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Ballew, R. M., Sabelko, J., & Gruebele, M. (1996). Direct observation of fast protein folding: the initial collapse of apomyoglobin. Proc. Natl. Acad. Sci. U.S.A., 93(12), 5759–5764.
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.
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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.
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Polverini, E., Cugini, G., Annoni, F., Abbruzzetti, S., Viappiani, C., & Gensch, T. (2006). Molten globule formation in apomyoglobin monitored by the fluorescent probe Nile Red. Biochemistry, 45(16), 5111–5121.
Abstract: The interaction of nile red (NR) with apomyoglobin (ApoMb) in the native (pH 7) and molten globule (pH 4) states was investigated using experimental and computational methods. NR binds to hydrophobic locations in ApoMb with higher affinity (K(d) = 25 +/- 5 microM) in the native state than in the molten globule state (K(d) = 52 +/- 5 microM). In the molten globule state, NR is located in a more hydrophobic environment. The dye does not bind to the holoprotein, suggesting that the binding site is located at the heme pocket. In addition to monitoring steady-state properties, the fluorescence emission of NR is capable of tracking submillisecond, time-resolved structural rearrangements of the protein, induced by a nanosecond pH jump. Molecular dynamics simulations were run on ApoMb at neutral pH and at pH 4. The structure obtained for the molten globule state is consistent with the experimentally available structural data. The docking of NR with the crystal structure shows that the ligand binds into the binding pocket of the heme group, with an orientation bringing the planar ring system of NR to overlap with the position of two of the heme porphyrin rings in Mb. The docking of NR with the ApoMb structure at pH 4 shows that the dye binds to the heme pocket with a slightly less favorable binding energy, in keeping with the experimental K(d) value. Under these conditions, NR is positioned in a different orientation, reaching a more hydrophobic environment in agreement with the spectroscopic data.
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Arnold, W., Ruf, T., & Kuntz, R. (2006). Seasonal adjustment of energy budget in a large wild mammal, the Przewalski horse (Equus ferus przewalskii) II. Energy expenditure. J Exp Biol, 209(Pt 22), 4566–4573.
Abstract: Many large mammals show pronounced seasonal fluctuations of metabolic rate (MR). It has been argued, based on studies in ruminants, that this variation merely results from different levels of locomotor activity (LA), and heat increment of feeding (HI). However, a recent study in red deer (Cervus elaphus) identified a previously unknown mechanism in ungulates--nocturnal hypometabolism--that contributed significantly to reduced energy expenditure, mainly during late winter. The relative contribution of these different mechanisms to seasonal adjustments of MR is still unknown, however. Therefore, in the study presented here we quantified for the first time the independent contribution of thermoregulation, LA and HI to heart rate (f(H)) as a measure of MR in a free-roaming large ungulate, the Przewalski horse or Takhi (Equus ferus przewalskii Poljakow). f(H) varied periodically throughout the year with a twofold increase from a mean of 44 beats min(-1) during December and January to a spring peak of 89 beats min(-1) at the beginning of May. LA increased from 23% per day during December and January to a mean level of 53% per day during May, and declined again thereafter. Daily mean subcutaneous body temperature (T(s)) declined continuously during winter and reached a nadir at the beginning of April (annual range was 5.8 degrees C), well after the annual low of air temperature and LA. Lower T(s) during winter contributed considerably to the reduction in f(H). In addition to thermoregulation, f(H) was affected by reproduction, LA, HI and unexplained seasonal variation, presumably reflecting to some degree changes in organ mass. The observed phase relations of seasonal changes indicate that energy expenditure was not a consequence of energy uptake but is under endogenous control, preparing the organism well in advance of seasonal energetic demands.
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Witte, T. H., Knill, K., & Wilson, A. M. (2004). Determination of peak vertical ground reaction force from duty factor in the horse (Equus caballus). J Exp Biol, 207(Pt 21), 3639–3648.
Abstract: Measurement of peak vertical ground reaction force (GRFz) from multiple limbs simultaneously during high-speed, over-ground locomotion would enhance our understanding of the locomotor mechanics of cursorial animals. Here, we evaluate the accuracy of predicting peak GRFz from duty factor (the proportion of the stride for which the limb is in contact with the ground). Foot-mounted uniaxial accelerometers, combined with UHF FM telemetry, are shown to be practical and accurate for the field measurement of stride timing variables, including duty factor. Direct comparison with the force plate produces a mean error of 2.3 ms and 3.5 ms for the timing of foot on and foot off, respectively, across all gaits. Predictions of peak GRFz from duty factor show mean errors (with positive values indicating an overestimate) of 0.8+/-0.04 N kg(-1) (13%; N=42; mean +/- S.E.M.) at walk, -0.3+/-0.06 N kg(-1) (3%; N=75) at trot, -2.3+/-0.27 N kg(-1) (16%; N=18) for the non-lead limb at canter and +2.1+/-0.7 N kg(-1) (19%; N=9) for the lead limb at canter. The substantial over- and underestimate seen at canter, in the lead and non-lead limbs, respectively, is attributed to the different functions performed by the two limbs in the asymmetrical gaits. The difference in load experienced by the lead and non-lead limbs decreased with increasing speed.
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Dunn, M. F., & Branlant, G. (1975). Roles of zinc ion and reduced coenzyme in horse liver alcohol dehydrogenase catalysis. The mechanism of aldehyde activation. Biochemistry, 14(14), 3176–3182.
Abstract: 1,4,5,6-Tetrahydronicotinamide adenine dinucleotide (H2NADH) has been investigated as a reduced coenzyme analog in the reaction between trans-4-N,N-dimethylaminocinnamaldehyde (I) (lambdamax 398 nm, epsilonmax 3.15 X 10-4 M-minus 1 cm-minus 1) and the horse liver alcohol dehydrogenase-NADH complex. These equilibrium binding and temperature-jump kinetic studies establish the following. (i) Substitution of H2NADH for NADH limits reaction to the reversible formation of a new chromophoric species, lambdamax 468 nm, epsilonmax 5.8 x 10-4 M-minus 1 cm-minus 1. This chromophore is demonstrated to be structurally analogous to the transient intermediate formed during the reaction of I with the enzyme-NADH complex [Dunn, M. F., and Hutchison, J. S. (1973), Biochemistry 12, 4882]. (ii) The process of intermediate formation with the enzyme-NADH complex is independent of pH over the range 6.13-10.54. Although studies were limited to the pH range 5.98-8.72, a similar pH independence appears to hold for the H2NADH system. (iii) Within the ternary complex, I is bound within van der Waal's contact distance of the coenzyme nicotinamide ring. (iv) Formation of the transient intermediate does not involve covalent modification of coenzyme. Based on these findings, we conclude that zinc ion has a Lewis acid function in facilitating the chemical activation of the aldehyde carbonyl for reduction, and that reduced coenzyme plays a noncovalent effector role in this substrate activating step.
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Ordakowski-Burk, A. L., Quinn, R. W., Shellem, T. A., & Vough, L. R. (2006). Voluntary intake and digestibility of reed canarygrass and timothy hay fed to horses. J. Anim Sci., 84(11), 3104–3109.
Abstract: Thousands of hectares of timothy (Phleum pretense L.) grown in the Mid-Atlantic region are infected by cereal rust mite (Abacarus hysterix) that causes discoloration and curling of leaves, decreased nutritional quality, and substantial decreases in yield. A decline in production of timothy hay can lower income for hay producers and cause horse owners to search for alternative hays. Low alkaloid reed canarygrass (Phalaris arundinacea L.) hay has potential as an alternative to timothy hay because it grows well in the Mid-Atlantic region, is believed to have a similar nutrient quality to timothy, and is not as susceptible to cereal rust mite. Eleven mature, stalled Thoroughbred geldings (549 +/- 12.1 kg) that were exercised daily were used to compare voluntary DMI and apparent nutrient DM digestibility of timothy and low-alkaloid Chiefton variety reed canarygrass hay. Horses were paired by age and BW and randomly assigned to timothy or reed canarygrass hay during a 14-d period to measure voluntary DMI followed by a 4-d period to measure apparent DM digestibility. Both hays met the minimum requirements for DE, CP, Ca, P, K, Fe, and Mn, but they did not meet the minimum requirements for Cu, Zn, and Na for horses at maintenance and averaging 550 kg of BW. Timothy hay seemed to have a lower CP concentration (14.4%) compared with reed canarygrass hay (17.1%) and a more desirable Ca:P ratio at 1.6:1 compared with 0.8:1 for reed canarygrass hay. Horses fed timothy consumed more hay (P <0.001) during the voluntary DMI period compared with horses fed reed canarygrass. Greater voluntary DMI of timothy occurred on d 1, 3, and 5 (P <0.05), but DMI was similar for other days. Apparent DM digestibility was greater in horses fed timothy hay by 9.6% compared with horses fed reed canarygrass hay (P <0.05). Horses fed timothy had greater DM digestibility of ADF (P = 0.001), NDF (P = 0.001), sugar (P = 0.05), and Ca (P = 0.001) but lower apparent DM digestibility of CP (P = 0.012) and crude fat (P = 0.004). Timothy hay was superior in voluntary DMI and apparent DM digestibility compared with low-alkaloid reed canarygrass hay fed to horses.
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Skedros, J. G., Dayton, M. R., Sybrowsky, C. L., Bloebaum, R. D., & Bachus, K. N. (2006). The influence of collagen fiber orientation and other histocompositional characteristics on the mechanical properties of equine cortical bone. J Exp Biol, 209(Pt 15), 3025–3042.
Abstract: This study examined relative influences of predominant collagen fiber orientation (CFO), mineralization (% ash), and other microstructural characteristics on the mechanical properties of equine cortical bone. Using strain-mode-specific (S-M-S) testing (compression testing of bone habitually loaded in compression; tension testing of bone habitually loaded in tension), the relative mechanical importance of CFO and other material characteristics were examined in equine third metacarpals (MC3s). This model was chosen since it had a consistent non-uniform strain distribution estimated by finite element analysis (FEA) near mid-diaphysis of a thoroughbred horse, net tension in the dorsal/lateral cortices and net compression in the palmar/medial cortices. Bone specimens from regions habitually loaded in tension or compression were: (1) tested to failure in both axial compression and tension in order to contrast S-M-S vs non-S-M-S behavior, and (2) analyzed for CFO, % ash, porosity, fractional area of secondary osteonal bone, osteon cross-sectional area, and population densities of secondary osteons and osteocyte lacunae. Multivariate multiple regression analyses revealed that in S-M-S compression testing, CFO most strongly influenced total energy (pre-yield elastic energy plus post-yield plastic energy); in S-M-S tension testing CFO most strongly influenced post-yield energy and total energy. CFO was less important in explaining S-M-S elastic modulus, and yield and ultimate stress. Therefore, in S-M-S loading CFO appears to be important in influencing energy absorption, whereas the other characteristics have a more dominant influence in elastic modulus, pre-yield behavior and strength. These data generally support the hypothesis that differentially affecting S-M-S energy absorption may be an important consequence of regional histocompositional heterogeneity in the equine MC3. Data inconsistent with the hypothesis, including the lack of highly longitudinal collagen in the dorsal-lateral ;tension' region, paradoxical histologic organization in some locations, and lack of significantly improved S-M-S properties in some locations, might reflect the absence of a similar habitual strain distribution in all bones. An alternative strain distribution based on in vivo strain measurements, without FEA, on non-Thoroughbreds showing net compression along the dorsal-palmar axis might be more characteristic of the habitual loading of some of the bones that we examined. In turn, some inconsistencies might also reflect the complex torsion/bending loading regime that the MC3 sustains when the animal undergoes a variety of gaits and activities, which may be representative of only a portion of our animals, again reflecting the possibility that not all of the bones examined had similar habitual loading histories.
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A. Lanata, A. Guidi, G. Valenza, P. Baragli, & E. P. Scilingo. (2016). Quantitative heartbeat coupling measures in human-horse interaction. In 2016 38th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC) (pp. 2696–2699). 2016 38th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (E.
Abstract: Abstract— We present a study focused on a quantitative estimation of a human-horse dynamic interaction. A set of measures based on magnitude and phase coupling between heartbeat dynamics of both humans and horses in three different conditions is reported: no interaction, visual/olfactory interaction and grooming. Specifically, Magnitude Squared Coherence (MSC), Mean Phase Coherence (MPC) and Dynamic Time Warping (DTW) have been used as estimators of the amount of coupling between human and horse through the analysis of their heart rate variability (HRV) time series in a group of eleven human subjects, and one horse. The rationale behind this study is that the interaction of two complex biological systems go towards a coupling process whose dynamical evolution is modulated by the kind and time duration of the interaction itself. We achieved a congruent and consistent
statistical significant difference for all of the three indices. Moreover, a Nearest Mean Classifier was able to recognize the three classes of interaction with an accuracy greater than 70%. Although preliminary, these encouraging results allow a discrimination of three distinct phases in a real human-animal interaction opening to the characterization of the empirically proven relationship between human and horse.
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Kida, H. (1997). [Ecology of influenza viruses in animals and the mechanism of emergence of new pandemic strains]. Nippon Rinsho, 55(10), 2521–2526.
Abstract: Ecological studies on influenza viruses revealed that the hemagglutinin genes are introduced into new pandemic strains from viruses circulating in migratory ducks through domestic ducks and pigs in southern China. Experimental infection of pigs with 38 avian influenza virus strains with H1-H13 hemagglutinins showed that at least one strain of each HA subtype replicated in the upper respiratory tract of pigs. Co-infection of pigs with a swine virus and with an avian virus generated reassortant viruses. The results indicate that avian viruses of any subtype can contribute genes in the generation of reassortants. Virological surveillance revealed that influenza viruses in waterfowl reservoir are perpetuated year-by-year in the frozen lake water while ducks are absent.
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