[Functional MRI for schizophrenia: importance of the type of task being scanned]. / Functionele MRI bij schizofrenie: belang van aangepaste taak in de scanner.

BACKGROUND: Functional magnetic resonance imaging (fMRI) is an important technique for detecting neural network problems in patients with schizophrenia. Very often, however, the professionals involved are insufficiently aware of the fact that when fMRI scans are used for patients with schizophrenia, it is the type of task that patients are performing or failing to perform which is of vital importance for the correct interpretation of the results. AIM: To demonstrate that in scans of patients with schizophrenia the choice of task can influence neuroimaging results, particularly when the neural problems under study are performance-related. METHOD: We begin by presenting a brief history of functional neuroimaging techniques. This provides a context for the study of the potential role of fMRI in detecting dysfunctional brain networks in schizophrenia. In this way we demonstrate more clearly why the rapidly developing scanning techniques and analysis methods are becoming more and more important for measuring specific differences between psychiatric disorders. Then, we discuss the complex relationship between cognitive deficits in patients with schizophrenia, problems in task performance, and disease-related effects on brain activation measured by fMRI. RESULTS: This is illustrated by our own recent work, in which we demonstrate the complex relationship between cognitive deficits in the task performance of patients with schizophrenia and differences in brain activity measured with fMRI. We stress the importance of task-independent neural networks for the interpretation of results. These networks may play a role similar to that of the potentially confounding effects of task choice. Finally, we consider challenges for the future and comment on how fMRI needs to be developed so that it can be used successfully in clinical practice in order to assist with the diagnosis, prognosis and treatment of schizophrenia. CONCLUSION: It is crucial for neuroimaging research into schizophrenia, and for potential clinical applications, that new types of tasks are developed that avoid the confounding effects on neural activity which are caused by performance differences stemming from aspecific factors such as demotivation or task-disengagement.

An ab initio quantum-chemical study of the blue-copper site of azurin.

The electronic structure of the blue-copper site of Pseudomonas aeruginosa azurin has been investigated by ab initio multireference determinantal configuration interaction (MRD-CI) calculations. A truncated site consisting of copper and its three equatorial ligands has been studied with emphasis on the g tensor and the nitrogen hyperfine tensors of the coordinating histidines. In the ground state the singly occupied molecular orbital (SOMO) involves a copper 3d orbital pi antibonded to the cysteine sulfur and sigma antibonded to the histidine nitrogens. A proper description of the electron-paramagnetic-resonance parameters has been achieved through the use of an effective core potential for copper up to and including the 3s electrons. Both the complete g tensor and the anisotropic hyperfine tensors at the nitrogens are essentially reproduced. Mulliken spin densities of 35 and 59% on copper and sulfur, respectively, and 2.1 and 1.7% on the respective coordinating nitrogens reflect the delocalized character of the SOMO and the inequivalence of the histidines.

Pretransplantation assessment of renal viability with NADH fluorimetry.

BACKGROUND: A pathophysiologic feature possibly involved in ischemic injury in transplant kidneys is mitochondrial dysfunction caused by disintegration of oxidative metabolic pathways. Because the ability to synthesize ATP by respiratory activity determines the organ's capacity to recover from ischemic injury, an assessment of respiratory activity may provide information related to graft viability. METHODS: NADH fluorimetry can be used to monitor kidney cortex metabolism noninvasively. During perfusion with (an)-aerobic perfusate, NADH fluorescence images were recorded. We evaluated the NADH oxidation kinetics of 20 rat kidneys, which were divided over four experimental groups. For six minimally damaged kidneys and six kidneys that had been stored for one hour at 37 degrees C, perfusion was followed by transplantation. We related the kinetic parameters of these kidneys with their post-transplantation function and histology. The transplant function was monitored by serum creatinine and urea levels. RESULTS: Storage of transplant kidneys for one hour at 37 degrees C significantly reduced the post-transplantation function. Isolated perfusion of grafts, however, was not detrimental for renal function. The rate of NADH oxidation decreased with decreasing graft quality, and a good correlation between NADH oxidation kinetics and post-transplantation function was found. CONCLUSIONS: A reduction of NADH oxidation rates as a consequence of warm ischemia supports the view that mitochondrial respiratory activity is impaired by ischemic injury. The correlation between NADH oxidation kinetics in perfused grafts and their post-transplantation function indicates that NADH fluorimetry may be useful in predicting the viability of preserved grafts prior to transplantation.

The binding of imidazole in an azurin-like blue-copper site.

Frozen solutions of the azurin mutant His117Gly in the presence of excess of methyl-substituted imidazoles have been investigated by electron spin-echo envelope modulation (ESEEM) spectroscopy at 9 GHz. The addition of imidazole is known to reconstitute a blue-copper site and variation of the non-protein bound ligand [N-methyl-, 2-methyl-, 4(5)-methylimidazole] has allowed the study of the copper-imidazole binding as a model for histidine binding in such sites. Quadrupole and hyperfine tensors of the remote nitrogen of the imidazoles have been determined. The quadrupole tensors indicate that the methyl-substituted imidazoles in the mutant adopt the same orientation relative to copper as the histidine-117 in the wild-type protein. Analysis of the hyperfine tensors in terms of spin densities reveals that the spin density on the remote nitrogen of the substituted imidazole has sigma and a variable pi character, depending on the position of the methyl group. For azurin the corresponding spin density is of virtually pure sigma character. In conclusion, blue-copper sites show subtle variations as regards the histidine/imidazole centred part of the wavefunction of the unpaired electron.

(Semi-)quantitative analysis of reduced nicotinamide adenine dinucleotide fluorescence images of blood-perfused rat heart.

In vivo analysis of the metabolic state of tissue by means of reduced nicotinamide adenine dinucleotide (NADH) fluorimetry is disturbed by tissue movements and by hemodynamic and oximetric effects. These factors cause changes in the absorption of ultraviolet (UV) excitation light by the tissue. Many different methods have been used in the literature to compensate measured NADH fluorescence intensities for these effects. In this paper we show on theoretical grounds that the ratio of NADH fluorescence intensity and UV diffuse reflectance intensity provides a (semi-)quantitative measure of tissue NADH concentrations. This result is corroborated by experiments with tissue phantoms in which absorption and back-scattering properties were varied. Furthermore, we have verified the validity of this compensation method in isolated Langendorff-perfused rat heart preparations. In this preparation oximetric effects (of blood and tissue) are the major determinants of the metabolism-dependent UV diffuse reflectance change. Hemodynamic effects accompanying compensatory vasodilation are negligible. Movement artifacts were eliminated by simultaneously recording fluorescence and reflectance images, using a CCD camera with a biprism configuration. The results show that the NADH fluorescence/UV reflectance ratio can be used to monitor the mitochondrial redox state of the surface of intact blood-perfused myocardium.

A comparison between different imaging strategies for diffusion measurements with the centric phase-encoded turboFLASH sequence.

The study compared the results of three centrally reordered phase-encoded turboFLASH sequences for diffusion-weighted imaging (DWI). The sequences were conventional turboFLASH, turboFLASH with subtraction of T1-related effects, and turboFLASH with correction for T1-related effects during the imaging period only. The relative merits were studied with respect to image quality and accuracy by computer simulation and by experimental validation on phantoms and on in vivo rat brain. A T1-related underestimation of the diffusion coefficient ranging from -30% (T1 approximately 200 ms) to -5% (T1 approximately 1 s) was found to exist for the conventional sequence. Image artifacts, caused by longitudinal relaxation during the imaging period, are reflected in calculated diffusion maps. When the correction sequence is used, the artifacts and the systematic errors are reduced but longitudinal relaxation during the delay between preparation and imaging periods remains large enough to induce significant errors (-15% for T1 approximately 200 ms to -3% for T1 approximately 1 s). The subtraction sequence eliminates the influence of T1 effects on the calibrations, but leads to identical artifacts for all diffusion-weighted images.

Increase of cardiac work is associated with decrease of mitochondrial NADH.

In this study we investigated the effect of work and substrate supply on mitochondrial NADH/NAD+ using epicardial autofluorescence in rat hearts perfused according to Langendorff. To avoid vasoconstrictor effects during high work output, nitroprusside-containing Tyrode solution was used. Photobleaching was avoided by using discontinuous ultraviolet excitation for NADH fluorescence measurements. To increase work, heartbeat rate was raised from 5 to 7 Hz, and concomitantly left ventricular pressure was raised stepwise from 0 to +/- 90 mmHg. During substrate-limited (5.5 mM glucose) perfusions, increase in O2 consumption (3.5 +/- 0.4 mumol.min-1.g-1, mean +/- SE, n = 6) caused by increase of heartbeat rate was associated with a significant decrease of NADH fluorescence (-31 +/- 2.5%, mean +/- SE, n = 6). During perfusions with 10 mM pyruvate increase of O2 consumption (3.6 +/- 0.7 mumol.min-1.g-1, mean +/- SE, n = 6) was associated with significant decrease of NADH fluorescence (-20 +/- 2.6%, mean +/- SE, n = 6). These results suggest that a rise in mitochondrial NADH/NAD+ is not the primary stimulus for increase in respiration and that changes of mitochondrial NADH/NAD+ are secondary to changes in O2 consumption.

A method for myelin fiber orientation mapping using diffusion-weighted MR images.

In the past, the anisotropic diffusion of water molecules in white matter in the brain has been correlated to the basic symmetry of the myelin fibers: water diffuses more readily along the fiber direction than perpendicular to it. As a consequence, diffusion sensitized magnetic resonance imaging can be expected to be useful for studying the fiber orientation. In this work, we present a method for exploiting this type of information to map the fiber orientations in the image plane. It makes use of three diffusion-weighted images with sensitizing gradients along x, y and u, an axis at 45 degrees with respect to x and y. The orientation information contained in these images is summarized in a single image representing the angle between the fiber direction and a fixed axis, making use of a cyclic color scale. The method is evaluated using computer simulations for a variety of diffusion weighting strengths and signal-to-noise ratios, tested on a phantom and illustrated on an in vivo example. An extension to the determination of the fiber orientation in three dimensions is also described.

Absolute quantification of 31P liver metabolites in rat using an external reference and a surface spoiling magnetic field gradient.

A simple method for measuring absolute concentrations of 31P metabolites in rat liver using an external reference is described. It neutralizes systematic errors due to conductive losses by calibration of the matching capacitor and selects liver signals via an electrically driven, surface spoiling magnetic field gradient. The technique avoids the chemical shift problems associated with linear field gradient localization methods at 4.7 T and allows combination with the double standard method for absolute metabolite concentration determination. Application of the method to the in vivo measurement of the absolute concentrations of ATP and P(i) in eight rat livers yields results that are in good agreement with literature values. Absolute phosphomonoester concentrations were also obtained, but no literature data were available.

On the redox equilibrium between H2 and hydrogenase.

Redox titrations of the nickel ion in active hydrogenase from Methanobacterium thermoautotrophicum and Chromatium vinosum were performed in the absence of artificial redox mediators, by variation of the H2-partial pressure. These experiments revealed a redox behaviour of the nickel ion which differed remarkably from previous redox titrations in the presence of redox mediators. Notably the EPR signal of the species earlier characterized as monovalent nickel with bound hydrogen, behaved as an n = 2 redox component upon reduction under varying H2-partial pressures. The EPR signal was not a transient one and persisted upon removal of hydrogen. Possible redox processes to explain these observations are discussed. A similar behaviour of nickel was also observed in enzyme as present in intact cells of M. thermoautotrophicum. These results suggest that nickel hydrogenases possess a second site for reaction with H2.

Distinct redox behaviour of prosthetic groups in ready and unready hydrogenase from Chromatium vinosum.

The redox behaviour of the Ni(III)/Ni(II) transition in hydrogenase from Chromatium vinosum is described and compared with the redox behaviour of the nickel ion in the F420-nonreducing hydrogenase from Methanobacterium thermoautotrophicum. Analogous to the situation in the oxidised hydrogenase of Desulfovibrio gigas (Fernandez, V.M., Hatchikian, E.C., Patil, D.S. and Cammack, R. (1986) Biochim. Biophys. Acta 883, 145-154), the C. vinosum enzyme can also exist in two forms: the 'unready' form (EPR characteristics of Ni(III): gx,y,z = 2.32, 2.24, 2.01) and the 'ready' form (EPR characteristics Ni(III): gx,y,z = 2.34, 2.16, 2.01). Like in the oxidised enzyme of M. thermoautotrophicum the Ni(III)/Ni(II) transition for the unready form titrated completely reversible (both at pH 6.0 and pH 8.0). In contrast, the reversibility of the Ni(III)/Ni(II) transition in the ready enzyme was strongly dependent on pH and temperature. At pH 6.0 and 2 degrees C reduction of Ni(III) in ready enzyme was completely irreversible, whereas at pH 8.0 and 30 degrees C Ni(III) in both ready and unready enzyme titrated with E0' = -115 mV (n = 1). Hampered redox equilibration between the ready enzyme and the mediating dyes is interpreted in terms of an obstruction of the electron transfer from nickel at the active site to the artificial electron acceptors in solution. The origin of this obstruction might be related to possible changes in the protein structure induced by the activation process. The E0'-value of the Ni(III)/Ni(II) equilibrium was pH sensitive (-60 mV/delta pH) indicating that reduction of nickel is coupled to a protonation. A similar pH-dependence was observed for the titration of the spin-spin interaction of Ni(III) and a special form of the [3Fe-4S]+ cluster (E0' = +150 mV, pH 8.0, 30 degrees C). Redox equilibration of this coupling was extremely sensitive to pH and temperature. The uncoupled [3Fe-4S]+ cluster titrated pH-independently with E0' = -10 mV (pH 8.0, 30 degrees C).

Absolute quantification of 31P muscle metabolites using NMRS with an internal standard and a high-Q, double-tuned coil.

31P-nuclear magnetic resonance spectroscopy (NMRS) is used to determine absolute concentrations of 31P metabolites in rat muscle. A technique exploiting tissue water 1H as internal concentration reference and a highly sensitive, double-tuned coil is described and evaluated experimentally. On KH2PO4 solutions of known concentration and varying coil loading it is shown to allow neutralization of systematic errors due to conductive losses, which normally range up to 20% or more. In vivo application in the determination of the absolute concentrations of ATP, PCr and Pi in eight rat thighs yields results that are in good agreement with literature values.

Effect of 17O2 and 13CO on EPR spectra of nickel in hydrogenase from Chromatium vinosum.

Oxygen, either molecular oxygen or a reduction adduct, can tightly bind in the vicinity of the two forms of trivalent nickel occurring in hydrogenase from Chromatium vinosum, as evident from studies with 17O-enriched O2. This oxygen is not in the first coordination sphere of nickel. As has been reported earlier for hydrogenase from Desulfovibrio gigas (Fernandez, V.M., Hatchikian, A.C., Patil, D.S. and Cammack, R. (1986) Biochim. Biophys. Acta 883, 145-154), also the relative activity of the C.vinosum enzyme correlates well with the presence of only one of the two Ni(III) forms in the oxidized preparation. These results make it less likely that a specific oxygenation of only one of the Ni(III) forms would be the reason for the reversible inactivation of nickel hydrogenases by oxygen. Reaction of H2-reduced enzyme with 13CO now demonstrated beyond doubt that: (i) One 13CO molecule is a direct ligand to nickel in axial position; and (ii) hydrogen binds at the same coordination site as CO. It can also be concluded that hydrogen is not bound as a hydride ion, but presumably as molecular hydrogen. A simple way to explain the EPR spectra from the 13CO-adduct of the enzyme is to assume a monovalent state for the nickel.

Phagocytosis by human macrophages is accompanied by changes in ionic channel currents.

The present study has shown that changes in ionic channel currents accompany the phagocytosis of particles by mononuclear phagocytes. The patch-clamp technique in the cell-attached configuration was applied to human monocyte-derived macrophages to measure the activity of single transmembrane ionic channels in intact cells. During such measurements, IgG-opsonized and non-opsonized latex particles were offered for phagocytosis under continuous video-microscopical observation. Single particles were presented to the phagocytes at a membrane location some distance from that of the patch electrode. After a lag period following particle attachment, enhanced inward and outward time-variant single channel currents coinciding with particle engulfment were observed. On the basis of current-voltage characteristics and membrane potential measurements, the outward-directed channels were identified as K+ channels. Phagocytosis was also accompanied by slow transient changes in background membrane currents, probably due to changes in the membrane potential of the phagocytosing cell. Phagocytosis of IgG-coated latex particles differed from phagocytosis of uncoated or albumin-coated particles by a shorter lag time between particle attachment and the onset of enhanced ionic channel activity.

Purification and some properties of the corrinoid-containing membrane protein from Methanobacterium thermoautotrophicum.

The cytoplasmic membrane of the methanogenic archaebacterium Methanobacterium thermoautotrophicum does not contain cytochromes, but did contain a corrinoid protein of molecular mass about 33 kDa which, after treatment with 10 mg Triton X-100/mg protein, was contained in a protein complex of about 500 kDa. Washed membranes from 1 g dry cells contained about 70 nmol of the cobamide factor III (5-hydroxybenzimidazolyl cobamide) as the sole corrinoid. The corrinoid-containing protein complex was purified and some of its properties were studied. According to several criteria it is an integral membrane protein complex. The corrinoid-protein complex, after about 100-fold purification, gave a single band on native PAGE and still had molecular mass of about 500 kDa. In SDS-PAGE several subunits were observed: in addition to the corrinoid-carrying subunit of about 33 kDa, other polypeptides of approximately 28 kDa, 26 kDa, and possibly 23 kDa were present. One mole of the purified 500-kDa protein complex contained greater than or equal to eight moles of the cobamide factor III. It was estimated that the corrinoid-protein complex accounts for 8% of the membrane protein of M. thermoautotrophicum. The visible spectrum of the oxidized protein exhibited absorbance maxima at 547 nm, 511 nm, and a shoulder at 468 nm, which disappeared upon reduction with dithionite. The midpoint potential of this transition was around -145 mV (pH 7). With EPR a Co2+ signal was observed within -50 mV and -350 mV with a maximum around -200 mV. Possible reasons for the disappearance of the Co2+ signal at low redox potentials are discussed. The line shape of the Co2+ signal was similar to that of Co2+ in free corrinoids. The signal of Co2+ could also be evoked by reduction with 5 mM dithiothreitol. From the redox properties of the corrinoid membrane protein it may be expected that in vivo the cobalt may become reduced and reoxidized. Its possible function as an electron-mediating membrane protein in the metabolism of methanogenic bacteria is discussed.