Determination of fast proton exchange rates of biomolecules by NMR using water selective diffusion experiments

We present a new water selective pulse sequence allowing rapid determination of exchange rates of labile protons on the millisecond time scale. Using diffusion measurements, exchange rates of resolved protons can be determined without prior knowledge of relaxation parameters in a short overnight experiment. The use of a sensitive, highly selective and easy to implement water excitation scheme allows for its straightforward application to a wide range of biomolecules. The results obtained for the imino proton exchange rates of a 16 bp DNA are in strong agreement with values obtained by the classical approach of two-dimensional exchange spectroscopy.     

Localized T2 measurements using an OSIRIS-CPMG method. Application to measurements of blood oxygenation and transverse relaxation free of diffusion effect

This work presents a new method allowing localized T2 measurements, based upon the OSIRIS scheme. A train of 180 degrees pulses is applied after the OSIRIS preparation cycle, recording directly the transverse magnetization decay. The method was verified for two nuclei, 1H and 19F, with phantoms and in vivo on rats. The accuracy of the T2 values is discussed, as well as possible applications of the OSIRIS-CPMG method to proton transverse spin relaxation measurements, free of diffusion effects, and to non-invasive in vivo blood oxygenation measurements, through the use of an emulsion of perfluorooctylbromide, a blood substitute containing fluorine.     

Strong gradients for spatially resolved diffusion measurements

A new multilayer approach to gradient coil design, which allows the production of very strong gradient coils with reasonable resistance and consequent power dissipation, has been developed. Using this approach we have designed and built a strong z-gradient coil that will accommodate vertically mounted samples contained in 5-mm nuclear magnetic resonance tubes. The coil has an efficiency of 1.73 Tm-1A-1, an inductance of 49 microH, and a resistance of 1.8 omega, with a homogeneous volume consisting of a central cylinder of 4.5-mm length and diameter. This coil has been used to monitor the diffusion of water in Nylon 6.6 at room temperature, during desorption. This system is difficult to monitor via nuclear magnetic resonance (NMR), because the diffusion coefficients are typically less than 10(-13) m2s-1, while the T2 relaxation time is less than 1 ms even when the sample is fully saturated. The resulting measurements show a strong concentration dependence of the T2 relaxation time and self-diffusion coefficient of the absorbed water. The measured concentration profiles are consistent with a Fickian diffusion process with a concentration-dependent diffusion coefficient. The measured self-diffusion values are in reasonable agreement with those inferred from the variation of the concentration profiles as a function of time, using the one-dimensional Fickian diffusion equation.     

Diffusion elastic measurements of hydrogen diffusion in nickel

The diffusion coefficient of hydrogen in nickel has been determined at room temperature using a combination of electrochemical charging and measurements of the diffusion-elastic phenomenon. This technique has the advantage of allowing a determination of the dilitation of nickel due to interstitially diffusing hydrogen in addition to a determination of the diffusion coefficient in metals where diffusion coefficients are low. The results obtained indicate that reasonable diffusion coefficients can be easily measured and, in the absence of hydride formation, lattice dilitation was in the range of 10⁻⁴/at.% hydrogen.     

Quantitation of lipid in biological tissue by chemical shift magnetic resonance imaging

A method combining several previously used approaches is described for the rapid, accurate quantitation of the fat content of biological tissue based on chemical shift images (CSI) corrected for magnetic field inhomogeneity, and compensated for T1 and T2 effects. The gravimetrically determined lipid content of fatty tissues (pork fat, rabbit and human liver) that had been differentially depleted of lipid by chloroform extraction correlated well (r = 0.99) with the lipid image intensities of the respective tissues. This multi-point CSI method was used to quantitate lipid in fresh fatty human liver tissue (wet and dry) containing varying amounts of lipid. Plots of integrated lipid intensity versus tissue lipid content gave straight parallel lines for hydrated (r = 0.94) and dehydrated (r = 0.98) tissues, permitting determination of a proportionality constant for measuring absolute amounts of lipid present in a specific biological tissue. These results suggest the feasibility of using the method in vivo for absolute quantitation of lipid in tissues of agricultural (e.g. pork, beef) and medical (e.g. human liver) interest.     

Determination of diffusion coefficients by measurements of electrical resistivity

The evolution of the electrical resistivity of ribbons and wires in which a transient diffusion process occurs is calculated. From these results the optimal experimental procedure is inferred for determining the diffusion coefficient without the use of correction procedures.     

Amelioration of bacterial genomes: rates of change and exchange

Although bacterial species display wide variation in their overall GC contents, the genes within a particular species' genome are relatively similar in base composition. As a result, sequences that are novel to a bacterial genome-i.e., DNA introduced through recent horizontal transfer-often bear unusual sequence characteristics and can be distinguished from ancestral DNA. At the time of introgression, horizontally transferred genes reflect the base composition of the donor genome; but, over time, these sequences will ameliorate to reflect the DNA composition of the new genome because the introgressed genes are subject to the same mutational processes affecting all genes in the recipient genome. This process of amelioration is evident in a large group of genes involved in host-cell invasion by enteric bacteria and can be modeled to predict the amount of time required after transfer for foreign DNA to resemble native DNA. Furthermore, models of amelioration can be used to estimate the time of introgression of foreign genes in a chromosome. Applying this approach to a 1.43-megabase continuous sequence, we have calculated that the entire Escherichia coli chromosome contains more than 600 kb of horizontally transferred, protein-coding DNA. Estimates of amelioration times indicate that this DNA has accumulated at a rate of 31 kb per million years, which is on the order of the amount of variant DNA introduced by point mutations. This rate predicts that the E. coli and Salmonella enterica lineages have each gained and lost more than 3 megabases of novel DNA since their divergence.     

Measurements of diffusion rates of Au in metal-metal and metal-metalloid glasses

Diffusion rates of Au in Ni_33.3Zr_66.7, Ni₆₄Zr₃₆, Fe₄₀Ni₄₀B₂₀ and Pd_77.5Cu₆Si_16.5 metallic glasses have been measured over a range of temperatures by a Rutherford α-particle backscattering technique. Experimental procedures and methods of analysis are described briefly and possibilities of experimental error are discussed. The measured diffusion rates obey Arrhenius relationships, yielding activation energies of approximately 2eV. Some diffusivity values measured for Fe₄₀Ni₄₀B₂₀ and Pd_77.5Cu₆Si_16.5 above the glass transition temperature, Tg, also lie on the same Arrhenius plots. In each alloy, pre-annealing of the glass has virtually no effect on the diffusivity or activation energy values. Diffusion rates of Au are between two and three orders of magnitude lower than the diffusion rates of a metalloid atom at the same temperatures in the same glass. At a given temperature, the diffusion rate of Au is lower in a glass of higher Tg. However, normalisation with Tg does not normalise the diffusivity values. Thus, at a given fraction of Tg, the diffusion rate is higher, in a glass with higher Tg.     

Study of the mechanism of fast diffusion of cobalt in thorium by diffusion and internal friction measurements

The activation energy for diffusion,Q _d, of cobalt in fcc thorium was determined to be 83.7±4 kJ/mol from chemical diffusion measurements between 900 °C and 1332 °C. An internal friction peak associated with cobalt was identified in thorium at 45°C, and the activation energy for relaxation,Q _R, was found to be 76.0±1.5 kJ/mol by a frequency variation technique. The nearly equal values forQ _D andQ _R eliminate the interstitial-vacancy pair mechanism for fast diffusion in this system. The host-solute diplon and substitutional-interstitial (S-I) pair models for fast diffusion are considered, however, and preference is given to the latter mechanism for cobalt in thorium. S.C. AXTELL, formerly Graduate Student, Ames Laboratory, Iowa State University     

Measurements of exit rates to distinguish between facilitated and simple diffusion

A modification of the method of Sen and Widdas (J. Physiol. London 160:392-403, 1962) was used to measure the rate of exit of several nonelectrolytes from erythrocytes of various species. In spite of additional errors introduced by the larger half-saturation values of the carriers (phi) and concentrations, it was possible to distinguish between systems with small values of phi, systems with relatively large values of phi, and systems involving only simple diffusion. Approximate values of phi in millimoles and of maximum transfer rate (K) in isotonic units per minute were obtained using times and initial slopes measured on experimental curves. The following values in the foregoing units were obtained: human glucose, phi = 1.8, 1.0, K = 0.8, 1.1; human glycerol, phi = 178, 94, K = 4.3, 3.3; sheep thiourea, phi = 56, 56, K = 0.9, 0.6; and rabbit glycerol, phi = 328, 64, K = 2.2, 1.0. Simple diffusion was demonstrated for the following systems: ox-ethylene glycol; ox-glycerol; sheep-ethylene glycol; and sheep glycerol.     

Quantitation of in vitro activity of synthetic trans-acting ribozymes using HPLC

The cleavage activity of synthetic ribozymes needs to be characterized by reliable and rapid methods. A chromatographic method to simultaneously quantitate the amounts of substrate, cleavage fragments and ribozyme is described. The method allows the rapid normalization of analytical data because the sum of the 260-nm peak areas of remaining substrate and obtained fragments is essentially equal to the initial substrate peak area. Moreover, the simultaneous determination of the ribozyme content improves the accuracy of the evaluation of kinetic parameters compared with conventional densitometric methods. Finally, the characterization of two different hammerhead motifs indicated that the method is suitable for a rapid screening of synthetic ribozyme activity.     

Axial strain measurements in skeletal muscle at various strain rates

A noncontact optical system using high speed image analysis to measure local tissue deformations and axial strains along skeletal muscle is described. The spatial resolution of the system was 20 pixels/cm and the accuracy was +/- 0.125 mm. In order to minimize the error associated with discrete data used to characterize a continuous strain field, the displacement data were fitted with a third order polynomial and the fitted data differentiated to measure surface strains using a Lagrangian finite strain formulation. The distribution of axial strain along the muscle-tendon unit was nonuniform and rate dependent. Despite a variation in local strain distribution with strain rate, the maximum axial strain, Exx = 0.614 +/- 0.045 mm/mm, was rate insensitive and occurred at the failure site for all tests. The frequency response of the video system (1000 Hz) and the measurement of a continuous strain field along the entire length of the structure improve upon previous noncontact optical systems for measurement of surface strains in soft tissues.