The role of magnetization transfer in the observed contrast in T1 weighted imaging under clinical setups.

In T1 weighted magnetic resonance imaging of brain and spinal cord in the clinical setting, the white matter (WM) appears with greater intensity than the gray matter (GM). This contrast has been assigned to differences in T1 values. In these experiments the RF pulses are too long to excite both the water and the species with restricted motion of the protons (SRMP). In in vitro studies using short RF pulses, the contrast is reversed, with greater intensity for the GM. These results raise the question of whether magnetization transfer (MT) plays a role in the contrast observed in the T1 weighting experiments. In the present work we implemented selective saturation recovery alone and together with the conventional magnetization transfer contrast (MTC) method. The results confirm that a major factor that determines the characteristic WM/GM averaged intensity ratio observed in T1 weighted imaging under clinical conditions is MT between the SRMP and water. When selective saturation recovery is combined with MTC, the SRMP yields spectral widths ranging from a few to tens of kilohertz, indicating that more than one type of SRMP is involved in the MT. The z-spectrum obtained with this combination is free of the effect of direct saturation of the water peak. Selective saturation recovery enables an independent measurement of the exchange time and T1 , while the combination with MTC with complete saturation of the SRMP enables measurement of T1 without the effect of MT. The latter measurement can be carried out on a timescale much shorter than T1.

Sodium NMR/MRI for anisotropic systems.

Sodium ((23)Na) plays a central role in many physiological processes, and its high NMR sensitivity makes it an attractive nucleus for biomedical NMR and MRI research. Many biological tissues contain structures such as fibers and membranes that impose anisotropic translational and rotational motions on the sodium ions. Translational motion can be studied by diffusion measurements. Anisotropic rotational motion results in non-vanishing quadrupolar interaction that it is best studied by exploiting multiple quantum coherences for (23)Na NMR spectroscopy and MRI. The current review covers the application of the various NMR techniques to the study of (23)Na in anisotropic compartments in cartilage, tendon, intervertebral discs, red blood cells, nervous system and muscles.

Bypassing absorbing objects in focused ultrasound using computer generated holographic technique.

PURPOSE: Focused ultrasound (FUS) technology is based on heating a small volume of tissue, while keeping the temperature outside the focus region with minimal heating only. Several FUS applications, such as brain and liver, suffer from the existence of ultrasound absorbers in the acoustic path between the transducer and the focus. These absorbers are a potential risk for the FUS therapy since they might cause to unwanted heating outside the focus region. An acoustic simulation based solution for reducing absorbers' heating is proposed, demonstrated, and compared to the standard geometrical solution. The proposed solution uses 3D continuous acoustic holograms, generated by the Gerchberg-Saxton (GS) algorithm, which are described and demonstrated for the first time using ultrasound planar phased-array transducer. METHODS: Holograms were generated using the iterative GS algorithm and fast Fourier transform (FFT) acoustic simulation. The performances of the holograms are demonstrated by temperature elevation images of the absorber, acquired by GE 1.5T MRI scanner equipped with InSightec FUS planar phased-array transducer built out of 986 transmitting elements. RESULTS: The acoustic holographic technology is demonstrated numerically and experimentally using the three letters patterns, "T," "A," and "U," which were manually built into 1 × 1 cm masks to represent the requested target fields. 3D holograms of a focused ultrasound field with a hole in intensity at the absorber region were generated and compared to the standard geometrical solution. The proposed holographic solution results in 76% reduction of heating on absorber, while keeping similar heating at the focus. CONCLUSIONS: In the present work we show for the first time the generation of efficient and uniform continuous ultrasound holograms in 3D. We use the holographic technology to generate a FUS beams that bypasses an absorber in the acoustic path to reduce unnecessary heating and potential clinical risk. The developed technique is superior in performance and flexibility compared to the intuitive geometrical technique that is being used in clinical practice.

Ultrasound focusing using magnetic resonance acoustic radiation force imaging: application to ultrasound transcranial therapy.

PURPOSE: Magnetic resonance guided ultrasonic therapy is a promising minimally invasive technology for constantly growing variety of clinical applications. Delivery of focused ultrasound (FUS) energy to the targeted point with optimal intensity is highly desired; however, due to tissue aberrations, optimal focal intensity is not always achieved. Especially in transcranial applications, the acoustic waves are shifted and distorted mainly by the skull. In order to verify that magnetic resonance acoustic radiation force imaging (MR-ARFI) can be used as a focusing tool in transcranial treatments, such an imaging was applied in vivo on a porcine brain via ex vivo human skull. Then, by the use of MR-ARFI technique, an improved ultrasound focusing algorithm is proposed and demonstrated for both transcranial and none brain applications. METHODS: MR-ARFI images were acquired on a GE 1.5 T scanner equipped with InSightec FUS systems ExAblate 2000 and ExAblate 4000. Imaging was performed with MR-ARFI sequences of line-scan spin-echo and single-shot gradient-echo echo-planar. The in-plane resolution of both acquisitions was 0.9 x 0.9 mm2. The total acquisition time of MR-ARFI image was 31 s by the line-scan sequence and 1 s by the echo-planar sequence. An in vivo experiment was performed using FUS transducer, which is built out of 1024 ultrasound transmitting piezoelectric elements at 220 kHz frequency. The transducer was focused into the brain of a pig, which was wrapped in a human skull, in degassed water environment to resemble human treatments. The pig underwent a wide bilateral craniectomy to prevent a bone heating from the ultrasound beams. Two focusing experiments were performed in phantoms using 1 MHz and 710 kHz FUS transducers working with 208 and 225 elements, respectively. In the first experiment, aberration was added virtually to the apparatus by adding random phases to the phase map of the transducer. A simple focusing correction scheme was used, in which the corrected phase of a group of elements was chosen such that it maximizes the radiation force at the focal point. In the second experiment, aberrations made by a human skull were corrected using geometrical and phase based adjustments on segments of the transducer. RESULTS: A maximum displacement of 10 microm was obtained using 1.4 kW acoustic power on a live pig's head that its skull was removed and replaced by ex vivo human skull. Aberration correction using MR-ARFI resulted in near optimal focus, as the radiation force was similar to the nonaberration case. Transcranial, MR-ARFI based aberration correction performed better than CT based aberration correction, a technique that is currently used in brain FUS treatments. CONCLUSIONS: In the present work, the authors show for the first time a result of MR-ARFI in a live brain through ex vivo human skull. They have demonstrated that aberration correction could be done using MR-ARFI by measuring the radiation force at the focal point. Aberration correction using MR-ARFI is a promising noninvasive technique for transcranial focusing, which may result in near optimal focus and more reliable and safer brain FUS treatments.

Study of order and dynamic processes in tendon by NMR and MRI.

Tendons are composed of a parallel arrangement of densely packed collagen fibrils that results in unique biomechanical properties of strength and flexibility. In the present review we discuss several advanced magnetic resonance spectroscopy (MRS) and imaging (MRI) techniques that have allowed us to better understand the biophysical properties of tendons and ligaments. The methods include multiple quantum and T(2) filtering combined with NMR and MRI techniques. It is shown in detail how these techniques can be used to extract a number of useful parameters: 1) the (1)H-(1)H and (1)H-(2)H dipolar interactions; 2) the proton exchange rates between water and collagen, and between water molecules; 3) the distribution of fibril orientations; and 4) the anisotropy of diffusion. It is shown that relaxation data as a function of angular dependence can be obtained in vivo using mobile NMR sensors. Finally, this article describes how double quantum filtered (DQF) MRI can be used to image and monitor the healing process in injured tendons.

Self-diffusion anisotropy of water in sheep Achilles tendon.

The principal values of the diffusion tensor of free water in the pores of sheep Achilles tendon were determined. For this purpose, the azimuthally angular dependence of the self-diffusion coefficient was measured using a radiofrequency tilt coil and pulsed-field-gradient stimulated-echo (PFGSE) NMR. Combining the PFGSE with multiple acquisitions of Hahn echoes using the Carr-Purcell-Meiboom-Gill pulse sequence reduced the measuring time. The diffusion measurements revealed two diffusion process characterized by a fast and a slow effective diffusion coefficient. A model which describes the stimulated-echo amplitude, encoded by the water diffusion and magnetization transfer, was used for evaluation of the fast diffusion coefficients. The fast diffusion process characterizes the water molecules in pores surrounding the collagen fibrils. The diffusion coefficients characterizing the fast process show a well-defined anisotropy. The principal values of the diffusion tensors were determined assuming the elongated pores to be oriented parallel to the tendon fibrils and thus the orientation distribution function of the pores followed that of the collagen fibrils. The average aspect ratio of pores was estimated from the principal values of the water diffusion tensor and is suitable to characterize quantitatively the changes in tendon morphology due to healing or aging. The methods in this investigation can also be applied to measurements of the diffusion anisotropy using ex situ NMR sensors.

Anisotropy of collagen fiber orientation in sheep tendon by 1H double-quantum-filtered NMR signals.

The anisotropy of the angular distribution of collagen fibrils in a sheep tendon was investigated by 1H double-quantum (DQ) filtered NMR signals. Double-quantum build-up curves generated by the five-pulse sequence were measured for different angles between the direction of the static magnetic field and the axis of the tendon plug. Proton residual dipolar couplings determined from the DQ build-up curves in the initial excitation/reconversion time regime which mainly represent the bound water are interpreted in terms of a model of spin-1/2 pairs with their internuclear axes oriented on average along the fibril direction in the presence of proton exchange. The angular distribution of collagen fibrils around the symmetry axis of the tendon measured by the anisotropy of the residual dipolar couplings was described by a Gaussian function with a standard deviation of 12 degrees +/-1 degrees and with the center of the distribution at 4 degrees +/-1 degrees. The existence of this distribution is directly reflected in the finite value of the residual dipolar couplings at the magic angle, the value of the angular contrast, and the oscillatory behavior of the DQ build-up curves. The 1H residual dipolar couplings were also measured from the doublets recorded by the DQ-filtered signals. From the angular dependence of the normalized splitting the angular distribution of the collagen fibrils was evaluated using a Gaussian function with a standard deviation of 19 degrees +/-1 degrees and with the center of distribution at 2 degrees +/-1 degrees. The advantages and disadvantages of these approaches are discussed.

NMR spectroscopic characterization of sarcolemmal permeability during myocardial ischemia and reperfusion.

This study aims to characterize the pattern of membrane disintegration during myocardial ischemia and reperfusion. Intracellular volumes were measured by 1H and 59Co NMR in isolated rat hearts during 10, 30 and 60 min of total ischemia and 30 min of reperfusion at normothermia. Perfusion with hypo-osmotic medium (210 mosm/l) increased intracellular water from 2.50+/-0.06 to 3.07+/-0.07 ml/g dry weight (P<0.001) during pre-ischemia. Hypo-osmotic swelling decreased by 16+/-3, 32+/-6 and 44+/-11% of the pre-ischemic value after 10, 30 and 60 min of ischemia (n.s., P<0.005, P<0.001) respectively, indicating that membrane permeabilization facilitated efflux of osmolytes and counterbalanced the osmotic driving force for water influx. Hypo-osmotic swelling decreased during 30 min of reperfusion by 18+/-5% in all groups (P<0.0.005 v post-ischemia). The volume of distribution of the extracellular marker cobalticyanide increased by more than 3.2+/-0.4 and 5.8+/-0.5% of the intracellular space after 30 and 60 min of ischemia respectively (P<0.001), and by an additional 2% after reperfusion. During 30 min of reperfusion, hearts released 1.6+/-0.2 and 3.2+/-0.4% of the intracellular creatine kinase contents after 30 and 60 min of ischemia, respectively (P<0.001). In addition to the correlation between ischemia duration and membrane permeability, evident from the analysis of each probe, the data showed a progressive increase in severity of membrane injury over time and permeabilization to larger molecules. 23Na NMR spectroscopy in conjunction with an extracellular shift reagent (SR) showed formation of a resonance at an intermediate chemical shift in between the intra and extracellular Na+ peaks, suggesting penetration of SR into cells with disrupted membranes. The constant chemical shift and narrow line shape of this resonance, characteristic of a homogeneous chemical environment, suggested that the distribution of SR was contained within the cytosol of cardiomyocytes. We propose that sarcolemmal membranes are gradually permeabilized to larger molecules by ischemia, and the evolving chemical instability is spatially contained within the myocyte.

Multiquantum filters and order in tissues.

In ordered systems, where the molecular motion is anisotropic, quadrupolar and dipolar interactions are not averaged to zero. In such cases, double quantum (DQ) coherences can be formed. This review deals mainly with the effect of anisotropic motion of water molecules and sodium ions in intact biological tissues on (2)H, (1)H and (23)Na NMR spectroscopy and its application to NMR imaging (MRI). Double quantum filtered (DQF) spectra of water molecules and sodium ions were detected in a variety of ordered biological tissues. In collagen-containing tissues such as ligaments, tendons, cartilage, skin, blood vessels and nerves, the DQ coherences are formed as a result of the interaction with the collagen fibers. In red blood cells and presumably also in nerve axons it stems from the interaction with the cytoskeleton. For (23)Na, an I = 3/2 nucleus, the DQ coherences can also be formed in isotropic media. By a judicial choice of the pulse angle in the DQ pulse sequence only the DQ coherences arising from anisotropic motion are detected. For I = 1 nuclei such as 2H, DQF spectra can be observed only in ordered structures. Thus, the observation of 2H DQF spectra is an indication of order. The same is true for pairs of equivalent 1H nuclei. The dependence of the DQF signal on the creation time of the double quantum coherences is characteristic to each tissue and allows signals to be resolved from different tissues by performing the measurements at different creation times. In this way, the 2H DQF signals of the different compartments of sciatic nerve were resolved and water diffusion in each compartment was studied independently. In the axon, the diffusion was heavily restricted perpendicular to the axon's long axis, a result from which the axon diameter could be deduced. In blood vessel walls, this characteristic enabled the different layers of the vessel to be viewed and studied under strain. For 2H, a DQF spectroscopic imaging sequence was used to study the orientation of the collagen fibers in the different zones of articular cartilage and bone plug. The effect of pressure on the fibers and their return to equilibrium was studied as well. In blood vessels, a DQF image was obtained and strain maps of the different layers were calculated. The efficiency of the 1H DQF imaging technique was demonstrated on a phantom of rat tail where only the four tendons were detected at short creation times. 1H DQF imaging and spectroscopy followed the healing of a rabbit's ruptured Achilles tendon and the results were far more sensitive to the process than conventional imaging. Finally, the method was implemented on a commercial whole body MRI spectrometer. Images of human wrist and ankle showed a positive contrast for the tendons and ligaments, indicating the potential of the method for clinical imaging. (c) 2001 John Wiley & Sons, Ltd.

Excretion of a phosphorus-containing carbohydrate by Streptomyces sp. A50.

A new phosphorus-containing compound (1) was detected by (31)P NMR spectroscopy in Streptomyces sp. A50. Compound 1, 1(alpha)-O-methyl-2-(N-acetyl)glucoseamine-6-O-phosphate-1(alpha)-2-(N-acetyl)glucosamine, exhibited a pK(a) value around zero. The compound was found both in the extracellular culture broth and in the cells. While very low concentrations of 1 were found in the culture broth of other species of Streptomyces, its presence in high concentrations was specific to Streptomyces sp. A50. The highly acidic compound was isolated from the broth, and its structure was elucidated by a combination of 1D-, 2D-homonuclear, and inverse heteronuclear NMR techniques and mass spectroscopy.

HGF/SF activates glycolysis and oxidative phosphorylation in DA3 murine mammary cancer cells.

Hepatocyte growth factor/scatter factor (HGF/SF) is a paracrine growth factor which increases cellular motility and has also been implicated in tumor development and progression and in angiogenesis. Little is known about the metabolic alteration induced in cells following Met-HGF/SF signal transduction. The hypothesis that HGF/SF alters the energy metabolism of cancer cells was investigated in perfused DA3 murine mammary cancer cells by nuclear magnetic resonance (NMR) spectroscopy, oxygen and glucose consumption assays and confocal laser scanning microscopy (CLSM). 31P NMR demonstrated that HGF/SF induced remarkable alterations in phospholipid metabolites, and enhanced the rate of glucose phosphorylation (P < .05). 13C NMR measurements, using [13C1]-glucose-enriched medium, showed that HGS/SF reduced the steady state levels of glucose and elevated those of lactate (P < .05). In addition, HGF/SF treatment increased oxygen consumption from 0.58+/-0.02 to 0.71+/-0.03 micromol/hour per milligram protein (P < .05). However, it decreased CO2 levels, and attenuated pH decrease. The mechanisms of these unexpected effects were delineated by CLSM, using NAD(P)H fluorescence measurements, which showed that HGF/SF increased the oxidation of the mitochondrial NAD system. We propose that concomitant with induction of ruffling, HGF/SF enhances both the glycolytic and oxidative phosphorylation pathways of energy production.

In vivo (1)H double quantum filtered MRI of the human wrist and ankle.

Proton double quantum filtered (DQF) NMR imaging was applied in vivo to the human wrist and ankle with a clinical 1.5 T MR scanner. Water molecules having anisotropic motion were detected from tendons and ligaments. Images of Achilles tendon were obtained for a voxel size of 1.25 x 1.25 x 20 mm with three values of TR = 1.0, 0.5, and 0.2 sec, resulting in total acquisitions time of 17, 8.5, and 3.4 mins, respectively. Some degradation of the signal-to-noise ratio was observed at the shortest TR value and the contrast was significantly reduced due to SQ coherence leakage. The in vivo DQF images showed structure within the tendon that is otherwise not visible by conventional gradient-recalled echo (GRE) methods. Published 2000 Wiley-Liss, Inc.

Slice-selective proton double quantum filtered MRI of joint connective tissues.

1H double quantum filtered (DQF) imaging has been shown to highlight tendons. In this work the DQF magnetic resonance imaging pulse sequence is extended to include slice selection. The short transverse relaxation time of the 1H nuclear magnetic resonance in connective tissues, presents a stringent demand on the application of gradients and soft radiofrequency pulse lengths needed for slice selection. In the present work a slice selection pulse sequence is implemented by postponing the application of the slice refocusing gradient to the period after the last pulse just before the acquisition. Slice-selective DQF images of rat lower leg and knee are given to demonstrate the efficacy of the technique.

Detection and characterization of boric acid and borate ion binding to cytochrome c using multiple quantum filtered NMR.

The application of multiple quantum filtered (MQF) NMR to the identification and characterization of the binding of ligands containing quadrupolar nuclei to proteins is demonstrated. Using relaxation times measured by MQF NMR multiple binding of boric acid and borate ion to ferri and ferrocytochrome c was detected. Borate ion was found to have two different binding sites. One of them was in slow exchange, k(diss) = 20 +/- 3 s(-1) at 5 degrees C and D(2)O solution, in agreement with previous findings by (1)H NMR (G. Taler et al., 1998, Inorg. Chim. Acta 273, 388-392). The triple quantum relaxation of the borate in this site was found to be governed by dipolar interaction corresponding to an average B-H distance of 2.06 +/- 0.07 A. Other, fast exchanging sites for borate and boric acid could be detected only by MQF NMR. The binding equilibrium constants at these sites at pH 9.7 were found to be 1800 +/- 200 M(-1) and 2.6 +/- 1.5 M(-1) for the borate ion and boric acid, respectively. Thus, detection of binding by MQF NMR proved to be sensitive to fast exchanging ligands as well as to very weak binding that could not be detected using conventional methods.

1H double-quantum-filtered MR imaging as a new tool for assessment of healing of the ruptured Achilles tendon.

1H double-quantum-filtered magnetic resonance imaging (DQF MRI) was applied to monitor the healing process of the Achilles tendons in rabbits after tenotomy. DQF MRI provides a new contrast, which is based on the non-zero average of the dipolar interaction caused by anisotropic motion of water molecules, determined mainly by their interaction with the ordered collagen fibers. Tissues are characterized by the dependence of their DQF signal on the DQ creation time, tau. With the use of DQF MRI, higher tissue contrast is obtained between tendon, bone, skin, and muscle. The tendons, which give weak signals in standard MRI techniques, are highlighted in the (1)H DQF image. The image changed dramatically during the healing process of the injured Achilles tendon. These changes matched the phases of the healing process. By using a tau-weighted contrast, the DQF images indicate the part of tendon that has not completely healed, even after the conventional MRI appeared normal. Magn Reson Med 42:884-889, 1999.

Efficient limitation of intracellular edema and sodium accumulation by cardioplegia is dissociated from recovery of rat hearts from cold ischemic storage.

Energy deficiency and disturbances of sodium and water homeostasis are considered as mechanisms of injury during hypothermic preservation of cardiac muscle. The present study attempts to characterize the effect of potassium (K+) and magnesium (Mg2+) cardioplegia on these mechanisms. Cellular parameters were measured by multinuclear NMR spectroscopy in isolated rat hearts during 12 h of ischemia at 4 degrees C and 2 h of normothermic reperfusion with an isoosmotic Krebs-Henseleit (KH) solution. Potassium and magnesium cardioplegia (a) reduced the rate of ATP hydrolysis and cellular acidification during early stages of ischemia; (b) caused an early cessation of the phase of fast sodium influx after 40 min (P<0.001 vs 120 min with KH); (c) reduced intracellular sodium accumulation to 148-165 micromol/gdw after 12 h (P<0.01 vs 268+/-15 micromol/gdw with KH); (d) decreased ischemic volumes to 2.7+/-0.1 and 2.8+/-0.1 ml/gdw after 8 and 12 h of storage, respectively (P<0.005 v 3.0 and 3.3 ml/gdw with KH). Quantitative analysis of these parameters showed that both hypothermia and cardioplegia increased the relative contribution of sodium to intracellular water accumulation by a factor of 2-2.5. In view of the marked reduction in absolute sodium and water contents, the data indicate that cold cardioplegia limits the increase in intracellular osmolarity. Myocardial mechanical and metabolic recoveries, and cellular viability deteriorated during prolongation of the ischemic period from 8 to 12 h in all experimental groups (P<0.005). Reperfusion was efficient in reversing intracellular sodium and water accumulation in hearts stored with cardioplegia, in contrast to hearts stored in KH. Magnesium, but not potassium cardioplegia, lowered interstitial water contents (P<0.01 v KH), increased intracellular magnesium concentrations (P<0.001), improved mechanical and metabolic recoveries (P<0.01) and cellular viability (P<0.001). These results indicate (a) cardioplegia reduces intracellular sodium (by approximately 46%) and water accumulation (by 66%) during cold ischemia; (b) both hypothermia and cardioplegia limit the rise in intracellular osmolarity and increase the contribution of sodium to cellular swelling; (c) intracellular sodium and water contents were dissociated from myocardial viability and recovery from cold ischemia in potassium and magnesium cardioplegic solutions. It is concluded that intracellular sodium and water accumulation are not dominant factors in determination of cardiac outcome from ischemia.

Anisotropic and restricted diffusion of water in the sciatic nerve: A (2)H double-quantum-filtered NMR study.

The signals of water in the different compartments of rat sciatic nerve are resolved in the (2)H double-quantum-filtered NMR spectrum, due to their different quadrupolar splittings and relaxation rates. This resolution allowed the independent measurement of the water diffusion coefficients in the different compartments. The water diffusion in all three compartments, the endoneurium, the epineurium and the axon was found to be anisotropic. Parallel to the nerve fiber the average intraxonal water diffusion coefficient was 1.11 x 10(-5) cm(2)/sec, while in the perpendicular direction the diffusion is heavily restricted. The average perpendicular diffusion coefficient ranged from 0.29 x 10(-5) cm(2)/sec to 0.05 x 10(-5) cm(2)/sec for diffusion times of 7 msec and 50 msec, respectively. Assuming restricted diffusion in nonpermeable cylinders, intra-axonal mean diameters of 6.0, 7.4 and 9.0 microm were obtained for nerves taken from three different rats. Magn Reson Med 42:461-466, 1999.

The source of heterogeneity in the heme vicinity of ferricytochrome c.

Heterogeneity in the heme vicinity of ferricytochrome c was reported to be detectable by a split of the NMR signal of the heme methyl 3 group [P.D. Burns and G.N. La Mar, J. Am. Chem. Soc. 101 (1979) 5844]. Using cytochrome c mutants and computer simulations of the native and mutated cytochromes, the source of this heterogeneity is found to originate from the His-33 residue motions. The H33F mutation abolished the NMR split and computer simulations of the H33F mutant revealed a narrower distribution of fluctuations of the radius of gyration, suggesting a more rigid structure due to the mutation. The stabilization of the mutant was further demonstrated by a reduction in the H33F mutant of 4 Kcal/mol in the calculated interaction energy between residue 33 and the rest of the cytochrome, in keeping with known experimental results.

A study of dipolar interactions and dynamic processes of water molecules in tendon by 1H and 2H homonuclear and heteronuclear multiple-quantum-filtered NMR spectroscopy.

The effect of proton exchange on the measurement of 1H-1H, 1H-2H, and 2H-2H residual dipolar interactions in water molecules in bovine Achilles tendons was investigated using double-quantum-filtered (DQF) NMR and new pulse sequences based on heteronuclear and homonuclear multiple-quantum filtering (MQF). Derivation of theoretical expressions for these techniques allowed evaluation of the 1H-1H and 1H-2H residual dipolar interactions and the proton exchange rate at a temperature of 24 degrees C and above, where no dipolar splitting is evident. The values obtained for these parameters at 24 degrees C were 300 and 50 Hz and 3000 s-1, respectively. The results for the residual dipolar interactions were verified by repeating the above measurements at a temperature of 1.5 degrees C, where the spectra of the H2O molecules were well resolved, so that the 1H-1H dipolar interaction could be determined directly from the observed splitting. Analysis of the MQF experiments at 1.5 degrees C, where the proton exchange was in the intermediate regime for the 1H-2H dipolar interaction, confirmed the result obtained at 24 degrees C for this interaction. A strong dependence of the intensities of the MQF signals on the proton exchange rate, in the intermediate and the fast exchange regimes, was observed and theoretically interpreted. This leads to the conclusion that the MQF techniques are mostly useful for tissues where the residual dipolar interaction is not significantly smaller than the proton exchange rate. Dependence of the relaxation times and signal intensities of the MQF experiments on the orientation of the tendon with respect to the magnetic field was observed and analyzed. One of the results of the theoretical analysis is that, in the fast exchange regime, the signal decay rates in the MQF experiments as well as in the spin echo or CPMG pulse sequences (T2) depend on the orientation as the square of the second-rank Legendre polynomial.

The interaction of borate ions with cytochrome c surface sites: a molecular dynamics study.

Ionic interactions of cytochrome c play an important role in the electron transfer process. Molecular dynamics simulations of the binding of borate ion, which serves as a model ion, at three different cytochrome c surface sites are performed. This work is motivated by previous NMR studies of cytochrome c in borate solution, which indicate the existence of two types of binding sites, a slow exchange site and a fast exchange site. These two types of binding behavior were observed in the dynamic simulations, offering a molecular interpretation of "loose" and "tight" binding. At the "loose" binding sites (near Lys25/Lys27 and Lys55/Lys73) the ion forms two to three hydrogen bonds to the nearest lysine residue. This binding is transient on the time scale of the simulation, demonstrating the feasibility of fast exchange. At the "tight" binding site (near Lys13/Lys86), on the other hand, the ion becomes integrated into the protein hydrogen bond network and remains there for the duration of the simulation (exemplifying slow exchange). Binding simulations of the ion at the "tight" site of H26Q mutant cytochrome c also showed integration of the ion into the protein's hydrogen bond network. However, this integration differs in details from the binding of the ion to the native protein, in agreement with previous NMR observations.