Biomedical subjects

C T Moonen

Publications and source records attributed to C T Moonen.

87 records · Page 5Linked to original sources

PubMed1986

Acute renal failure in hemorrhagic hypotension: cellular energetics and renal function.

In male Wistar rats, renal adenosine triphosphate (ATP), inorganic phosphate (Pi) and intracellular pH were measured by 31phosphorus nuclear magnetic resonance (31P NMR) and correlated with renal function before, during, and for one hour after a period of 30 to 40 minutes hemorrhagic hypotension. In animals which suffered no change in these metabolites during hypotension, retransfusion immediately restored normal renal function. When metabolite changes were observed during hypotension, they occurred suddenly with severe ATP depletion, Pi accumulation, and intracellular acidosis occurring almost concurrently. Metabolic changes of this magnitude were always associated with renal dysfunction in the post-hypotensive period, which occurred even when the period of biochemical change was only 10 to 15 minutes. The abnormalities in post-hypotensive renal function resemble the pattern of change seen in human acute tubular necrosis (ATN): depressed glomerular filtration rate (GFR), urine output varying from polyuria to oliguria, decreased urine to plasma inulin ratio, increased urinary sodium concentration, increased fractional excretion of sodium, and increased fractional excretion of potassium. It is postulated that changes in renal cellular energy status during hemorrhagic hypotension distinguish pre-renal failure from early or incipient ATN.

Acute Kidney Injury↗

PubMed1984

Reinvestigation of the structure of oxidized and reduced flavin: carbon-13 and nitrogen-15 nuclear magnetic resonance study.

Several chemically substituted flavins are investigated in the oxidized and the reduced state by 13C and 15N NMR techniques. The dependence on the polarity of the solvent and on the concentration is studied. In combination with already published results, a semiempirical theory is developed to interpret the chemical shifts in terms of the solution structure of flavins. Where possible, the results are compared with crystallographic and light absorption data. In contrast to common ideas, the solution structure of the oxidized state is not fully coplanar, but the N(10) atom is situated out of plane to a certain degree. Polarizing the flavin by hydrogen bonds in a high dielectric medium moves the N(10) atom into the molecular plane, and the flavin molecule becomes coplanar. In the coplanar molecule, pi electrons are delocalized from the N(10) atom mainly to O(2 alpha) and O(4 alpha). The NMR results show that the solution structure of reduced flavin is mainly governed by sterical hindrance and hydrogen bonds. The findings are in contrast to commonly accepted ideas that reduced flavin is strongly bent. In an apolar solvent, the reduced neutral isoalloxazine is only slightly bent. The formation of hydrogen bonds in a protic solvent of a high dielectric constant decreases the bend. The N(10) atom is now almost fully sp2 hybridized, and the N(5) atom has an endocyclic angle of 115-117 degrees, indicating its predominant sp2 character. The results have several important implications for flavin catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Flavins↗

PubMed1984

Carbon-13 nuclear magnetic resonance study on the dynamics of the conformation of reduced flavin.

Several flavin model compounds in the reduced state have been investigated by 13C NMR techniques. The NMR spectra were recorded in dependence of temperature, in the range of 30 to -100 degrees C. The results show that the activation barrier for the ring inversion ("butterfly" motion) is too low to be observed directly. In order to be able to detect the barrier of the ring inversion, it was coupled with a side-chain rotation. In this way, the intrinsic barrier for the ring inversion is increased by the barrier of the side-chain rotation, which allowed detection of the former barrier. It is shown that the intrinsic barrier for the ring inversion is less than 20 kJ/mol. Moreover, it is shown that previous results of Tauscher et al. [Tauscher, L., Ghisla, S., & Hemmerich, P. (1973) Helv. Chim. Acta 56, 630-649] are incorrect and nitrogen inversion is not observed. Symmetry arguments in the dynamic processes are discussed. From the low activation barrier for the ring inversion, it can be concluded that the conformation of the reduced flavin can be easily influenced upon binding to apoflavoproteins. This aspect might be of importance in the regulation of the function of the flavin prosthetic group in biological systems.

Animals↗

PubMed1984

The use of two-dimensional nuclear-magnetic-resonance spectroscopy and two-dimensional difference spectra in the elucidation of the active center of Megasphaera elsdenii flavodoxin.

1H-1H 'through bond' correlated (COSY) and 1H-1H 'through space' (NOESY) two-dimensional NMR techniques were applied to study the structure of Megasphaera elsdenii flavodoxin in the oxidized and reduced state. It is shown that two-dimensional NOESY difference spectra between spectra of flavodoxin in the reduced and semiquinone state are sensitive to the active center of the fully reduced state. The sphere of the active center observed in the difference spectra can be varied easily by changing the relative amount of flavodoxin semiquinone in the second sample. The difference NOESY spectra simplified the analysis of the complex spectra. Resonances could be assigned to Ala-56, Tyr-89 and Trp-91, which are located in the direct vicinity of the protein-bound flavin. The relative positions and side-chain dihedral angles of these residues are compared for the two redox states. Ala-56 and Tyr-89 show identical relative positions and dihedral angles in the two redox states, although the rotational motion of Tyr-89 is enhanced in the oxidized state. In both redox states Trp-91 is immobilized and extremely close to the prosthetic group. However, a small displacement of Trp-91 towards the (N(5) atom of the flavin occurs upon reduction. The results obtained for Trp-91 are in excellent agreement with crystallographic results of the related flavodoxin from Clostridium MP. However, the latter studies showed a somewhat different position of the tyrosine residue compared with our results.

Amino Acids↗

PubMed1984

On the intermolecular electron transfer between different redox states of flavodoxin from Megasphaera elsdenii. A 500-MHz 1H NMR study.

The electron transfer reactions between molecules of flavodoxin from Megasphaera elsdenii in different redox states have been investigated by proton nuclear magnetic resonance techniques at 500 MHz. The electron transfer between molecules in the oxidized and semiquinone state is shown to be at least 350-times slower than that between molecules in the semiquinone and hydroquinone state. The latter reaction was studied at different ionic strengths and temperatures. The rate of electron transfer increases with increasing ionic strength, as expected for a reaction between molecules of identical charges. The electron transfer reaction is only slightly dependent on temperature suggesting an outer sphere reaction mechanism. The results indicate that the activation energy for the electron transfer reaction between the semiquinone and hydroquinone state is negligible in contrast to that between the oxidized and semiquinone state. It is suggested that this feature renders M. elsdenii flavodoxin to an exclusive one-electron donor/acceptor in the cell, thereby shuttling between the semiquinone and the hydroquinone state. Mechanistic implications of the findings are briefly discussed.

Bacterial Proteins↗

PubMed1984

A proton-nuclear-magnetic-resonance study at 500 MHz on Megasphaera elsdenii flavodoxin. A study on the stability, proton exchange and the assignment of some resonance lines.

1H NMR studies were performed on the three redox states of Megasphaera elsdenii flavodoxin. The results show that the protein is remarkably stable, as concluded from amide proton exchange studies. Some amide protons are still present in the 1H NMR spectrum even after one month in 2H2O at 33 degrees C (pH 8.3). The reactivity of the exchangeable protons can be grouped into three categories, i.e. t1/2 much greater than 5 min, 10 s approximately less than t1/2 approximately less than 5 min, and t1/2 much less than 10 s. The amide proton exchange reactions are hardly dependent on the redox state. Optimal resolution of 1H NMR spectra is obtained at 33 degrees C, independent of the redox state. No conformational change of the protein is observed in the pH range between 6 and 8.5. Assignments of resonances to protons of flavin and of some amino acid residues are established in both the oxidized and the hydroquinone state using chemically and isotopically substituted flavins and the driven nuclear Overhauser technique. Preliminary two-dimensional 1H-1H correlated spectra show that the protein is amenable to two-dimensional NMR techniques. Previous assignments are confirmed by this technique.

Amides↗

PubMed1983

On the mobility of riboflavin 5'-phosphate in Megasphaera elsdenii flavodoxin as studied by 13C-nuclear-magnetic-resonance relaxation.

The mobility of the isoalloxazine ring of the prosthetic group of Megasphaera elsdenii flavodoxin was investigated by a 13C relaxation study of the non-protonated ring atoms 2, 4, 4a and 10a. In this study a selectively enriched (greater than 90% 13C) prosthetic group was bound to the apoprotein. T1 and T2 values were determined at two magnetic field strengths, i.e. 8.46 T (90.5 MHz) and 5.88 T (62.8 MHz). Values of nuclear Overhauser effects (NOE) were determined at 5.88 T. It is shown that both the dipole-dipole interaction and the chemical shift anisotropy are important relaxation sources for all the carbon atoms investigated. The results are in agreement with a spectral density function of the isoalloxazine ring in which only the overall reorientational motion of the protein is accounted for. From this it is concluded that the isoalloxazine ring is tightly associated with the apoprotein. The protein-bound isoalloxazine ring does not exhibit large fluctuations on the nanosecond time scale, although small amplitude fluctuations cannot be excluded. This information was obtained by a combination of field-dependent T1 and NOE measurements. T2 values are in agreement with these results. On the basis of the dipolar part of the overall T1 values, the distance between the carbon investigated and the nearest proton was calculated and found to be in fair agreement with the crystallographic results of the related flavodoxin from Clostridium MP. In addition, it is shown that, based on the chemical shift anisotropy as a relaxation source, information on the internal mobility is difficult to obtain. The main reason for this is the low precision in the determination of the chemical shift anisotropy tensor.

Chemical Phenomena↗

PubMed1982

A photo-CIDNP study of the active sites of Megasphaera elsdenii and Clostridium MP flavodoxins.

Megasphaera elsdenii and Clostridium MP flavodoxins have been investigated by photo-CIDNP techniques. Using time-resolved spectroscopy and external dyes carrying different charges it was possible to assign unambiguously the resonance lines in the NMR-spectra to tyrosine, tryptophan and methionine residues in the two proteins. The results show that Trp-91 in M.elsdenii and Trp-90 in Cl.MP flavodoxin are strongly immobilized and placed directly above the benzene subnucleus of the prosthetic group. The data further indicate that the active sites of the two flavodoxins are extremely similar.

Binding Sites↗

PubMed1982

Structural and dynamic information on the complex of Megasphaera elsdenii apoflavodoxin and riboflavin 5'-phosphate. A phosphorus-31 nuclear magnetic resonance study.

It is shown that commercial FMN contains a considerable amount of the 4', 3', and 2' isomers and other phosphorus-containing compounds. These impurities can easily be analyzed and quantified by the 31P NMR technique. The phosphate group of FMN bound to Megasphaera elsdenii apoflavodoxin is probably in the dianionic form. Its chemical shift is almost independent of pH in the range 5.5-9.2 and of the redox state of the protein. The phosphate group of bound FMN is buried in the protein. Protons of the apoprotein and of bound water located in the vicinity of the phosphate group in native flavodoxin are not exchangeable with deuteron of the bulk solvent. These protons are, however, easily exchangeable in apoflavodoxin, as shown by reconstitution experiments. The distance between the phosphorus atom of bound FMN and the N(10) atom of the isoalloxazine moiety of FMN is calculated to be about 7.8 A. This result is in good agreement with X-ray data published for the related flavodoxin from Clostridium MP. The electron exchange between the oxidized and semiquinone state of M. elsdenii flavodoxin is rather slow (kexc' much less than 2 s-1) whereas that between the semiquinone and hydroquinone form is much more favored (kexc' much greater than 100 s-1). This indicates that the activation energy for the transition between the semiquinone and hydroquinone states must be smaller than that for the transition between the oxidized and semiquinone states. These results offer a reasonable explanation for the one-electron transfer reaction of flavodoxins in biological reactions.

Apoproteins↗

PubMedDate not supplied

Imaging of diffusion and microcirculation with gradient sensitization: design, strategy, and significance.

Recent developments in the use of magnetic resonance (MR) to measure and image diffusion and blood microcirculation ("perfusion") are summarized. After a brief description of the effects of diffusion and perfusion on the MR signal, the different methods (conventional spin-echo, stimulated-echo, gradient-echo, and echo-planar imaging) that have been proposed and used to image and measure diffusion and perfusion by gradient sensitization are presented, along with their advantages and limitations. The difficulties of diffusion/perfusion imaging related to both hardware and software are then discussed. Special attention is given to specific problems encountered with in vivo studies and data analysis. Finally, the potential biologic and clinical applications are outlined, and some examples are presented.

Brain↗

PubMedDate not supplied

On the feasibility of MRI-guided focused ultrasound for local induction of gene expression.

Gene therapy is a promising approach to the treatment of many forms of disease, including cancer. Of critical concern in its implementation is the ability to control the location, duration, and level of expression of the therapeutic gene. Here, we propose the use of local heat in combination with a heat-sensitive promoter to help accomplish this. Certain members of the family of heat shock protein (hsp) promoters display a regulation that depends strongly on temperature. We present a study of natural hsp70 induction in rat leg by MRI-guided focused ultrasound to investigate the hsp70 promoter as a possible candidate for use in control of gene expression with local heat. A temperature increase of 5-8 degrees C in the focal region for 45 minutes led to a differential expression of the hsp70 mRNA between the focal region and the surrounding tissue ranging from a factor of 3 to 67.

Animals↗

PubMedDate not supplied

Measuring random microscopic motion of water in tissues with MR imaging: a cat brain study.

Cat brain images sensitized to incoherent motion by additional gradient pulses were obtained on a 4.7 T magnetic resonance unit equipped with shielded gradient coils. The apparent diffusion coefficient of water in gray and white matter was accurately determined and imaged from the signal attenuation curve obtained as a function of gradient strength. Contrast in calculated diffusion images differed from typical T2-weighted contrast. Furthermore, in gray matter and in areas containing flowing CSF the attenuation curve was found to be biexponential. These results are interpreted in terms of a simple voxel model with microcirculation and diffusion contributions.

Animals↗

PubMedDate not supplied

Short echo time proton MR spectroscopic imaging.

Proton spectroscopic imaging at short TEs (20-30 ms) in human brain requires volume preselection inside the brain to suppress overwhelming lipid and water signals from surrounding tissue. In this article we discuss limitations of conventional volume preselection using stimulated echoes that lead to spectral contamination from surrounding tissue. Improved volume preselection was obtained by adding a complete outer volume suppression (presaturation). The performance of the method is illustrated on normal volunteers and on clinical cases with brain tumors and multiple sclerosis (MS) plaques. In normal human brain, we detected resonances with short T2 values and complex J-coupling, including rather broad methyl/methylene resonances in the chemical shift range between 0 and 2 ppm. Spectroscopic images obtained on patients with intracranial tumors and on one patient with several MS plaques demonstrate the possibility of detecting regional distributions of increased methyl/methylene resonances between 0 and 2 ppm in brain lesions.

Adult↗

PubMedDate not supplied

3D bolus tracking with frequency-shifted BURST MRI.

OBJECTIVE: Our goal was to develop and test a 3D bolus-tracking MR technique for perfusion imaging of normal and pathological (infarcted) human brain. MATERIALS AND METHODS: All experiments were performed on standard 1.5 T GE/Signa clinical scanners. Five normal volunteers and one patient with a subacute brain infarct were studied. Modified [frequency-shifted (FS)] BURST MRI was performed during injection of a bolus of Gd-DTPA (0.13 mmol/kg) in the antecubital vein. The 3D datasets were acquired with a time resolution of 2.2 s and an effective spatial resolution of 4.3 x 4.3 x 6.4 mm. Three-dimensional maps of blood volume and bolus arrival time were determined by fitting a synthetic curve to the intensity time course on a voxel-by-voxel basis. RESULTS: Both relative cerebral blood volume and arrival time maps demonstrated sensitivity to regional differences in blood supply in both normal brain and in the subacute brain infarction. The transit time maps showed arrival time delays of 5-7 s within and around the infarct and confirmed the diagnosis of left middle cerebral artery occlusion. CONCLUSION: The results of the measurements on both normal and diseased human brain demonstrated the ability to acquire valuable 3D information about brain perfusion using FS BURST MRI.

Adult↗

PubMedDate not supplied

Reproducibility of proton MR spectroscopic imaging findings.

PURPOSE: To study the intraindividual, interindividual, and intraregional reproducibility of multisection proton MR spectroscopic imaging in healthy adults. METHODS: Six subjects were studied three times with proton MR spectroscopic imaging. Multisection long-echo-time proton MR spectroscopic imaging permits simultaneous acquisition of N-acetylaspartate (NAA), choline-containing compounds (Cho), and creatine plus phosphocreatine (Cr) signal intensities from four 15-mm-thick sections divided into 0.84-mL single-volume elements. Regions of interest were the frontal cortex, the occipital cortex, the parietal cortex, the insular cortex, the cingulate gyrus, the centrum semiovale, the thalamus, and the caudate. Statistical evaluation was performed by analyses of variance and components of variance method. RESULTS: The ratio NAA/Cr showed the lowest overall coefficient of variation (CV, %) in most of the regions of interest (range, 8.9 to 26.1). Interregional differences in the overall CV were present. Interindividual CVs ranged from 4.2 to 8.7 for NAA/Cr, from 6.8 to 17.4 for NAA/Cho, and from 5.0 to 13.6 for Cho/Cr. Intraindividual CVs ranged from 8.2 to 22.2 for NAA/Cr, from 12.8 to 25.8 for NAA/Cho, and from 4.5 to 21.0 for Cho/Cr. Intraregional CVs ranged from 12.3 to 21.2 for NAA/Cr, from 13.0 to 20.4 for NAA/Cho, and from 12.2 to 18.9 for Cho/Cr. CONCLUSIONS: Proton MR spectroscopic imaging showed good overall reproducibility. The finding of interregional variations of CV indicates that care is needed when using this imaging technique for follow-up studies.

Adult↗