulating heme oxygenase (HO)-1 protein expression in activated HSCs. In the current study, we tested whether cotreatment with BDMC and D2O can modulate the AQP11-dependent inhibition of cell proliferation effectively. We observed that D2O cotreatment with BDMC significantly decreased cell proliferation compared to treatment with D2O alone, and this effect was accompanied by downregulation of HO-1 and an increase in p53 levels.

Deuterium Oxide Enhances Escherichia coli SOS Response Induced by Genotoxicants.

It has been demonstrated that deuterium oxide enhances the SOS response of Escherichia coli cells induced by chemical genotoxicants and mutagens. This demonstrates that the heavy nonradioactive hydrogen isotope deuterium can be considered to be a comutagen.

Determination of (2)H-enrichment of rat brain interstitial fluid and rat plasma by headspace-gas-chromatography - quadrupole-mass-spectrometry.

(2)H2O as nonradioactive, stable marker substance is commonly used in preclinical and clinical studies and the precise determination of (2)H2O concentration in biological samples is crucial. However, aside from isotope ratio mass spectrometry (IRMS), only a very limited number of methods to accurately measure the (2)H2O concentration in biological samples are routinely established until now. In this study, we present a straightforward method to accurately measure (2)H-enrichment of rat brain interstitial fluid (ISF) and rat plasma to determine the relative recovery of a cerebral open flow microperfusion (cOFM) probe, using headspace-gas-chromatography - quadrupole-mass-spectrometry. This method is based on basic-catalyzed hydrogen/deuterium exchange in acetone and detects the (2)H-labelled acetone directly by the headspace GC-MS. Small sample volumes and limited number of preparation steps make this method highly competitive. It has been fully validated. (2)H enriched to 8800 ppm in plasma showed an accuracy of 98.9% and %Relative Standard Deviation (RSD) of 3.1 with n = 18 over three days and with two operators. Similar performance was obtained for cerebral ISF enriched to 1100 ppm (accuracy: 96.5%, %RSD: 3.1). With this highly reproducible method we demonstrated the successful employment of (2)H2O as performance marker for a cOFM probe.

Modulation of inhibitory and excitatory fast neurotransmission in the rat CNS by heavy water (D2O).

The effects of heavy water (deuterium oxide, D2O) on GABAergic and glutamatergic spontaneous and evoked synaptic transmission were investigated in acute brain slice and isolated "synaptic bouton" preparations of rat hippocampal CA3 neurons. The substitution of D2O for H2O reduced the frequency and amplitude of GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) in a concentration-dependent manner but had no effect on glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs). In contrast, for evoked synaptic responses in isolated neurons, the amplitude of both inhibitory and excitatory postsynaptic currents (eIPSCs and eEPSCs) was decreased in a concentration-dependent manner. This was associated with increases of synaptic failure rate (Rf) and paired-pulse ratio (PPR). The effect was larger for eIPSCs compared with eEPSCs. These results clearly indicate that D2O acts differently on inhibitory and excitatory neurotransmitter release machinery. Furthermore, D2O significantly suppressed GABAA receptor-mediated whole cell current (IGABA) but did not affect glutamate receptor-mediated whole cell current (IGlu). The combined effects of D2O at both the pre- and postsynaptic sites may explain the greater inhibition of eIPSCs compared with eEPSCs. Finally, D2O did not enhance or otherwise affect the actions of the general anesthetics nitrous oxide and propofol on spontaneous or evoked GABAergic and glutamatergic neurotransmissions, or on IGABA and IGlu. Our results suggest that previously reported effects of D2O to mimic and/or modulate anesthesia potency result from mechanisms other than modulation of GABAergic and glutamatergic neurotransmission.

Investigating the relationship between changes in collagen fiber orientation during skin aging and collagen/water interactions by polarized-FTIR microimaging.

Upon chronological aging, human skin undergoes structural and molecular modifications, especially at the level of type I collagen. This macromolecule is one of the main dermal structural proteins and presents several age-related alterations. It exhibits a triple helical structure and assembles itself to form fibrils and fibers. In addition, water plays an important role in stabilizing the collagen triple helix by forming hydrogen-bonds between collagen residues. However, the influence of water on changes of dermal collagen fiber orientation with age has not been yet understood. Polarized-Fourier Transform Infrared (P-FTIR) imaging is an interesting biophotonic approach to determine in situ the orientation of type I collagen fibers, as we have recently shown by comparing skin samples of different ages. In this work, P-FTIR spectral imaging was performed on skin samples from two age groups (35- and 38-year-old on the one hand, 60- and 66-year-old on the other hand), and our analyses were focused on the effect of H2O/D2O substitution. Spectral data were processed with fuzzy C-means (FCM) clustering in order to distinguish different orientations of collagen fibers. We demonstrated that the orientation was altered with aging, and that D2O treatment, affecting primarily highly bound water molecules, is more marked for the youngest skin samples. Collagen-bound water-related spectral markers were also highlighted. Our results suggest a weakening of water/collagen interactions with age. This non-destructive and label-free methodology allows us to understand better the importance of bound water in collagen fiber orientation alterations occurring with skin aging. Obtaining such structural information could find benefits in dermatology as well as in cosmetics.

[PECULIARITIES OF THE CELLULAR COMPOSITION OF SPLENIC LYMPHOID TISSUE IN MICE AFTER LONG-TERM USE OF LIGHT WATER AND IRRADIATION].

The changes of the cellular composition of splenic lymphoid tissue were studied 7, 15 and 30 days after irradiation with a dose of 50 rad, in BALB/c mice which received either distilled water or light (deuterium-depleted) water for a long time prior to and after irradiation. The irregular pattern of changes of splenic cellular composition was observed during the experiment. It was found that at day 7 after irradiation, the splenic structural zones in mice demonstrated a sharp decrease in the number of blast forms and mitotic cells, reflecting a lower level of lymphocytopoiesis, as well as an increased cellular destruction in mice consuming light water. By day 30 of the experiment, different responses of lymphoid structures were observed in the organ. In the periarteriolar lymphoid sheaths, the processes of cellular composition regeneration were more pronounced than in the germinal centers of lymphoid nodules, indicating the enhancement of body cell-mediated immunity and immunomodulating properties of light water in mice at later dates of post-irradiation period.

A validation of the application of D(2)O stable isotope tracer techniques for monitoring day-to-day changes in muscle protein subfraction synthesis in humans.

Quantification of muscle protein synthesis (MPS) remains a cornerstone for understanding the control of muscle mass. Traditional [(13)C]amino acid tracer methodologies necessitate sustained bed rest and intravenous cannulation(s), restricting studies to ~12 h, and thus cannot holistically inform on diurnal MPS. This limits insight into the regulation of habitual muscle metabolism in health, aging, and disease while querying the utility of tracer techniques to predict the long-term efficacy of anabolic/anticatabolic interventions. We tested the efficacy of the D2O tracer for quantifying MPS over a period not feasible with (13)C tracers and too short to quantify changes in mass. Eight men (22 ± 3.5 yr) undertook one-legged resistance exercise over an 8-day period (4 × 8-10 repetitions, 80% 1RM every 2nd day, to yield "nonexercised" vs. "exercise" leg comparisons), with vastus lateralis biopsies taken bilaterally at 0, 2, 4, and 8 days. After day 0 biopsies, participants consumed a D2O bolus (150 ml, 70 atom%); saliva was collected daily. Fractional synthetic rates (FSRs) of myofibrillar (MyoPS), sarcoplasmic (SPS), and collagen (CPS) protein fractions were measured by GC-pyrolysis-IRMS and TC/EA-IRMS. Body water initially enriched at 0.16-0.24 APE decayed at ~0.009%/day. In the nonexercised leg, MyoPS was 1.45 ± 0.10, 1.47 ± 0.06, and 1.35 ± 0.07%/day at 0-2, 0-4, and 0-8 days, respectively (~0.05-0.06%/h). MyoPS was greater in the exercised leg (0-2 days: 1.97 ± 0.13%/day; 0-4 days: 1.96 ± 0.15%/day, P < 0.01; 0-8 days: 1.79 ± 0.12%/day, P < 0.05). CPS was slower than MyoPS but followed a similar pattern, with the exercised leg tending to yield greater FSRs (0-2 days: 1.14 ± 0.13 vs. 1.45 ± 0.15%/day; 0-4 days: 1.13 ± 0.07%/day vs. 1.47 ± 0.18%/day; 0-8 days: 1.03 ± 0.09%/day vs. 1.40 ± 0.11%/day). SPS remained unchanged. Therefore, D2O has unrivaled utility to quantify day-to-day MPS in humans and inform on short-term changes in anabolism and presumably catabolism alike.

[Isotopic effects of low concentration of deuterium in water on biological systems].

Isotopic effects of deuterium in water are studied in a broad range of concentrations on a number of biological objects of different organization levels. The results obtained show that biological objects are sensitive to variations of isotope composition in water. A decrease or increase in deuterium concentrations in water may cause activation or inhibition of biological functions. The values of biological isotopic effects of low deuterium concentration may even be higher than those of high deuterium concentration. No regularity in response for all the objects studied failed to find out in a range of deuterium concentration in water from 4 ppm to 1%.

Important Constituents of Heavy Water-containing Solution for Cold Storage and Subsequent Reperfusion on an Isolated Perfused Rat Liver.

The University of Wisconsin (UW) solution is the most effective preservation solution currently used; however, to safely use expanded-criteria donor grafts, a new cold storage solution that alleviates graft injury more effectively is required. We prepared a heavy water (D2O)-containing buffer, Dsol, and observed strong protective effects during extended cold storage of rat hearts and livers. In the current study, we modified Dsol (mDsol) and tested its efficacy. The aim of the present study was to determine whether mDsol could protect the rat liver more effectively than the UW solution and to clarify the roles of D2O and deferoxamine (DFX). Rat livers were subjected to cold storage for 48 hours in test solutions: UW, mDsol, mDsol without D2O or DFX (mDsol-D2O[-], mDsol-DFX[-]), and subsequently reperfused on an isolated perfused rat liver for 90 minutes at 37°C. In the UW group, the liver was dehydrated during cold storage and rapidly expanded during reperfusion. Accordingly, the cumulative weight change was the highest in the UW group, together with augmented portal veinous resistance and ALT leakage and decreased oxygen consumption rate and bile production. These changes were significantly suppressed in the mDsol-treated group. In the mDsol-D2O(-) and mDsol-DFX(-) groups offered partial protection. In conclusion, mDsol appeared to be superior to the UW solution for simple cold storage of the rat liver, presumably due to improved microcirculation in the early phase of reperfusion. Both heavy water and deferoxamine are essential for alleviating seamless organ swelling that occurs during cold storage and subsequent reperfusion.

A large solvent isotope effect on protein association thermodynamics.

Solvent reorganization can contribute significantly to the energetics of protein-protein interactions. However, our knowledge of the magnitude of the energetic contribution is limited, in part, by a dearth of quantitative experimental measurements. The biotin repressor forms a homodimer as a prerequisite to DNA binding to repress transcription initiation. At 20 °C, the dimerization reaction, which is thermodynamically coupled to binding of a small ligand, bio-5'-AMP, is characterized by a Gibbs free energy of -7 kcal/mol. This modest net dimerization free energy reflects underlying, very large opposing enthalpic and entropic driving forces of 41 ± 3 and -48 ± 3 kcal/mol, respectively. The thermodynamics have been interpreted as indicating coupling of solvent release to dimerization. In this work, this interpretation has been investigated by measuring the effect of replacing H2O with D2O on the dimerization thermodynamics. Sedimentation equilibrium measurements performed at 20 °C reveal a solvent isotope effect of -1.5 kcal/mol on the Gibbs free energy of dimerization. Analysis of the temperature dependence of the reaction in D2O indicates enthalpic and entropic contributions of 28 and -37 kcal/mol, respectively, considerably smaller than the values measured in H2O. These large solvent isotope perturbations to the thermodynamics are consistent with a significant contribution of solvent release to the dimerization reaction.

Impact of vitrification on the meiotic spindle and components of the microtubule-organizing center in mouse mature oocytes.

Cryopreservation of oocytes is becoming a valuable method for fertility preservation in women. However, various unphysiological alterations occur in the oocyte during the course of cryopreservation, one of which is the disappearance of the meiotic spindle. Fortunately, the meiotic spindle does regenerate after thawing the frozen oocytes, which enables completion of meiosis and further development after fertilization. Nonetheless, the mechanistic understanding of the meiotic spindle regeneration after cryopreservation is still scarce. Here, to gain insight into the mechanisms of the spindle disappearance and regeneration, we examined the status of spindle microtubules as well as the key components of the microtubule-organizing center (MTOC), specifically gamma-Tubulin, NEDD1, and Pericentrin, in mature (metaphase II) mouse oocytes at different steps of vitrification, a major cryopreservation technique. We found that the configuration of the spindle microtubules dynamically changed during the process of vitrification and that spindle regeneration was preceded by excessive microtubule polymerization, followed by reduction into the normal size and shape. Also, all three MTOC components exhibited disappearance and reappearance during the vitrification process, although Pericentrin appeared to regenerate in earlier steps compared to the other components. Furthermore, we found that the localization of the MTOC components to the spindle poles persisted even after depolymerization of spindle microtubules, suggesting that the MTOC components are impacted by vitrification independently from the integrity of the microtubules. The present study would set the stage for future investigations on the molecular mechanisms of the meiotic spindle regeneration, which may contribute to further improving protocols for oocyte cryopreservation.

Combination of Cold Storage in a Heavy Water-Containing Solution and Post-Reperfusion Hydrogen Gas Treatment Reduces Ischemia-Reperfusion Injury in Rat Livers.

We previously reported the efficacy of cold storage (CS) using a heavy water-containing solution (Dsol) and post-reperfusion hydrogen gas treatment separately. This study aimed to clarify the combined effects of these treatments. Rat livers were subjected to 48-hour CS and a subsequent 90-minute reperfusion in an isolated perfused rat liver system. The experimental groups were the immediately reperfused control group (CT), the CS with University of Wisconsin solution (UW) group, the CS with Dsol group, the CS with UW and post-reperfusion H2 treatment group (UW-H2), and the CS with Dsol and post-reperfusion H2 group (Dsol-H2). We first compared the Dsol-H2, UW, and CT groups to evaluate this alternative method to conventional CS. The protective potential of the Dsol-H2 group was superior to that of the UW group, as evidenced by lower portal venous resistance and lactate dehydrogenase leakage, a higher oxygen consumption rate, and increased bile production. Multiple comparison tests among the UW, Dsol, UW-H2, and Dsol-H2 groups revealed that both treatments, during CS and after reperfusion, conferred a similar extent of protection and showed additive effects in combination therapy. Furthermore, the variance in all treatment groups appeared smaller than that in the no-treatment or no-stress groups, with excellent reproducibility. In conclusion, combination therapy with Dsol during CS and hydrogen gas after reperfusion additively protects against graft injury.

Temperature- and hydration-dependent internal dynamics of stripped human erythrocyte vesicles studied by incoherent neutron scattering.

BACKGROUND: We focus on temperature- and hydration-dependence of internal molecular motions in stripped human red blood cell (RBC) vesicles, widely used as a model system for more complex biomembranes. METHODS: We singled out picosecond local motions of the non-exchangeable hydrogen atoms of RBC vesicles by performing elastic and quasielastic incoherent neutron scattering measurements in dry and heavy water (D2O)-hydrated RBC powders. RESULTS: In dry stripped RBCs, hydrogen motions remained harmonic all along the measured temperature range (100-310K) and mean-square displacements (MSDs) exhibited no temperature transition up to 310K. In contrast, MSDs of hydrated stripped RBCs (h ≈ 0.38g D2O/g dry powder) exhibited a pronounced transition near 260K, with the sharp rise of anharmonic diffusive motions of hydrogen atoms. This transition at ~260K was correlated with both the onset of nonvibrational (harmonic and nonharmonic) motions and the melting of crystallized hydration water. GENERAL SIGNIFICANCE: In conclusion, we have shown that MSDs in human RBC vesicles are temperature-and hydration-dependent. These results provide insight into biomembrane internal dynamics at picosecond timescale and nanometer length scale. Such motions have been shown to act as the "lubricant" of larger conformational changes on a slower, millisecond timescale that are necessary for important biological processes.

Deuteration as a tool in investigating the role of protons in cell signaling.

BACKGROUND: The mechanisms underlying the inhibitory effects of deuterium oxide (D2O; heavy water) are likely to provide insight into the fundamental significance of hydrogen bonds in biological functions. Previously, to begin elucidating the effect of D2O on physiological functions in living cells, we studied the effects of D2O on voltage-sensitive Ca²(+) channels in AtT 20 cells and showed that actin distribution, Ca²(+) currents, and ß-endorphin release were affected. However, the molecular mechanisms underlying the inhibitory effects of D2O in whole animals and living cells remain obscure, especially in the effects of D2O on the cell signaling. METHODS: We investigated the molecular mechanisms underlying the inhibitory effects of D2O on the IP3-mediated Ca²(+) signaling pathway using Ca²(+) imaging and micro-calorimetric measurements in mGluR1-expressing CHO cells. RESULTS: DHPG-induced Ca²(+) elevations were markedly reduced in D2O. Moreover, the Ca²(+) elevations were completely suppressed in H2O after receptor activation with DHPG in D2O, recovering gradually in H2O medium. Without prior stimulation in D2O, however, DHPG-induced Ca²(+) elevations in H2O were not affected. Micro-calorimetric measurements showed reduced total DHPG-evoked heat generation in D2O, while initial heat production and absorption associated with receptor activation were found to be larger. The reduction of DHPG-induced Ca²(+) elevation and heat generation in D2O medium may be due to decreased amount of IP3 by the reduced hydrolysis of PIP2. GENERAL SIGNIFICANCE: Protein structure changes due to the replacement of hydrogen with deuterium will induce the inhibitory effects of D2O by reduction of the frequency of -OH bonds.