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.

Abstract: The most universal approach to the studies of metal binding properties of single-site metal binding proteins, i.e., construction of a “phase diagram” in coordinates of free metal ion concentration-temperature, has been applied to equine lysozyme (EQL). EQL has one relatively strong calcium binding site and shows two thermal transitions, but only one of them is Ca(2+)-dependent. It has been found that the Ca(2+)-dependent behavior of the low temperature thermal transition (I) of EQL can be adequately described based upon the simplest four-states scheme of metal- and temperature-induced structural changes in a protein. All thermodynamic parameters of this scheme were determined experimentally and used for construction of the EQL phase diagram in the pCa-temperature space. Comparison of the phase diagram with that for alpha-lactalbumin (alpha-LA), a close homologue of lysozyme, allows visualization of the differences in thermodynamic behavior of the two proteins. The thermal stability of apo-EQL (transition I) closely resembles that for apo-alpha-LA (mid-temperature 25 degrees C), while the thermal stabilities of their Ca(2+)-bound forms are almost indistinguishable. The native state of EQL has three orders of magnitude lower affinity for Ca(2+) in comparison with alpha-LA while its thermally unfolded state (after the I transition) has about one order lower (K = 15M(-1)) affinity for calcium. Circular dichroism studies of the apo-lysozyme state after the first thermal transition show that it shares common features with the molten globule state of alpha-LA.

Abstract: Heart rate, arterial blood pressures, respiratory rate, body temperature, and central nervous system excitement were compared before and after epidural administration of morphine (0.1 mg/kg), butorphanol (0.08 mg/kg), alfentanil (0.02 mg/kg), tramadol (1.0 mg/kg), the k-opioid agonist U50488H (0.08 mg/kg), or sterile water using an incomplete Latin square crossover design in five conscious adult horses. Treatments were administered into the first intercoccygeal epidural space. Significant (P <.05) reductions in respiratory rate were detected after epidural administration of morphine, alfentanil, U50488H, and sterile water. Additionally, significant (P <.05) head ptosis was observed within the first hour after administration of morphine, U50488H, and tramadol, but neither of these changes appeared to be of clinical significance. No treatment-related changes in motor activity or behavior were observed.

Abstract: The acidic transition of porcine plasminogen, prepared by affinity chromatography, was studied by non-destructive methods. These methods are based on the analysis of the behaviour of the tryptophyls under various conditions. The perturbation of the absorption and emission spectra by pH or temperature and the dynamic quenching of the intrinsic fluorescence are used to obtain information on structural changes which affect the environment of these residues. It is shown that by decreasing pH the fluorescence emission spectra are shifted toward the long wavelengths, with a broadening of the fluorescence band. The same effect can be obtained at constant pH by heating the protein solution. In order to analyze these phenomena, it is assumed that the fluorescence intensities at 355 nm and 328 nm reflect the proportion of the tryptophans which are exposed to the solvent, and buried, respectively. The plot of the ratio of the fluorescence intensities at these wavelengths versus pH or temperature leads to a titration curve showing an unmasking of tryptophans. The proportion of exposed tryptophans is measured by the dynamic fluorescence quenching technique and the data analyzed according to Lehrer. The plot of the fraction of exposed tryptophyls versus pH also shows the unmasking of these chromophores. Thermal perturbation of a solution of plaminogen at neutral pH induces a difference absorption spectrum whose amplitudes at the maxima are proportional to the number of exposed aromatic residues. The comparison with a solution of fully denatured plasminogen in 6 M guanidium chloride, where all the tryptophyls are exposed, shows that the percentage of exposure is equal to 59%. This number is significantly higher than the percentage found by the fluorescence quenching technique (20%), indicating that some tryptophyls are located in crevices, exposed to the solvent but not to the iodide. At acidic pH the absorption difference spectra induced by thermal perturbation are not classical, since they show an inversion and a new band between 300 nm and 305 nm. This band is mentioned in the literature as a minor band of tryptophan which appears when this chromophore is located in an asymmetric environment. On plotting the maximum amplitude of these spectra obtained at acidic pH versus temperature, we obtain a curve indicating that two types of antagonistic interactions are involved in the perturbation of the chromophores spectra. The spectrophotometric titration of plasminogen gives classical absorption difference spectra. By plotting the maximum amplitude at 292 nm versus pH, we obtain a titration curve with an apparent pK of 2.9 units. This pK is acidic which respect to the pK value of a normal carboxyl. This low value can be due to a positively charged group in the neighbourhood of a carboxyl, which interacts with one or more chromophores. When the carboxyl becomes protonated, this positively charged group is free and available to perturb the environment of some chromophores...