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Miksovska, J., & Larsen, R. W. (2003). Photothermal studies of pH induced unfolding of apomyoglobin. J Protein Chem, 22(4), 387–394.
Abstract: Conformational dynamic and enthalpy changes associated with pH induced unfolding of apomyoglobin were studied using photoacoustic calorimetry and photothermal beam deflection methods. The transition between the native state and the I intermediate was induced by a nanosecond pH jump from o-nitrobenzaldehyde photolysis. Deconvolution of photoacoustic waves indicates two kinetic processes. The fast phase (T < 50 ns) is characterized by a volume expansion of 8.8 ml mol(-1). This process is followed by a volume contraction of about -22 ml mol(-1) (tau approximately 500 ns). Photothermal beam deflection measurements do not reveal any volume changes on the time scale between approximately 100 micros and 5 ms. We associate the volume contraction with structural changes occurring during the transition between the native state and the I intermediate. The lack of any processes on the ms time scale may indicate the absence of structural events involving larger conformational changes of apomyoglobin after the pH jump.
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Uzawa, T., Akiyama, S., Kimura, T., Takahashi, S., Ishimori, K., Morishima, I., et al. (2004). Collapse and search dynamics of apomyoglobin folding revealed by submillisecond observations of alpha-helical content and compactness. Proc. Natl. Acad. Sci. U.S.A., 101(5), 1171–1176.
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.
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Hirota, S., Suzuki, M., & Watanabe, Y. (2004). Hydrophobic effect of trityrosine on heme ligand exchange during folding of cytochrome c. Biochem Biophys Res Commun, 314(2), 452–458.
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.
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Kasashima, Y., Takahashi, T., Smith, R. K. W., Goodship, A. E., Kuwano, A., Ueno, T., et al. (2004). Prevalence of superficial digital flexor tendonitis and suspensory desmitis in Japanese Thoroughbred flat racehorses in 1999. Equine Vet J, 36(4), 346–350.
Abstract: REASONS FOR PERFORMING STUDY: Overstrain injuries to the superficial digital flexor tendon (SDFT) and suspensory ligament (SI) are among the most common musculoskeletal injuries which contribute to the considerable wastage of racing Thoroughbreds. Many epidemiological studies have demonstrated the prevalence of and risk factors for tendon injury when racing but have not included those injuries sustained during training. However, since tendon injury during training is seen commonly in clinical practice, it is appropriate to determine the overall prevalence of tendon injury sustained during both training and racing. OBJECTIVE: To determine the prevalence of overstrain injury to the SDFT and SL during training and racing among Thoroughbred flat racehorses in Japan in 1999. METHODS: A retrospective study was performed using a sample population of 10,262 Thoroughbred racehorses. The medical information database of Thoroughbred racehorses registered by the Japan Racing Association (JRA) in 1999 was analysed for SDFT and SL overstrain injury diagnosed by a veterinarian employed by JRA during training and racing. Jump racehorses were excluded from this study. RESULTS: The prevalence of forelimb SDFT tendonitis and SL desmitis was 11.1% (1130 cases) and 3.61% (370 cases) of the population, respectively. In the hindlimb, there were 0.06% (6 cases) and 0.14% (14 cases), respectively. Risks of SDF tendonitis in the forelimb in 3-year-olds or older horses were significantly higher than in 2-year-olds. In contrast, the risk of SL desmitis in the forelimb at age 3 and 4 years was 2.23 and 2.11 times higher, respectively, than in 2-year-olds, but this increased to 5.07 times in those age > or = 5 years. Entire males were at greater risk in comparison to females and geldings. CONCLUSIONS: The results suggest that the prevalence of SDF tendonitis and SL desmitis in the forelimb was associated with the horse's age and sex. The prevalence of SL desmitis increased further with age compared with SDF tendonitis, possibly reflecting a more rapid accumulation of degeneration in this structure. POTENTIAL RELEVANCE: The age-related risk demonstrated in this study provides further support that overstrain injuries are associated with accumulated degeneration. These data provide a valuable resource for further research into the aetiology of tendon injury in the racehorse.
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Pinchbeck, G. L., Clegg, P. D., Proudman, C. J., Morgan, K. L., & French, N. P. (2004). Case-control investigation of the factors affecting the risk of horses falling during steeplechase racing in the UK. Vet. Rec., 155(1), 11–15.
Abstract: A concurrent case-control study of 12 UK racecourses was made between March 1, 2000, and August 31, 2001, to identify and quantify the factors associated with the risk of horses falling in steeplechase races. Cases were defined as a jumping effort at a steeplechase fence that resulted in a fall and controls were defined as a successful jumping effort over any steeplechase fence at any of the 12 racecourses within 14 days before or after the case fall. Information on the horse, the jockey and the race were collected and all the fences on all the courses were surveyed. Conditional logistic regression was used to examine the relationships between the predictor variables and the risk of falling. There was one fall per 254 jumping efforts. The risk of a horse falling decreased the more times it had raced on a particular racecourse. The number of fences, the distance from the previous fence and the nature of the previous fence also affected the risk of falling. If the previous fence was a water jump the risk of falling increased; fences that were sited on flat or slight uphill gradients (up to approximately 1 in 25) were associated with a lower risk of horses falling than downhill fences, and higher takeoff boards were associated with a higher risk of falling.
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Santamaria, S., Bobbert, M. E., Back, W., Barneveld, A., & van Weeren, P. R. (2004). Variation in free jumping technique within and among horses with little experience in show jumping. Am J Vet Res, 65(7), 938–944.
Abstract: OBJECTIVE: To quantify variation in the jumping technique within and among young horses with little jumping experience, establish relationships between kinetic and kinematic variables, and identify a limited set of variables characteristic for detecting differences in jumping performance among horses. ANIMALS: Fifteen 4-year-old Dutch Warmblood horses. PROCEDURE: The horses were raised under standardized conditions and trained in accordance with a fixed protocol for a short period. Subsequently, horses were analyzed kinematically during free jumping over a fence with a height of 1.05 m. RESULTS: Within-horse variation in all variables that quantified jumping technique was smaller than variation among horses. However, some horses had less variation than others. Height of the center of gravity (CG) at the apex of the jump ranged from 1.80 to 2.01 m among horses; this variation could be explained by the variation in vertical velocity of the CG at takeoff (r, 0.78). Horses that had higher vertical velocity at takeoff left the ground and landed again farther from the fence, had shorter push-off phases for the forelimbs and hind limbs, and generated greater vertical acceleration of the CG primarily during the hind limb push-off. However, all horses cleared the fence successfully, independent of jumping technique. CONCLUSIONS AND CLINICAL RELEVANCE: Each horse had its own jumping technique. Differences among techniques were characterized by variations in the vertical velocity of the CG at takeoff. It must be determined whether jumping performance later in life can be predicted from observing free jumps of young horses.
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Abbruzzetti, S., Viappiani, C., Sinibaldi, F., & Santucci, R. (2004). Kinetics of histidine dissociation from the heme Fe(III) in N-fragment (residues 1-56) of cytochrome c. Protein J, 23(8), 519–527.
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.
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Rollot, Y., Lecuyer, E., Chateau, H., & Crevier-Denoix, N. (2004). Development of a 3D model of the equine distal forelimb and of a GRF shoe for noninvasive determination of in vivo tendon and ligament loads and strains. Equine Vet J, 36(8), 677–682.
Abstract: REASONS FOR PERFORMING STUDY: As critical locomotion events (e.g. high-speed and impacts during racing, jump landing) may contribute to tendinopathies, in vivo recording of gaits kinematic and dynamic parameters is essential for 3D reconstruction and analysis. OBJECTIVE: To propose a 3D model of the forelimb and a ground reaction force recording shoe (GRF-S) for noninvasively quantifying tendon and ligament loads and strains. METHODS: Bony segments trajectories of forelimbs placed under a power press were recorded using triads of ultrasonic kinematic markers linked to the bones. Compression cycles (from 500-6000 N) were applied for different hoof orientations. Locations of tendon and ligament insertions were recorded with regard to the triads. The GRF-S recorded GRF over the hoof wall and used four 3-axis force sensors sandwiched between a support shoe and the shoe to be tested. RESULTS: Validation of the model by comparing calculated and measured superficial digital flexor tendon strains, and evaluation of the role of proximal interphalangeal joint in straight sesamoidean ligament and oblique sesamoidean ligament strains, were successfully achieved. Objective comparisons of the 3 components of GRF over the hoof for soft and hard grounds could be recorded, where the s.d. of GRF norm was more important on hard ground at walk and trot. CONCLUSIONS: Soft grounds (sand and rubber) dissipate energy by lowering GRF amplitude and diminish bounces and vibrations at impact. At comparable speed, stance phase was longer on soft sand ground. POTENTIAL RELEVANCE: The conjugate use of the GRF-S and the numerical model would help to quantify and analyse ground/shoe combination on comfort, propulsion efficiency or lameness recovery.
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Young, L. E., Rogers, K., & Wood, J. L. N. (2005). Left ventricular size and systolic function in Thoroughbred racehorses and their relationships to race performance. J Appl Physiol, 99(4), 1278–1285.
Abstract: Cardiac morphology in human athletes is known to differ, depending on the sports-specific endurance component of their events, whereas anecdotes abound about superlative athletes with large hearts. As the heart determines stroke volume and maximum O(2) uptake in mammals, we undertook a study to test the hypothesis that the morphology of the equine heart would differ between trained horses, depending on race type, and that left ventricular size would be greatest in elite performers. Echocardiography was performed in 482 race-fit Thoroughbreds engaged in either flat (1,000-2,500 m) or jump racing (3,200-6,400 m). Body weight and sex-adjusted measures of left ventricular size were largest in horses engaged in jump racing over fixed fences, compared with horses running shorter distances on the flat (range 8-16%). The observed differences in cardiac morphologies suggest that subtle differences in training and competition result in cardiac adaptations that are appropriate to the endurance component of the horses' event. Derived left ventricular mass was strongly associated with published rating (quality) in horses racing over longer distances in jump races (P < or = 0.001), but less so for horses in flat races. Rather, left ventricular ejection fraction and left ventricular mass combined were positively associated with race rating in older flat racehorses running over sprint (<1,408 m) and longer distances (>1,408 m), explaining 25-35% of overall variation in performance, as well as being closely associated with performance in longer races over jumps (23%). These data provide the first direct evidence that cardiac size influences athletic performance in a group of mammalian running athletes.
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Bykov, S., Lednev, I., Ianoul, A., Mikhonin, A., Munro, C., & Asher, S. A. (2005). Steady-state and transient ultraviolet resonance Raman spectrometer for the 193-270 nm spectral region. Appl Spectrosc, 59(12), 1541–1552.
Abstract: We describe a state-of-the-art tunable ultraviolet (UV) Raman spectrometer for the 193-270 nm spectral region. This instrument allows for steady-state and transient UV Raman measurements. We utilize a 5 kHz Ti-sapphire continuously tunable laser (approximately 20 ns pulse width) between 193 nm and 240 nm for steady-state measurements. For transient Raman measurements we utilize one Coherent Infinity YAG laser to generate nanosecond infrared (IR) pump laser pulses to generate a temperature jump (T-jump) and a second Coherent Infinity YAG laser that is frequency tripled and Raman shifted into the deep UV (204 nm) for transient UV Raman excitation. Numerous other UV excitation frequencies can be utilized for selective excitation of chromophoric groups for transient Raman measurements. We constructed a subtractive dispersion double monochromator to minimize stray light. We utilize a new charge-coupled device (CCD) camera that responds efficiently to UV light, as opposed to the previous CCD and photodiode detectors, which required intensifiers for detecting UV light. For the T-jump measurements we use a second camera to simultaneously acquire the Raman spectra of the water stretching bands (2500-4000 cm(-1)) whose band-shape and frequency report the sample temperature.
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