A study of gramicidin using deuterium oxide

The single channel conductivity of the gramicidin channel has been measured for all the alkali ions using both H₂O and ²H₂O as a medium. Significant changes in conductivity with medium have been observed in all cases except lithium.     

Dynamics in synthetic oligonucleotides. A solid-state deuterium NMR study

Solid-state 2H NMR spectroscopy was used to investigate dynamics in the [methyl-2H]thymidine-labeled oligonucleotide [d(CGCGAAT*T*CGCG)]2. Quadrupole echo line shapes, spin-lattice relaxation, and quadrupolar echo decay rates were investigated as a function of hydration W (moles of water/moles of nucleotide) between 0 and approximately 30. The amplitude of the base motion, modeled as a fast four-site libration, or diffusion in a cone, increased slightly with higher levels of hydration. A slower component of motion about the helix axis appears at W approximately 10 and increases in rate and amplitude, leading to the intermediate rate line shape observed at W approximately 21.     

A study of gramicidin using deuterium oxide.

The single channel conductivity of the gramicidin channel has been measured for all the alkali ions using both H2O and 2H2O as a medium. Significant changes in conductivity with medium have been observed in all cases except lithium.     

A deuterium NMR study of labelled n-alkanol anesthetics in a model membrane

The 2H-NMR spectra of 50 wt.% aqueous multilamellar dispersions of dipalmitoylphosphatidylcholine (DPPC) containing either selectively deuterated 1-decanol (25 mol%) or [2H17]-1-octanol (25 mol%) have been measured as a function of temperature. Both alkanols are potent anesthetics. A detailed carbon-deuterium bond order parameter profile of 1-decanol in liquid crystalline phospholipid dispersions at 50 degrees C was determined from the quadrupolar splittings of 1-decanols deuterated at eight different positions. A maximum order parameter SCD = 0.20 was obtained for [5,5-2H2]-1-decanol, with labels at both ends of the 1-decanol exhibiting reduced order parameters. Explanations for the reduced order towards the hydroxyl group of 1-decanol are discussed in terms of either increased amplitudes of motion or geometric effects due to hydrogen bonding. By comparing the order parameter profile of sn-2 chain deuterated phosphatidylcholine dispersions containing 25 mol% 1-decanol (J.L. Thewalt, S.R. Wassall, H. Gorrissen and R.J. Cushley, Biochim. Biophys. Acta, 817 (1985) 355) with the profile of deuterated 1-decanol in DPPC, we estimate that decanol is approximately parallel to the C-3 to C-13 region of the phosphatidylcholine's sn-2 chain. Variation of the spectral moments M1 with temperature indicates that both 1-decanol and 1-octanol are sensitive to the packing of the lipid in which they are dissolved. Below the phase transition temperature, the 2H-NMR spectra of either 1-decanol (selectively deuterated) or 1-octanol (perdeuterated) are broad powder patterns, characteristic of axially symmetric rotation about the alcohol's long axis. This is in contrast to the 2H-NMR spectra obtained from deuterated phosphatidylcholine under similar conditions, which implies that the phospholipid acyl chain conformations are more restricted than those of the alcohol at these temperatures. From the M1 behavior of the various alkanol chain segments with temperature, the gel to liquid crystalline phase transition is seen to initiate in the middle of the DPPC/1-alkanol bilayer.     

The molecular dynamics of cholesterol in bilayer membranes: A deuterium NMR study

The ²H-NMR spectra of selectively deuterated cholesterol, intercalated in egg phosphatidyl-choline, were examined. The orientation of the axis of motional averaging was calculated using the observed quadrupole splittings and the atomic coordinates. With the known orientation of the rotation axis, quadrupole splittings observed for deuterium labels on cholesterol can be related to the molecular order parameter of the sterol. In addition, knowledge of the axis orientation allows prediction of the magnitudes of quadrupole splittings for deuterium at other positions, which is useful in the choice of labelling for particular applications. Finally, preliminary relaxation time measurements yield information on the rates of anisotropic motion of cholesterol in bilayer membranes.     

A novel approach to estimate trabecular bone anisotropy using a database approach

Continuum finite element (FE) models of bones have become a standard pre-clinical tool to estimate bone strength. These models are usually based on clinical CT scans and material properties assigned are chosen as isotropic based only on the density distribution. It has been shown, however, that trabecular bone elastic behavior is best described as orthotropic. Unfortunately, the use of orthotropic models in FE analysis derived from CT scans is hampered by the fact that the measurement of a trabecular orientation (fabric) is not possible from clinical CT images due to the low resolution of such images. In this study, we explore the concept of using a database (DB) of high-resolution bone models to derive the fabric information that is missing in clinical images. The goal of this study was to investigate if models with fabric derived from a relatively small database can already produce more accurate results than isotropic models.     

A novel approach to the study of kinetically changing cell populations

A method is described for estimating changes in cell cycle times during periods of rapid change in proliferation rate. This method, which depends upon the interpretation of pre- and post-velocity sedimentation fractionation continuous thymidine labelling patterns, exploits the relationship between sedimentation rate and cell cycle location. By this means, cycle times can be estimated under conditions that are difficult (if not impossible) to analyse by FLM methods.     

The effect of hydration on the dynamics of trimethoprim bound to dihydrofolate reductase. A deuterium NMR study.

To determine the effect of hydration on the dynamics of a protein complex, we used deuterium nuclear magnetic resonance (NMR) techniques to examine a trimethoprim (TMP)/E. coli dihydrofolate reductase (DHFR) complex in its lyophilized, partially hydrated, polycrystalline, and ammonium sulfate-precipitated states. The results indicate that TMP is rigid in the lyophilized powder state. The dynamic behavior could be restored by partial rehydration. At 30 wt% hydration the deuterium spectrum of the partially hydrated sample was indistinguishable from that of the polycrystalline and ammonium sulfate-precipitated samples, suggesting that the structure of the protein/TMP complex is similar in the three physical states. Furthermore, we found that the para- and meta-methoxyl groups have very different dynamical behavior.     

Application of quantitative deuterium NMR to the study of isotope fractionation in the conversion of saccharides to ethanols

³H-PAF-acether (Alkyl-[1′,2′-³H]-2-acetyl-sn-glyceryl-3-phosphorylcholine) was time-dependently transformed by plasma-free rabbit platelets into an unknown product, “PX”, which was distinct from lyso-PAF-acether. This effect was specific for platelets since plasma only converted ³H-PAF-acether into lyso-³H-PAF-acether and platelets were not effective in metabolizing lyso-³H-PAF-acether. Platelet aggregation did not interfere with the formation of “PX”. The latter is a novel platelet metabolite of PAF-acether with as yet unknown biological properties.     

The temperature dependence of chain disorder in potassium palmitate-water. A deuterium NMR study

Quadrupolar echo deuterium magnetic resonance spectroscopy is used to study the hydrocarbon chain disorder in a 70% potassium palmitate-30% water mixture. The C-D bond order parameters are measured as a function of temperature and position along the chain. In the liquid crystalline, L_α, phase the C-D bond order parameters of the methylene segments near the polar head group are found to increase with increasing temperature to a maximum at about 100°C and then decrease on further increasing the temperature. The order parameters for the remaining methylenes and the methyl group decrease monotonically with increasing temperature. The C–D bond order parameter for the α position undergoes a discontinuous change at $\text{～55°}\text{C}$ and, in fact, for a 10°C range two resolvable splittings are observed for the α posittion. The low temperature, coagel, phase gives a well resolved spectrum where all methylenes are equivalent yielding a value of 168 kHz for e²qQ/h, the quadrupole coupling constant.     

A deuterium surface coil NMR study of the metabolism of D-methionine in the liver of the anesthetized rat

The hepatic metabolism of deuteriated D-methionine has been studied in the intact, anesthetized rat using 2H NMR spectroscopy. The rate of formation of the principal labeled metabolite, [methyl-2H3]sarcosine, from the D-[methyl-2H3]methionine precursor was found to be as rapid as the rate observed previously in NMR studies of the hepatic metabolism of L-methionine. Similarly, rates of clearance of labeled methionine from the liver, formation of N-trimethyl-labeled metabolites, and labeling of the HDO pool were all found to be similar to the rates observed in the L-methionine studies. In contrast, all of these metabolic transformations are strongly inhibited by pretreatment of the rats with sodium benzoate, an inhibitor of D-amino acid oxidase. In vivo 2H NMR studies of sodium benzoate treated rats given L-[methyl-2H3]-methionine exhibit a much more rapid formation of [methyl-2H3]sarcosine than rats given the D enantiomer, consistent with the expectation that the sodium benzoate does not interfere with either the formation of S-adenosylmethionine or the subsequent transmethylation of glycine. However, the rates of methionine clearance and formation of deuteriated water are markedly reduced in this study relative to rats receiving the labeled D- or L-methionine without sodium benzoate pretreatment. These results indicate that subsequent to the initial oxidative deamination of the labeled D-methionine, the reamination to give L-methionine is rapid compared with the further degradation of the alpha-keto acid. Thus, the results are consistent with a dominant contribution of the glycine/sarcosine shuttle to the metabolism of excess D- or L-methionine.     

A novel transfection method for mammalian cells using gas plasma

Introduction of foreign genes into target cells is a crucial step for achievement of gene therapy. We have recently developed a novel transfection system for eukaryotic cells, namely the electric pulse-activated gas plasma generator. To measure the transfection efficiency and mortality by flow-cytometry, we employed enhanced green fluorescent protein and propidium iodide staining, respectively. One day after the 1-3s plasma exposures with DNA concentration at 0.5 microg/microl, favorable transfection efficiencies (17.8-21.6%) and mortalities (0.65-2.86%) were obtained for HeLa-S3, HT-1080 and MCF-7 cells. The recipient cells became transiently permeable for plasmid DNA during the plasma exposure, suggesting that plasma-mediated transfection may involve similar mechanisms that accounts for electroporation. The relatively low mortality rates are encouraging in our attempt to apply this system to the various cell lines including the primary cell cultures.     

A rapid approach to lipid profiling of mycobacteria using 2D HSQC NMR maps

Mycobacteria, including Mycobacterium tuberculosis, are characterized by a unique cell wall rich in complex lipids, glycolipids, polyketides, and terpenoids. Many of these metabolites have been shown to play important roles in mycobacterial virulence and their inherent resistance to many antibiotics. Here, we report the development of a new simple method for global analysis of these metabolites using two-dimensional (1)H-(13)C heteronuclear single quantum coherence nuclear magnetic resonance. The major advantages of this method are as follows: the small amount of sample and the minimal sample manipulation required; a relatively short procedural time; and the ability to rapidly attain a qualitative and quantitative lipid profile of a mycobacterial sample in which the majority of the clinically relevant lipids can be observed simultaneously. The effectiveness of this method is demonstrated in four different areas of major concern to the mycobacterial research community: i) adaptive changes in cell wall lipids as a result of drug treatment; ii) analysis of gene function; iii) characterization of new mycobacterial species; and iv) analysis of the production of virulence factors in clinical isolates of M. tuberculosis. This method is complementary to mass spectrometry-based lipidomic technologies and provides an urgently needed tool to gain a better understanding of the role of lipids in mycobacteria pathogenesis.     

The study of diabetic cataractogenesis in the intact rabbit lens by deuterium NMR spectroscopy

The polyol pathway has been implicated in the process of diabetic cataractogenesis. We report the use of deuterium (2H) spectroscopy for dynamically monitoring the polyol and glycolytic pathways in the single intact rabbit lens. Using 2H labeled C-1 D-glucose, the formation of sorbitol from glucose and the metabolism of sorbitol to fructose was dynamically monitored at 5.5 mM and 35.5 mM glucose concentrations. The accumulation of sorbitol at 35.5 mM glucose concentration was prevented by the inhibition of aldose reductase using an inhibitor (Sorbinil). 2H spectra were obtained in short acquisition times because of the short T1's of deuterated metabolites. A further advantage of 2H spectroscopy is that the natural abundance resonance of water (HDO) can be used as an internal reference standard. These findings confirm previous studies and demonstrate for the first time by NMR spectroscopy activity in the polyol pathway at low glucose concentrations.     

The effect of ceramide on phosphatidylcholine membranes: a deuterium NMR study

Biological membranes contain domains having distinct physical properties. We study defined mixtures of phosphoglycerolipids and sphingolipids to ascertain the fundamental interactions governing these lipids in the absence of other cell membrane components. By using (2)H-NMR we have determined the temperature and composition dependencies of membrane structure and phase behavior for aqueous dispersions of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the ceramide (Cer) N-palmitoyl-sphingosine. It is found that gel and liquid-crystalline phases coexist over a wide range of temperature and composition. Domains of different composition and phase state are present in POPC/Cer membranes at physiological temperature for Cer concentrations exceeding 15 mol %. The acyl chains of liquid crystalline phase POPC are ordered by the presence of Cer. Moreover, Cer's chain ordering is greater than that of POPC in the liquid crystalline phase. However, there is no evidence of liquid-liquid phase separation in the liquid crystalline region of the POPC/Cer phase diagram.     

A mammalian functional-genetic approach to characterizing cancer therapeutics

Identifying mechanisms of drug action remains a fundamental impediment to the development and effective use of chemotherapeutics. Here we describe an RNA interference (RNAi)-based strategy to characterize small-molecule function in mammalian cells. By examining the response of cells expressing short hairpin RNAs (shRNAs) to a diverse selection of chemotherapeutics, we could generate a functional shRNA signature that was able to accurately group drugs into established biochemical modes of action. This, in turn, provided a diversely sampled reference set for high-resolution prediction of mechanisms of action for poorly characterized small molecules. We could further reduce the predictive shRNA target set to as few as eight genes and, by using a newly derived probability-based nearest-neighbors approach, could extend the predictive power of this shRNA set to characterize additional drug categories. Thus, a focused shRNA phenotypic signature can provide a highly sensitive and tractable approach for characterizing new anticancer drugs.