Abstract: The most universal approach to the studies of metal binding properties of single-site metal binding proteins, i.e., construction of a “phase diagram” in coordinates of free metal ion concentration-temperature, has been applied to equine lysozyme (EQL). EQL has one relatively strong calcium binding site and shows two thermal transitions, but only one of them is Ca(2+)-dependent. It has been found that the Ca(2+)-dependent behavior of the low temperature thermal transition (I) of EQL can be adequately described based upon the simplest four-states scheme of metal- and temperature-induced structural changes in a protein. All thermodynamic parameters of this scheme were determined experimentally and used for construction of the EQL phase diagram in the pCa-temperature space. Comparison of the phase diagram with that for alpha-lactalbumin (alpha-LA), a close homologue of lysozyme, allows visualization of the differences in thermodynamic behavior of the two proteins. The thermal stability of apo-EQL (transition I) closely resembles that for apo-alpha-LA (mid-temperature 25 degrees C), while the thermal stabilities of their Ca(2+)-bound forms are almost indistinguishable. The native state of EQL has three orders of magnitude lower affinity for Ca(2+) in comparison with alpha-LA while its thermally unfolded state (after the I transition) has about one order lower (K = 15M(-1)) affinity for calcium. Circular dichroism studies of the apo-lysozyme state after the first thermal transition show that it shares common features with the molten globule state of alpha-LA.

Abstract: We have here investigated the dissociation kinetics of the His side chains axially ligated to the heme-iron in the ferric (1-56 residues) N-fragment of horse cyt c. The ligand deligation induced by acidic pH-jump occurs as a biexponential process with different pre-exponential factors, consistent with a structural heterogeneity in solution and the presence of two differently coordinated species. In analogy with GuHCl-denatured cyt c, our data indicate the presence in solution of two ferric forms of the N-fragment characterized by bis-His coordination, as summarized in the following scheme: His18-Fe(III)-His26 <==> His18-Fe(III)-His33. We have found that the pre-exponential factors depend on the extent of the pH-jump. This may be correlated with the different pKa values shown by His26 and His33; due to steric factors, His26 binds to the heme-Fe(III) less strongly than His33, as recently shown by studies on denatured cyt c. Interestingly, the two lifetimes are affected by temperature but not by the extent of the pH-jump. The lower pKa for the deligation reaction required the use of an improved laser pH-jump setup, capable of inducing changes in H+ concentration as large as 1 mM after the end of the laser pulse. For the ferric N-fragment, close activation entropy values have been determined for the two histidines coordinated to the iron; this result significantly differs from that for GuHCl-denatured cyt c, where largely different values of activation entropy were calculated. This underlines the role played by the missing segment (residues 57-104) peptide chain in discriminating deligation of the “nonnative” His from the sixth coordination position of the metal.

Abstract: The characterization of protein folding dynamics in terms of secondary and tertiary structures is important in elucidating the features of intraprotein interactions that lead to specific folded structures. Apomyoglobin (apoMb), possessing seven helices termed A-E, G, and H in the native state, has a folding intermediate composed of the A, G, and H helices, whose formation in the submillisecond time domain has not been clearly characterized. In this study, we used a rapid-mixing device combined with circular dichroism and small-angle x-ray scattering to observe the submillisecond folding dynamics of apoMb in terms of helical content (f(H)) and radius of gyration (R(g)), respectively. The folding of apoMb from the acid-unfolded state at pH 2.2 was initiated by a pH jump to 6.0. A significant collapse, corresponding to approximately 50% of the overall change in R(g) from the unfolded to native conformation, was observed within 300 micros after the pH jump. The collapsed intermediate has a f(H) of 33% and a globular shape that involves >80% of all its atoms. Subsequently, a stepwise helix formation was detected, which was interpreted to be associated with a conformational search for the correct tertiary contacts. The characterized folding dynamics of apoMb indicates the importance of the initial collapse event, which is suggested to facilitate the subsequent conformational search and the helix formation leading to the native structure.

Abstract: The design of a captive environment should facilitate the expression of the natural behavioural repertoire of the species concerned. The domestic horse (Equus caballus) is a social, herd dwelling species that is often housed in isolation from con-specifics or kept in groups in paddock enclosures. Although the latter allows for social interaction and does not restrict movement per se, it may not promote natural activity patterns or group cohesion. The aim of the current study was to assess the impact of two different paddock designs on the behaviour of a stable group of horses (n=6: 2 mares, 4 geldings).
The paddock designs tested were adjacent and grassed similarly, but configured differently. A central paddock (75 x 75m) designated NT, was surrounded by a track 1.6-5.5m wide, designated T. The horses were turned out in their group into T or NT for 3 consecutive days. Their behaviour was recorded for one hour three times each day (10.00, 13.00, 16.00 hrs). Within each observation period of one hour focal sampling was used, each individual horse being observed for a 10 minute period. They were then moved to the other enclosure type for a further 3 consecutive days, followed by a repeat of each condition. When turned out the horses were fitted with a global positioning system device (Garmin Forerunner 305) to monitor distance travelled and speed for the period 10.00-17.00 hrs. The mean percentage of time spent in each behavioural state (standing alert, standing resting, walk/trot, grazing, lying, social interaction) during the periods observed was calculated. Behaviour during social interactions was classified as either affiliative (approach, follow, friendly contacts, mutual grooming) or agonistic (approach and retreat, bite, chase, head threat).
The horses travelled significantly further in T than in NT (paired samples t-test: t (5) = 11.74, p<0.001) and moved significantly faster (Wilcoxon signed rank test: z = -2.21, p=0.03). See Table 1. When the percentage of time spent in each behavioural state in T and NT was compared some significant differences were found. A significantly higher percentage of time was spent active (walking /trotting) in T than in NT (paired samples t-test: t (5) = 5.74, p=0.002). Standing alert was only recorded in T (paired samples t-test: t (5) = 3.48, p=0.02). A significantly higher percentage of time was spent grazing in NT than in T (paired samples t-test: t (5) = -3.58, p=0.016). Significantly more social interaction occurred in T than in NT (paired samples t-test: t (5) = 5.93, p=0.002). See Figure 1. In T, 91% of social interactions were affiliative and 9% agonistic, whereas in NT 29% were affiliative and 71% agonistic. No difference was found in the percentage of time spent standing resting or lying down in T and NT.
The benefits of housing horses in groups as opposed to individually have been demonstrated in previous studies. In addition to better satisfying the behavioural needs of the horse it has been found that group housed horses adapt more easily to training and display less undesirable behaviour than those housed individually (Rivera et al. 2002; Søndergaard and Ladewig 2004; Visser et al. 2008). However, individual housing is frequently selected by horse owners in preference to group housing to avoid the risk of injury during agonistic encounters. Fureix et al. (2012) suggest that management practices may well contribute to aggressiveness in horses and that the conditions under which we keep horses should be reviewed. The findings of the present study indicate that the design of the enclosure in which groups of horses are kept affects the nature of social interactions. The T paddock design resulted in reduced intra-group aggression. However, this paddock design also reduced the time spent grazing and increased vigilant behaviour. Although the results demonstrate that a paddock system including tracks may facilitate group cohesion and more natural movement patterns, the long-term impact on behaviour and welfare requires further investigation.
References:
Fureix C, Bourjade M, Henry S, Sankey C, Hausberger M. (2012). Exploring aggression regulation in managed groups of horses (Equus caballus). Applied Animal Behaviour Science 138: 216-228.
Rivera E, Benjamin S, Nielsen B, Shelle J, Zanella AJ. (2002). Behavioural and physiological response of horses to initial training: the comparison between pastured versus stalled horses. Applied Animal Behaviour Science 78: 235–252.
Søndergaard E, Ladewig J. (2004). Group housing exerts a positive effect on the behaviour of young horses during training. Applied Animal Behaviour Science 87: 105-118.

Abstract: The objective of the study was to analyze certain factors influencing the condition of free-ranging Konik Polski horses in a fenced area measuring 200 ha located in the Biebrza National Park (Biebrza NP). The Body Condition Scoring system (BCS) was used not only as means of assessment of health and fitness of the horses but also for evaluation of the quality and productivity of the grazing area selected for horses, as well as stocking rate. Body Condition Scoring was determine with a 9-point scale by averaging the scores that were assigned to each of the six body areas that are most responsive to changes of body fat (Hennecke, 1983). The condition of horses was assessed 7 times from February 2009 to December 2011 on a total of 59 horses (3 stallions (n= 12 observations), 21 females 3–8 year olds (n=81) and 35 youngsters (up to 2 year olds) (n=88). The number (from 1 to 3) and composition of family bands varied throughout the period of investigation. The horse population in the Biebrza NP consist of horses from 3 different breeding centers. The analysis of variance (GLM) was used to investigate the effects of the fixed factors – weather conditions, family band, breeder of the horses, age and sex, on the mean of BCS points of horses (SPSS, version 19.0). The most important factor influencing the condition of free-ranging Konik Polski horses in the Biebrza NP were weather conditions, what is shown by the data concerning temperature from 3-month period preceding condition scoring (P < 0.01). This factor influenced not only animals, but also vegetation characteristics. The highest BCS means of horses (5.09) were achieved when average temperature during the 3-month period preceding condition scoring amounted between +1 to +8°C, and was better than obtained when average temperature amounted +18°C (4.80). The last temperature seems too high for horses and can be connected also with other factors, like high incidence of insects which could deteriorate grazing activity of animals. The lowest BCS means (4.56) were obtained when mean temperature was below 0°C. Other factors had no significant effect on the BCS means. As concern to breeder, the highest BCS means achieved mares from Popielno (5.08) – the oldest Polish free-ranging population of Konik Polski horses, which confirms that horses from this breeding center are justly considered to be the toughest and well adapting to difficult environmental conditions. The horses from the Biebrza NP achieved also high BCS means (4.79) which prove that they were well suited to the specific conditions of this habitat, which is overgrown mostly with forest – 73,7%, and willow and birch shrubs – 13,3% of area. The grazing area measures about 40 hectares and consists of wetlands dominated by sedges (Carex sp.). The horses moved from the Seven Island Reservation to Biebrza NP achieved the poorest results as far as adaptation to the conditions of the new habitat, and reached the lowest BCS means (4.57). The youngest horses (up to 2 year olds), 3-5 year olds, and 6-8 year olds had similar BCS means (respectively 4.94, 4.83, 4.68) which indicates good acclimatization of horses from different age categories to the grazing resources in the Biebrza NP. The highest means achieved by youngest horses indicate important trait of Konik Polski horses – mare`s careful care of offspring. The BCS means of horses from different family groups show the highest value of horses from family group of the most experienced stallion – Mrok (4.97). In every BCS scoring, stallions achieved the highest means, which was the reason to exclude their results from analysis. The results of this study suggest that the Body Condition Scoring of horses in the Biebrza National Park depends primarily on the acclimatization of horses to harsh environmental conditions and less importantly to belonging to a specific breeder, family band, age or sex.