Biomedical subjects

R G Bryant

Publications and source records attributed to R G Bryant.

At least 37 records · Page 2Linked to original sources

PubMed1997

Bound ligand motion in crystalline carboxypeptidase A.

Deuterium NMR spectra for the phenyl ring deuterons have been obtained for D-phenylalanine, L-phenylalanine, phenylacetic acid, and phenyl propionic acid in randomly oriented crystals of carboxypeptidase A as a function of water content. The spectra are analyzed using a two-site jump model for phenyl ring pi-flips when the ligand is bound to the protein, and the model includes the possibility that the ligand may exchange with isotropic or unbound environments within the crystal. Although the binding pocket may impose local dynamical constraints, a complete pi-flip motion is consistent with the spectra of all ligands at all water contents. The rate constants for the pi-flip at 298 K are found to be 7.5 x 10(5) S-1, 1.9 x 10(6) S-1, 4.0 x 10(6) S-1, and 4.0 x 10(6) S-1 for L-phenylalanine, D-phenylalanine, phenyl propionic acid, and phenylacetic acid, respectively, at water activity of 0.98. The pi-flip rate for the ligand bound to the enzyme increases with water content. Assuming that the activation barrier may be written, delta G+2 = delta G+2o + baw, where aw is the water activity, and the value of b is -1.9 kcal/mol for phenylacetic acid and phenyl propionic acid, -1.3 kcal/mol for L-phenylalanine, and -2.1 kcal/mol for D-phenylalanine. Phenylacetic acid crystals were studied as an example of a phenyl ring motion that is highly constrained by a known and symmetrical packing environment. The deuterium spectra are complex and are not consistent with pi-flip motions, but they are consistent with a superposition of ring jump motions of 24 degrees, 34 degrees, and 72 degrees, with probabilities in the ratio of 1:1:2. Because of the limited space for motion imposed by the tight packing in the crystal, these motions must be highly cooperative and probably locally coherent; however, the spectra by themselves do not prove this intuitively reasonable hypothesis.

Carboxypeptidases↗

PubMed1997

Cholera from raw seaweed transported from the Philippines to California.

In March 1994, a California woman without any recent travel developed acute, profuse, watery diarrhea. Her astute physician diagnosed cholera after ordering the appropriate stool culture, and the patient improved on an oral antibiotic. Epidemiologic investigation implicated seaweed from the Philippines that was transported by a friend to California and subsequently eaten raw as the vehicle of infection.

Adult↗

PubMed1996

Noninvasive measurement of protein concentration.

At selected magnetic field strengths, protein and water proton spin-lattice relaxation rates are sensitive to the concentration of rotationally immobilized peptide nitrogen because of field dependent heteronuclear cross relaxation coupling between protein proton and nitrogen-14 spins that is carried to the water by proton homonuclear cross-relaxation. Measurement of the water proton spin-lattice relaxation time, or a signal amplitude proportional to it, may provide a noninvasive measure of peptide bond concentration, which provides a direct measure of immobilized protein content in most tissues. The approach using protein gels in two magnetic field strengths is demonstrated. At 66.7 mT; the proton Zeeman energy matches one of the peptide nitrogen transitions dominated by the unaveraged nuclear electric quadrupole interaction; cross-relaxation between the protons and nitrogen-14 is efficient. At 77.5 mT, the proton Zeeman energy is not matched with the nitrogen energy and proton-nitrogen cross-relaxation is not efficient. It is shown that the difference in the water proton spin-lattice relaxation rates on and off the energy level match condition is a linear function of the rotationally immobilized protein concentration.

Animals↗

PubMed1996

1H magnetic cross-relaxation between multiple solvent components and rotationally immobilized protein.

Magnetic cross-relaxation spectra or Z-spectra are presented for water, acetone, methanol, dimethylsulfoxide, and acetonitrile in cross-linked bovine serum albumin gels. Each solvent studied, reports the same Z-spectrum linewidth and shape for the solid component that follows from solutions of the coupled relaxation equations. The Z-spectra demonstrate competition among solvents for specific protein binding sites. The rate of magnetization transfer in the rotationally immobilized protein environment is approximated by 1/T2 for the solid component, which is shown to account for the observed magnetization transfer rates in the systems studied. The temperature dependence of the Z-spectra are different for water compared with the organic solvents. The cross-relaxation efficiency in the organic solvents decreases with increasing temperature because molecules bind less well at high temperature. For water, the hydrogen exchange path becomes increasingly important relative to the whole molecule path with increasing temperature, which improves the net cross-relaxation efficiency.

Animals↗

PubMed1996

Ionic association and electron spin relaxation rates in aquo gadolinium (III) complexes.

The electron paramagnetic resonance linewidth of aquo gadolinium(III) ion changes with the counter-ion identity and concentration in aqueous solutions. The EPR linewidth of 2 mM gadolinium(III) chloride increases from 49.2 to 89.0 mT when carbonate ion is added and decreases to 17.3 mT when nitrite ion is added. These observations suggest association reactions between aquo gadolinium(III) ion and anions that change the electron spin relaxation rates of the aquo ion. The concentration dependence of the gadolinium(III) EPR linewidth is consistent with binding constants for nitrite and nitrate ion with the aquo gadolinium(III) ion of 37 +/- 6 and 2.3 +/- 0.3 L mol-1 respectively. The decreases in the EPR linewidth factors with these association reactions are difficult to understand unless the anion reactions increase the symmetry of the metal center. Although first-coordination reactions may not be ruled out, the decrease in EPR linewidth is more consistent with an outer-sphere association reaction that also reduces the coordination number of the metal center from 9 to 8.

Animals↗

PubMed1996

Water molecule binding and lifetimes on the DNA duplex d(CGCGAATTCGCG)2.

Measurements of the water proton spin-lattice relaxation rate for aqueous solutions of the palindromic dodecamer, d(CGCGAATTCGCG)2, are reported as a function of the magnetic field strength. The magnitude of the relaxation rates at low magnetic field strengths and the shape of the relaxation dispersion curve permit assessment of the number of water molecules which may be considered bound to the DNA for a time equal to or longer than the rotational correlation time of the duplex. The data are examined using limiting models that arbitrarily use the measured rotational correlation time of the polynucleotide complex as a reference point for the water molecule lifetime. If it is assumed that water molecules are bound at DNA sites for times as long as or longer than the rotational correlation time of the duplex, then the magnitude of the relaxation rates at low field require that there may be only two or three such water sites. However, if the lifetime constraint is relaxed, and we assume that the number of water molecules bound to the DNA is more nearly the number identified in the X-ray structures, then the average water molecule lifetime is on the order of 1 ns. Measurements of 1H NOESY spectra demonstrate that some water molecules must have lifetimes sufficiently long that negative Overhauser effects are observed. Taken together, these results suggest a distribution of water molecule lifetimes in which most of the DNA-bound water molecule lifetimes are shorter than the rotational correlation time of the duplex, but where some have lifetimes of at least 1 ns under these concentrated conditions.

Binding Sites↗

PubMed1996

Magnetic cross-relaxation and chemical exchange between microporous solid and mobile liquid phases.

Nuclear magnetic spin-lattice relaxation rates are reported as a function of magnetic field strength, pH, and temperature for water protons in Sephadex gels. The water proton rates report the relaxation profile characteristic of the solid at high pH values. At lower pH values, the relaxation rate is limited by a slow chemical exchange process that makes the relaxation rate independent of magnetic field at low field strengths. In cases where the proton exchange rate does not limit the total relaxation rate, the magnetic field dependence of the relaxation rate is given by a power law of the type R = Av-B, where B is 0.75 except at pH 11 where the data are better represented with B = 0.5. The exchange events important for the water proton relaxation are represented as a two-stage process in which water within the pores of the gel exchanges protons and magnetization with the solid spin system rapidly compared with the rate at which the water within the pores exchanges with the bulk water phase.

Dextrans↗

PubMed1996

The dynamics of water-protein interactions.

The magnetic field and temperature dependence of the water proton nuclear spin-lattice relaxation rate requires that the motion timescale for water molecules in contact with proteins is close to that for pure water at room temperature. Nevertheless, there are a few water molecules, which may be detected by high-resolution, cross-relaxation spectroscopy, that must have relatively long protein-bound lifetimes and that carry the bulk of the relaxation coupling between the protein and the water. The water-protein magnetic coupling affects the interpretation of water relaxation rates in heterogeneous protein systems, such as tissues, and provides new ways to extract useful information about the immobilized components through the effects on the water NMR spectrum. The discussion shows that the conclusions concerning the rapid water molecule motions at the interface are not in conflict with the observations of many water oxygen atom positions in protein crystal structures.

Gels↗

PubMed1995

Characterization of enzyme-bound ligand dynamics by solid-state NMR in the presence of ligand exchange: L-phenylalanine on carboxypeptidase A.

Deuterium NMR spectra were obtained for L-phenylalanine-d5, deuterated on the phenyl ring, in cross-linked polycrystalline samples of carboxypeptidase A containing different amounts of water. The deuterium powder pattern line shapes are simulated by extension of the theory to include both a local reorientational motion of the bound L-phenylalanine phenyl ring and exchange of the L-phenylalanine with an intracrystalline isotropic environment. The spectral simulations are consistent with the phenyl ring of the phenylalanine executing pi-flips in the bound environment at rates that vary from 3 x 10(4) Hz at 6% water content to 1 x 10(5) Hz at 21% water content. At all water contents studied, the ligand exchanges with an essentially isotropic environment in the crystal with a rate constant of approximately 2.5 x 10(-3) Hz. Although the dissociation constant for the L-phenylalanine is only 18 mM, the spectral simulations that reproduce the experimental line shape well do not require significant wobble of the phenyl ring rotation axis, which is consistent with the binding interactions identified by x-ray crystallography.

Binding Sites↗

PubMed1995

Nuclear magnetic resonance spectra for l > 1 spins in dynamically heterogeneous systems with chemical exchange among environments.

Nuclear magnetic resonance spectra for nuclei with spin l > 1 are considered in cases in which the observed nucleus may sample a rotationally immobilized and an isotropic environment that are coupled by a chemical exchange process. Spectra are simulated for the central (1/2, -1/2) transition for a 3/2 nucleus as a function of the concentrations of the two environments and as a function of the exchange rate between them. It is shown that a crucial feature determining the shape of the observable spectra is the spatial extent or the local order in the immobilized phase. In the case for which all rotationally immobilized sites sampled by the exchanging nucleus are identically oriented but where there is a distribution of these microdomain orientations with respect to the magnetic field direction, a powder pattern for the central transition is observed that carries whatever dynamic information may be derived from it. In the fast exchange limit, the width of the powder pattern scales inversely with the concentration of the isotropic environment as usual. In the intermediate exchange regimes, a complex line shape results that may mask the anisotropic character of the spectrum. In the slow exchange limit, superposition of the spectral contributions results; however, if the isotropic environment concentration is significantly larger than the anisotropic environment concentration, the anisotropic contribution is very difficult to detect because of the dynamic range problem and the possibly large difference in the effective line widths. In the case for which the exchanging nucleus samples a considerable distribution of rotationally immobilized site orientations, the anisotropic character of the spectrum is lost and a super-Lorentzian line shape results. These effects are demonstrated experimentally by 35Cl nuclear magnetic resonance spectra obtained on a lamellar liquid crystal that is modified with the addition of a thiolmercurate to provide a site of large quadrupole coupling constant and with cross-linked bovine serum albumin gels.

Chlorides↗

PubMed1994

Quantitative MRI of Gd-DTPA uptake in tumors: response to photodynamic therapy.

A partial saturation method is described for obtaining rapid images of tissue 1H spin-lattice relaxation rates following administration of the paramagnetic contrast agent gadolinium-diethylenetriaminepentaacetate. The paramagnetic contribution to the relaxation rates is proportional to the concentration of contrast agent, making possible quantitative studies of paramagnetic contrast agent uptake or vessel leakage. Snapshot imaging capabilities are not required. Maps of contrast agent uptake rates are made in rat borne tumors before and following photodynamic therapy, which is known to cause vascular damage. Uptake efficiency is spatially heterogeneous before and after therapy. Decreases in uptake rate are observed after two photo-irradiation protocols, which differ by a factor of four in fluence rate but deliver the same total fluence. There is no apparent fluence rate dependence for changes in the uptake rates within 5 h after therapy. Whole tumor measurements of nucleotide triphosphates, inorganic phosphate, pH, and lactate made with NMR spectroscopy indicate that, while net ATP production is inhibited, lactate concentrations are not strongly affected by photodynamic therapy. The ratio of nucleotide triphosphates to inorganic phosphate falls to 0.21 +/- 0.02 of initial values 5 h after tumors are treated with the lower fluence rate protocol, and falls to 0.40 +/- 0.06 in tumors treated with the higher fluence rate.

Adenocarcinoma↗

PubMed1994

Magnetization transfer, cross-relaxation, and chemical exchange in rotationally immobilized protein gels.

Water proton spin-lattice relaxation rates are reported as a function of the magnetic field strength for cross-linked bovine serum albumin samples. The relaxation dispersion profile is analyzed using a relaxation model where the solid components have the magnetic field dependence proportional to v-0.5 which may result from a defect diffusion model with two degrees of freedom. If the cross-linking agent concentration is not sufficiently high, the relaxation dispersion curve may have significant contributions from freely rotating protein. The magnetic field dependence of the relaxation rates studied as a function of the proton mole fraction in the sample show that approximately 30% of the magnetization transfer rate is directly proportional to the proton mole fraction. This contribution is identified with the magnetization transfer from exchange of whole water molecules with buried binding sites on the protein. The second order magnetization transfer rate constant is 388 s-1 assuming unit water spin concentration. The solid component relaxation obeys an Arrhenius activation law, but the overall temperature dependence of the cross-relaxation is complicated by chemical exchange processes which enter with opposite sign.

Animals↗

PubMed1994

Molecular flexibility demonstrated by paramagnetic enhancements of nuclear relaxation. Application to alamethicin: a voltage-gated peptide channel.

A nitroxide spin label attached to the C-terminus of the channel forming peptide alamethicin produces an enhancement of the nuclear spin-lattice relaxation rates of peptide protons as a result of both intermolecular and intramolecular magnetic dipole-dipole interactions. The intermolecular contribution provides evidence that alamethicin monomers collide preferentially in a C-terminal-to-N-terminal configuration in methanol. From the intramolecular paramagnetic enhancement of nuclear spin-lattice relaxation times, effective distances between the unpaired electron on the nitroxide at the C-terminus of alamethicin and protons along the peptide backbone were calculated. These distances are much shorter than distances based on the reported crystal structure of alamethicin, and cannot be accounted for by motion in the bonds that attach the nitroxide to the peptide. In addition, the differences between distances deduced from the nuclear spin relaxation and the distances seen in the crystal structure increase toward the N-terminal end of the peptide. The simplest explanation for these data is that the alamethicin backbone suffers large structural fluctuations that yield shorter effective distances between the C-terminus and positions along the backbone. This finding can be interpreted in terms of a molecular mechanism for the voltage-gating of the alamethicin channel. When the distances between a paramagnetic center and a nucleus fluctuate, paramagnetic enhancements are expected to yield distances that are weighted by r-6, and distances calculated using the Solomon-Bloembergen equations may more nearly represent a distance of closest approach than a time average distance. Therefore, the use of paramagnetic centers such as spin labels or metal ions with long electron T1 values provides a distance measurement that reflects a dynamically averaged structure where the averaging process heavily weights short distances. The results of such measurements, when combined with other structural information, may provide particularly clear evidence for the magnitude of structural fluctuations involving distances greater than 10 A.

Alamethicin↗

PubMed1994

Flow through clots determines the rate and pattern of fibrinolysis.

Thrombolytic therapy depends on penetration of plasminogen activator into clots which occurs through diffusion and flow. An in vitro system has been developed to characterize the rate and pattern of fibrinolysis in relation to flow through occlusive clots exposed to a pressure gradient. Whole blood clots formed in plastic tubes were perfused with plasma containing 1 microgram/ml tissue plasminogen activator (t-PA) and 0.5 or 1 mmol/l gadolinium-diethylentriamine pentaacetic acid (Gd-DTPA), a paramagnetic substance used as a contrast enhancer for magnetic resonance (MR) imaging. T1-weighted spin echo MR images were obtained during clot perfusion at 3-5 min intervals for 45 min. Characteristic signal intensities allowed identification of non-perfused, perfused but non-lysed, and completely lysed areas of clot. A spatially resolved time course of perfusion and subsequent lysis was constructed for 10 clots. Plasma flowed non-uniformly through clots forming asymmetric channels that left some areas non-perfused. The longitudinal velocity of flow through the dominant channel was 1.6 +/- 0.7 mm/min. The flow rate during the first five minutes was 7.5 +/- 6.5 microliters/min and 15.3 +/- 10 microliters/min between min 26-30 in clots that had not completely recanalized by that time. A sharp increase in flow was noted at the time of recanalization that occurred at 37 +/- 11 min. Clot lysis followed the pattern of perfusion through the dominant channel after a lag time of 13 +/- 4 min, representing the time required for enzymatic processes. The delay time between perfusion and lysis was longer in regions with slower flow indicating that the rate of t-PA delivery influenced the rate of fibrinolysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood Flow Velocity↗

PubMed1992

Magnetically coupled paramagnetic relaxation agents.

Measurements of the spin-lattice relaxation rates of water protons made over a wide frequency range have demonstrated that the effects of paramagnetic relaxation agents may be considerably enhanced when the paramagnetic center is incorporated into a compact structure macromolecule such as a protein that is rotationally constrained. The immobilization of the macromolecule profoundly changes the nature of the magnetic field dependence of the relaxation rate for both the diamagnetic and the paramagnetic samples. The immobilization also amplifies the effect of the paramagnetic center as a water-proton relaxation agent. The direct exchange of labile water molecules or protons is not a requirement for the high efficiency of this class of magnetic relaxation agents.

Animals↗

PubMed1992

Clinical and laboratory comparison of botulism from toxin types A, B, and E in the United States, 1975-1988.

Cases of adult botulism (n = 309) were studied to identify clinical differences between toxin types and to evaluate the sensitivity of diagnostic laboratory testing. Patients with illness from type E toxin had the shortest incubation periods. Sporadic case-patients were more severely ill: 85% required intubation compared with only 42% in multiperson outbreaks. Of patients with type A botulism, 67% required intubation compared with 52% with type B and 39% with type E. Toxin testing was positive for 40%-44% of serum and stool specimens obtained within 3 days of toxin ingestion and for 15%-23% of specimens obtained thereafter, while 37% of stool specimens obtained > 3 days after toxin ingestion were positive by culture. Patients with type A botulism have more severe illness. In general, specimens obtained early are more likely to be positive by toxin assay, and stool cultures are more sensitive than toxin detection for specimens obtained later in the illness.

Botulinum Toxins↗

PubMed1991

The hydration response of poly (L-lysine) dynamics measured by 13C-NMR spectroscopy.

13C-nmr measurements are reported for samples of poly (L-lysine) both static and spinning at the magic angle in the beta-sheet form as a function of water content. The addition of water decreases the side-chain line widths considerably. Measurements of the cross-polarization time constants indicate that hydration by either H2O or D2O increases the time constant. Measurements of spin-lattice relaxation times in the laboratory frame and the rotating frame indicate that hydration does not change the dynamics of the backbone carbon atoms in the beta-sheet structure appreciably, but the side-chain atoms experience considerable increase in local mobility with increasing hydration. Deuteration of the exchangeable protons or the water has only small effects on the carbon relaxation times, indicating that relaxation is driven by intramolecular dipole-dipole interactions.

Magnetic Resonance Spectroscopy↗

PubMed1991

Applications of nuclear magnetic cross-relaxation spectroscopy to tissues.

Magnetic cross-relaxation in aqueous heterogeneous systems is a long established phenomenon that makes the observable decay constants for the system mixtures of more fundamental relaxation times which characterize the relaxation of the coupled components. By exploiting the magnetic relaxation coupling between the water spins and the immobilized spins in a tissue, the water-proton-signal intensity may be used to map indirectly a frequency response that is directly related to the 1H NMR spectrum of the immobilized components of the tissue. This method is applied to a number of rat tissues to determine whether there are significant differences among tissues that might be exploited in applications of this experiment to diagnostic magnetic imaging. Significant differences are found among nine rat tissues studied, which suggests that the experimental approach may be used to super-impose fundamentally new information, the dynamic character of the usually unobservable immobilized macromolecular components of the tissue, on a magnetic image.

Adrenal Glands↗