Scanning electrochemical microscopy. 44. Imaging of horseradish peroxidase immobilized on insulating substrates

Scanning electrochemical microscopy (SECM) was used to study horseradish peroxidase (HRP) immobilized with copolymer on insulating substrates (glass slide or polycarbonate membrane filter). Two methods were used to immobilize HRP: In the first, HRP was coimmobilized by cross-linking on a glass slide with a copolymer swelled in water to form a hydrogel; in the second, the same copolymer and avidin were coimmobilized on the glass slide and biotin-labeled HRP was conjugated to the avidin of the film. SECM was then used to detect the presence of the bound enzyme by observing the feedback current in a solution of benzoquinone and hydrogen peroxide, when hydroquinone was generated at the tip. A detection limit less than 7 x 10(5) HRP molecules within a approximately 7-microm-diameter area was demonstrated.     

Quantification of haemodynamic response to auditory stimulus in intensive care

Measuring effects of sensory stimuli on haemodynamics could provide information about the interplay between central and autonomous nervous system (ANS). However, ANS response to sensory stimulus has received little attention. In this paper we present a signal processing scheme to extract the responses of heart rate and systemic arterial pressure on auditory stimulus in intensive care patients (N=5). In short, the effect of mechanical ventilation is rejected by optimal linear modelling. Other disturbances are attenuated by filtering and efficient rejection of outlying sweeps of data. The results show identifiable responses on three out of five cases. The response characteristics may be explained by synchronisation of spontaneous variability in systemic arterial pressure to auditory stimulus.     

Solid-state NMR studies of the prion protein H1 fragment

Conformational changes in the prion protein (PrP) seem to be responsible for prion diseases. We have used conformation-dependent chemical-shift measurements and rotational-resonance distance measurements to analyze the conformation of solid-state peptides lacking long-range order, corresponding to a region of PrP designated H1. This region is predicted to undergo a transformation of secondary structure in generating the infectious form of the protein. Solid-state NMR spectra of specifically 13C-enriched samples of H1, residues 109-122 (MKHMAGAAAAGAVV) of Syrian hamster PrP, have been acquired under cross-polarization and magic-angle spinning conditions. Samples lyophilized from 50% acetonitrile/50% water show chemical shifts characteristic of a beta-sheet conformation in the region corresponding to residues 112-121, whereas samples lyophilized from hexafluoroisopropanol display shifts indicative of alpha-helical secondary structure in the region corresponding to residues 113-117. Complete conversion to the helical conformation was not observed and conversion from alpha-helix back to beta-sheet, as inferred from the solid-state NMR spectra, occurred when samples were exposed to water. Rotational-resonance experiments were performed on seven doubly 13C-labeled H1 samples dried from water. Measured distances suggest that the peptide is in an extended, possibly beta-strand, conformation. These results are consistent with the experimental observation that PrP can exist in different conformational states and with structural predictions based on biological data and theoretical modeling that suggest that H1 may play a key role in the conformational transition involved in the development of prion diseases.     

Selenium concentration and activity of glutathione peroxidase in lysate of human erythrocytes

The concentration of selenium in human red cells was measured by a fluorometric determination method after wet digestion of the biological material. In the red cells from the same blood samples the activity of the selenoenzyem glutathione peroxidase (EC 1.11.1.9) was determined with tertiary butyl hydroperoxide as acceptor substrate. The results show that only 10% of the total selenium content of human red cells is fixed to the enzyme. No correlation between the enzyme activity and the selenium concentration could be found. On the basis of these results can be presumed that the protective effect of selenium in glutathione peroxidase against oxidant damage of cells is not the only biological function of this element.     

Altered electrodermal responsivity associated with clinical pain

We used certain physiologic maneuvers to perturb the autonomic nervous system (ANS) in an attempt to detect a link between the ANS and pain. In the unperturbed state, we found no difference in the electrodermal response among normal controls, preoperative patients (increased stress without pain) and postoperative patients (increased stress and pain). The electrodermal response elicited by autonomic maneuvers was significantly attenuated in postoperative patients but not in preoperative patients or in normal control subjects.