Use of magnetic resonance imaging for noninvasive characterization and follow-up of an experimental injury to normal mouse muscles.

A magnetic resonance imaging protocol was tested in a cardiotoxin-induced myonecrosis of hindlimb muscles of three normal mice to assess the usefulness of data provided by longitudinal follow-up of a few individuals. Magnetic resonance imaging examinations were performed sequentially at 4 T between days 1 and 11 post-injury. Axial T1-weighted images after injection of a paramagnetic contrast agent were used to determine the volume of lesions from regions of increased signal intensity. T2 measurements were performed from a single-slice ten-echo acquisition centered upon the largest section of lesion. Early after injury, a very large T2 increase was observed. As recovery proceeded, T2 values progressively decreased toward normal values. Similarly, the volumes of lesions decreased to virtually zero by days 10-11. The evolution of these indices followed the same time scheme observed in histological studies. The use of a volume probe allowed accurate measurement of T2 values, and the acquisition of volumetric data. Such magnetic resonance imaging follow-up should help gather valuable information using few animals.

Magnetic susceptibility shift selected imaging (MESSI) and localized (1)H(2)O spectroscopy in living plant tissues.

Maize root segments permeated with aqueous solutions of the paramagnetic agents GdDTPA(2-) or DyDTPA-BMA display two well-resolved NMR peaks corresponding to the signals from intracellular and extracellular (1)H(2)O, which arise from well-understood bulk magnetic susceptibility effects. This allows each component to be studied separately. Images obtained at each frequency with MESSI editing, and single- and multiple-voxel ('spectroscopic imaging') localized spectra, clearly indicate that the agents permeate into the interstitial spaces, and into the longitudinal (xylem/phloem) channels in the stele (core) of the root, confirming earlier assessments. We believe these are the first images of a multicellular tissue acquired in vivo exclusively from the intracellular water proton resonance. This method can be further exploited to study water transport in similar systems.

In vivo MR imaging and spectroscopy using hyperpolarized 129Xe.

Hyperpolarized 129Xe has been used to obtain gas phase images of mouse lung in vivo, showing distinct ventilation variation as a function of the breathing cycle. Spectra of 129Xe in the thorax show complex structure in both the gas phase (-4 to 3 ppm) and tissue-dissolved (190-205 ppm) regions. The alveolar gas peak shows correlated intensity and frequency oscillations, both attributable to changes in lung volume during breathing. The two major dissolved peaks near 195-200 ppm are attributed to lung parenchyma and to blood; they reach maximum intensity in 5-10 s and decay with an apparent T1 of 30 s. Another peak at 190 ppm takes 20-30 s to reach maximum; this must represent other well-vascularized tissue (e.g., heart and other muscles) in the thorax. The maximum integrated area of the tissue components reaches 30-80% of the maximum alveolar gas area, indicating that imaging at tissue frequencies can be achieved.

Biological magnetic resonance imaging using laser-polarized 129Xe.

As currently implemented, magnetic resonance imaging (MRI) relies on the protons of water molecules in tissue to provide the NMR signal. Protons are, however, notoriously difficult to image in some biological environments of interest, notably the lungs and lipid bilayer membranes such as those in the brain. Here we show that 129Xe gas can be used for high-resolution MRI when the nuclear-spin polarization of the atoms is increased by laser optical pumping and spin exchange. This process produces hyperpolarized 129Xe, in which the magnetization is enhanced by a factor of about 10(5). By introducing hyperpolarized 129Xe into mouse lungs we have obtained images of the lung gas space with a speed and a resolution better than those available from proton MRI or emission tomography. As xenon (a safe general anaesthetic) is rapidly and safely transferred from the lungs to blood and thence to other tissues, where it is concentrated in lipid and protein components, images of the circulatory system, the brain and other vital organs can also be obtained. Because the magnetic behaviour of 129Xe is very sensitive to its environment, and is different from that of 1H2O, MRI using hyperpolarized 129Xe should involve distinct and sensitive mechanisms for tissue contrast.

25Mg NMR studies of magnesium binding to erythrocyte constituents.

The binding of Mg2+ ion to ATP, ADP, AMP, 2,3-bisphosphyoglycerate (DPG), and hemoglobin has been studied by 25Mg NMR spectroscopy at 9.4 T. Addition of any of these ligands to a solution of 2 mM 25MgCl2 at pH 7.2 caused a progressive increase in linewidth, with no discernible chemical shift. ATP and ADP, which form tight 1:1 complexes with Mg2+, did not cause maximal broadening until present in several-fold excess, implying that bis(nucleotide) complexes also form. The studies showed progressively weaker Mg2+ binding to ATP, ADP, DPG, and AMP, consistent with published binding constants. Hemoglobin cause fairly little broadening, consistent with its known weak affinity for Mg2+. Competition studies determined ATP affinities for Ca2+ and H+ that were also in good agreement with published values. 25Mg NMR spectra of 2 mM bound 25Mg2+ were obtained with good signal to noise in less than 1 hr. The technique may now be a practical means for studying the binding of Mg2+ within erythrocytes and other cells.

Pressure dependence of equilibria and kinetics of Escherichia coli ribosomal subunit association.

Equilibria and rates were observed over the ranges 1-1600 atm, 3-10 mM Mg2+, at 60 mM NH4Cl, pH 7.5, 20 degrees C, by light scattering. The main reaction is accurately represented at all conditions by the following phenomenological equations. 30 S + 50 S = 70 S, KA70 = ka/kd = [70 S]/[30 S][50 S] The equilibrium constants obey simple rules: the volume of association, delta VA0, has the constant value 242 +/- 9 ml/mol, independent of pressure, at all Mg2+ concentrations; the derived values of log KA70 at 1 atm increase linearly with log [Mg2+] at a slope of 7.5. In contrast, the rate constants show a clear break at 6 mM Mg2+: below 6 mM, log ka decreases with pressure with a delta Va of 105 +/- 9 ml/mol and increases with log [Mg2+] at a slope of 4.9; above 6 mM, these values are halved; a split can actually be seen at 6 mM Mg2+, near 500 atm. The usual two-step mechanism for second order reactions in solution, which would insert a 70 S' species, either an encounter complex or a true low concentration steady state intermediate, into the above equation can accommodate these results: as [Mg2+] increases, the rate of transformation of 70 S' into 70 S finally predominates over the rate of dissociation of 70 S' into subunits. The bulk of the pressure effects and all of the [Mg2+] dependence arise from the progressive increase in delta GA0 (electrostatic) that occurs when 30, 50, and 70 S particles all lose equivalent fractions of their internal Mg2+ in response to increases in pressure or decreases in [Mg2+].

Characterization of the fluorescent bimane derivative of E. coli initiator transfer RNA (tRNAfMet).

The invariant modified base 4-thiouridine of the E. Coli initiator tRNA was chemically modified using a sulfhydryl specific fluorogenic probe, monobromobimane. The modified tRNAfMet is virtually indistinguishable biochemically from the native form in the aminoacylation and formylation reactions, and in its binding behavior to the ribosomal P site. Fluorescence quenching by I- increases 40% when the modified tRNA is charged with formylmethionine, even at this relatively well-shielded position in the tRNA elbow. Most important, the fluorescence polarization increases by a factor of 2, to almost the irrotational value, when fMet-tRNAfMet binds to the ribosomal P site, providing a useful tool for studying fMet-tRNAfMet-ribosome interaction equilibria and kinetics.

The effect of initiation factor IF-3 on Escherichia coli ribosomal subunit association kinetics.

IF-3 strikingly inhibits the rate of association of 30 S and 50 S subunits to form 70 S ribosome, at all Mg2+ concentrations between 1 and 18 mM, in 60 mM KCl, pH 7.8, at 26 degrees C. The rate of formation of 70 S is determined from the increase in light scattering intensity in stopped flow experiments in which, typically, largely dissociated 30 S-50S mixtures in 2 mM Mg2+ buffer are jumped against high [Mg2+] buffers containing either no or 1-2 eq of IF-3. The curves can be analyzed using two reactions (the anti-association model): 30 S + 50 S (formula see text) Both forward rates are large: in the physiologically important range, 3-6 mM Mg2+, k1 increases from 0.4 to 15 X 10(6) M-1 s-1, while k2 ranges from 3 to 18 X 10(6) M-1 s-1. The kinetic curves have a characteristic shape: an early spurt of 70 S particle formation while IF-3 and 50 S particles are competing for the large initial pool of free 30 S subunits, and a slower phase in which 70 S formation is controlled by the release of 30 S subunits from the 30 S.IF-3 complexes (k-2 runs between 0.05 and 0.17 s-1). The addition of a third reaction, 30 S.IF-3 + 50 S (formula see text) 70 S + IF-3, is not required for an adequate fit, changes the values of k2 slightly, and of k-2 not too much, and yields values of k-3 (0-20 X 10(3) M-1 s-1) probably too small to play a significant physiological role in initiation of protein synthesis unaided by other interactions. The "anti-association" effect of IF-3 on ribosomal subunits is clear-cut. The "pro-dissociation" effect on 70 S ribosomes remains to be demonstrated.

Binding of iodomercurates to sulfhydryl-blocked beta-lactoglobulin-A, -B, and -C.

Sulfhydryl-blocked beta-lactoglobulins (beta-LG-S-SCH2CH2OH)-A, -B, and -C bind only one iodomercurate species, HgI3-, at only one site, with a dissociation constant of 4.0 X 10(-5) M at 25 degrees, pH 5.0, 0.10 ionic strength. (Binding to native beta-LG-SH-A, -B, and -C is more complex, involving the sulfhydryl and two other sites and several iodomercurates.) The red shift of the HgI3- spectrum on binding would ordinarily suggest a hydrophobic site, but the HgI3- site is distinct from, and independent of, the alkane-binding site of native and blocked beta-LG; HgI3- may bind a group that shifts its trigonal planar structure toward the tetrahedron of HgI4(2-). Binding of HgI3- to blocked beta-LG interferes with the well-known association of beta-LG-A to octamers at pH 4.6 and low temperature. The relation of the HgI3- site to the crystallographic iodomercurate-binding sites of beta-LG-SH is examined. To facilitate these and future studies of iodomercurate binding, the 200-400 nm spectra of HgI2, HgI3-, and HgI4(2-) in aqueous solutions and the thermodynamic formation constants at 25 degrees for the equilibria HgI2 + I- = HgI3- (4.9 X 10(3) M-1) and HgI3- + I- = HgI4(2-) (0.118 X 10(3) M-1) were obtained.

The intrinsic structural asymmetry of highly curved phospholipid bilayer membranes.

Phosphorus-31 NMR studies of solutions of small L-alpha-dipalmitoyl phosphatidylcholine bilayer vesicles containing sodium dimethyl phosphate uniformly distributed between the continuous external and the intravesicular aqueous spaces, with the paramagnetic shift reagent Pr3+ present only in the external space, are reported. These studies give the distribution both of dipalmitoyl phosphatidylcholine in the vesicle inner and outer monolayers and of dimethyl phosphate in the aqueous spaces. With the third necessary parameter obtained from the vesicle sedimentation coefficient, the very different packing parameters of dipalmitoyl phosphatidylcholine in inner and outer monolayers can be determined. The vesicle outer radius is 109 A. Although the total bilayer thickness is virtually identical to that of planar bilayers, the outer monolayer is thicker (20 A) and the inner monolayer thinner (15 A). The area per head group at the inner surface, 68 A2, is like the planar value, but the tails are much more folded, so as to decrease the radial lengths and increase the tangential spreat (to 94A2). The reverse is true in the outer layer: the surface per head group is 76 A2, tapering to 51 A2 in the tail region, so that outer layer tails are relatively extended. The difference is equivalent to a shift of about two 2g1 kinks from outer to inner layers; the uneven packing certainly affects fluidity, and may have important biological consequences.