Abstract: Effect of a hydrophobic peptide on folding of oxidized cytochrome c (cyt c) is studied with trityrosine. Folding of cyt c was initiated by pH jump from 2.3 (acid-unfolded) to 4.2 (folded). The Soret band of the 2-ms transient absorption spectrum during folding decreased its intensity and red-shifted from 397 to 400 nm by interaction with trityrosine, whereas tyrosinol caused no significant effect. The change in the transient absorption spectrum by interaction with trityrosine was similar to that obtained with 100 mM imidazole, which showed that the population of the intermediate His/His coordinated species increased during folding of cyt c by interaction with trityrosine. The absorption change was biphasic, the fast phase (82+/-9s(-1)) corresponding to the transition from the His/H(2)O to the His/Met coordinated species, whereas the slow phase (24+/-3s(-1)) from His/His to His/Met. By addition of trityrosine, the relative ratio of the slow phase increased, due to increase of the His/His species at the initial stage of folding. According to the resonance Raman spectra of cyt c, the high-spin 6-coordinate and low-spin 6-coordinate species were dominated at pH 2.3 and 4.2, respectively, and these species were not affected by addition of trityrosine. These results demonstrated that the His/His species increased by interaction with trityrosine at the initial stage of cyt c folding, whereas the heme coordination structure was not affected by trityrosine when the protein was completely unfolded or folded. Hydrophobic peptides thus may be useful to study the effects of hydrophobic interactions on protein folding.

Abstract: IgA antibodies reflecting airways or intestinal mucosal immune responses can be found in saliva and feces, respectively, and IgG antibodies reflecting serum antibodies can be found in saliva. In this study, antibodies were detected in samples of saliva and feces which had been air-dried at room temperature (+20°C) or +37°C, and stored at these temperatures for up to 6 months. In saliva the antibody levels increased, while the antibodies in feces decreased upon storage. The individual IgA antibody concentrations which were adjusted by using the ratios of specific IgA/total IgA were relatively stable in both saliva and feces, and correlated with corresponding antibody levels in samples which had been stored at -20°C. The results indicate that air-dried saliva and feces can be used for semiquantitative measurements of mucosal antibodies, even after prolonged storage at high temperatures and lack of refrigeration.

Abstract: Two related studies on injuries sustained by event horses during competition and during training are reported. During the cross-country phase of competition, the most common injuries were lacerations and abrasions to the carpus and stifle. Superficial digital flexor tendonitis and exertional rhabdomyolysis were significantly more common during Cours Complete Internationale (CCI) competitions compared to one-day event (ODE) competitions. The difference in injury types at ODEs and CCI competitions probably relates to the increased athletic demands of the CCI and the closer veterinary observation at these competitions. The results of the training study indicate that 21% of horses intending to compete in a CCI did not start due to injury. Forty-three percent of these injuries involved soft tissue structures with injuries to the superficial digital flexor tendon and the suspensory ligament each accounting for 33%. The most important area for future research is investigation of the risk factors for these career-threatening soft tissue injuries.

Abstract: The ability of horses to learn through social observation may ease the implementation of new management systems, because the use of automatic feeders etc. by naive horses could be facilitated by observation of experienced horses. However, previous studies found no documentation for observational learning abilities in horses. This study aimed to investigate the ability of horses to learn an instrumental task from a familiar conspecific when social interaction was allowed during the demonstration. Two similar experiments were performed. In the first experiment, Observer horses (n=11) participated in ten successive demonstrations, where a trained Demonstrator opened an operant device by pushing a sliding lid aside with the muzzle in order to obtain a food reward. Immediately after the demonstrations the Observer horses were given the opportunity to operate the device alone. Control horses (n=11) were aware that the device contained food but were presented to the operant device without demonstration of the task. The learning criterion was at least two openings. Accomplishment of and latency to accomplish the learning criterion, and investigative behaviour towards the operant device were recorded. Five Observers and one Control, out of the eleven horses in each treatment group, accomplished the learning criterion. Even though this presents a high odds ratio (OR) in favour of the Observer treatment (OR=7.6), there was no significant difference between the treatment groups (P=0.15). Analysis of investigative behaviour showed, however, that the demonstrations increased the motivation of the Observer horses to investigate the device. Subsequently, a similar experiment was performed in a practical setting with 44 test horses (mixed age, gender and breed). We used the same operant device and the same number and type of demonstrations, although the horses were held on a loose rope to minimise aggression. In this second experiment, six of 23 Observer horses and five of 21 Control horses learned the instrumental task, representing no influence of the demonstration. Thus, this study did not demonstrate an ability of horses to learn an instrumental task through observation.

Abstract: Equilibrium constants for coenzyme binding to liver alcohol dehydrogenase have been determined over the pH range 10--12 by pH-jump stop-flow techniques. The binding of NADH or NAD+ requires the protonated form of an ionizing group (distinct from zinc-bound water) with a pKa of 10.4. Complex formation with NADH exhibits an additional dependence on the protonation state of an ionizing group with a pKa of 11.2. The binding of trans-N,N-dimethylaminocinnamaldehyde to the enzyme . NADH complex is prevented by ionization of the latter group. It is concluded from these results that the pKa-11.2-dependence of NADH binding most likely derives from ionization of the water molecule bound at the catalytic zinc ion of the enzyme subunit. The pKa value of 11.2 thus assigned to zinc-bound water in the enzyme . NADH complex appears to be typical for an aquo ligand in the inner-sphere ligand field provided by the zinc-binding amino acid residues in liver alcohol dehydrogenase. This means that the pKa of metal-bound water in zinc-containing enzymes can be assumed to correlate primarily with the number of negatively charged protein ligands coordinated by the active-site zinc ion.