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PubMed1999

Slug is a mediator of epithelial-mesenchymal cell transformation in the developing chicken heart.

An epithelial-mesenchymal cell transformation occurs during the development of the endocardial cushions in the atrioventricular (AV) canal of the heart. We hypothesized that the transcription factor Slug is required for this epithelial-mesenchymal cell transformation since Slug is required for similar transformations during gastrulation and neural crest differentiation in chicken embryos. We found by RT-PCR and immunostaining that the temporal and spatial localization of Slug in the embryonic chicken heart is consistent with a role for Slug in endocardial cushion formation. Moreover, we found that Slug expression by AV canal endothelial cells is induced by a signal provided by AV canal myocardium. Slug appears to be required for epithelial-mesenchymal cell transformation in the chicken heart since treatment of AV canal explants with antisense Slug oligodeoxynucleotides inhibited mesenchymal cell formation in vitro. Antisense Slug oligodeoxynucleotides prevented endothelial cell-cell separation, suggesting that Slug acts early in the transformation pathway.

Animals↗
PubMed2002

It takes guts: the Drosophila hindgut as a model system for organogenesis.

The Drosophila hindgut is fruitful territory for investigation of events common to many types of organogenesis. The development of the Drosophila hindgut provides, in microcosm, a genetic model system for studying processes such as establishment (patterning) of an epithelial primordium, its internalization by gastrulation, development of left--right asymmetric looping, patterning in both the anteroposterior and dorsoventral axes, innervation, investment of an epithelium with mesoderm, reciprocal epitheliomesenchymal interactions, cell shape change, and cell rearrangement. We review the genetic control of these processes during development of the Drosophila hindgut, and compare these to related processes in other bilaterians, particularly vertebrates. We propose that caudal/Cdx, brachyenteron/Brachyury, fork head/HNF-3, and wingless/Wnt constitute a conserved "cassette" of genes expressed in the blastopore and later in the gut, involved in posterior patterning, cell rearrangement, and gut maintenance. Elongation of the internalized Drosophila hindgut primordium is similar to elongation of the archenteron and also of the entire embryonic axis (both during and after gastrulation), as well as of various tubules (e.g., nephric ducts, Malpighian tubules), as it is driven by cell rearrangement. The genes drumstick, bowl, and lines (which encode putative transcriptional regulators) are required for this cell rearrangement, as well as for spatially localized gene expression required to establish the three morphologically distinct subregions of the hindgut. Expression of signaling molecules regulated by drumstick, bowl, and lines, in particular of the JAK/STAT activator Unpaired at the hindgut anterior, may play a role in controlling hindgut cell rearrangement. Other cell signaling molecules expressed in the hindgut epithelium are required to establish its normal size (Dpp and Hh), and to establish and maintain the hindgut visceral mesoderm (Wg and Hh). Both maternal gene activity and zygotic gene activity are required for asymmetric left--right looping of the hindgut. Some of the same genes (caudal and brachyenteron) required for embryonic hindgut development also act during pupation to construct a new hindgut from imaginal cells. Application of the plethora of genetic techniques available in Drosophila, including forward genetic screens, should identify additional genes controlling hindgut development and thus shed light on a variety of common morphogenetic processes.

Animals↗
PubMed1997

Regulation of cellular tyrosine phosphorylation by stimulatory and inhibitory muscarinic acetylcholine receptors.

Tyrosine phosphorylation is a key signaling event in transmembrane and cytoplasmic signal transduction. The m5 muscarinic receptor (m5AChR) responds to ligand stimulation with calcium influx and protein phosphorylation. In contrast, neither of these responses has been associated with m4AChR signaling. We hypothesized that activation of the m5AChR would alter tyrosine phosphorylation patterns spatially within the cell and in a calcium influx-sensitive manner. CHO cells stably transfected with m4- or m5AChRs were assessed for spatial localization and quantity of phosphotyrosylated proteins in response to receptor activation. Results were confirmed by immunoblot of whole cell lysates and cytosol and membrane fractions. m5AChR activation increased tyrosine phosphorylation in all subcellular compartments; coincubation with CAI, a calcium influx inhibitor, reduced phosphorylation below basal levels. Western blot confirmed the change of phosphotyrosylated proteins of M(r) 70, 85, 120, and 180 kDa in whole and fractionated cells. PLC-gamma, used as a marker of m5AChR activity, was increased in quantity and degree of phosphorylation in CHOm5 cell membranes and microvilli in response to receptor activation. Both the quantitative increase and tyrosine phosphorylation of PLC-gamma in membrane fractions was inhibited by CAI. In contrast, CC treatment of CHOm4 cells reduced tyrosine phosphorylation throughout the cell. CC-stimulation of m5AChR cells caused a calcium influx-sensitive increase in phosphotyrosylated proteins throughout the cell, though predominantly in the membrane and microvilli. Activation of the m5AChR induces tyrosine phosphorylation, whereas activation of the m4AChR inhibited tyrosine phosphorylation below baseline, further demonstrating the dichotomy between signaling of these two AChRs.

Animals↗
PubMed1994

Enzyme-linked fluorescent detection for automated multiplex DNA sequencing.

Initiatives to sequence DNA on a large scale have created a need for increased throughput and decreased costs. One scheme for increasing throughput, multiplex sequencing, involves the processing of a mixture of sequencing templates followed by sequential hybridization to reveal the individual sequence ladders on a membrane. Because multiplex sequencing has not been fully automated, and has not seemed automatable, few sequencing efforts have attempted to exploit it. We describe here a scheme for the automation of multiplex sequencing. Probe hybridized to target DNA is detected via spatially localized enzyme-linked fluorescence. Light output is high enough that imaging is possible with simple instrumentation. Direct imaging within an automated hybridization apparatus is made feasible so that the entire process will be automatic once a multiplex membrane is produced. The technique has the potential to increase severalfold the throughput of automated sequencing instruments required for sequencing the human genome.

Base Sequence↗
PubMed1994

Multiple-quantum filters of spin-3/2 with pulses of arbitrary flip angle.

The influence of inhomogeneous RF fields on the double- and triple-quantum filtering of spin-3/2 nuclei in the presence of biexponential relaxation is analyzed. In this analysis, spherical tensor operators have been used for density-matrix calculations. In the presence of inhomogeneous RF fields, it is shown that the three-pulse triple-quantum filter (TQF) without a refocusing 2 theta pulse (with delta omega = 0) is on average about 100% more sensitive than the corresponding double-quantum filter (DQF), and in the case of four-pulse DQF and TQF with a refocusing 2 theta pulse (with delta omega not equal to 0), two relaxation coefficients f(1)11(tau) and f(1)33(tau) also contribute to the observed DQ and TQ coherences. It is also shown that the three-pulse filters are more sensitive than the corresponding four-pulse filters. In both three- and four-pulse cases, when used with surface coils, these filters act as depth pulses and thus yield spatial localization. The experimental results obtained with homogeneous RF coils are in excellent agreement with the theoretical results.

Equipment Design↗
PubMed1996

Measuring Diffusion in the Presence of Material Strain

Material strain during the course of diffusion encoding by MRI will in general change the observed diffusional signal losses. These changes will occur even when the material returns cyclically to its initial location during the diffusion-evolution period. This effect derives from the modification of the local spatial modulation k of spin phase within a sample by a material deformation \font\serif=cmss10 at 10pt $\hbox{\serif F}$ as k --> $\hbox{\serif F}^{\rm 1T}$ k, resulting in an observed diffusion tensor $\hbox{\serif D}^{\rm obs} = {{1}\over{\Delta}} \int^{\Delta}_0 \hbox{\serif U}(t)^{-1} \hbox{\serif DU}(t)^{-1}dt,$ where $\hbox{\serif{U}}$ is the material stretch tensor. For example, when a material is compressed during pulsed-gradient diffusion encoding, the compression acts to increase the attenuation due to diffusion just as if a larger gradient were used. By using a simple gelatin phantom, the existence of this effect is demonstrated, and an effective method for its correction based on an MRI mapping of the material strain is presented. This correction is particularly relevant for inferring myofiber structure in the beating in vivo human heart, since the measurement of $\hbox{\serif{D}}$ is perturbed by the deformation of myocardium during the heart's contraction.

Journal Article↗
PubMed1996

Observing N-acetyl aspartate via both its N-acetyl and its strongly coupled aspartate groups in in vivo proton magnetic resonance spectroscopy.

The approximately 2.6 ppm aspartate multiplet of N-acetyl aspartate (NAA) is considered a potential source of additional information on N-acetyl aspartate in vivo. Because the aspartate multiplet is the AB part of a strongly coupled ABX system it gives rise, as is shown in the analysis presented, to a significant field-strength dependence in the echo-time-dependent modulations of the response to typical spatial-localization sequences. The echo-time dependence of this response is developed analytically, not only for the STEAM and the PRESS localization sequences, but also for a spin-echo sequence. It is then verified experimentally at 2.35 T. The field-strength dependence of the response is demonstrated by evaluating the changes in the echo-time-dependent responses to each of the three sequences at field strengths of 1.5, 2.35, and 4.0 T. By means of these results, the preferred sequence (PRESS) can be optimized for the NAA aspartate multiplet at each field strength, as is illustrated with the human brain spectra obtained in vivo at 1.5 T. These in vivo spectra compare the optimal, long TE timing (163 ms) with a suboptimal TE (70 ms), for the observation of the approximately 2.6 ppm aspartate resonances of NAA.

Aspartic Acid↗
PubMed1998

In vivo multiple spin echoes.

The demagnetizing field produced by the nuclear polarization can induce refocusing of multiple spin echoes. We show that multiple spin echoes can be observed in vivo with a clinical MR system at 1.5 T. Strategies for the spatial localization of the multiple spin echo signals are considered. Multiple spin echo studies in brain white matter and skeletal muscle in healthy volunteers are reported. The dependence of the signal amplitudes on the experimental parameters is compared with the theory. The sources of contrast for MRI and the perspectives for medical applications are discussed.

Brain↗
PubMed1999

Spectral simulations incorporating gradient coherence selection.

Computer-aided methods can considerably simplify the use of the product operator formalism for theoretical analysis of NMR phenomena, which otherwise becomes unwieldy for anything but simple spin systems and pulse sequences. In this report, two previously available programming approaches using symbolic algebra (J. Shriver, Concepts Magn. Reson. 4, 1-33, 1992) and numerical simulation using object-oriented programming (S. A. Smith, T. O. Levante, B. H. Meier, and R. R. Ernst, J. Magn. Reson. A 106, 75-105, 1994) have been extended to include the use of gradient operators for simulation of spatially localized NMR spectroscopy and gradient coherence selection. These methods are demonstrated using an analysis of the response of an AX(3) spin system to the STEAM pulse sequence and verified with experimental measurements on lactate.

Computer Simulation↗
PubMed2001

Improving resolution in fast rotating-frame experiments.

The rapid rotating-frame technique allows significant reduction in data-acquisition time compared with the two-dimensional method by stroboscopic observation of the nuclear magnetization during its evolution in the rotating frame. A onefold reduction in the dimensionality of the original rotating-frame experiment is achieved by using a train of strong radiofrequency pulses separated by short acquisition windows. The penalty for shortening experimental time is a reduction in spectral resolution compared with the two-dimensional method due to relaxation of transverse magnetization components during the observation windows. A variant of the rapid-rotating frame technique for improving spectral resolution based on undersampling and self-phase encoding is presented. An M-fold resolution improvement requires M experiments, thus, making possible a tradeoff between spectral resolution and experimental time. The technique was applied for spatial localization of quadrupole nuclei in powder solids, and resolution improvement is demonstrated on one- and two-dimensional NQR images.

Journal Article↗
PubMed2001

Detection of gamma-aminobutyric acid (GABA) by longitudinal scalar order difference editing.

Two novel spectral editing techniques for the in vivo detection of gamma-aminobutyric acid (GABA) are presented. The techniques rely on the generation of longitudinal scalar order (LSO) coherences, which in combination with J-difference editing results in the selective detection of GABA. The utilization of LSO coherences makes the editing sequences insensitive to phase and frequency instabilities. Furthermore, the spectral editing selectivity can be increased independent of the echo time, thereby opening the echo time for state-of-the-art water suppression and/or spatial localization techniques. The performance of the LSO editing techniques is theoretically demonstrated with product operator calculations and density matrix simulations and experimentally evaluated on phantoms in vitro and on human brain in vivo.

Brain↗
PubMed2002

Sample restriction using radiofrequency field selective pulses in high-resolution solid-state NMR.

In this article a method is suggested for restricting a sample (spatial localization) by preparing the magnetization with a phase-modulated radiofrequency pulse which inverts magnetization only over a very narrow range of radiofrequency field strengths. This is the most efficient method, in terms of sensitivity, of restricting the sample to improve rf homogeneity. The method is demonstrated by using it to improve the resolution obtained in a homonuclear dipolar decoupling experiment.

Magnetic Resonance Spectroscopy↗
PubMed2002

A rotational approach to localized SPAMM 1-1 tagging.

Magnetic resonance tagging usually relies on controlling the phase dispersion of the transverse magnetization component. Phase dispersion is, however, affected by the inherent phase of selective excitation pulses, thus limiting their combination with tagging sequences to the application of refocusable pulses, as in the localized spatial modulation of magnetization (L-SPAMM) technique. In this study, we examine the effect of selective excitation pulses on a L-SPAMM 1-1 sequence, showing that in the case of two identical pulses the phase component is canceled out, and thus preemphasis and refocus gradients are not needed, allowing us to take advantage of a constant gradient throughout the tagging sequence, and also that one might choose nonrefocusable maximum and minimum phase pulses.

Journal Article↗
PubMed1998

Model studies of the role of mechano-sensitive currents in the generation of cardiac arrhythmias.

Mechano-electrical feedback is studied by incorporating linear, instantaneously activating mechano-sensitive conductances into single cardiac cell models, as well as one- and two-dimensional cardiac network models. The models qualitatively reproduce effects of maintained mechanical stretch on experimentally measured action potential characteristics such as amplitude, maximum diastolic potential, peak upstroke velocity, and conduction velocity. Models are also used to simulate stretch-induced depolarizations, action potentials, and arrhythmias produced by pulsatile volume changes in left ventricle of dog. The mechano-sensitive conductance threshold for a stretch-induced action potential is closely related to the magnitude of the time-independent K+ current, IK1, which offsets inward mechano-sensitive current. Activation of mechano-sensitive conductances in small, spatially localized region of cells can evoke graded depolarizations, propagating ectopic beats, and if timed appropriately, spiral reentrant waves. Mechano-sensitive conductance changes required to evoke these responses are well within the physiologically plausible range. Results therefore indicate that many mechano-electrical feedback effects can be modeled using linear, instantaneously activating mechano-sensitive conductances. As an example of how stretch can occur in real human hearts, magnetic resonance images with saturation tagging are used to reconstruct the three-dimensional left ventricular wall motion. In patients with infarcts or recent ischemic events, "paradoxical deformation" is observed in that regions of myocardium are stretched rather than contracted during systole. In contrast, normal hearts contract uniformly with no stretch during systole. Paradoxical deformations in ischemic hearts may therefore present one possible substrate for the mechanically induced arrhythmias modeled above.

Action Potentials↗
PubMed2001

Assessment of sensorimotor and cognitive deficits induced by a moderate traumatic injury in the right parietal cortex of the rat.

The purpose of this study was to set-up a battery of behavioral tests to assess sensorimotor and cognitive deficits following a moderate traumatic brain injury (TBI) in rats. Coordinated walking ability was evaluated in an accelerated rotarod test. Vestibulomotor function and fine motor coordination were assessed by using a beam-walking task. Rotarod and beam-walking performances were both altered in injured rats compared to sham-operated and control rats. A more pronounced and longer-lasting deficit was measured in the beam-walking test. Cognitive function was studied by using the Lashley maze paradigm. A spatial localization deficit was significant for 4 weeks posttrauma in TBI rats. The beam-walking task and the Lashley maze are robust and sensitive methods in detecting sensorimotor and cognitive impairment after TBI in rats, respectively. These tests are proposed for evaluating the ability of new pharmacological agents to improve the functional recovery after a TBI in rats.

Animals↗
PubMed1998

Event-related brain potential imaging of semantic encoding during processing single words.

Functional brain imaging studies with positron emission tomography (PET) have identified blood flow changes in widely separated areas of brain during the performance of word processing tasks. In the present study we have utilized event-related brain potentials (ERPs) to investigate the temporal relationships among cortical areas previously identified by PET to be differentially activated when performing semantic tasks with visual words. ERPs revealed task-related differences over the central and left inferior frontal regions around 170 and 220 ms, respectively, over a left occipital region around 200 ms, over a large left parietotemporal region around 600 ms, and finally over the right temporal lobe around 800 ms after the word presentation. Analysis of topographic maps and dipole sources as well as PET data allowed relating frontal midline positivity around 170 ms to the anterior cingulate activation, and left inferior frontal positivity around 220 ms to the PET activation of the left inferior prefrontal cortex. The left parieto-temporal positivity around 600 ms seems to reflect the activity of Wernicke's area. The right anterior temporal negativity beginning around 800 ms and peaking around 1100 ms may reflect the activity of the right insula. The left occipital negativity around 200 ms is likely to reflect activation of a visual word-form area in the left occipital lobe. These results provide the time course for parts of the circuitry involved in semantic processing of words and also demonstrate how combining the spatial localization of PET with the temporal resolution of ERPs helps to understand the brain mechanisms involved in human cognition.

Adult↗
PubMed2002

An investigation of the value of spin-echo-based fMRI using a Stroop color-word matching task and EPI at 3 T.

This study examines the value of spin-echo-based fMRI for cognitive studies at the main magnetic field strength of 3 T using a spin-echo EPI (SE-EPI) sequence and a Stroop color-word matching task. SE-EPI has the potential advantage over conventional gradient-echo EPI (GE-EPI) that signal losses caused by dephasing through the slice are not present, and hence although image distortion will be the same as for an equivalent GE-EPI sequence, signal voids will be eliminated. The functional contrast in SE-EPI will be lower than for GE-EPI, as static dephasing effects do not contribute. As an auxiliary experiment interleaved diffusion-weighted and non-diffusion-weighted SE-EPI was performed in the visual cortex to further elucidate the mechanims of functional contrast. In the Stroop experiment activation was detected in all areas previously found using GE-EPI. Additional frontopolar and ventral frontomedian activations were also found, which could not be detected using GE-EPI. The experiments from visual cortex indicated that at 3 T the BOLD signal change has contributions from the extravascular space and larger blood vessels in roughly equal amounts. In comparison with GE-EPI the absence of static dephasing effects would seem to result in a superior intrinsic spatial resolution. In conclusion the sensitivity of SE-EPI at 3 T is sufficient to make it the method of choice for fMR studies that require a high degree of spatial localization or where the requirement is to detect activation in regions affected by strong susceptibility gradients.

Adult↗
PubMed2002

A fully automated method for tissue segmentation and CSF-correction of proton MRSI metabolites corroborates abnormal hippocampal NAA in schizophrenia.

In this report, we describe the implementation and application of a fully automated segmentation routine using SPM99 algorithms and MATLAB for clinical Magnetic Resonance Spectroscopic Imaging (MRSI) studies. By segmenting high-resolution 3-D image data and coregistering the results to the spatial localizer slices of a spectroscopy examination, the program offers the possibility to easily calculate segmentation maps for a large variety of MRSI experiments. The segmented data are corrected for the individual point-spread function, slice and VOI profiles for measurement sequences with selective pulses as well as for the chemical shifts of different metabolites. The new method was applied to investigate discrete hippocampal metabolite abnormalities in a small sample of schizophrenic patients in comparison to healthy controls (15 patients, 15 controls). Only after correction was the N-acetyl-aspartate (NAA) signal significantly lower in patients compared to controls. No differences were found for the corrected signals from the creatine/phosphocreatine (Cr) or choline-containing compounds (Ch). These results are in good agreement with neuropathological and previous MR spectroscopy studies of the hippocampus in schizophrenic patients.

Adult↗