Mutagenesis analysis of the membrane-proximal ligand binding site of the TGF-beta receptor type III extracellular domain

There are two TGF-beta binding subdomains in the extracellular domain of receptor type III (proximal and distal in relation to the transmembrane domain). Here we present an extension of our analysis of the proximal binding site of receptor type III. Due to the original deletion mutagenesis strategy, our proximal binding site contained 19 amino acids from the N-terminal part of the receptor. By deleting these, we demonstrated that they did not contribute to the binding ability of the proximal binding site. We also produced a soluble, secreted form of the proximal binding site and demonstrated that it was able to bind TGF-beta. Finally, we analyzed the role of the three asparagine residues (580, 591, 595) that are located in the region of the receptor that is necessary for expression of a functional proximal binding site, and found that mutation of these residues individually to alanine did not affect ligand binding.     

Pressor response to isometric exercise in patients with multiple sclerosis

The purpose of this study was to determine whether patients with multiple sclerosis (MS) would show attenuated heart rate and/or pressor responses to isometric handgrip exercise. Patients with MS (30 males, 74 females, aged 23-61 yr) and control subjects (9 males, 16 females, aged 25-47 yr) performed isometric handgrip exercise at 30% of maximal voluntary contraction (MVC) to fatigue. Systolic, diastolic, and mean arterial pressure (MAP) increased linearly in both groups, but were significantly lower (P < 0.05) in patients with MS at 20%, 40%, 60%, 80%, and 100% of exercise duration. Mean change in MAP at fatigue was +47.9 mm Hg for controls and +28.2 mm Hg for patients with MS, with 18 patients with MS between -6 mm Hg and +15 mm Hg. Heart rate increased normally in patients with MS. To predict change in MAP at fatigue in patients with MS, stepwise regression analysis using six variables yielded an R2 of 0.26. These data suggest that in some patients MS lesions exist in areas of autonomic cardiovascular control that result in attenuated pressor responses to exercise. In 17% of patients tested, attenuation was profound. Data also suggest an abnormal dissociation between the heart rate and pressor response to static work in patients with MS.     

Reconceptualising comparative education: the case of international studies in mathematics education

International Comparisons in Mathematics Education GABRIELE KAISER, EDUARDO LUNA & IAN HUNTLEY (Eds), 1999 London: Falmer Press. 269 pp., £17.99, ISBN 0 7507 0902 2     

Purification and properties of the chloroplastic form of biotin holocarboxylase synthetase from Arabidopsis thaliana overexpressed in Escherichia coli

Holocarboxylase synthetases (HCSs) are key enzymes in biotin utilisation in both prokaryotes and eukaryotes. In a previous study, we demonstrated that, in plants, HCS activity is localised in cytosol, chloroplasts and mitochondria. We also described the cloning and sequencing of a full-length cDNA encoding an Arabidopsis thaliana HCS isoform with a putative organelle-transit peptide. In the study reported here, this cDNA was used to construct an overproducing Escherichia coli strain. The recombinant enzyme was isolated using an efficient three-step purification procedure. Polyclonal antibodies raised against pure HCS were produced to elucidate the subcellular localisation of this protein. Immunodetection carried out by Western blotting of isolated pea leaf subcellular compartments specifically revealed a single polypeptide that was ascribed to the chloroplast compartment. Immunocytochemistry of thin-cut sections from tobacco leaves, transformed by the complete coding sequence of A. thaliana HCS cDNA via Agrobacterium tumefaciens, confirmed that the enzyme encoded by this cDNA is the chloroplastic isoform. Moreover, physicochemical, biochemical and kinetic properties of the pure recombinant HCS were determined. The native recombinant enzyme is a 37-kDa monomer. In contrast to the major part of HCS activity measured in leaf extracts, the recombinant chloroplastic enzyme did not require addition of Mg2+ to be fully active, but was substantially inhibited by EDTA. This suggested that the chloroplastic HCS may contain a tightly-bound divalent cation required for enzyme activity. The recombinant enzyme was able to biotinylate efficiently apo-biotin carboxyl carrier protein (BCCP) from E. coli and apo-methylcrotonoyl-CoA carboxylase (MCCase) from A. thaliana. Apparent Km values for the enzyme substrates D-biotin, ATP and apo-MCCase were found to be 130 nM, 4.4 microM and 32 microM, respectively. Steady-state kinetic analyses of the HCS-catalysed reaction were investigated with respect to reaction mechanism and inhibition by AMP, one of the end-products of the enzyme-catalysed reaction. Substrate interaction and product inhibition patterns indicated that ATP and D-biotin bind sequentially, in an ordered manner, to the enzyme and that ATP or D-biotin and apo-BCCP bind in ping-pong fashion.     

Reliability of a handgrip test for evaluating heart rate and pressor responses in multiple sclerosis

PURPOSE: The purpose of this study was to determine the test-retest reliability of an isometric handgrip exercise protocol designed to evaluate heart rate and arterial pressure responses in individuals with multiple sclerosis (MS). METHODS Three males and eleven females, aged 24 through 51, performed isometric handgrip contractions at 30% of maximal voluntary contraction (MVC) to the point of fatigue (defined as inability to maintain the target force for three consecutive seconds). During this exercise, rate of perceived exertion (RPE) was recorded every 30 s. Heart rate and beat-to-beat systolic, diastolic, and mean arterial pressures were recorded continuously throughout the duration of exercise. Surface EMG was monitored continuously via loudspeaker to provide feedback on extraneous muscular activity. Each subject performed three trials. A repeated measures ANOVA was used to calculate interclass reliability estimates for each dependent variable. RESULTS: Reliability estimates for MVC and time to fatigue were 0.98 and 0.84, respectively. Reliability estimates for the following dependent variables at the point of fatigue were: RPE, 0.90; delta HR, 0.97; delta systolic pressure, 0.92; delta diastolic pressure, 0.87; and delta MAP, 0.88. CONCLUSIONS: We conclude that this isometric handgrip protocol is a reliable method for evaluating heart rate and blood pressure responses in MS patient.     

Silver-Palladium Thick-Film Conductors

The trends in integrated circuit packaging technology are toward high speed, high density, reliability, and low cost. These demand the improvement of material formulations and processing technology. Among the thick-film materials systems, conductor materials generally represent an important and the most expensive element. Therefore, attention has been centered on the performance of the fired metal film and its cost. Silver and palladium (Ag/Pd) conductors are important components of thick-film paste technology. Thick-film Ag/Pd conductors find applications in many aspects of electronics and electronic packaging, such as hybrid microcircuits, multichip modules, packaging for integrated microcircuits, and in passive electronic components such as multilayer capacitors, varistors, and inductors. In this paper, the performance and properties of fired Ag/Pd films are discussed through their physical and chemical aspects. The final film properties are correlated to a number of factors, including thermodynamics and kinetics of Pd oxidation during burnout and firing; chemical and physical reaction of the Ag/Pd with the ceramic substrate, organic vehicle, and solder; Ag diffusion and migration; inorganic and organic additives; powders characteristics; and paste properties.