Toward true 3D visualization of active catheters using compressed sensing.

A crucial requirement in MR-guided interventions is the visualization of catheter devices in real time. However, true 3D visualization of the full length of catheters has hitherto been impossible given scan time constraints. Compressed sensing (CS) has recently been proposed as a method to accelerate MR imaging of sparse objects. Images acquired with active interventional devices exhibit a high CNR and are inherently sparse, therefore rendering CS ideally suited for accelerating data acquisition. A framework for true visualization of active catheters in 3D is proposed employing CS to gain high undersampling factors making real-time applications feasible. Constraints are introduced taking into account prior knowledge of catheter geometry and catheter motion over time to improve and accelerate image reconstruction. The potential of the method is demonstrated using computer simulations and phantom experiments and in vivo feasibility is demonstrated in a pig experiment.

Dental photography in support of patient documentation and communication.

Intraoral conditions, which in the course of dental treatment are subject to change, can be recorded in detail by means of photography. Pictures provide an improved documentation and the option of monitoring particular situations over longer periods of time. We propose a standardized preoperative 35-mm photographic series. For intraoral photography, special intraoral mirrors and lip-and-cheek retractors are modified as needed. Macro 35-mm cameras with macro flash are by now well-established. Such equipment combined with the technique outlined in this report make standardized photographic documentation possible without mouth-angle retractors and mirror rims obstructing the view.

Early postnatal switch in magnesium sensitivity of NMDA receptors in rat CA1 pyramidal cells.

1. Whole-cell patch-clamp recordings of iontophoretically induced N-methyl-D-aspartate (NMDA) receptor-mediated currents (INMDA) in CA1 pyramidal cells in hippocampal slices from 1- to 40-day-old rats were used to characterize developmental changes in the Mg2+ sensitivity of NMDA receptors. 2. The dose-response relations for extracellular Mg2+ blockade of INMDA indicated a high affinity binding of Mg2+ to NMDA receptors at membrane potentials more negative than -60 mV, independent of postnatal age. 3. Depolarizing the cells unblocked NMDA receptors by decreasing their affinity for Mg2+. The efficacy of depolarization in unblocking NMDA receptors markedly increased after postnatal day 4 (P4), endowing the receptors with a greater voltage dependence. 4. The NR2B subunit-specific NMDA antagonist ifenprodil reduced INMDA in pyramidal cells of all ages. The sensitivity of INMDA to ifenprodil was greatest during the first postnatal week and decreased thereafter, indicating an enhanced contribution of NR2B subunit-containing NMDA receptors to INMDA in the first week after birth. 5. In the first postnatal week, the ifenprodil-insensitive INMDA component had a lower voltage dependence than the total INMDA. In older pyramidal cells, the voltage dependence of the ifenprodil-insensitive component and the total INMDA were similar. 6. In another set of CA1 pyramidal cells, single-cell reverse transcription and polymerase chain reaction (RT-PCR) were used to characterize concomitant developmental changes in NMDA subunit mRNA expression. The mRNA for the NR2D subunit was detected during the first postnatal week in 50 % of the cells and disappeared thereafter. The proportion of cells expressing the NR2A and NR2B subunits remained relatively constant throughout the first five postnatal weeks. 7. We conclude that NMDA receptors in hippocampal CA1 pyramidal cells are effectively blocked by Mg2+ at all ages. After 4 days they become much less sensitive to Mg2+ at depolarized membrane potentials. This postnatal switch in voltage control of Mg2+ binding to NMDA receptors may be due to the downregulation of NR2D subunit expression in developing CA1 pyramidal cells.

Role of region C in regulation of the heat shock gene-specific sigma factor of Escherichia coli, sigma32.

Expression of heat shock genes is controlled in Escherichia coli by the antagonistic action of the sigma32 subunit of RNA polymerase and the DnaK chaperone system, which inactivates sigma32 by stress-dependent association and mediates sigma32 degradation by the FtsH protease. A stretch of 23 residues (R122 to Q144) conserved among sigma32 homologs, termed region C, was proposed to play a role in sigma32 degradation, and peptide analysis identified two potential DnaK binding sites central and peripheral to region C. Region C is thus a prime candidate for mediating stress control of sigma32, a hypothesis that we tested in the present study. A peptide comprising the central DnaK binding site was an excellent substrate for FtsH, while a peptide comprising the peripheral DnaK binding site was a poor substrate. Replacement of a single hydrophobic residue in each DnaK binding site by negatively charged residues (I123D and F137E) strongly decreased the binding of the peptides to DnaK and the degradation by FtsH. However, introduction of these and additional region C alterations into the sigma32 protein did not affect sigma32 degradation in vivo and in vitro or DnaK binding in vitro. These findings do not support a role for region C in sigma32 control by DnaK and FtsH. Instead, the sigma32 mutants had reduced affinities for RNA polymerase and decreased transcriptional activities in vitro and in vivo. Furthermore, cysteines inserted into region C allowed cysteine-specific cross-linking of sigma32 to RNA polymerase. Region C thus confers on sigma32 a competitive advantage over other sigma factors to bind RNA polymerase and thereby contributes to the rapidity of the heat shock response.

Adhesive protocol for the utilization of aluminum oxide crown restorations.

The ultimate objective of anterior tooth restoration is to achieve an aesthetic appearance that is in harmony with the natural dentition. It is often challenging to replicate the optical characteristics (i.e., transparency and translucency) of a natural tooth without utilizing all-ceramic crown restorations, which may provide enhanced aesthetics and biocompatibility in comparison to metal-based treatment. This article demonstrates the use of all-ceramic technology with a contemporary adhesive procedure as a means to achieve improved aesthetic results in the restoration of the anterior dentition.

Cell-type specific expression of ATP-sensitive potassium channels in the rat hippocampus.

1. The distribution of ATP-sensitive K+ channels (KATP channels) was investigated in four cell types in hippocampal slices prepared from 10- to 13-day-old rats: CA1 pyramidal cells, interneurones of stratum radiatum in CA1, complex glial cells of the same area and granule cells of the dentate gyrus. The neuronal cell types were identified visually and characterized by the shapes and patterns of their action potentials and by neurobiotin labelling. 2. The patch-clamp technique was used to study the sensitivity of whole-cell currents to diazoxide (0.3 mM), a KATP channel opener, and to tolbutamide (0.5 mM) or glibenclamide (20 microM), two KATP channel inhibitors. The fraction of cells in which whole-cell currents were activated by diazoxide and inhibited by tolbutamide was 26% of pyramidal cells, 89 % of interneurones, 100% of glial cells and 89% of granule cells. The reversal potential of the diazoxide-induced current was at the K+ equilibrium potential and a similar current activated spontaneously when cells were dialysed with an ATP-free pipette solution. 3. Using the single-cell RT-PCR method, the presence of mRNA encoding KATP channel subunits (Kir6.1, Kir6.2, SUR1 and SUR2) was examined in CA1 pyramidal cells and interneurones. Subunit mRNA combinations that can result in functional KATP channels (Kir6.1 together with SUR1, Kir6.2 together with SUR1 or SUR2) were detected in only 17% of the pyramidal cells. On the other hand, KATP channels may be formed in 75% of the interneurones, mainly by the combination of Kir6.2 with SUR1 (58% of all interneurones). 4. The results of these combined analyses indicate that functional KATP channels are present in principal neurones, interneurones and glial cells of the rat hippocampus, but at highly different densities in the four cell types studied.

Single-cell RT-PCR and functional characterization of Ca2+ channels in motoneurons of the rat facial nucleus.

Voltage-dependent Ca2+ channels are a major pathway for Ca2+ entry in neurons. We have studied the electrophysiological, pharmacological, and molecular properties of voltage-gated Ca2+ channels in motoneurons of the rat facial nucleus in slices of the brainstem. Most facial motoneurons express both low voltage-activated (LVA) and high voltage-activated (HVA) Ca2+ channel currents. The HVA current is composed of a number of pharmacologically separable components, including 30% of N-type and approximately 5% of L-type. Despite the dominating role of P-type Ca2+ channels in transmitter release at facial motoneuron terminals described in previous studies, these channels were not present in the cell body. Remarkably, most of the HVA current was carried through a new type of Ca2+ channel that is resistant to toxin and dihydropyridine block but distinct from the R-type currents described in other neurons. Using reverse transcription followed by PCR amplification (RT-PCR) with a powerful set of primers designed to amplify all HVA subtypes of the alpha1-subunit, we identified a highly heterogeneous expression pattern of Ca2+ channel alpha1-subunit mRNA in individual neurons consistent with the Ca2+ current components found in the cell bodies and axon terminals. We detected mRNA for alpha1A in 86% of neurons, alpha1B in 59%, alpha1C in 18%, alpha1D in 18%, and alpha1E in 59%. Either alpha1A or alpha1B mRNAs (or both) were present in all neurons, together with various other alpha1-subunit mRNAs. The most frequently occurring combination was alpha1A with alpha1B and alpha1E. Taken together, these results demonstrate that the Ca2+ channel pattern found in facial motoneurons is highly distinct from that found in other brainstem motoneurons.

Molecular determinants of NMDA receptor function in GABAergic neurones of rat forebrain.

1. The functional and molecular properties of NMDA receptors (NMDA-Rs) were studied in single, visually identified GABAergic medial septal neurones of the rat forebrain using patch clamp, fluorometric Ca2+ measurements and the single-cell reverse transcription-polymerase chain reaction (RT-PCR) technique. 2. Large neurones close to the mid-line of the medial septal region were shown by the expression of mRNA for a form of glutamate decarboxylase (GAD65) to be almost exclusively GABAergic. A variety of NR2 subunit combinations were detected in the same population of neurones. When tested for NR2A-C, all but one neurone were shown to express mRNA for NR2B. The NR2B subunit mRNA was usually detected together with NR2A or NR2C. mRNA for NR2D was detected in most neurones from a separate batch of cells tested only for this subunit. 3. Single channel measurements in outside-out patches combined with RT-PCR on the same cell showed that NMDA-R channels from these neurones had main single channel conductance levels of 42 pS in 2 mM Ca2+ and 49 pS in 1 mM Ca2+. In addition, a number of other conductance levels were observed, with values in 2 mM Ca2+ of 51, 31, 19 and 13 pS. No clear difference was observed in the pattern of conductance levels displayed by neurones in which different subunit combinations were detected. 4. Whole-cell agonist-induced currents were strongly reduced by the NMDA-R antagonist ifenprodil, at a concentration that mainly affects receptors containing NR2B in recombinant systems. Currents activated by NMDA had a high sensitivity to extracellular Mg2+. 5. The fraction of the total cation current through NMDA-R that was carried by Ca2+, measured using a combination of patch clamp and fluorometry in neurones loaded with a high concentration of the Ca2+ indicator fura-2, was found to be approximately 12%. 6. NMDA-R-mediated excitatory synaptic currents (EPSCs) had similar time courses to those in neurones in other brain regions. The decay kinetics were biexponential, with respective mean values for the fast (tau f) and slow (tau 8) time constants of 79 and 300 ms at -60 mV, and 66 and 284 ms at +40 mV. EPSCs were greatly reduced by ifenprodil (3 microM). 7. In conclusion, NMDA receptors in GABAergic medial septal neurones display a characteristic functional profile. The NR2 subunit mRNA detected and the single channel conductance levels observed suggest that, in addition to NR2B, which is present in nearly all cells, NR2A, NR2C and NR2D are also expressed. However, most of the functional properties of NMDA-Rs in these neurones, including the strong inhibition by ifenprodil and Mg2+, the high fractional Ca2+ current, and the time course of the synaptic currents, are more consistent with those known for NR2B than for the other NR2 subunits. These results suggest that the NR2B subunit dominates over other NR2 subunits in determining the functional properties of NMDA-Rs in these neurones.

Fractional Ca2+ currents through somatic and dendritic glutamate receptor channels of rat hippocampal CA1 pyramidal neurones.

1. The Ca2+ permeability of non-NMDA and NMDA receptor channels was studied using a fluorometric flux measurement approach in somata and dendrites of CA1 pyramidal neurones in rat hippocampal slices. For this purpose, the Ca2+ fraction of the total cation current (named 'fractional Ca2+ current') was measured directly from the change in the Ca(2+)-sensitive fura-2 fluorescence at 380 nm excitation wavelength. 2. The fractional Ca2+ current through the somatic NMDA receptor channels was 10.69 +/- 2.13% (mean +/- S.D.) and that through dendritic receptor channels was 10.70 +/- 1.96%. The fractional Ca2+ current was not dependent on the extracellular Mg2+ concentration and its voltage dependence was in agreement with the Goldman-Hodgkin-Katz current equation. 3. AMPA (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate) or kainate applications produced small but significant Ca2+ entry. Fractional Ca2+ currents of 0.58 +/- 0.34% were measured for somatic AMPA applications, 0.68 +/- 0.20% for somatic kainate applications, 0.66 +/- 0.25% for dendritic AMPA applications and 0.61 +/- 0.16% for dendritic kainate applications. 4. The expression pattern of glutamate receptor subunits encoding messenger ribonucleic acids (mRNAs) was analysed with the single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) approach applied to CA1 pyramidal neurones. The AMPA receptor subunits GluR-A, GluR-B and GluR-C, and the NMDA receptor subunits NR2A and NR2B were found to be abundantly expressed in all CA1 pyramidal neurones tested. 5. This study establishes the fractional Ca2+ current through somatic and dendritic NMDA and non-NMDA receptor channels in CA1 pyramidal neurones. The dendritic, presumably synaptic, NMDA receptor channels are highly Ca2+ permeable and have a fractional Ca2+ current closely resembling that of somatic extrasynaptic NMDA receptor channels. Both somatic and dendritic non-NMDA receptor channels are of the 'low Ca2+ permeable' type and have a fractional Ca2+ current that is about twenty times smaller than that of NMDA receptor channels.

A cycle of binding and release of the DnaK, DnaJ and GrpE chaperones regulates activity of the Escherichia coli heat shock transcription factor sigma32.

The chaperone system formed by DnaK, DnaJ and GrpE mediates stress-dependent negative modulation of the Escherichia coli heat shock response, probably through association with the heat shock promoter-specific sigma32 subunit of RNA polymerase. Interactions of the DnaK system with sigma32 were analysed. DnaJ and DnaK bind free, but not RNA polymerase-bound, sigma32 with dissociation constants of 20 nM and 5 muM respectively. Association and dissociation rates of DnaJ-sigma32 complexes are 5900- and 20-fold higher respectively than those of DnaK-sigma32 complexes in the absence of ATP. ATP destabilizes DnaK-sigma32 interactions. DnaJ, through rapid association with sigma32 and stimulation of hydrolysis of DnaK-bound ATP, mediates efficient binding of DnaK to sigma32 in the presence of ATP, resulting in DnaK-DnaJ-sigma32 complexes containing ADP. GrpE binding to these complexes stimulates nucleotide release and subsequent complex dissociation by ATP. We propose that the principles of this cycle also operate in other chaperone activities of the DnaK system. DnaK and DnaJ cooperatively inhibit sigma32 activity in heat shock gene transcription and GrpE partially reverses this inhibition. These data indicate that reversible inhibition of sigma32 activity through transient association of DnaK and DnaJ is a central regulatory element of the heat shock response.

Fractional calcium current through neuronal AMPA-receptor channels with a low calcium permeability.

The Ca(2+)-permeation properties of AMPA-receptor (AMPA-R) channels in Purkinje neurons in rat cerebellar slices were studied using a combination of whole-cell patch-clamp recordings, Fura-2 fluorometry, and single-cell reverse-transcription (RT)-PCR. Several lines of evidence indicate that Purkinje neurons, at both early and late stages of postnatal development, express exclusively AMPA-R channels with a low Ca2+ permeability. First, no Ca2+ signal was detected during application of either AMPA or kainate to Purkinje neurons loaded with the Ca2+ indicator Fura-2 AM. In contrast, kainate application induced large Ca2+ transients in Bergmann glia cells. Second, in ion substitution experiments, when Ca2+ is the only extracellular permeant cation, the reversal potential corresponds to that expected for AMPA-R channels with a low permeability for Ca2+. Third, using a fluorometric flux-measurement approach (Schneggenburger et al., 1993a), we found that the Ca2+ fraction of the total cation current through AMPA-R channels is approximately 0.6%. This value is approximately sixfold lower than that found for recombinant AMPA-R lacking the AMPA-R subunit GluR2. Furthermore, single-cell RT-PCR experiments revealed the presence of the AMPA-R subunits GluR1, GluR2, and GluR3 in Purkinje neurons in cerebellar slices at developmental stages corresponding to those studied electrophysiologically. The expression of GluR2 in all cells tested (n = 14) is consistent with the subunit composition predicted from studies of recombinant AMPA-R channels with a low permeability for Ca2+ (Burnashev et al., 1992b). In conclusion, this study establishes that cerebellar Purkinje neurons at all postnatal developmental stages possess AMPA-R channels with a low permeability for Ca2+.