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.

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.

Abstract: A wireless acceleration measurement system was applied to free-moving cows and horses. Sensors were available as a collar and a flat box for measuring leg or trunk movements. Results were transmitted simultaneously by radio or stored in an 8-MB internal memory. As analytical procedures, frequency distributions with standard deviations, spectral analyses, and fractal analyses were applied. Bymeans of the collar sensor, basic behavior patterns (standing, grazing, walking, ruminating, drinking, and hay uptake) could be identified in cows. Lameness could be detected in cows and horses by means of the leg sensor. The portion of basic and harmonic spectral components was reduced; the fractal dimension was reduced. The system can be used for the detection and analysis of even small movements of free-moving humans or animals over several hours. It is convenient for the analysis of basic behaviors, emotional reactions, or events causing flight or fright or for comparing different housing elements, such as floors or fences.

Abstract: OBJECTIVE: To describe clinical findings, ultrasonographic features and outcome of injury to the branches of insertion of the superficial digital flexor (SDF) tendon in horses. DESIGN: Retrospective study of 14 cases. PROCEDURE: Fourteen Thoroughbred horses with tendonitis affecting the branches of insertion of the SDF tendon were examined for lameness, location and amount of swelling, and the presence of other musculoskeletal abnormalities. The flexor tendons were assessed by ultrasonographic examination, and recommendations were made for management of the cases. Outcome was assessed by re-examination of some horses, direct communication with the owner or trainer, and examination of race records. RESULTS: The lateral SDF branch was affected in 10 horses; the medial branch in three, and both branches in one horse. Two horses had concurrent injuries to the SDF tendon in the metacarpal region of the contralateral limb. Ultrasonographic findings included swelling of the affected SDF branch, peritendinous fluid accumulation, disruption of normal fibre alignment on sagittal scan, and variable loss of echogenicity. As healing occurred, there was return of normal echogenicity, but normal fibre alignment did not return completely and apparent adhesions formed between the affected SDF branch and adjacent structures. Seven of 10 horses which returned to their previous use were able to compete without further tendon injury. Recurrence of injury occurred in one case, and another two horses developed tendonitis in the metacarpal region. One horse was retired from racing but was able to compete at dressage without recurrence of injury. Two horses were retired for breeding without returning to training, and one horse was sold and lost to follow up but did not race. CONCLUSION: The prognosis is fair for return to previous use following injury to the branches of insertion of the SDF tendon in athletic horses.

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.