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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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Abbruzzetti, S., Crema, E., Masino, L., Vecli, A., Viappiani, C., Small, J. R., et al. (2000). Fast events in protein folding: structural volume changes accompanying the early events in the N-->I transition of apomyoglobin induced by ultrafast pH jump. Biophys J, 78(1), 405–415.
Abstract: Ultrafast, laser-induced pH jump with time-resolved photoacoustic detection has been used to investigate the early protonation steps leading to the formation of the compact acid intermediate (I) of apomyoglobin (ApoMb). When ApoMb is in its native state (N) at pH 7.0, rapid acidification induced by a laser pulse leads to two parallel protonation processes. One reaction can be attributed to the binding of protons to the imidazole rings of His24 and His119. Reaction with imidazole leads to an unusually large contraction of -82 +/- 3 ml/mol, an enthalpy change of 8 +/- 1 kcal/mol, and an apparent bimolecular rate constant of (0.77 +/- 0.03) x 10(10) M(-1) s(-1). Our experiments evidence a rate-limiting step for this process at high ApoMb concentrations, characterized by a value of (0. 60 +/- 0.07) x 10(6) s(-1). The second protonation reaction at pH 7. 0 can be attributed to neutralization of carboxylate groups and is accompanied by an apparent expansion of 3.4 +/- 0.2 ml/mol, occurring with an apparent bimolecular rate constant of (1.25 +/- 0.02) x 10(11) M(-1) s(-1), and a reaction enthalpy of about 2 kcal/mol. The activation energy for the processes associated with the protonation of His24 and His119 is 16.2 +/- 0.9 kcal/mol, whereas that for the neutralization of carboxylates is 9.2 +/- 0.9 kcal/mol. At pH 4.5 ApoMb is in a partially unfolded state (I) and rapid acidification experiments evidence only the process assigned to carboxylate protonation. The unusually large contraction and the high energetic barrier observed at pH 7.0 for the protonation of the His residues suggests that the formation of the compact acid intermediate involves a rate-limiting step after protonation.
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Pere, M. C. (1995). Maternal and fetal blood levels of glucose, lactate, fructose, and insulin in the conscious pig. J. Anim Sci., 73(10), 2994–2999.
Abstract: To study nutrition and metabolism in the fetal pig, a chronic catheterization method was developed that allows blood sampling in arteries and veins, at both the umbilical and uterine sources, in the conscious, unstressed animal. A catheter was inserted in the fetal aorta through a femoral artery, and another one was introduced in the umbilical vein. A catheter was put in a femoral artery of the sow so that its end was in the abdominal aorta. A fourth catheter was placed in a uterine vein draining the fetoplacental unit studied. This procedure was applied to 18 Large White primiparous sows at 99 d of gestation. Blood samples were drawn simultaneously using the four catheters before a meal at 103 d of pregnancy, and glucose, insulin, lactate, and fructose were determinated. Glycemia was 2.5 times higher in the sow than in the fetus. The extraction coefficient of glucose by the fetus amounted to 14% of the umbilical supply. The insulin level in the fetal pig was very low ( < 5 microU/mL). Lactate and fructose seemed to originate from the placenta. Blood lactate was 2.6 times lower in the sow than in the fetus, and its extraction coefficient by the fetus amounted to 8%. Fructose in the fetal blood was 2.3 times higher than that of glucose. Fructose was not utilized by the pig fetus. The present results obtained in the fetal pig are comparable to the conclusions drawn from studies with other species.
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Heistermann, M., Palme, R., & Ganswindt, A. (2006). Comparison of different enzyme-immunoassays for assessment of adrenocortical activity in primates based on fecal analysis. Am. J. Primatol., 68(3), 257–273.
Abstract: Most studies published to date that used fecal glucocorticoid measurements to assess adrenocortical activity in primate (and many nonprimate) species applied a specific cortisol or corticosterone assay. However, since these native glucocorticoids are virtually absent in the feces of most vertebrates, including primates, the validity of this approach has recently been questioned. Therefore, the overall aim of the present study was to assess the validity of four enzyme-immunoassays (EIAs) using antibodies raised against cortisol, corticosterone, and reduced cortisol metabolites (two group-specific antibodies) for assessing adrenocortical activity using fecal glucocorticoid metabolite (GCM) measurements in selected primate species (marmoset, long-tailed macaque, Barbary macaque, chimpanzee, and gorilla). Using physiological stimulation of the hypothalamo-pituitary-adrenocortical (HPA) axis by administering exogenous ACTH or anesthesia, we demonstrated that at least two assays detected the predicted increase in fecal GCM levels in response to treatment in each species. However, the magnitude of response varied between assays and species, and no one assay was applicable to all species. While the corticosterone assay generally was of only limited suitability for assessing glucocorticoid output, the specific cortisol assay was valuable for those species that (according to high-performance liquid chromatography (HPLC) analysis data) excreted clearly detectable amounts of authentic cortisol into the feces. In contrast, in species in which cortisol was virtually absent in the feces, group-specific assays provided a much stronger signal, and these assays also performed well in the other primate species tested (except the marmoset). Collectively, the data suggest that the reliability of a given fecal glucocorticoid assay in reflecting activity of the HPA axis in primates clearly depends on the species in question. Although to date there is no single assay system that can be used successfully across species, our data suggest that group-specific assays have a high potential for cross-species application. Nevertheless, regardless of which GC antibody is chosen, our study clearly reinforces the necessity of appropriately validating the respective assay system before it is used.
Keywords: 11-Hydroxycorticosteroids/*analysis; Adrenocorticotropic Hormone/pharmacology; Anesthesia; Animals; Corticosterone/analysis; Feces/*chemistry; Glucocorticoids/*analysis; Haplorhini/*metabolism; Hydrocortisone/analysis; Hypothalamo-Hypophyseal System/drug effects/physiology; Immunoenzyme Techniques/*methods; Pituitary-Adrenal System/drug effects/physiology; Species Specificity
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Permyakov, S. E., Khokhlova, T. I., Nazipova, A. A., Zhadan, A. P., Morozova-Roche, L. A., & Permyakov, E. A. (2006). Calcium-binding and temperature induced transitions in equine lysozyme: new insights from the pCa-temperature “phase diagrams”. Proteins, 65(4), 984–998.
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.
Keywords: Animals; Apoproteins/chemistry/metabolism; Binding Sites; Calcium/chemistry/*metabolism; Cattle; Edetic Acid/metabolism; Horses/metabolism; Hydrogen-Ion Concentration; Lactalbumin/chemistry/metabolism; Muramidase/*chemistry/*metabolism; Protein Denaturation; Spectrometry, Fluorescence; *Temperature; Thermodynamics; Tryptophan/chemistry/metabolism
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Petter-Puchner, A. H., Froetscher, W., Krametter-Froetscher, R., Lorinson, D., Redl, H., & van Griensven, M. (2007). The long-term neurocompatibility of human fibrin sealant and equine collagen as biomatrices in experimental spinal cord injury. Exp Toxicol Pathol, 58(4), 237–245.
Abstract: INTRODUCTION: While fibrin sealant (FS) and equine collagen (EC) have been used as scaffold materials in experimental spinal cord injury (SCI), questions concerning neurocompatibility still remain. In this study, we assessed potential adverse effects, as well as functional and histological impact of FS and EC in subtotal hemisection of the thoracic spinal cord (SC) in rats. METHODS: 124 male rats were randomly assigned to four main groups (n=31): Sham (SH), Lesion only (L), fibrin sealant (GFS) and equine collagen group (GEC). SH animals received laminectomy only; all other animals underwent subtotal lateral hemisection at T9. Treatment consisted of application of FS or EC into the lesion gap in GFS and GEC, which was left empty in L. GFS, GEC, L and SH were each further divided into 4 subgroups: One subgroup, consisting of 10 rats was subjected to behavioural and reflex testing before surgery and followed up on days 1,7, 14, 21, 28 post op and then sacrificed. Haemalaun or cresyl violet (CV) was used to identify neutrophils in parasagittal cord sections which were obtained on day 1 (n=7). Sections stained for quantification of microglia/macrophages using ED-1 on day 3 (n=7), day 7 (n=7) and day 28 (n=7 out of 10). Additionally, neural filament (NF) staining was chosen to detect axonal regeneration and the length of ingrowth into FS and EC, Luxol blue for myelination, Von Willebrand factor for vascularisation, and glial fibrillary acidic protein (GFAP) staining for detection of astrocytes in glial scars on day 28. RESULTS: No adverse effects were observed in the treatment groups. Compared to L, GFS and GEC performed significantly better in the Basso, Beattie, Bresnahan (BBB) score and hopping responses. Proprioceptive placing was markedly improved in FS and EC compared to L. Axonal regrowth was found in GFS and GEC--the regrowth in the GFS was accompanied by myelination and vascularisation. Glial scarring occurred in all groups. Discussion Both biomatrices improved functional recovery compared to L and no adverse effects were perceived.
Keywords: Animals; Axotomy; Biocompatible Materials/*therapeutic use; Collagen/*therapeutic use; Fibrin Tissue Adhesive/*therapeutic use; Horses; Humans; Immunohistochemistry; Male; Motor Activity/physiology; Nerve Regeneration/*physiology; Rats; Recovery of Function; Spinal Cord/pathology/physiology; Spinal Cord Injuries/pathology/*therapy; Thoracic Vertebrae
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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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Touma, C., Palme, R., & Sachser, N. (2004). Analyzing corticosterone metabolites in fecal samples of mice: a noninvasive technique to monitor stress hormones. Horm Behav, 45(1), 10–22.
Abstract: In small animals like mice, the monitoring of endocrine functions over time is constrained seriously by the adverse effects of blood sampling. Therefore, noninvasive techniques to monitor, for example, stress hormones in these animals are highly demanded in laboratory as well as in field research. The aim of our study was to evaluate the biological relevance of a recently developed technique to monitor stress hormone metabolites in fecal samples of laboratory mice. In total, six experiments were performed using six male and six female mice each. Two adrenocorticotropic hormone (ACTH) challenge tests, two dexamethasone (Dex) suppression tests and two control experiments [investigating effects of the injection procedure itself and the diurnal variation (DV) of glucocorticoids (GCs), respectively] were conducted. The experiments clearly demonstrated that pharmacological stimulation and suppression of adrenocortical activity was reflected accurately by means of corticosterone metabolite (CM) measurements in the feces of males and females. Furthermore, the technique proved sensitive enough to detect dosage-dependent effects of the ACTH/Dex treatment and facilitated to reveal profound effects of the injection procedure itself. Even the naturally occurring DV of GCs could be monitored reliably. Thus, our results confirm that measurement of fecal CM with the recently established 5alpha-pregnane-3beta,11beta,21-triol-20-one enzyme immunoassay is a very powerful tool to monitor adrenocortical activity in laboratory mice. Since mice represent the vast majority of all rodents used for research worldwide and the number of transgenic and knockout mice utilized as animal models is still increasing, this noninvasive technique can open new perspectives in biomedical and behavioral science.
Keywords: Adrenal Cortex/drug effects; Adrenal Cortex Function Tests; Adrenocorticotropic Hormone/pharmacology; Analysis of Variance; Animals; Circadian Rhythm; Corticosterone/*analysis/metabolism; Dexamethasone/pharmacology; Feces/*chemistry; Female; Immunoenzyme Techniques/*methods; Male; Mice; Mice, Inbred C57BL; Models, Animal; Reproducibility of Results; Stress, Psychological/*metabolism
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Schwarzenberger, F., Mostl, E., Bamberg, E., Pammer, J., & Schmehlik, O. (1991). Concentrations of progestagens and oestrogens in the faeces of pregnant Lipizzan, trotter and thoroughbred mares. J Reprod Fertil Suppl, 44, 489–499.
Abstract: Faecal samples were collected at weekly intervals from pregnant Lipizzan mares during Weeks 7-16 following mating and from Lipizzan, Trotter and Thoroughbred mares during the last 3 months of gestation. After parturition, samples were taken daily from the Thoroughbred mares for another 6 days. Non-pregnant mares served as controls. The concentrations of unconjugated oestrogens (Eg), 20 alpha-OH-progestagens (20 alpha-G) and 20 beta-OH-progestagens (20 beta-G) were measured by enzyme immunoassay. In the faeces of Lipizzan mares, immunoreactive progestagens were significantly (P less than 0.01) elevated above the levels in non-pregnant mares by Week 11, and Eg by Week 13 of pregnancy onwards. During the last 3 months of gestation, concentrations of Eg were significantly higher in Trotter mares than in Lipizzan and Thoroughbred mares. Concentrations of 20 alpha-G and 20 beta-G increased to maximal values in the last month of gestation. There was no significant difference among the 3 breeds with respect to 20 alpha-G but, during the 10 weeks before parturition, concentrations of 20 beta-G in the Lipizzan mares were significantly lower (P less than 0.05) than those in the Thoroughbred mares. They were also significantly lower than those of the Trotter mares during the last 4 weeks of gestation. After parturition, the concentrations of Eg and progestagens had declined to baseline values by Days 3 and 4 respectively. From these results we conclude that high concentrations of progestagens with 20 alpha- and 20 beta-hydroxyl groups are present in the faeces of pregnant mares, especially during the last month of gestation.
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Lucas, Z., Raeside, J. I., & Betteridge, K. J. (1991). Non-invasive assessment of the incidences of pregnancy and pregnancy loss in the feral horses of Sable Island. J Reprod Fertil Suppl, 44, 479–488.
Abstract: Field observations of 400 totally unmanaged feral horses on Sable Island, Nova Scotia, were complemented by oestrogen determinations in faecal samples from 154 identified females over a 4-year period (454 mare-years). Of mares that were sampled throughout the year and subsequently produced foals, 92.1% exhibited elevated faecal oestrogens between 15 October and 30 March. The results confirm that faecal oestrogens are a useful indicator of pregnancy after approximately 120 days gestation. Distribution of foaling resembled that seen in other feral populations, with 95% of births occurring from April through July. The foaling rate for mares aged 3 years or older was 62.0%, with 50.7% of mares foaling in 3 or 4 years. Foaling rates were low (4.1%) in mares bred as yearlings and rose with age to 70.8% in those bred as 4-year-olds. Fetal loss after Day 120 was deduced from faecal oestrogens to be 26.0% overall, with marked variation from year to year (9.6-37.3%) and with age (70.0% in those bred as yearlings, decreasing to 5.6% in those bred as 4-year-olds). Of 58 mares aged 2 years or older that were sampled every year, about half (49.6%) the barren years were attributable to fetal loss after 120 days gestation. All mares conceived in at least 2 of the 4 years, suggesting that pregnancy loss, even after Day 120, is as important as failure to conceive in causing barren years.
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