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We detected very strong coupling between the oscillating concentration of ATP and the dynamics of intracellular water during glycolysis in Saccharomyces cerevisiae. Our results indicate that: i) dipolar relaxation of intracellular water is heterogeneous within the cell and different from dilute conditions, ii) water dipolar relaxation oscillates with glycolysis and in phase with ATP concentration, iii) this phenomenon is scale-invariant from the subcellular to the ensemble of synchronized cells and, iv) the periodicity of both glycolytic oscillations and dipolar relaxation are equally affected by D2O in a dose-dependent manner. These results offer a new insight into the coupling of an emergent intensive physicochemical property of the cell, i.e. cell-wide water dipolar relaxation, and a central metabolite (ATP) produced by a robustly oscillating metabolic process.

Indirect evidence suggests that impaired triglyceride storage in the subcutaneous fat depot contributes to the development of insulin resistance via lipotoxicity. We directly tested this hypothesis by measuring, in vivo, TG synthesis, de novo lipogenesis (DNL), adipocyte proliferation, and insulin suppression of lipolysis in subcutaneous adipose tissue of BMI-matched individuals classified as insulin resistant (IR) or insulin sensitive (IS). Nondiabetic, moderately obese subjects with BMI 25-35 kg/m(2), classified as IR or IS by the modified insulin suppression test, consumed deuterated water ((2)H2O) for 4 weeks. Deuterium incorporation into glycerol, palmitate, and DNA indicated TG synthesis, DNL, and adipocyte proliferation, respectively. Net TG synthesis and DNL in adipose cells were significantly lower in IR as compared with IS subjects, whereas adipocyte proliferation did not differ significantly. Plasma FFAs measured during an insulin suppression test were 2.5-fold higher in IR subjects, indicating resistance to insulin suppression of lipolysis. Adipose TG synthesis correlated directly with DNL but not with proliferation. These results provide direct in vivo evidence for impaired TG storage in subcutaneous adipose tissue of IR as compared with IS. Relative inability to store TG in the subcutaneous depot may represent a mechanism contributing to the development of insulin resistance in the setting of obesity., (Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.)

Microbial communities are essential to the function of virtually all ecosystems and eukaryotes, including humans. However, it is still a major challenge to identify microbial cells active under natural conditions in complex systems. In this study, we developed a new method to identify and sort active microbes on the single-cell level in complex samples using stable isotope probing with heavy water (D2O) combined with Raman microspectroscopy. Incorporation of D2O-derived D into the biomass of autotrophic and heterotrophic bacteria and archaea could be unambiguously detected via C-D signature peaks in single-cell Raman spectra, and the obtained labeling pattern was confirmed by nanoscale-resolution secondary ion MS. In fast-growing Escherichia coli cells, label detection was already possible after 20 min. For functional analyses of microbial communities, the detection of D incorporation from D2O in individual microbial cells via Raman microspectroscopy can be directly combined with FISH for the identification of active microbes. Applying this approach to mouse cecal microbiota revealed that the host-compound foragers Akkermansia muciniphila and Bacteroides acidifaciens exhibited distinctive response patterns to amendments of mucin and sugars. By Raman-based cell sorting of active (deuterated) cells with optical tweezers and subsequent multiple displacement amplification and DNA sequencing, novel cecal microbes stimulated by mucin and/or glucosamine were identified, demonstrating the potential of the nondestructive D2O-Raman approach for targeted sorting of microbial cells with defined functional properties for single-cell genomics.

Background: Biochemical evidence has linked the coordinate control of fatty acid (FA) synthesis with the activity of stearoyl-CoA desaturase-1 (SCD1). The ratio of 16:1n-7 to 16:0 [SCD1₁₆] in plasma triacylglycerol FA has been used as an index to reflect liver SCD1₁₆ activity and has been proposed as a biomarker of FA synthesis, although this use has not been validated by comparison with isotopically measured de novo lipogenesis (DNL(Meas))., Objective: We investigated plasma lipid 16:1n-7 and FA indexes of elongation and desaturation in relation to lipogenesis., Design: In this cross-sectional investigation of metabolism, 24 overweight adults, who were likely to have elevated DNL, consumed D2O for 10 d and had liver fat (LF) measured by magnetic resonance spectroscopy. Very-low-density lipoprotein (VLDL)-triacylglycerols and plasma free FA [nonesterified fatty acids (NEFAs)] were analyzed by using gas chromatography for the FA composition (molar percentage) and gas chromatography-mass spectrometry and gas chromatography-combustion isotope ratio mass spectrometry for deuterium enrichment., Results: In all subjects, VLDL-triacylglycerol 16:1n-7 was significantly (P < 0.01) related to DNL(Meas) (r = 0.56), liver fat (r = 0.53), and adipose insulin resistance (r = 0.56); similar positive relations were shown with the SCD1₁₆ index, and the pattern in NEFAs echoed that of VLDL-triacylglycerols. Compared with subjects with low LF (3.1 ± 2.7%; n = 11), subjects with high LF (18.4 ± 3.6%; n = 13) exhibited a 45% higher VLDL-triacylglycerol 16:1n-7 molar percentage (P < 0.01), 16% of subjects had lower 18:2n-6 (P = 0.01), and 27% of subjects had higher DNL as assessed by using a published DNL index (ratio of 16:0 to 18:2n-6; P = 0.03), which was isotopically confirmed by DNL(Meas) (increased 2.5-fold; P < 0.01). Compared with 16:0 in the diet, the low amount of dietary 16:1n-7 in VLDL-triacylglycerols corresponded to a stronger signal of elevated DNL., Conclusion: The current data provide support for the use of the VLDL-triacylglycerol 16:1n-7 molar percentage as a biomarker for elevated liver fat when isotope use is not feasible; however, larger-scale confirmatory studies are needed., (© 2015 American Society for Nutrition.)

The objective of this study was to evaluate the validity of total energy expenditure (TEE) provided by Actiheart and Armband. Normal-weight adult volunteers wore both devices either for 17 hours in a calorimetric chamber (CC, n = 49) or for 10 days in free-living conditions (FLC) outside the laboratory (n = 41). The two devices and indirect calorimetry or doubly labelled water, respectively, were used to estimate TEE in the CC group and FLC group. In the CC, the relative value of TEE error was not significant (p > 0.05) for Actiheart but significantly different from zero for Armband, showing TEE underestimation (-4.9%, p < 0.0001). However, the mean absolute values of errors were significantly different between Actiheart and Armband: 8.6% and 6.7%, respectively (p = 0.05). Armband was more accurate for estimating TEE during sleeping, rest, recovery periods and sitting-standing. Actiheart provided better estimation during step and walking. In FLC, no significant error in relative value was detected. Nevertheless, Armband produced smaller errors in absolute value than Actiheart (8.6% vs. 12.8%). The distributions of differences were more scattered around the means, suggesting a higher inter-individual variability in TEE estimated by Actiheart than by Armband. Our results show that both monitors are appropriate for estimating TEE. Armband is more effective than Actiheart at the individual level for daily light-intensity activities.

Slowing the aging process can reduce the risk for multiple chronic diseases simultaneously. It is increasingly recognized that maintaining protein homeostasis (or proteostasis) is important for slowing the aging process. Since proteostasis is a dynamic process, monitoring it is not a simple task and requires use of appropriate methods. This review will introduce methods to assess protein and DNA synthesis using deuterium oxide (D2O), and how protein and DNA synthesis outcomes provide insight into proteostatic mechanisms. Finally, we provide a discussion on how these assessments of protein and DNA synthesis are "mechanistic" investigations and provide an appropriate framework for the further development of slowed aging treatments., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: In clinical practice, global oxygen delivery (DO2) is often considered as a whole; however pathological and adaptive responses after a decrease in individual constituents of the DO2 equation (cardiac output, haemoglobin, oxyhaemoglobin saturation) are likely to be diverse. We hypothesized that an equivalent decrease in DO2 after reductions in each separate component of the equation would result in different haemodynamic, tissue oxygenation, and stress hormonal responses., Methods: Anaesthetized, fluid-resuscitated male Wistar rats were subjected to circulatory, anaemic, or hypoxic hypoxia (by haemorrhage, isovolaemic haemodilution, and breathing a hypoxic gas mix, respectively), produced either rapidly over 5 min or graded over 30 min, to a targeted 50% decrease in global oxygen delivery. Sham-operated animals acted as controls. Measurements were made of haemodynamics, skeletal muscle tissue oxygen tension, blood gas analysis, and circulating stress hormone levels., Results: Whereas haemorrhage generated the largest decrease in cardiac output, and the greatest stress hormone response, haemodilution had the most marked effect on arterial pressure. In contrast, rapid hypoxaemia produced a minor impact on global haemodynamics yet induced the greatest decrease in regional oxygenation. A greater degree of hyperlactataemia was observed with graded insults compared with those administered rapidly., Conclusions: Decreasing global oxygen delivery, achieved by targeted reductions in its separate components, induces varying circulatory, tissue oxygen tension, and stress hormone responses. We conclude that not all oxygen delivery is the same; this disparity should be emphasized in classical teaching and re-evaluated in patient management., (© The Author [2014]. Published by Oxford University Press on behalf of the British Journal of Anaesthesia. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

We recently developed a method to measure mitochondrial proteome dynamics with heavy water ((2)H2O)-based metabolic labeling and high resolution mass spectrometry. We reported the half-lives and synthesis rates of several proteins in the two cardiac mitochondrial subpopulations, subsarcolemmal and interfibrillar (SSM and IFM), in Sprague Dawley rats. In the present study, we tested the hypothesis that the mitochondrial protein synthesis rate is reduced in heart failure, with possible differential changes in SSM versus IFM. Six to seven week old male Sprague Dawley rats underwent transverse aortic constriction (TAC) and developed moderate heart failure after 22weeks. Heart failure and sham rats of the same age received heavy water (5% in drinking water) for up to 80days. Cardiac SSM and IFM were isolated from both groups and the proteins were separated by 1D gel electrophoresis. Heart failure reduced protein content and increased the turnover rate of several proteins involved in fatty acid oxidation, electron transport chain and ATP synthesis, while it decreased the turnover of other proteins, including pyruvate dehydrogenase subunit in IFM, but not in SSM. Because of these bidirectional changes, the average overall half-life of proteins was not altered by heart failure in both SSM and IFM. The kinetic measurements of individual mitochondrial proteins presented in this study may contribute to a better understanding of the mechanisms responsible for mitochondrial alterations in the failing heart., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Purpose: High-throughput quantification of human protein turnover via in vivo administration of deuterium oxide ((2) H2 O) is a powerful new approach to examine potential disease mechanisms. Its immediate clinical translation is contingent upon characterizations of the safety and hemodynamic effects of in vivo administration of (2) H2 O to human subjects., Experimental Design: We recruited ten healthy human subjects with a broad demographic variety to evaluate the safety, feasibility, efficacy, and reproducibility of (2) H2 O intake for studying protein dynamics. We designed a protocol where each subject orally consumed weight-adjusted doses of 70% (2) H2 O daily for 14 days to enrich body water and proteins with deuterium. Plasma proteome dynamics was measured using a high-resolution MS method we recently developed., Results: This protocol was successfully applied in ten human subjects to characterize the endogenous turnover rates of 542 human plasma proteins, the largest such human dataset to-date. Throughout the study, we did not detect physiological effects or signs of discomfort from (2) H2 O consumption., Conclusions and Clinical Relevance: Our investigation supports the utility of a (2) H2 O intake protocol that is safe, accessible, and effective for clinical investigations of large-scale human protein turnover dynamics. This workflow shows promising clinical translational value for examining plasma protein dynamics in human diseases., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Hyperventilation is known to decrease cerebral blood flow (CBF) and to impair cerebral metabolism, but the threshold in patients undergoing intravenous anesthesia is unknown. The authors hypothesized that reduced CBF associated with moderate hyperventilation might impair cerebral aerobic metabolism in patients undergoing intravenous anesthesia., Methods: Thirty male patients scheduled for coronary surgery were included in a prospective, controlled crossover trial. Measurements were performed under fentanyl-midazolam anesthesia in a randomized sequence aiming at partial pressures of carbon dioxide of 30 and 50 mmHg. Endpoints were CBF, blood flow velocity in the middle cerebral artery, and cerebral metabolic rates for oxygen, glucose, and lactate. Global CBF was measured using a modified Kety-Schmidt technique with argon as inert gas tracer. CBF velocity of the middle cerebral artery was recorded by transcranial Doppler sonography. Data were presented as mean (SD). Two-sided paired t tests and one-way ANOVA for repeated measures were used for statistical analysis., Results: Moderate hyperventilation significantly decreased CBF by 60%, blood flow velocity by 41%, cerebral oxygen delivery by 58%, and partial pressure of oxygen of the jugular venous bulb by 45%. Cerebral metabolic rates for oxygen and glucose remained unchanged; however, net cerebral lactate efflux significantly increased from -0.38 (2.18) to -2.41(2.43) µmol min 100 g., Conclusions: Moderate hyperventilation, when compared with moderate hypoventilation, in patients with cardiovascular disease undergoing intravenous anesthesia increased net cerebral lactate efflux and markedly reduced CBF and partial pressure of oxygen of the jugular venous bulb, suggesting partial impairment of cerebral aerobic metabolism at clinically relevant levels of hypocapnia.

The alkanesulfonate monooxygenase system catalyzes the desulfonation of alkanesulfonates through proposed acid-base mechanistic steps that involves the abstraction of a proton from the alkane peroxyflavin intermediate and protonation of the FMN-O(-) intermediate. Both solvent and kinetic isotope studies were performed to define the proton transfer steps involved in the SsuD reaction. Substitution of the protium at the C1 position of octanesulfonate with deuterium resulted in an observed primary isotope effect of 3.0 ± 0.2 on the kcat parameter, supporting abstraction of the α-proton from the alkane peroxyflavin as the rate-limiting step in catalysis. Previous studies implicated Arg226 as the acid involved in the reprotonation of the hydroxyflavin intermediate. Solvent isotope kinetic studies gave an inverse isotope effect on (D2O)kcat of 0.75 ± 0.04 with no observable effect on (D2O)kcat/Km. This resulted in equivalent solvent isotope effects on (D2O)kcat and (D2O)(kcat)D, suggesting a solvent equilibrium isotope effect on a step occurring after the first irreversible step through product release. Data from proton inventory studies on kcat were best fit to a dome-shaped curve consistent with a conformational change to an open conformation during product release. The solvent isotope effect data coupled with the corresponding proton inventory results support and extend our previous observations that Arg226 donates a proton to the FMN-O(-) intermediate, triggering a conformational change that opens the enzyme to solvation and promotes product release.

The stratum corneum is a strong barrier that must be overcome to achieve successful transdermal delivery of a pharmaceutical agent. Many strategies have been developed to enhance the permeation through this barrier. Traditionally, drug penetration through the stratum corneum is evaluated by employing tape-stripping protocols and measuring the content of the analyte. Although effective, this method cannot provide a detailed information regarding the penetration pathways. To address this issue various microscopic techniques have been employed. Raman microscopy offers the advantage of label free imaging and provides spectral information regarding the chemical integrity of the drug as well as the tissue. In this paper we present a relatively simple method to obtain XZ-Raman profiles of human stratum corneum using confocal Raman microscopy on intact full thickness skin biopsies. The spectral datasets were analysed using a spectral unmixing algorithm. The spectral information obtained, highlights the different components of the tissue and the presence of drug. We present Raman images of untreated skin and diffusion patterns for deuterated water and beta-carotene after Franz-cell diffusion experiment., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Human aromatase (CYP19A1) is a steroidogenic cytochrome P450 converting androgens into estrogens. No ligand-free crystal structure of the enzyme is available to date. The crystal structure in complex with the substrate androstenedione and the steroidal inhibitor exemestane shows a very compact conformation of the enzyme, leaving unanswered questions on the conformational changes that must occur to allow access of the ligand to the active site. As H/D exchange kinetics followed by FTIR spectroscopy can provide information on the conformational changes in proteins where solvent accessibility is affected, here the amide I region was used to measure the exchange rates of the different elements of the secondary structure for aromatase in the ligand-free form and in the presence of the substrate androstenedione and the inhibitor anastrozole. Biphasic exponential functions were found to fit the H/D exchange data collected as a function of time. Two exchange rates were assigned to two populations of protons present in different flexible regions of the protein. The addition of the substrate androstenedione and the inhibitor anastrozole lowers the H/D exchange rates of the α-helices of the enzyme when compared to the ligand-free form. Furthermore, the presence of the inhibitor anastrozole lowers exchange rate constant (k1) for β-sheets from 0.22±0.06 min(-1) for the inhibitor-bound enzyme to 0.12±0.02 min(-1) for the free protein. Dynamics effects localised in helix F were studied by time resolved fluorescence. The data demonstrate that the fluorescence lifetime component associated to Trp224 emission undergoes a shift toward longer lifetimes (from ≈5.0 to ≈5.5 ns) when the substrate or the inhibitor are present, suggesting slower dynamics in the presence of ligands. Together the results are consistent with different degrees of flexibility of the access channel and therefore different conformations adopted by the enzyme in the free, substrate- and inhibitor-bound forms.

Human steroid hormone biosynthesis is the result of a complex series of chemical transformations operating on cholesterol, with key steps mediated by members of the cytochrome P450 superfamily. In the formation of the male hormone dehydroepiandrosterone, pregnenolone is first hydroxylated by P450 CYP17A1 at the 17-carbon, followed a second round of catalysis by the same enzyme that cleaves the C17-C20 bond, releasing acetic acid and the 17-keto product. In order to explore the mechanism of this C-C "lyase" activity, we investigated the kinetic isotope effect on the steady-state turnover of Nanodisc-incorporated CYP17A1. Our experiments revealed the expected small positive (~1.3) isotope effect for the hydroxylase chemistry. However, a surprising result was the large inverse isotope effect (~0.39) observed for the C-C bond cleavage activity. These results strongly suggest that the P450 reactive intermediate involved in this latter step is an iron-bound ferric peroxoanion.

Where and how fast does water flow from soil into roots? The answer to this question requires direct and in situ measurement of local flow of water into roots of transpiring plants growing in soil. We used neutron radiography to trace the transport of deuterated water (D₂O) in lupin (Lupinus albus) roots. Lupins were grown in aluminum containers (30 × 25 × 1 cm) filled with sandy soil. D₂O was injected in different soil regions and its transport in soil and roots was monitored by neutron radiography. The transport of water into roots was then quantified using a convection-diffusion model of D₂O transport into roots. The results showed that water uptake was not uniform along roots. Water uptake was higher in the upper soil layers than in the lower ones. Along an individual root, the radial flux was higher in the proximal segments than in the distal segments. In lupins, most of the water uptake occurred in lateral roots. The function of the taproot was to collect water from laterals and transport it to the shoot. This function is ensured by a low radial conductivity and a high axial conductivity. Lupin root architecture seems well designed to take up water from deep soil layers., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

Transaldolase (TA) exchange overestimates gluconeogenesis measured with deuterated water (²H₂O). However, it is unknown whether TA differs in people with type 2 diabetes (T2DM). ²H₂O was ingested, and [1-¹³C]acetate and [3-³H]glucose were infused in T2DM (n = 10) and healthy nondiabetic (ND; n = 8) subjects. TA was assessed from the ratio of ¹³C3 to ¹³C4 glucose enrichment (¹³C3/¹³C4) measured by ¹³C NMR. Glucose turnover was measured before (~16-h fast) and during hyperglycemic (~10 mM) moderate-dose insulin (~0.35 mU·kg⁻¹·min⁻¹) clamp. ¹³C3/¹³C4 in T2DM vs. ND was <1.0 and not different at baseline and clamp, indicating equivalent TA. To determine whether incomplete triose phosphate isomerase (TPI) exchange contributed to asymmetric ¹³C3/¹³C4, [U-¹³C]glycerol was infused in lieu of [1-¹³C]acetate during a separate visit in a subset of ND (n = 7) subjects. Ratio of ¹³C3/¹³C4 obtained following either tracer was <1.0 at baseline and during clamp, indicating that TPI exchange was essentially complete and did not contribute to asymmetric glucose enrichment. Uncorrected and corrected rates of gluconeogenesis were no different (P = not significant) in T2DM vs. ND both at baseline and during clamp. TA correction resulted in equivalent estimates of corrected gluconeogenesis in T2DM and ND that were ~25-35% lower than uncorrected gluconeogenesis both at baseline and during the clamp. The asymmetric enrichment of glucose from ¹³C-gluconeogenic tracers is attributable to TA exchange and can be utilized to correct for TA exchange. In conclusion, TA exchange does not differ between T2DM and ND under fasting or hyperglycemic clamp conditions, and the ²H₂O method continues to provide an accurate estimation of gluconeogenesis.

Heavy water (D2O) has a distinct molecular vibration spectrum, and this has been used to analyze the water content, distribution, and movement in single dormant Bacillus cereus spores using confocal Raman microspectroscopy and Raman imaging. These methods have been used to measure the kinetics of D2O release from spores suspended in H2O, the spatial distribution of D2O in spores, and the kinetics of D2O release from spores during dehydration in air at room temperature. The results obtained were as follows. (1) The Raman spectrum of single D2O-loaded dormant spores suggests that D2O in spores is in a relatively weak hydrogen-bonded mode, compared to the strong hydrogen-bonded mode in pure D2O. (2) The D2O content of individual spores in a population was somewhat heterogeneous. (3) The spatial distribution of D2O in single dormant spores is uneven, and is less dense in the central core region. Raman images of different molecular components indicate that the water distribution is somewhat different from those of proteins and Ca-dipicolinic acid. (4) Exchange of spore D2O with external H2O took place in less than 1 s. (5) However, release of spore D2O during air dehydration at room temperature was slow and heterogeneous and took 2-3 h for complete D2O release.

A lysine instead of the usual carboxyl group is in place of the internal proton donor to the retinal Schiff base in the light-driven proton pump of Exiguobacterium sibiricum (ESR). The involvement of this lysine in proton transfer is indicated by the finding that its substitution with alanine or other residues slows reprotonation of the Schiff base (decay of the M intermediate) by more than 2 orders of magnitude. In these mutants, the rate constant of the M decay linearly decreases with a decrease in proton concentration, as expected if reprotonation is limited by the uptake of a proton from the bulk. In wild type ESR, M decay is biphasic, and the rate constants are nearly pH-independent between pH 6 and 9. Proton uptake occurs after M formation but before M decay, which is especially evident in D2O and at high pH. Proton uptake is biphasic; the amplitude of the fast phase decreases with a pKa of 8.5 ± 0.3, which reflects the pKa of the donor during proton uptake. Similarly, the fraction of the faster component of M decay decreases and the slower one increases, with a pKa of 8.1 ± 0.2. The data therefore suggest that the reprotonation of the Schiff base in ESR is preceded by transient protonation of an initially unprotonated donor, which is probably the ε-amino group of Lys-96 or a water molecule in its vicinity, and it facilitates proton delivery from the bulk to the reaction center of the protein.

To understand the effect of any biomolecules in specific metabolic pathways in humans, bioavailability and for other basic understanding, stable isotopically-labelled biomolecules (preferably deuterated) is the fundamental pre-requisite. Production of deuterated biomolecules such as, astaxanthin, β-carotene, lutein, chlorophyll-a, and eicosapentaenoic acid (EPA, 20:5n-3) by metabolic tagging have been shown in commercially important microalgae, Haematococcus pluvialis and Phaeodactylum tricornutum. These microalgae were grown in appropriate optimized medium supplemented with 25% (v/v) deuterated water. LC-MS analysis showed a maximum of 20, 25, 23, 24, and 27% replacement of hydrogen by deuterium atoms respectively in astaxanthin, β-carotene, lutein, chlorophyll-a, and EPA. To our knowledge, this is the first report on the production of deuterated astaxanthin, chlorophyll-a and EPA by these microalgae.

CD4(+) memory cell development is dependent upon T cell receptor (TCR) signal strength, antigen dose and the cytokine milieu, all of which are altered in type 1 diabetes (T1D). We hypothesized that CD4(+) T cell turnover would be greater in type 1 diabetes subjects compared to controls. In vitro studies of T cell function are unable to evaluate dynamic aspects of immune cell homoeostasis. Therefore, we used deuterium oxide ((2) H(2)O) to assess in vivo turnover of CD4(+) T cell subsets in T1D (n = 10) and control subjects (n = 10). Serial samples of naive, memory and regulatory (T(reg)) CD4(+) T cell subsets were collected and enrichment of deoxyribose was determined by gas chromatography-mass spectrometry (GC-MS). Quantification of T cell turnover was performed using mathematical models to estimate fractional enrichment (f, n = 20), turnover rate (k, n = 20), proliferation (p, n = 10) and disappearance (d*, n = 10). Although turnover of T(regs) was greater than memory and naive cells in both controls and T1D subjects, no differences were seen between T1D and controls in T(reg) or naive kinetics. However, turnover of CD4(+) memory T cells was faster in those with T1D compared to control subjects. Measurement and modelling of incorporated deuterium is useful for evaluating the in vivo kinetics of immune cells in T1D and could be incorporated into studies of the natural history of disease or clinical trials designed to alter the disease course. The enhanced CD4(+) memory T cell turnover in T1D may be important in understanding the pathophysiology and potential treatments of autoimmune diabetes., (© 2013 British Society for Immunology.)

Femtosecond mid-infrared (mid-IR) spectroscopy was used to probe geminate rebinding (GR) dynamics of photo-released nitric oxide (NO) to ferric hemoglobin (Hb(III)) in D2O solution at room temperature. Time-resolved vibrational spectra exhibit two overlapping NO bands for NO-bound Hb(III) (Hb(III)NO), a major band at 1925 cm(-1) (89%) and a minor one at 1905 cm(-1) (11%), suggesting that Hb(III)NO has at least two conformational substates. Both bands decay nonexponentially, each with a different time scale, and the decays are described by a stretched exponential function; the major band's decay is described by 0.96 exp(-t/40 ps)(0.86) + 0.04 and the minor band's decay is described by exp(-t/85 ps)(0.75). These decays arise mainly from the GR of the photo-released NO to Hb(III), indicating that the bound state's conformer influences the NO binding. In particular, the His64 residue, known to have inward conformation in the major band and outward conformation in the minor band, plays a significant role in controlling the binding of NO to Hb(III). The GR of NO to ferric Hb is slower than that to ferrous Hb, which shows fast and efficient GR due to the high reactivity of NO to the heme Fe(ii). The slower GR of NO to Hb(III) may be caused by the lower reactivity of NO to the heme Fe(iii).

Oxygen homeostasis plays a critical role in angiogenesis, erythropoiesis, and cell metabolism. Oxygen homeostasis is set by the hypoxia inducible factor-1α (HIF-1α) pathway, which is controlled by factor inhibiting HIF-1α (FIH). FIH is a non-heme Fe(II), α-ketoglutarate (αKG)-dependent dioxygenase that inhibits HIF-1α by hydroxylating the C-terminal transactivation domain (CTAD) of HIF-1α at HIF-Asn(803). A tight coupling between CTAD binding and O2 activation is essential for hypoxia sensing, making changes in the coordination geometry of Fe(II) upon CTAD encounter a crucial feature of this enzyme. Although the consensus chemical mechanism for FIH proposes that CTAD binding triggers O2 activation by causing the Fe(II) cofactor to release an aquo ligand, experimental evidence of this has been absent. More broadly, this proposed coordination change at Fe(II) has not been observed during steady-state turnover in any αKG oxygenase to date. In this work, solvent isotope effects (SIEs) were used as a direct mechanistic probe of substrate-triggered aquo release in FIH, as inverse SIEs (SIE < 1) are signatures for pre-equilibrium aquo release from metal ions. Our mechanistic studies of FIH have revealed inverse solvent isotope effects in the steady-state rate constants at limiting concentrations of CTAD or αKG [(D2O)kcat/KM(CTAD) = 0.40 ± 0.07, and (D2O)kcat/KM(αKG) = 0.32 ± 0.08], providing direct evidence of aquo release during steady-state turnover. Furthermore, the SIE at saturating concentrations of CTAD and αKG was inverse ((D2O)kcat = 0.51 ± 0.07), indicating that aquo release occurs after CTAD binds. The inverse kinetic SIEs observed in the steady state for FIH can be explained by a strong Fe-OH2 bond. The stable Fe-OH2 bond plays an important part in FIH's regulatory role over O2 homeostasis in humans and points toward a strategy for tightly coupling O2 activation with CTAD hydroxylation that relies on substrate triggering.

Dietary fructose can benefit or hinder glycemic control, depending on the quantity consumed, and these contrasting effects are reflected by alterations in postprandial hepatic glycogen synthesis. Recently, we showed that ²H enrichment of glycogen positions 5 and 2 from deuterated water (²H₂O) informs direct and indirect pathway contributions to glycogenesis in naturally feeding rats. Inclusion of position 6(S) ²H enrichment data allows indirect pathway sources to be further resolved into triose phosphate and Krebs cycle precursors. This analysis was applied to six rats that had fed on standard chow (SC) and six rats that had fed on SC plus 35% sucrose in their drinking water (HS). After 2 wk, hepatic glycogenesis sources during overnight feeding were determined by ²H₂O administration and postmortem analysis of glycogen ²H enrichment at the conclusion of the dark period. Net overnight hepatic glycogenesis was similar between SC and HS rodents. Whereas direct pathway contributions were similar (403 ± 71 μmol/g dry wt HS vs. 578 ± 76 μmol/g dry wt SC), triose phosphate contributions were significantly higher for HS compared with SC (382 ± 61 vs. 87 ± 24 μmol/g dry wt, P < 0.01) and Krebs cycle inputs lower for HS compared with SC (110 ± 9 vs. 197 ± 32 μmol/g dry wt, P < 0.05). Analysis of plasma glucose ²H enrichments at the end of the feeding period also revealed a significantly higher fractional contribution of triose phosphate to plasma glucose levels in HS vs. SC. Hence, the ²H enrichment distributions of hepatic glycogen and glucose from ²H₂O inform the contribution of dietary fructose to hepatic glycogen and glucose synthesis.

d-Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the most abundant enzyme on Earth and is responsible for the fixation of atmospheric CO(2) into biomass. The reaction consists of incorporation of CO(2) and solvent H(2)O into d-ribulose 1,5-bisphosphate (RuBP) to yield 3-phospho-d-glycerate. The reaction involves several proton-dependent events: abstraction and protonation during enolization of RuBP and hydrolysis and reprotonation of the six-carbon reaction intermediate (carboxyketone). Although much is known about Rubisco structure and diversity, fundamental aspects of the reaction mechanism are poorly documented. How and when are protons exchanged among substrate, amino acid residues, and solvent water, and could alterations of proton exchange influence catalytic turnover? What is the energy profile of the reaction? To answer these questions, we measured catalytic rates and the (12)CO(2)/(13)CO(2) isotope effect in isotopic waters. We show that with increasing D(2)O content, the maximal carboxylation velocity (k(cat)(c)) decreased linearly and was 1.7 times lower in pure D(2)O. By contrast, the isotope effect on the apparent Michaelis constant for CO(2) (K(c)) was unity, suggesting that H/D exchange might have occurred with the solvent in early steps thereby slowing the overall catalysis. Calculations of kinetic commitments from observed isotope effects further indicate that (1) enolization and processing of the carboxyketone are similarly rate-limiting and (2) the tendency of the carboxyketone to go backward (decarboxylation) is likely exacerbated upon deuteration. Our results thus suggest that Rubisco catalysis is achieved by a rather equal distribution of energy barriers along the reaction.

The goal of this work was to determine the time-dependent changes in fractional hepatic gluconeogenesis (GNG) during conditions of hindlimb suspension unloading (HSU), a 'ground-based' method for inducing muscular atrophy to simulate space flight. We hypothesized that GNG would increase in HSU conditions as a result of metabolic shifts in the liver and skeletal muscle. A significant and progressive atrophy was observed in the soleus (30, 47 and 55%) and gastrocnemius muscles (0, 15 and 17%) after 3, 7 and 14 days of HSU, respectively. Fractional hepatic GNG was determined following the incorporation of deuterium from deuterated water ((2)H(2)O) into C-H bonds of newly synthesized glucose after an 8 h fast. Enrichment of plasma glucose with (2)H was measured using the classic method of Landau et al. (the 'hexamethylenetetramine (HMT) method'), based on specific (2)H labelling of glucose carbons, and the novel method of Chacko et al. ('average method'), based on the assumption of equal (2)H enrichment on all glucose carbons (except C2). After 3 days of HSU, fractional GNG was significantly elevated in the HSU group, as determined by either method (∼13%, P < 0.05). After 7 and 14 days of HSU, gluconeogenesis was not significantly different. Both analytical methods yielded similar time-dependent trends in gluconeogenic rates, but GNG values determined using the average method were consistently lower (∼30%) than those found by the HMT method. To compare and validate the average method against the HMT method further, we starved animals for 13 h to allow for hepatic GNG to contribute 100% to endogenous glucose production. The HMT method yielded 100% GNG, while the average method yielded GNG of ∼70%. As both methods used the same values of precursor enrichment, we postulated that the underestimation of gluconeogenic rate was as a result of differences in the measurements of product enrichment ((2)H labelling of plasma glucose). This could be explained by the following factors: (i) loss of deuterium via exchange between acetate and glucose; (ii) interference caused by fragment m/z 169, representing multiple isobaric species; and (iii) interference from other sugars at m/z 169. In conclusion, HSU caused a time-dependent increase in hepatic gluconeogenesis, irrespective of the analytical methods used.

Anthropometrics and body mass index are only proxies in the evaluation of adiposity in the pediatric population. Air displacement plethysmography technology was not available for children aged 6 months to 9 years until recently. Our study was designed to test the precision of air displacement plethysmography (ADP) in measuring body fat mass in children at ages 3 to 5 years compared with a criterion method, deuterium oxide dilution (D(2)O), which estimates total body water and a commonly used methodology, dual-energy x-ray absorptiometry (DXA). A prospective, cross-sectional cohort of 66 healthy children (35 girls) was recruited in the central Arkansas region between 2007 and 2009. Weight and height were obtained using standardized procedures. Fat mass (%) was measured using ADP, DXA, and D(2)O. Concordance correlation coefficient and Bland-Altman plots were used to investigate the precision of the ADP techniques against D(2)O and DXA in children at ages 3 to 5 years. ADP concordance correlation coefficient for fat mass was weak (0.179) when compared with D(2)O. Bland-Altman plots revealed a low accuracy and large scatter of ADP fat mass (%) results (mean=-2.5, 95% CI -20.3 to 15.4) compared with D(2)O. DXA fat mass (%) results were more consistent although DXA systematically overestimated fat mass by 4% to 5% compared with D(2)O. Compared with D(2)O, ADP does not accurately assess percent fat mass in children aged 3 to 5 years. Thus, D(2)O, DXA, or quantitative nuclear magnetic resonance may be considered better options for assessing fat mass in young children., (Copyright © 2012 Academy of Nutrition and Dietetics. Published by Elsevier Inc. All rights reserved.)

In ion channels, 'rings' of ionized side chains that decorate the walls of the permeation pathway often lower the energetic barrier to ion conduction. Using single-channel electrophysiological recordings, we studied the poorly understood ring of four glutamates (and one glutamine) that dominates this catalytic effect in the muscle nicotinic acetylcholine receptor ('the intermediate ring of charge'). We show that all four wild-type glutamate side chains are deprotonated in the range of 6.0-9.0 pH, that only two of them contribute to the size of the single-channel current, that these side chains must be able to adopt alternate conformations that either allow or prevent their negative charges from increasing the rate of cation conduction and that the location of these glutamate side chains squarely at one of the ends of the transmembrane pore is critical for their largely unshifted pK(a) values and for the unanticipated impact of their conformational flexibility on cation permeation.

Dietary β-carotene is the most important precursor of vitamin A. However, the determination of the efficiency of in vivo conversion of β-carotene to vitamin A requires sensitive and safe techniques. It presents the following challenges: 1) circulating β-carotene concentration cannot be altered by eating a meal containing ≤6 mg β-carotene; 2) because retinol concentrations are homeostatically controlled, the conversion of β-carotene into vitamin A cannot be estimated accurately in well-nourished humans by assessing changes in serum retinol after supplementation with β-carotene. In the past half-century, techniques using radioisotopes of β-carotene and vitamin A, depletion-repletion with vitamin A and β-carotene supplements, measurement of postprandial chylomicron fractions after consumption of a β-carotene dose, and finally, stable isotopes as tracers to follow the absorption and conversion of β-carotene in humans have been developed. The reported values for β-carotene to vitamin A conversion showed a wide variation from 2 μg β-carotene to 1 μg retinol (for synthetic pure β-carotene in oil) and 28 μg β-carotene to 1 μg retinol (for β-carotene from vegetables). In recent years, a stable isotope reference method (IRM) was developed that used labeled synthetic β-carotene. The IRM method provided evidence that the conversion of β-carotene to vitamin A is likely dose dependent. With the development of intrinsically labeled plant foods harvested from a hydroponic system with heavy water, vitamin A activity of stable isotope-labeled biosynthetic β-carotene from various foods consumed by humans was studied. The efficacy of plant foods rich in β-carotene, such as natural (spinach, carrots, spirulina), hybrid (high-β-carotene yellow maize), and bioengineered (Golden Rice) foods, to provide vitamin A has shown promising results. The results from these studies will be of practical importance in recommendations for the use of pure β-carotene and foods rich in β-carotene in providing vitamin A and ultimately in preventing either overconsumption or poor intake of vitamin A by humans.

Background/objectives: Obesity and undernutrition co-exist in many regions of Mexico. However, accurate assessments are difficult because epidemiological data on body composition are not available. The aim of this study was to facilitate assessments of body composition in Mexican school children of different geographical regions and ethnicity by developing equations for bioelectrical impedance and anthropometry based on deuterium oxide dilution., Subjects/methods: We evaluated 336 subjects (143 belonged to six major indigenous groups) from Northern, Central and Southern Mexico. We measured height (Ht), weight (Wt), tricipital skinfold (Tricp-SKF) and resistance (R) based on a bioimpedance analysis (BIA). Fat-free mass (FFM) and fat mass (FM) were estimated from measurements of total body water with the deuterium dilution technique., Results: The final BIA equation was FFM (kg)=0.661 × Ht²/R+0.200 × Wt-0.320. The R² was 0.96; the square root of the mean square error (SRMSE) was 1.39 kg. The final anthropometric equation was FM (kg)=-1.067 × sex+0.458 × Tricp-SKF+0.263 × Wt-5.407. The R² was 0.91; SRMSE was 1.60 kg. The BIA equation had a bias of 0.095 kg and precision of 1.43 kg. The anthropometric equation had a bias of 0.047 kg and precision of 1.58 kg., Conclusions: We validated two equations for evaluating body composition in Mexican indigenous and non-indigenous children and youth from three main regions of the country. These equations provided reliable estimates and will promote a better understanding of both obesity and undernutrition.

Background: Golden Rice (GR) has been genetically engineered to be rich in β-carotene for use as a source of vitamin A., Objective: The objective was to compare the vitamin A value of β-carotene in GR and in spinach with that of pure β-carotene in oil when consumed by children., Design: Children (n = 68; age 6-8 y) were randomly assigned to consume GR or spinach (both grown in a nutrient solution containing 23 atom% ²H₂O) or [²H₈]β-carotene in an oil capsule. The GR and spinach β-carotene were enriched with deuterium (²H) with the highest abundance molecular mass (M) at M(β-C)+²H₁₀. [¹³C₁₀]Retinyl acetate in an oil capsule was administered as a reference dose. Serum samples collected from subjects were analyzed by using gas chromatography electron-capture negative chemical ionization mass spectrometry for the enrichments of labeled retinol: M(retinol)+4 (from [²H₈]β-carotene in oil), M(retinol)+5 (from GR or spinach [²H₁₀]β-carotene), and M(retinol)+10 (from [¹³C₁₀]retinyl acetate)., Results: Using the response to the dose of [¹³C₁₀]retinyl acetate (0.5 mg) as a reference, our results (with the use of AUC of molar enrichment at days 1, 3, 7, 14, and 21 after the labeled doses) showed that the conversions of pure β-carotene (0.5 mg), GR β-carotene (0.6 mg), and spinach β-carotene (1.4 mg) to retinol were 2.0, 2.3, and 7.5 to 1 by weight, respectively., Conclusions: The β-carotene in GR is as effective as pure β-carotene in oil and better than that in spinach at providing vitamin A to children. A bowl of ~100 to 150 g cooked GR (50 g dry weight) can provide ~60% of the Chinese Recommended Nutrient Intake of vitamin A for 6-8-y-old children.

Background/objectives: In this study, we aimed to validate the accuracy of single-frequency bioelectrical impedance analysis (SF-BIA) at 50 kHz to assess total body water (TBW) against the reference technique deuterium dilution (D(2)O) and to explore if the simple clinical parameters extracellular fluid (ECF) composition and body shape explain individual differences between D(2)O and SF-BIA (Diff(BIA-D(2)O))., Subjects/methods: We assessed TBW with D(2)O and SF-BIA in 26 women and 26 men without known disease or anomalous body shapes. In addition, we measured body shape with anthropometry and ECF composition (osmolality, albumin, glucose, urea, creatinine, sodium and potassium)., Results: On group average, SF-BIA to predict TBW agreed well with D(2)O (SF-BIA, 39.8 ± 10.1 l; D(2)O, 40.4 ± 10.2 l; and Diff(BIA-D(2)O) -0.7 l). In four individuals ('outliers'; 15% of the study population), Diff(BIA-D(2)O) was high (-6.8 to +3.8 l). Diff(BIA-D(2)O) was associated with individual variations in body shape rather than ECF composition. Using gender-specific analysis, we found that individual variability of waist circumference in men and arm length in women significantly contributed to Diff(BIA-D(2)O). When removing the four 'outliers', these associations were lost., Conclusions: In the majority of our sample, BIA agreed well with D(2)O. Adjusting for individual variability in body shape by anthropometrical assessment could possibly improve the accuracy of SF-BIA for individuals who deviate from mean values with respect to body shape. However, further studies with higher subject numbers are needed to confirm our findings.

Understanding the pathologies related to the regulation of protein metabolism requires methods for studying the kinetics of individual proteins. We developed a (2)H(2)O metabolic labeling technique and software for protein kinetic studies in free living organisms. This approach for proteome dynamic studies requires the measurement of total body water enrichments by GC-MS, isotopic distribution of the tryptic peptide by LC-MS/MS, and estimation of the asymptotical number of deuterium incorporated into a peptide by software. We applied this technique to measure the synthesis rates of several plasma lipoproteins and acute phase response proteins in rats. Samples were collected at different time points, and proteins were separated by a gradient gel electrophoresis. (2)H labeling of tryptic peptides was analyzed by ion trap tandem mass spectrometry (LTQ MS/MS) for measurement of the fractional synthesis rates of plasma proteins. The high sensitivity of LTQ MS in zoom scan mode in combination with (2)H label amplification in proteolytic peptides allows detection of the changes in plasma protein synthesis related to animal nutritional status. Our results demonstrate that fasting has divergent effects on the rate of synthesis of plasma proteins, increasing synthesis of ApoB 100 but decreasing formation of albumin and fibrinogen. We conclude that this technique can effectively measure the synthesis of plasma proteins and can be used to study the regulation of protein homeostasis under physiological and pathological conditions.

Exclusive breastfeeding during the first 6 months of infant's life is a public health recommendation and important factor for the promotion of optimal growth, health and behavioural development of each child. The accuracy of the mothers' self-reported past infant-feeding events was examined and compared with the isotopic dilution technique. Breastfeeding practices were assessed in a sample of 44 Cameroonian mother-infant pairs using dietary recall since birth. Intakes of breast milk and non-breast milk water were measured in the same sample using the dose-to-the-mother deuterium-oxide turnover technique and compared with questionnaire. Results showed that mothers' self-reported behaviour overestimates the exclusive breastfeeding rate. Seventy-five per cent of the mothers who claimed to be exclusively breastfeeding were found to be predominantly or partially breastfeeding by the dose-to-the-mother deuterium-oxide turnover technique. Only 11% of the infants were exclusively breastfed, and the breast milk output was not significantly affected (P ≤ 0.05) by the mother's body composition. Mean intakes of breast milk and non-breast milk water were 701 mL day(-1) and 268 mL day(-1), respectively. Introduction of non-breast milk foods is associated with a reduction in the level of breast milk intake, but the difference in breast milk intake was not significant between exclusively and predominantly breastfed infants. In conclusion, the dose-to-the-mother deuterium-oxide turnover technique can be applied to validate the mother's reports of infant-feeding practices, but non-breast milk water intake by breastfeeding category still needs to be normalized., (© 2011 Blackwell Publishing Ltd.)

The active site of photosynthetic water oxidation by Photosystem II (PSII) is a manganese-calcium cluster (Mn(4)CaO(5)). A postulated catalytic base is assumed to be crucial. CP43-Arg357, which is a candidate for the identity of this base, is a second-sphere ligand of the Mn(4)-Ca cluster and is located near a putative proton exit pathway, which begins with residue D1-D61. Transient absorption spectroscopy and time-resolved O(2) polarography reveal that in the D1-D61N mutant, the transfer of an electron from the Mn(4)CaO(5) cluster to Y(Z)(OX) and O(2) release during the final step of the catalytic cycle, the S(3)-S(0) transition, proceed simultaneously but are more dramatically decelerated than previously thought (t(1/2) of up to ~50 ms vs a t(1/2) of 1.5 ms in the wild type). Using a bare platinum electrode to record the flash-dependent yields of O(2) from mutant and wild-type PSII has allowed the observation of the kinetics of release of O(2) from extracted thylakoid membranes at various pH values and in the presence of deuterated water. In the mutant, it was possible to resolve a clear lag phase prior to the appearance of O(2), indicating formation of an intermediate before the onset of O(2) formation. The lag phase and the photochemical miss factor were more sensitive to isotope substitution in the mutant, indicating that proton efflux in the mutant proceeds via an alternative pathway. The results are discussed in comparison with earlier results obtained from the substitution of CP43-Arg357 with lysine and in regard to hypotheses concerning the nature of the final steps in photosynthetic water oxidation. These considerations led to the conclusion that proton expulsion during the initial phase of the S(3)-S(0) transition starts with the deprotonation of the primary catalytic base, probably CP43-Arg357, followed by efficient proton egress involving the carboxyl group of D1-D61 in a process that constitutes the lag phase immediately prior to O(2) formation chemistry.

We synthesized block copolymer composed of hyaluronic acid (HA) and poly(DL-lactide-co-glycolide) (PLGA) (HAbLG) for antitumor targeting. (1)H NMR was employed to confirm synthesis of block copolymer. At (1)H NMR study, HabLG nanoparticles showed HA intrinsic peaks only at D(2)O, indicating that they contained HA as a hydrophilic outer-shell and PLGA as a inner-core. Anti-tumor activity was studied using CD44-overexpressing HCT-116 human colon carcinoma cells. Addition of doxorubicin (DOX)-incorporated nanoparticles to tumor cells resulted in the expression of a strong red fluorescence color while they expressed very weak fluorescence when CD44 receptor was blocked with free HA. Flow cytometry data also showed similar results, indicating that the fluorescence intensity of tumor cells treated with nanoparticles was significantly decreased when CD44 receptor was blocked. These results indicate that HAbLG nanoparticles were able to target CD44-overexpressing tumor cells via receptor-mediated endocytosis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Leaf waxes protect terrestrial plants from biotic and abiotic stresses and are important sedimentary biomarkers for terrestrial plants. Thus, understanding the production and ablation of leaf waxes is critical in plant physiology and for geochemical studies. However, there have been no accurate approaches to quantify leaf wax production at different time scales. In this study, we demonstrate a novel approach to study leaf wax regeneration by irrigating plants with a pulse of deuterium-enriched water, followed by measurements of leaf wax D/H ratios by gas chromatography/isotope-ratio mass spectrometry (GC/IRMS). We demonstrate the efficacy of this approach using the grass species Phleum pratense in a greenhouse environment. Using a binary isotope mass balance model, we are able to quantify the regeneration rates of the C(16), C(18) acids and leaf waxes (C(23)-C(31) n-alkanes; C(22)-C(30) n-acids) over a diurnal cycle. Our results show that within one day 33-47% of C(16) and C(18) acids are regenerated, and thus the recycling time for these compounds is 2-3 days. For C(22)-C(26) n-alkyl lipids, 7-21% are regenerated within one day and thus they require 5-16 days to recycle. In comparison, the recycling time for long-chain n-alkyl lipids (C(27)-C(31)) is as long as 71-128 days. Our approach can be applied to different plants at shorter or longer time scales by adjusting the degree of isotopic labeling, sampling intervals and the amount of irrigation water., (Copyright © 2011 John Wiley & Sons, Ltd.)

Error-prone polymerase chain reactions (epPCRs) are often used to introduce mutations in random mutagenesis, which has been used as a tool in protein engineering. Here, we developed a new method of epPCR using heavy water as a solvent instead of normal water (H(2)O). Rhodopsin cDNA of the Ayu fish (Plecoglossus altivelis) was used as a template and was amplified using five different conditions: (A) 100% H(2)O with no Mn(2+), (B) 100% H(2)O/0.6mM Mn(2+), (C) 99% D(2)O with no Mn(2+), (D) 99% D(2)O/0.6mM Mn(2+) and (E) 99% H(2)(18)O with no Mn(2+). The 13,960 (for each of the conditions A to D) and 33,504 (for condition E) base pairs were sequenced. A maximum error rate of 1.8×10(-3)errors/bp was detected in condition D, without any particular hot-spot mutations. A high preference for AT→GC transitions was observed in condition D, whereas a high preference for transitions over transversions was observed in condition C. All of the mutations observed in condition E were transversions. When conditions A and C were applied to another template, the honeybee actin gene, the results were comparable to those for Ayu rhodopsin. Based on these results, the use of heavy water, instead of H(2)O, as a solvent for epPCR can introduce random mutations without positional bias, template dependency or decreased yield. Our new epPCR method, and possibly combining the use of D(2)O and H(2)(18)O, may be a powerful random mutagenesis technique., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The use of stable isotopes in ecological studies requires that we know the magnitude of discrimination factors between consumer and element sources. The causes of variation in discrimination factors for carbon and nitrogen have been relatively well studied. In contrast, the discrimination factors for hydrogen have rarely been measured. We grew cabbage looper caterpillars (Trichoplusia ni) on cabbage (Brassica oleracea) plants irrigated with four treatments of deuterium-enriched water (δD = -131, -88, -48, and -2‰, respectively), allowing some of them to reach adulthood as moths. Tissue δD values of plants, caterpillars, and moths were linearly correlated with the isotopic composition of irrigation water. However, the slope of these relationships was less than 1, and hence, discrimination factors depended on the δD value of irrigation water. We hypothesize that this dependence is an artifact of growing plants in an environment with a common atmospheric δD value. Both caterpillars and moths were significantly enriched in deuterium relative to plants by ∼45‰ and 23‰ respectively, but the moths had lower tissue to plant discrimination factors than did the caterpillars. If the trophic enrichment documented here is universal, δD values must be accounted for in geographic assignment studies. The isotopic value of carbon was transferred more or less faithfully across trophic levels, but δ(15)N values increased from plants to insects and we observed significant non-trophic (15)N enrichment in the metamorphosis from larvae to adult.

Water can pass through biological membranes via two pathways: simple diffusion through the lipid bilayer, or water-selective facilitated diffusion through aquaporins (AQPs). Although AQPs play an important role in osmotic water permeability (P(f)), the role of AQPs in diffusional water permeability remains unclear because of the difficulty of measuring diffusional water permeability (P(d)). Here, we report an accurate and instantaneous method for measuring the P(d) of a single HeLa S3 cell using coherent anti-Stokes Raman scattering (CARS) microscopy with a quick perfusion device for H(2)O/D(2)O exchange. Ultra-high-speed line-scan CARS images were obtained every 0.488 ms. The average decay time constant of CARS intensities (τ(CARS)) for the external solution H(2)O/D(2)O exchange was 16.1 ms, whereas the intracellular H(2)O/D(2)O exchange was 100.7 ± 19.6 ms. To evaluate the roles of AQP in diffusional water permeability, AQP4 fused with enhanced green fluorescent protein (AQP4-EGFP) was transiently expressed in HeLa S3 cells. The average τ(CARS) for the intracellular H(2)O/D(2)O exchange in the AQP4-EGFP-HeLa S3 cells was 43.1 ± 15.8 ms. We also assessed the cell volume and the cell surface area to calculate P(d). The average P(d) values for the AQP4-EGFP-HeLa S3 cells and the control EGFP-HeLa S3 cells were 2.7 ± 1.0 × 10(-3) and 8.3 ± 2.6 × 10(-4) cm/s, respectively. AQP4-mediated water diffusion was independent of the temperature but was dependent on the expression level of the protein at the plasma membrane. These results suggest the possibility of using CARS imaging to investigate the hydrodynamics of single mammalian cells as well as the regulation of AQPs., (Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The M2 proton channel is essential for the replication of the flu virus and is a known drug target. The functional mechanism of channel activation and conductance is key to both the basic biology of viral replication and the design of drugs that can withstand mutations. A quantitative model was previously developed for calculating the rate of proton transport through the M2 channel. The permeant proton was assumed to diffuse to the pore, obligatorily bind to the His37 tetrad, and then dissociate and be released to either side of the tetrad. Here the model is used to calculate the effect of a change in solvent from H(2)O to D(2)O on the rate of proton transport. The solvent substitution affects two parameters in the model: the proton diffusion constant and the pK (a) for proton binding to the His37 tetrad. When the known effects on these two parameters are included, the deuterium isotope effect calculated from the model is in quantitatively agreement with experimental results. This strict test of the theoretical model provides strong support for the hypothesis that the permeant proton obligatorily binds to and then unbinds from the His37 tetrad. This putatively essential role of the His37 tetrad in the functional mechanism of the M2 channel makes it a promising target for designing mutation-tolerant drugs.

Mangrove forest trees grow in severe conditions such as diurnal submersion and high salinity surface and subsurface waters. This study focuses on two species on Mayotte Island, i.e. Ceriops tagal and Rhizophora mucronata, living in the middle range of the coastal mangrove. The seedlings of these trees were planted in a tropical greenhouse with an original pump system built to reproduce the natural tidal effect. The water used by these saplings, in two contrasted salinity conditions, was measured by lysimetry. For adult species, the trees' water consumption was measured on the field side after being injected with heavy water (D(2)O). Our work shows that this isotopic technique also works in saline conditions, and a water consumption of around 1 ± 0.2 L per day and per centimeter of diameter was found. These values are discussed as follows: the techniques used, the distinctive features of the mangrove trees, and other factors affecting the water absorption., (Copyright © 2011 John Wiley & Sons, Ltd.)

A membrane cell for hydrogen and deuterium exchange on-line with mass spectrometry has been developed to monitor protein-protein interactions and protein conformations. It consists of two channels separated by a semipermeable membrane, where one channel carries the protein sample and the other deuterium oxide. The membrane allows transfer of deuterium oxide into the sample flow. The labeling time is controlled via the flow rate in the sample channel. This cell was validated against three models commonly used in hydrogen-deuterium exchange mass spectrometry: monitoring of folded and unfolded states in a protein, mapping the protein secondary structure at the peptide level, and detection of protein and antibody interactions. The system avoids the conventionally used sample dilution and handling, allowing for potential automation.

Rapid responses of bacteria to sudden changes in their environment can have important implications for the structure and function of microbial communities. In this study, we used heavy-water stable isotope probing (H2(18)O-SIP) to identify bacteria that respond to soil rewetting. First, we conducted experiments to address uncertainties regarding the H2(18)O-SIP method. Using liquid chromatography-mass spectroscopy (LC-MS), we determined that oxygen from H2(18)O was incorporated into all structural components of DNA. Although this incorporation was uneven, we could effectively separate 18O-labeled and unlabeled DNAs derived from laboratory cultures and environmental samples that were incubated with H2(18)O. We found no evidence for ex vivo exchange of oxygen atoms between DNA and extracellular H2O, suggesting that 18O incorporation into DNA is relatively stable. Furthermore, the rate of 18O incorporation into bacterial DNA was high (within 48 to 72 h), coinciding with pulses of CO2 generated from soil rewetting. Second, we examined shifts in the bacterial composition of grassland soils following rewetting, using H2(18)O-SIP and bar-coded pyrosequencing of 16S rRNA genes. For some groups of soil bacteria, we observed coherent responses at a relatively course taxonomic resolution. Following rewetting, the relative recovery of Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria increased, while the relative recovery of Chloroflexi and Deltaproteobacteria decreased. Together, our results suggest that H2(18)O-SIP is effective at identifying metabolically active bacteria that influence soil carbon dynamics. Our results contribute to the ecological classification of soil bacteria while providing insight into some of the functional traits that influence the structure and function of microbial communities under dynamic soil moisture regimes.

The advantages of using (2)H(2)O to quantify cholesterol synthesis include i) homogeneous precursor labeling, ii) incorporation of (2)H via multiple pathways, and iii) the ability to perform long-term studies in free-living subjects. However, there are two concerns. First, the t(1/2) of tracer in body water presents a challenge when there is a need to acutely replicate measurements in the same subject. Second, assumptions are made regarding the number of hydrogens (n) that are incorporated during de novo synthesis. Our primary objective was to determine whether a step-based approach could be used to repeatedly study cholesterol synthesis a subject. We observed comparable changes in the (2)H-labeling of plasma water and total plasma cholesterol in African-Green monkeys that received five oral doses of (2)H(2)O, each dose separated by one week. Similar rates of cholesterol synthesis were estimated when comparing data in the group over the different weeks, but better reproducibility was observed when comparing replicate determinations of cholesterol synthesis in the same nonhuman primate during the respective dosing periods. Our secondary objective was to determine whether n depends on nutritional status in vivo; we observed n of ∼25 and ∼27 in mice fed a high-carbohydrate (HC) versus carbohydrate-free (CF) diet, respectively. We conclude that it is possible to acutely repeat studies of cholesterol synthesis using (2)H(2)O and that n is relatively constant.

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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

While the water permeability of the plasma membranes of mammalian cells has been studied extensively, water transport across membranes of subcellular compartments (e.g., lysosomes, macropinosomes) has been difficult to study. Here we demonstrate a new method for measuring water flux in late endosomes and lysosomes of intact living cells using time-lapse fluorescence microscopy. Cells were loaded by fluid-phase uptake with a mixture of the Lucifer Yellow dextran (LY-dex), a D(2)O sensitive dye, and a D(2)O insensitive control dye, Alexa fluor 546 dextran (AF546-dex). LY-dex responded linearly to changes in D(2)O concentration and the LY-dex D(2)O sensitivity was not affected by changes in pH, physiological salt, and protein concentrations. The co-loaded control dye, AF546-dex, showed no signal changes as a function of D(2)O concentration. To measure membrane water flux, the LY-dex fluorescence in labeled organelles was recorded during rapid superfusion of cells with isotonic buffers prepared in D(2)O. The time constant of water exchange across the lysosomal membrane of intact cells was determined by fitting the data to a single exponential function. From these data, together with the measured area of the organelles, observed water permeability for intracellular CHO-K1 lysosomes was calculated to be 5.3 × 10(-3) ± 0.3 × 10(-3) cm/s. This work demonstrates the feasibility of measuring water flux into subcellular organelles in live cells using LY-dex.

Cerebral blood flow (CBF) quantification is a valuable tool in stroke research. Mice are of special interest because of the potential of genetic engineering. Magnetic resonance imaging (MRI) provides repetitive, noninvasive CBF quantification. Many MRI techniques require the knowledge of the brain-blood partition coefficient (BBPC) for water. Adopting an MRI protocol described by Roberts et al (1996) in humans, we determined the BBPC for water in 129S6/SvEv mice from proton density measurements of brain and blood, calibrated with deuterium oxide/water phantoms. The average BBPC for water was 0.89 ± 0.03 mL/g, with little regional variation within the mouse brain.

A description is given of the results of neutron diffraction studies of the structures of four different metal-ion complexes of deuterated D-xylose isomerase. These represent four stages in the progression of the biochemical catalytic action of this enzyme. Analyses of the structural changes observed between the various three-dimensional structures lead to some insight into the mechanism of action of this enzyme.

N-Ethylglutamate (NEG) was detected in Escherichia coli BL21 cells grown on LB broth, and it was found to occur at a concentration of ∼4 mM in these cells under these conditions. The same cells grown on M9 glucose medium contained no detectable amount of NEG. Analysis of the LB broth showed the presence of NEG, a compound never before reported as a natural product. Isotope dilution analysis showed that it occurred at a concentration of 160 μM in LB broth. Analyses of yeast extract and tryptone, the organic components of LB broth, both showed the presence NEG. It was demonstrated that NEG can be generated during the autolysis of the yeast used in the preparation of the yeast extract. Growth of these E. coli cells in LB broth prepared in deuterated water showed no incorporation of deuterium into NEG, demonstrating that E. coli cells did not generate the NEG. Cell growth rates were not affected by the addition of 5 mM NEG to either LB or M9 glucose medium. l-[ethyl-(2)H(4)]NEG was found to be readily incorporated into the cells and metabolized by the cells. From these results, it was concluded that all of the NEG present in the cells was taken up from the medium. NEG could serve as the sole nitrogen source for E. coli when grown on M9 glucose medium in the presence of glucose but could not serve as the sole carbon source on M9 medium in the absence of glucose.

• As described in the two-layer hypothesis, woody plants are often assumed to use deep soils to avoid competition with grasses. Yet the direct measurements of root activity needed to test this hypothesis are rare. • Here, we injected deuterated water into four soil depths, at four times of year, to measure the vertical and horizontal location of water uptake by trees and grasses in a mesic savanna in Kruger National Park, South Africa. • Trees absorbed 24, 59, 14 and 4% of tracer from the 5, 20, 50, and 120  cm depths, respectively, while grasses absorbed 61, 29, 9 and 0.3% of tracer from the same depths. Only 44% of root mass was in the top 20 cm. Trees absorbed tracer under and beyond their crowns, while 98% of tracer absorbed by grasses came from directly under the stem. • Trees and grasses partitioned soil resources (20 vs 5  cm), but this partitioning did not reflect, as suggested by the two-layer hypothesis, the ability of trees to access deep soil water that was unavailable to grasses. Because root mass was a poor indicator of root activity, our results highlight the importance of precise root activity measurements.