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Gulotta, M., Rogatsky, E., Callender, R. H., & Dyer, R. B. (2003). Primary folding dynamics of sperm whale apomyoglobin: core formation. Biophys J, 84(3), 1909–1918.
Abstract: The structure, thermodynamics, and kinetics of heat-induced unfolding of sperm whale apomyoglobin core formation have been studied. The most rudimentary core is formed at pH(*) 3.0 and up to 60 mM NaCl. Steady state for ultraviolet circular dichroism and fluorescence melting studies indicate that the core in this acid-destabilized state consists of a heterogeneous composition of structures of approximately 26 residues, two-thirds of the number involved for horse heart apomyoglobin under these conditions. Fluorescence temperature-jump relaxation studies show that there is only one process involved in Trp burial. This occurs in 20 micro s for a 7 degrees jump to 52 degrees C, which is close to the limits placed by diffusion on folding reactions. However, infrared temperature jump studies monitoring native helix burial are biexponential with times of 5 micro s and 56 micro s for a similar temperature jump. Both fluorescence and infrared fast phases are energetically favorable but the slow infrared absorbance phase is highly temperature-dependent, indicating a substantial enthalpic barrier for this process. The kinetics are best understood by a multiple-pathway kinetics model. The rapid phases likely represent direct burial of one or both of the Trp residues and parts of the G- and H-helices. We attribute the slow phase to burial and subsequent rearrangement of a misformed core or to a collapse having a high energy barrier wherein both Trps are solvent-exposed.
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Haruta, N., & Kitagawa, T. (2002). Time-resolved UV resonance Raman investigation of protein folding using a rapid mixer: characterization of kinetic folding intermediates of apomyoglobin. Biochemistry, 41(21), 6595–6604.
Abstract: The 244-nm excited transient UV resonance Raman spectra are observed for the refolding intermediates of horse apomyoglobin (h-apoMb) with a newly constructed mixed flow cell system, and the results are interpreted on the basis of the spectra observed for the equilibrium acid unfolding of the same protein. The dead time of mixing, which was determined with the appearance of UV Raman bands of imidazolium upon mixing of imidazole with acid, was 150 micros under the flow rate that was adopted. The pH-jump experiments of h-apoMb from pH 2.2 to 5.6 conducted with this device demonstrated the presence of three folding intermediates. On the basis of the analysis of W3 and W7 bands of Trp7 and Trp14, the first intermediate, formed before 250 micros, involved incorporation of Trp14 into the alpha-helix from a random coil. The frequency shift of the W3 band of Trp14 observed for this process was reproduced with a model peptide of the A helix when it forms the alpha-helix. In the second intermediate, formed around 1 ms after the start of refolding, the surroundings of both Trp7 and Trp14 were significantly hydrophobic, suggesting the formation of the hydrophobic core. In the third intermediate appearing around 3 ms, the hydrophobicity was relaxed to the same level as that of the pH 4 equilibrium intermediate, which was investigated in detail with the stationary state technique. The change from the third intermediate to the native state needs more time than 40 ms, while the appearance of the native spectrum after the mixing of the same solutions was confirmed separately.
Keywords: Animals; Apoproteins/*chemistry; Circular Dichroism; Holoenzymes/chemistry; Horses; Hydrochloric Acid/chemistry; Hydrogen-Ion Concentration; Imidazoles/chemistry; Kinetics; Models, Molecular; Myoglobin/*chemistry; Peptide Fragments/chemistry; *Protein Folding; Protein Structure, Secondary; Spectrum Analysis, Raman/*methods; Tryptophan/*chemistry; Ultraviolet Rays; Whales
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Gulotta, M., Gilmanshin, R., Buscher, T. C., Callender, R. H., & Dyer, R. B. (2001). Core formation in apomyoglobin: probing the upper reaches of the folding energy landscape. Biochemistry, 40(17), 5137–5143.
Abstract: An acid-destabilized form of apomyoglobin, the so-called E state, consists of a set of heterogeneous structures that are all characterized by a stable hydrophobic core composed of 30-40 residues at the intersection of the A, G, and H helices of the protein, with little other secondary structure and no other tertiary structure. Relaxation kinetics studies were carried out to characterize the dynamics of core melting and formation in this protein. The unfolding and/or refolding response is induced by a laser-induced temperature jump between the folded and unfolded forms of E, and structural changes are monitored using the infrared amide I' absorbance at 1648-1651 cm(-1) that reports on the formation of solvent-protected, native-like helix in the core and by fluorescence emission changes from apomyoglobin's Trp14, a measure of burial of the indole group of this residue. The fluorescence kinetics data are monoexponential with a relaxation time of 14 micros. However, infrared kinetics data are best fit to a biexponential function with relaxation times of 14 and 59 micros. These relaxation times are very fast, close to the limits placed on folding reactions by diffusion. The 14 micros relaxation time is weakly temperature dependent and thus represents a pathway that is energetically downhill. The appearance of this relaxation time in both the fluorescence and infrared measurements indicates that this folding event proceeds by a concomitant formation of compact secondary and tertiary structures. The 59 micros relaxation time is much more strongly temperature dependent and has no fluorescence counterpart, indicating an activated process with a large energy barrier wherein nonspecific hydrophobic interactions between helix A and the G and H helices cause some helix burial but Trp14 remains solvent exposed. These results are best fit by a multiple-pathway kinetic model when U collapses to form the various folded core structures of E. Thus, the results suggest very robust dynamics for core formation involving multiple folding pathways and provide significant insight into the primary processes of protein folding.
Keywords: Animals; Apoproteins/*chemistry; Computer Simulation; Horses; Hydrogen-Ion Concentration; Kinetics; Models, Molecular; Myoglobin/*chemistry; *Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Spectrometry, Fluorescence/instrumentation/methods; Thermodynamics; Tryptophan/chemistry
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Grandin, T. (1999). Safe handling of large animals. Occup Med, 14(2), 195–212.
Abstract: The major causes of accidents with cattle, horses, and other grazing animals are: panic due to fear, male dominance aggression, or the maternal aggression of a mother protecting her newborn. Danger is inherent when handling large animals. Understanding their behavior patterns improves safety, but working with animals will never be completely safe. Calm, quiet handling and non-slip flooring are beneficial. Rough handling and excessive use of electric prods increase chances of injury to both people and animals, because fearful animals may jump, kick, or rear. Training animals to voluntarily cooperate with veterinary procedures reduces stress and improves safety. Grazing animals have a herd instinct, and a lone, isolated animal can become agitated. Providing a companion animal helps keep an animal calm.
Keywords: Accidents, Occupational/*prevention & control/statistics & numerical data; Aggression/physiology/psychology; Animal Husbandry/*methods; Animals; *Behavior, Animal/physiology; Cattle; Conditioning, Operant/physiology; Crowding/psychology; Fear/physiology/psychology; Female; *Horses/physiology/psychology; Humans; Male; Movement/physiology; *Occupational Health; Risk Factors; *Ruminants/physiology/psychology
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Allen, D., & Tanner, K. (2007). Putting the horse back in front of the cart: using visions and decisions about high-quality learning experiences to drive course design. CBE Life Sci Educ, 6(2), 85–89.
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Takahashi, T., Kasashima, Y., Eto, D., Mukai, K., & Hiraga, A. (2006). Effect of uphill exercise on equine superficial digital flexor tendon forces at trot and canter. Equine Vet J Suppl, (36), 435–439.
Abstract: REASONS FOR PERFORMING STUDY: One cause of overstrain injury to the superficial digital flexor tendon (SDFT) in horses is the force loaded on the SDFT during repeated running. Therefore, decreasing this force may reduce SDFT injury. It has been reported that strain on the SDFT decreases with a toe-wedge shoe. Uphill courses are used for training of racehorses, and the angle of hoof-sole to the horizon during uphill running is similar to that of the toe-wedge shoe. OBJECTIVES: To determine the effects of uphill exercise on the force on the SDFT during trotting and cantering. METHODS: Arthroscopically implantable force probes (AIFP) were implanted into the SDFT of the left or right forelimb of 7 Thoroughbred horses and AIFP output recorded during trotting and cantering on a treadmill inclined at slopes of 0, 3 or 8%, and then 0% again. Superficial digital flexor tendon force was calculated as a relative value, with the amplitude of AIFP output voltage at initial 0% slope equal to 100. RESULTS: Out of 14 sets of experiments, AIFP data were analysed successfully in 9 at the trot, in 3 at the canter in the trailing forelimb on a slope of 3 and 8%, and in 2 at the canter in the leading forelimb on a slope of 3%. Increasing the incline from 0-8% tended to decrease peak force in the SDFT at the trot, and in the trailing forelimb at the canter. However, force in the SDFT was unchanged in the leading forelimb at the canter on the 3% incline. CONCLUSIONS: The force in the SDFT trotting or cantering uphill is unchanged or lower than that loaded at the same speed on a flat surface. Because at similar speeds the workload for uphill exercise is greater than on the flat, uphill running increases exercise intensity without increasing force in the SDFT. POTENTIAL RELEVANCE: Uphill exercise may reduce the risk of SDFT injury as both running speed and SDFT force are decreased on an incline as compared to the flat, even when exercise intensity is the same. Further study is needed to confirm these findings at canter in a larger population of horses.
Keywords: Animals; Biomechanics; Exercise Test/veterinary; Female; Forelimb/physiology; Hoof and Claw/physiology; Horses/*physiology; Male; Physical Conditioning, Animal/*methods/*physiology; Tarsal Joints/*physiology; Tarsus, Animal; Tendon Injuries/etiology/prevention & control/veterinary; Time Factors
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Peel, J. A., Peel, M. B., & Davies, H. M. S. (2006). The effect of gallop training on hoof angle in thoroughbred racehorses. Equine Vet J Suppl, (36), 431–434.
Abstract: REASONS FOR PERFORMING STUDY: The economic impact of soundness problems in racehorses is very high and low hoof angle at the toe has been associated with a lack of soundness. However, it is not clear what environmental and management factors might contribute to a low hoof angle. OBJECTIVES: To investigate the hypothesis that the hooves of racehorses become flatter when in gallop training, as well as to determine factors contributing to this trend. METHODS: Weekly hoof measurements were taken with a hoof gauge from 45 Thoroughbred racehorses; 4 Thoroughbred show horses kept in consistent conditions and shod by the same farrier as some of the racehorses; and 6 unshod free-ranging horses. A further 15 horses were measured twice in one day to determine the repeatability of the method. RESULTS: Repeatability coefficients were 0.31 degrees for the left hoof and 0.37 degrees for the right. Racehorses in training showed a significant decrease in hoof angle over time while free ranging horses and show horses did not. Free-ranging horses had a significantly lower angle in winter (wet) compared with summer (dry) in both left (P = 0.040) and right (P = 0.017). Show horses had no significant change in hoof angle. Racehorses that had a period of rest during the experiment (n = 11) showed a decrease in hoof angle during training and an increase over their rest period for both hooves (P = 0.005 for the left hoof, P = 0.0009 for the right). CONCLUSIONS: Training for fast exercise in Thoroughbred racehorses is associated with a reduction in hoof angle and wet pasture conditions may also be associated with a reduced hoof angle in free-ranging horses. Potential relevance: Gallop exercise has a potentially large effect on hoof angle and therefore, a change in angle should be expected to occur in racehorses starting fast exercise work. Hence management of horses with abnormally low hoof angles may require an adaptation to their training regime in order to minimise this effect.
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Winkelmayr, B., Peham, C., Fruhwirth, B., Licka, T., & Scheidl, M. (2006). Evaluation of the force acting on the back of the horse with an English saddle and a side saddle at walk, trot and canter. Equine Vet J Suppl, (36), 406–410.
Abstract: REASONS FOR PERFORMING STUDY: Force transmission under an English saddle (ES) at walk, trot and canter is commonly evaluated, but the influence of a side saddle (SS) on the equine back has not been documented. HYPOTHESIS: Force transmission under a SS, with its asymmetric construction, is different from an ES in walk, trot and canter, expressed in maximum overall force (MOF), force in the quarters of the saddle mat, and centre of pressure (COP). The biomechanics of the equine back are different under a SS compared to ES. METHODS: Thirteen horses without clinical signs of back pain ridden in an indoor riding school with both saddles were measured using an electronic saddle sensor pad. Synchronous kinematic measurements were carried out with tracing markers placed along the back in front of (withers, W) and behind the saddle (4th lumbar vertebra, L4). At least 6 motion cycles at walk, trot and canter with both saddles (ES, SS) were measured. Out of the pressure distribution the maximum overall force (MOF) and the location of the centre of pressure (COP) were calculated. RESULTS: Under the SS the centre of pressure was located to the right of the median and slightly caudal compared to the COP under the ES in all gaits. The MOF was significantly different (P<0.01) between saddles. At walk, L4 showed significantly larger (P<0.01) vertical excursions under the ES. Under the SS relative horizontal movement of W was significantly reduced (P<0.01) at trot, and at canter the transversal movement was significantly reduced (P<0.01) . In both trot and canter, no significant differences in the movement of L4 were documented. CONCLUSIONS AND POTENTIAL RELEVANCE: The results demonstrate that the load under a SS creates asymmetric force transmission under the saddle, and also influences back movement. To change the load distribution on the back of horses with potential back pain and as a training variation, a combination of both riding styles is suitable.
Keywords: Animals; Back/*physiology; Back Pain/etiology/veterinary; Biomechanics; Exercise Test/veterinary; Female; Gait/physiology; Horse Diseases/etiology; Horses/*physiology; Humans; Locomotion/physiology; Male; Movement/*physiology; *Physical Conditioning, Animal/instrumentation/methods/physiology; *Pressure; Weight-Bearing/*physiology
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Kinnunen, S., Laukkanen, R., Haldi, J., Hanninen, O., & Atalay, M. (2006). Heart rate variability in trotters during different training periods. Equine Vet J Suppl, (36), 214–217.
Abstract: REASONS FOR PERFORMING STUDY: Endurance training induces changes in autonomic nervous system functions. High intensity training includes the risk of overtraining, in man and horse. Heart rate variability (HRV) is a noninvasive measurement of the autonomic regulation of the heart rate, which is quick and easy to measure with modern telemetric technology. HYPOTHESIS: Since HRV is affected by changes in the autonomic nervous system, it might be an early stage indicator of poor recovery from a previous bout of exercise or overreaching or overtraining in horses in general. METHODS: The aim of the study was to monitor recovery and the possible overtraining status in horses by measuring HRV. The measurements reflected the responses of the previous day activities during different training periods including basic training, precompetition and competition during a one-year follow-up. RESULTS: HRV was at the highest during precompetition period (P<0.05) and it decreased significantly during competition period (P<0.05), indicating an increased stress load in the competition period. Walking increased HRV significantly compared to complete rest or jogging as previous day activities during basic training and precompetition periods (P<0.05). This finding suggests that horses are more relaxed during moderate exercise than standing still or anaerobic exercise. CONCLUSIONS: HRV can be used to monitor the cardiovascular responses to training in horses but confirmatory measures may also be required in addition to HRV to exclude other possible causes of underperformance.
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Cottin, F., Barrey, E., Lopes, P., & Billat, V. (2006). Effect of repeated exercise and recovery on heart rate variability in elite trotting horses during high intensity interval training. Equine Vet J Suppl, (36), 204–209.
Abstract: REASONS FOR PERFORMING STUDY: Interval training is a commonly used training method for trotting horses. In addition, trainers are provided with efficient and inexpensive heart rate monitor devices for the management of training. HYPOTHESIS: Since the high frequency (HF) frequency peak (fHF) of heart rate variability (HRV) corresponds to the breathing frequency in combination with stride frequency during trotting, it is hypothesised that modifications of breathing and stride frequencies induced by repeated exercise could be detected from fHF. METHODS: RR interval time series of 7 trotting horses were recorded during an interval training session. Interval training was made up of 5 successive 800 m high-velocity trotting runs (H1, H2...H5) separated by 1 min recovery bouts at low speed (R1, R2...R5). Fast Fourier transform (FFT) and Poincare plot analysis techniques were applied to RR series. RESULTS: Repeated exercise had significant effects on HRV components during interval training. Despite constant trotting velocities during high-speed and recovery, repetition induced a decrease in mean RR interval (H1: 295 +/- 19 vs. H5: 283 +/- 15 msec, P<0.05) and in the root mean square of successive differences in RR series (RMSSD; H1: 6.31 +/- 1.28 vs. H5: 5.31 +/- 1.31 msec, P<0.05). Furthermore, high-speed and recovery repetitions induced an increase in fHF (H1: 1.37 +/- 0.35 vs. H5: 1.62 +/- 0.40 Hz and R1: 0.22 +/- 0.02 vs. R4: 0.64 +/- 0.38 Hz, P<0.05). Hence, recovery induced a decrease in the s.d. of the successive RR series (SDRR; R3: 10.5 +/- 3.96 vs. R5: 6.17 +/- 2.65 msecs, P>0.05) and in the long term index of Poincare plot (SD2; R1: 43.29 +/- 28.90 vs. R5: 18.19 +/- 9.35 msecs, P<0.05). CONCLUSIONS: The observed increase in fHF during the interval training could be induced by alterations of the coupling between breathing and stride frequency linked to the emergence of fatigue. The decrease in SD2 and SDRR during successive recovery bouts could be linked with a deterioration of the recovery pattern. POTENTIAL RELEVANCE: HRV can provide breathing frequency data of Standardbreds during training without any respiratory device. Furthermore, HRV could provide useful makers of the emergence of fatigue states during training.
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