Abstract: The refolding kinetics of horse cyanometmyoglobin induced by concentration jump of urea was investigated by five optical probe stopped-flow methods: absorption at 422 nm, tryptophyl fluorescence at around 340 nm, circular dichroism (CD) at 222 nm, CD at 260 nm, and CD at 422 nm. In the refolding process, we detected three phases with rate constants of > 1 x 10(2) s-1, (4.5-9.3) s-1, and (2-5) x 10(-3) s-1. In the fastest phase, a substantial amount of secondary structure (approximately 40%) is formed within the dead time of the CD stopped-flow apparatus (10.7 ms). The kinetic intermediate populated in the fastest phase is shown to capture a hemindicyanide, suggesting that a “heme pocket precursor” recognized by hemindicyanide must be constructed within the dead time. In the middle phase, most of secondary and tertiary structures, especially around the captured hemindicyanide, have been constructed. In the slowest phase, we detected a minor structural rearrangement accompanying the ligand-exchange reaction in the fifth coordination of ferric iron. We present a possible model for the refolding process of myoglobin in the presence of the heme group.

Abstract: The place and date of the domestication of the horse has long been a matter for debate among archaeologists. To determine whether horses were domesticated from one or several ancestral horse populations, we sequenced the mitochondrial D-loop for 318 horses from 25 oriental and European breeds, including American mustangs. Adding these sequences to previously published data, the total comes to 652, the largest currently available database. From these sequences, a phylogenetic network was constructed that showed that most of the 93 different mitochondrial (mt)DNA types grouped into 17 distinct phylogenetic clusters. Several of the clusters correspond to breeds and/or geographic areas, notably cluster A2, which is specific to Przewalski's horses, cluster C1, which is distinctive for northern European ponies, and cluster D1, which is well represented in Iberian and northwest African breeds. A consideration of the horse mtDNA mutation rate together with the archaeological timeframe for domestication requires at least 77 successfully breeding mares recruited from the wild. The extensive genetic diversity of these 77 ancestral mares leads us to conclude that several distinct horse populations were involved in the domestication of the horse.

Abstract: The rapid refolding dynamics of apomyoglobin are followed by a new temperature-jump fluorescence technique on a 15-ns to 0.5-ms time scale in vitro. The apparatus measures the protein-folding history in a single sweep in standard aqueous buffers. The earliest steps during folding to a compact state are observed and are complete in under 20 micros. Experiments on mutants and consideration of steady-state CD and fluorescence spectra indicate that the observed microsecond phase monitors assembly of an A x (H x G) helix subunit. Measurements at different viscosities indicate diffusive behavior even at low viscosities, in agreement with motions of a solvent-exposed protein during the initial collapse.

Abstract: Mirror Self-Recognition (MSR) is an extremely rare capacity in the animal kingdom that reveals the emergence of complex cognitive capacities (de Waal 2008). So far, MSR has been reported only in humans, chimpanzees (Gallup, 1970), bottlenose dolphins (Reiss and Marino, 2001) and Asian elephants (Plotnik et al, 2006), all species characterized by a highly developed cognition. There is growing evidence that domestic horses posses high cognitive abilities, such as cross-modal individual recognition (Proops et al, 2009), triadic post-conflict reunion to maintain social homeostasis (Cozzi et al, 2010), complex communicative systems (Whatan and McComb, 2014), flexibility in problem-solving (Lovrovich et al, 2015), and long-term memory (Hanggi and Ingersoll, 2009). All these capacities make horses a good candidate to test the ability of MSR in a domestic species. Through a classical MSR experimental paradigm (de Waal 2008) we tested eight horses living in social groups under semi-natural conditions (from the Italian Horse Protection rescue centre). Animals showing MSR typically go through four stages (Plotnik et al, 2006): (i) social response, (ii) physical mirror inspection (e.g., looking behind the mirror), (iii) repetitive mirror-testing behaviour (i.e., the beginning of mirror understanding), and (iv) self-directed behaviour (i.e., recognition of the mirror image as self). The final stage, known as the “mark-test”, is verified when a subject spontaneously uses the mirror to check for a coloured artificial mark on its own body which it cannot perceive otherwise. The horses underwent a three-phase “mark-test”: 1) with sham mark (transparent ultrasound water gel) positioned on both side at jaw level, 2) mark (yellow eye shadow mixed with ultrasound water gel) positioned on left side of jaw (with sham mark on the right), 3) mark (yellow eye shadow mixed with ultrasound water gel) positioned on right side of jaw (with sham mark on the left)
The mirror was one 0.5-cm-thick piece of 140x220-cm plexiglass glue on wood. Each test lasted one hour, horses were tested once a day, in consecutive days and at the same time. Our preliminary result on 1 horse shows some changes in self-directed behaviours which can be attributed to presence of the coloured mark. Firstly, the presence of the coloured mark significantly increased the frequency of scratching on both sides of the muzzle (p < 0.0001). The most intriguing result (p < 0.0001) comes from the comparison of the scratching rates directed towards the coloured mark side (N = 41) and the sham mark side (N = 23). Under the control condition (i.e. sham mark on both sides) no statistical difference was found for the scratching rates directed to the muzzle sides (dx N = 8; sx N = 5). Although further analyses are needed to confirm these preliminary results, our finding opens new scenarios about the evolution of Mirror Self-Recognition. The capacity of horses to recognize themselves in a mirror may be the outcome of an evolutionary convergence process driven by the cognitive pressures imposed by a complex social system and maintained despite thousands years of domestication.
Keywords:
Domestic horse · Mark test · Socio-cognitive skills · Self-awareness
References
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Proops L, McComb K, Reby D. (2009) Cross-modal individual recognition in domestic horses (Equus caballus). Proc Nat Acad Sci USA;106:947-951.
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Lovrovich P, Sighieri C, Baragli P (2015) Following human-given cues or not? Horses (Equus caballus) get smarter and change strategy in a delayed three choice task. Appl Anim Behav Sci, in press.
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