Pig heart fumarase contains two distinct substrate-binding sites differing in affinity

A eukaryotic fumarase is for the first time unequivocally shown to contain two distinct substrate-binding sites. Pig heart fumarase is a tetrameric enzyme consisting of four identical subunits of 50 kDa each. Besides the true substrates L-malate and fumarate, the active sites (sites A) also bind their analogs D-malate and oxaloacetate, as well as the competitive inhibitor glycine. The additional binding sites (sites B) on the other hand also bind the substrates and their analogs D-malate and oxaloacetate, as well as L-aspartate which is not an inhibitor. Depending on the pH, the affinity of sites B for ligands (Kd being in the millimolar range) is 1-2 orders of magnitude lower than the affinity of sites A (of which Kd is in the micromolar range). However, saturating sites B results in an increase in the overall activity of the enzyme. The benzenetetracarboxyl compound pyromellitic acid displays very special properties. One molecule of this ligand is indeed able to bind into a site A and a site B at the same time. Four molecules of pyromellitic acid were found to bind per molecule fumarase, and the affinity of the enzyme for this ligand is very high (Kd = 0.6 to 2.2 microM, depending on the pH). Experiments with this ligand turned out to be crucial in order to explain the results obtained. An essential tyrosine residue is found to be located in site A, whereas an essential methionine residue resides in or near site B. Upon limited proteolysis, a peptide of about 4 kDa is initially removed, probably at the C-terminal side; this degradation results in inactivation of the enzyme. Small local conformational changes in the enzyme are picked up by circular dichroism measurements in the near-UV region. This spectrum is built up of two tryptophanyl triplets, the first one of which is modified upon saturating the active sites (A), and the second one upon saturating the low affinity binding sites (B).     

Imaging birds in a bird cage: in-vivo FSE 3D MRI of bird brain

An in-vivo magnetic resonance imaging (MRI) procedure is described that allows one to obtain three-dimensional high quality images of the entire brain of small birds such as the canary (20 g) and the starling (75 g) with an image resolution of 0.1 mm (58-113 μm, dependent on the size of the imaged bird). The entire imaging procedure took about 2 h after which the birds recovered from anaesthesia uneventfully and could be reused for subsequent additional imaging. This non invasive MRI technique enables to correlate brain measures with behavioural or physiological data that are dynamic in nature and could permit significant progress for bird neurological research. © 1998 Elsevier Science B.V. All rights reserved.     

Air-Bubble Method of Locking Central-Vein Catheters for Prevention of Hub Colonization: A Pilot Study

Background: The major source of catheter‐associated bacteremia is contamination of the catheter hub during connection–disconnection procedures. A new method of catheter locking has been developed wherein anticoagulant is injected first, followed by a 0.1‐mL air bubble and 0.9 mL of bactericidal solution. The anticoagulant is then located at the catheter tip and the bactericidal solution is located at the catheter hub. The air bubble prevents mixing of the two solutions. The bactericidal solution was acidified concentrated saline (ACS). The 27% saline solution has a pH of 2.0. ACS was chosen because it is theoretically harmless if injected in the amount used to lock the catheter lumens. The goals of this pilot study were to determine whether the new method of catheter locking is easy to perform with available syringes and whether eventual injection of the experimental solution is well tolerated. Methods: Ten patients were randomly assigned, either to heparin lock (5 patients, 62 treatments) or air‐bubble method (5 patients, 56 treatments). In the control group, the catheters were locked with heparin, 5000 U/mL. In the experimental group, the catheters were locked with heparin, air bubble, and ACS. Altogether, the lumens were overfilled by 0.2 mL. Results: Compared to the routine method, the experimental method required a 1‐ to 2‐min‐longer procedure time. There were no errors in proper sequence of injections into the lumina. There were no episodes of bacteremia related to hub contamination in either group. In the air‐bubble group, there was one case of bacteremia associated with purulent drainage from the exit and the same organism in both cultures. In three instances in each group, the locking solution could not be aspirated and was injected without any subjective symptoms or objective signs. Conclusion: We conclude that the air‐bubble method of locking central‐vein catheters is easy to perform. In three instances of air‐bubble and ACS injection, there were no adverse effects. A full‐scale prospective randomized study is feasible and warranted.     

Spectrally pure excitation signals: only a dream?

For accurate testing of a nonlinear device-under-test (DUT), the spectral purity of the input signal is utterly important. This paper describes a method to create very pure signals, such as sine waves, dual-tones, or multitones, using only power measurements. The signals are generated with an arbitrary waveform generator. Any unwanted spectral line is removed, independent of its exact origin. This allows to apply the correct signal to the DUT, even if the signal source is not perfect or is followed by filters or amplifiers with a small nonlinear distortion. Measurements are used to demonstrate the capabilities of the method.     

A model for the transverse strain response of unidirectional ceramic matrix composites during tensile testing

A comprehensive micromechanical model relating the longitudinal stress and transverse strain of unidirectional fibre toughened ceramic matrix composites (CMCs) is presented. The model uses different cylindrical unit-cells to describe the composite throughout a tensile test and considers all relevant damage mechanisms. The proposed model takes into account the Poisson contraction of fibre and matrix, the relief of thermal residual stresses upon damage development, and the build-up of compressive radial stresses at the interface due to mismatch between fibre and matrix after debonding and sliding. Thus the modelled transverse strain response depends on a wide range of microstructural and micromechanical parameters. The approach is checked by comparing the experimentally observed and simulated response of a unidirectional SiC/CAS composite of which all constituent properties were determined experimentally. The agreement between experiment and theory is excellent.     

Inhibition of N-methyl-D-aspartate glutamate receptor subunit expression by antisense oligonucleotides reveals their role in striatal motor regulation

N-methyl-D-aspartate (NMDA) glutamate receptors have an established role in the regulation of motor behavior by the basal ganglia. Recent studies have revealed that NMDA receptors are heteromeric assemblies of structurally related subunits from two families: NMDAR1, which is required for channel activity, and NMDAR2A-D, which modulate the properties of the channels. In the rat, the NMDA receptor subunits exhibit anatomically restricted patterns of expression, so that each component of the basal ganglia has a distinct NMDA receptor subunit mRNA phenotype. We have used in vivo intrastriatal injection of synthetic antisense oligodeoxynucleotides (ODNs) to examine the roles of particular NMDA receptor subunits in the regulation of motor behavior in rats. Injection of 15 nmol of a 20-mer ODN targeted to the NMDAR1 subunit induced spontaneous ipsilateral rotation. Smaller doses of NMDAR1 antisense ODN did not lead to spontaneous rotation, but prominent ipsilateral rotation was observed after systemic administration of D-amphetamine. An antisense ODN to NMDAR2A was also effective in eliciting amphetamine-inducible rotation, although the magnitude of the effect was less than that seen with NMDAR1, whereas ODNs targeted to NMDAR2B, NMDAR2C and an NMDAR1 sense strand ODN had no effect on behavior. In situ hybridization demonstrated that injection of the NMDAR1, NMDAR2A or NMDAR2B antisense ODNs produced specific reductions in target mRNA signal intensity in the injected striatum. After NMDAR1 antisense ODN injection, striatal binding of 3H-glutamate target mRNA signal intensity in the injected striatum. After NMDAR1 antisense ODN injection, striatal binding of 3H-glutamate to NMDA sites was not altered, although strychnine-insensitive 3H-glycine binding sites exhibited a small but significant reduction. These observations suggest that NMDA receptor complexes containing NMDAR1 and, to a lesser extent, NMDAR2A subunits play particularly important roles in the regulation of motor behavior by neostriatal neurons.     

An inequality for the empirical D.F. in the non–I.I.D. case

A generalization to the non-i.i.d. case of an inequality for the empirical df. due to Mason (1981) is proved