Biomedical subjects

R H Griffey

Publications and source records attributed to R H Griffey.

65 records · Page 4Linked to original sources

PubMed1987

Galactose neuropathy. Structural changes evaluated by nuclear magnetic resonance spectroscopy.

We examined the hypothesis that the polyol accumulation resulting from chronic galactose supplementation in the diet produces endoneurial edema that can be prevented by inhibition of aldose reductase. We explored the potential of nuclear magnetic resonance (NMR) spectroscopy to quantitate and characterize the water accumulation in the sciatic nerve in this "galactose neuropathy." The data demonstrate a 16% increase in gravimetrically determined total water content of nerve in the galactose-fed rat after 8 mo of this diet and a 50% increase in the T1 relaxation time for nerve water as determined by NMR spectroscopy. Prolongation of the T1 relaxation time reflects increased rotation of water in a magnetic field, consistent with an extracellular site of the additional water. Simultaneous feeding of sorbinil to inhibit aldose reductase resulted in normalization of both total nerve water and of the prolongation of T1 relaxation time. These data define the NMR-spectroscopic state of endoneurial edema in the galactose-fed rat and suggest specific application to the investigation of the role of aldose reductase in human diabetic neuropathy.

Animals↗

PubMed1986

15N-labeled tRNA. Identification of 4-thiouridine in Escherichia coli tRNASer1 and tRNATyr2 by 1H-15N two-dimensional NMR spectroscopy.

Uridine is uniquely conserved at position 8 in elongator tRNAs and binds to A14 to form a reversed Hoogsteen base pair which folds the dihydrouridine loop back into the core of the L-shaped molecule. On the basis of 1H NMR studies, Hurd and co-workers (Hurd, R. E., Robillard, G. T., and Reid, B. R. (1977) Biochemistry 16, 2095-2100) concluded that the interaction between positions 8 and 14 is absent in Escherichia coli tRNAs with only 3 base pairs in the dihydrouridine stem. We have taken advantage of the unique 15N chemical shift of N3 in thiouridine to identify 1H and 15N resonances for the imino units of S4U8 and s4U9 in E. coli tRNASer1 and tRNATyr2. Model studies with chloroform-soluble derivatives of uridine and 4-thiouridine show that the chemical shifts of the protons in the imino moieties move downfield from 7.9 to 14.4 ppm and from 9.1 to 15.7 ppm, respectively; whereas, the corresponding 15N chemical shifts move downfield from 157.5 to 162.5 ppm and from 175.5 to 180.1 ppm upon hydrogen bonding to 5'-O-acetyl-2',3'-isopropylidene adenosine. The large difference in 15N chemical shifts for U and s4U allows one to unambiguously identify s4U imino resonances by 15N NMR spectroscopy. E. coli tRNASer1 and tRNATyr2 were selectively enriched with 15N at N3 of all uridines and modified uridines. Two-dimensional 1H-15N chemical shift correlation NMR spectroscopy revealed that both tRNAs have resonances with 1H and 15N chemical shifts characteristic of s4UA pairs. The 1H shift is approximately 1 ppm upfield from the typical s4U8 resonance at 14.8 ppm, presumably as a result of local diamagnetic anisotropies. An additional s4U resonance with 1H and 15N shifts typical of interaction of a bound water or a sugar hydroxyl group with s4U9 was discovered in the spectrum of tRNATyr2. Our NMR results for tRNAs with 3-base pair dihydrouridine stems suggest that these molecules have an U8A14 tertiary interaction similar to that found in tRNAs with 4-base pair dihydrouridine stems.

Magnetic Resonance Spectroscopy↗

PubMed1986

15N-labeled tRNA. Identification of dihydrouridine in Escherichia coli tRNAfMet, tRNALys, and tRNAPhe by 1H-15N two-dimensional NMR.

The N3 imino units of dihydrouridine were identified in samples of 15N-labeled Escherichia coli tRNAfMet, tRNALys, and tRNAPhe by 1H-15N two-dimensional NMR. The peaks for dihydrouridine had high field 1H (9.7-9.8 ppm) and 15N (147.8-149.5 ppm) chemical shifts. Assignments were made by 1H-15N chemical shift correlation based on values obtained in model studies with tri-O-benzoyl- and tri-O-acetyldihydrouridine. The rates of exchange of the imino protons with water suggest that the D-loop in tRNAfMet is less stable than the D-loops in tRNALys or tRNAPhe. Closely spaced peaks were observed for the two dihydrouridines in tRNAPhe in a high resolution spectrum.

Escherichia coli↗

PubMed1986

Isotope-detected 1H NMR studies of proteins: a general strategy for editing interproton nuclear Overhauser effects by heteronuclear decoupling, with application to phage lambda repressor.

A strategy for editing interproton nuclear Overhauser effects (NOEs) in proteins is proposed and illustrated. Selective incorporation of 13C- (or 15N)-labeled amino acids into a protein permits NOEs involving the labeled residues to be identified by heteronuclear difference decoupling. Such heteronuclear editing simplifies the NOE difference spectrum and avoids ambiguities due to spin diffusion. Isotope-detected 1H NMR thus opens to study proteins too large for conventional one- and two-dimensional NMR methods (20-75 kDa). We have applied this strategy to the N-terminal domain of phage lambda repressor, a protein of dimer molecular mass 23 kDa. A tertiary NOE from an internal aromatic ring (Phe-51) to a beta-13C-labeled alanine residue (Ala-62) is demonstrated.

DNA-Binding Proteins↗

PubMed1985

15N-labeled Escherichia coli tRNAfMet, tRNAGlu, tRNATyr, and tRNAPhe. Double resonance and two-dimensional NMR of N1-labeled pseudouridine.

The N1 imino units in Escherichia coli tRNAfMet, tRNAGlu, tRNAPhe, and tRNATyr were studied by 1H-15N NMR using three different techniques to suppress signals of protons not attached to 15N. Two of the procedures, Fourier internuclear difference spectroscopy and two-dimensional forbidden echo spectroscopy permitted 1H and 15N chemical shifts to be measured simultaneously at 1H sensitivity. The tRNAs were labeled by fermentation of the uracil auxotroph S phi 187 on a minimal medium containing [1-15N]uracil. 1H and 15N resonances were detected for all of the N1 psi imino units except psi 13 at the end of the dihydrouridine stem in tRNAGlu. Chemical shifts for imino units in the tRNAs were compared with "intrinsic" values in model systems. The comparisons show that the A X psi pairs at the base of the anticodon stem in E. coli tRNAPhe and tRNATyr have psi in an anti conformation. The N1 protons of psi in other locations, including psi 32 in the anticodon loop of tRNAPhe, form internal hydrogen bonds to bridging water molecules or 2'-hydroxyl groups in nearby ribose units. These interactions permit psi to stabilize the tertiary structure of a tRNA beyond what is provided by the U it replaces.

Escherichia coli↗

PubMed1985

Nuclear magnetic resonance observation and dynamics of specific amide protons in T4 lysozyme.

We have produced T4 lysozyme using a bacterial expression system which allows efficient incorporation of isotopically labeled amino acids in lysozyme. By using conditions that repress the expression of various transaminases, we have incorporated 15N-labeled amino acid into the five phenylalanine residues of the protein. The relatively large spin--spin coupling (87 +/- 3 Hz) between the 15N nucleus and the phenylalanine amide protons may then be exploited in a variety of ways to selectively observe the five phenylalanine amide proton resonances. These include a simple "echo difference" technique which displays the amide proton resonances in one dimension and a "forbidden echo" technique [Bax, A., Griffey, R. H., & Hawkins, B.L. (1983) J. Magn. Reson. 55, 301-335] which gives two-dimensional information allowing the proton and 15N chemical shifts of each amide to be determined. With these approaches, all five phenylalanine amide protons give resolved resonances. Deuterium exchange experiments demonstrate that three of the five resonances are slow to exchange (half-times of about 1 week at pH 5.5 and 4 degrees C) while the other two are rapid with complete exchange in hours or less. These observations correlate well with the secondary structure of the protein which shows three residues in alpha-helical regions and two residues in surface-exposed environments. This approach of isotopic substitution on nitrogen or carbon atoms is of general utility and should allow virtually any proton on a protein of molecular weight 20 000 or thereabout to be selectively observed.

Amides↗

PubMed1983

Efficient syntheses of [3-15N]uracil and [3-15N]thymine.

Regiospecific syntheses of [3-15N]uracil and [3-15N]thymine are described using [15N]ammonium sulfate as a source of labeled nitrogen. The overall yields are excellent, and the reactions are amenable to production of multigram quantities of labeled material.

Ammonium Sulfate↗

PubMed1983

Multiple quantum two-dimensional 1H--15N nuclear magnetic resonance spectroscopy: chemical shift correlation maps for exchangeable imino protons of Escherichia coli tRNAMetf in water.

A procedure based on multiple quantum two-dimensional nuclear magnetic resonance spectroscopy is described for generation of 1H--15N chemical shift correlation maps. The method is used to obtain 15N chemical shifts for the exchangeable imino protons in 1H--15N units of site-specifically labeled Escherichia coli tRNAMetf in water. The high sensitivity and excellent chemical shift dispersion of the multiple quantum two-dimensional technique make it ideally suited for studying protonated nitrogens by NMR.

Escherichia coli↗

PubMedDate not supplied

MR image contrast at high field strength.

The T1 of soft tissues increases with magnetic field strength. Some tissue contrast may be diminished on high-field-strength magnetic resonance (MR) images when conventional TRs are used, because of altered T1 effects on the MR signals. This necessitates longer TRs in techniques that use long TRs, which prolongs the examination excessively. Behavior of macroscopic magnetization is governed by the Bloch equations. Therefore, T1 contributions to the MR signal can be modulated by means of both timing intervals and radio-frequency pulses. The analytic solution to the Block equations allowed calculation of white matter/gray matter and gray matter/cerebrospinal fluid contrast in both spin-echo and inversion-recovery (IR) imaging. Rabbit brains (normal and tumor-containing) were then imaged in vivo at 1.5 and 4.7 T. In addition, MR images of a human head were obtained at 4.0 T. Experimental results supported the theoretical predictions that brain contrast on long TR spin-echo or IR images increases with field strength. However, varying the excitation flip angle allowed optimization of the T1 contribution to the MR signals, improving image contrast and/or reducing examination time. Thus, the dependence of T1 on field strength determines the optimum choice of imaging techniques and parameters in a predictable fashion.

Animals↗

PubMedDate not supplied

19F 2-FDG NMR imaging of the brain in rat.

Images of the rat head reflecting glucose utilization were obtained using 2-fluoro-2-deoxy-D-glucose (2-FDG) and 19F nuclear magnetic resonance (NMR) imaging. Spatial heterogeneity of glucose utilization in the rat head was clearly demonstrated showing significantly higher glucose utilization in the brain as compared to the surrounding tissues. Although the potential adverse effects of the high doses of 2-FDG (400 mg/kg) needed to perform the study preclude immediate application of this technique to clinical quantitative glucose utilization studies, the present study shows potential for future development of glucose utilization imaging by NMR.

Animals↗

PubMedDate not supplied

Lactate observation in vivo by spectral editing in real time.

We have developed a novel in vivo proton MR spectroscopy magnetization transfer method for detection of lactate in ischemic tissue in the presence of interfering fat proton resonances. Pyruvate is magnetically labeled with a saturation pulse and, when converted to lactate, the lactate retains the label. Difference of spectra obtained with and without a saturation pulse contain no fat resonances. High-resolution spectra (determined with a GE 1.5 T Signa) of low lactate levels were obtained in vivo by water suppression using a 2662 composite RF pulse and slice-selective gradients. Spectral subtraction was performed in real time allowing the monitoring of a buildup of the intensity of the lactate peak. Pyruvate-lactate saturation transfer techniques should find wide applicability in the study of ischemia.

Humans↗