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.

Abstract: Association equilibria and association kinetics of the thiocyanate binding reaction to methemoglobin in single crystals and solution are studied using temperature-jump technique and polarized absorption spectroscopy. Different kinetic constants are found for the reaction in solution and crystal phase for the alpha- and beta-subunits of the methemoglobin tetramer. The reduction of the reactivity of the alpha- and beta-subunits in crystalline phase is 6-fold and 2.4-fold, respectively, compared to the values found in solution. The intramolecular binding reaction of the N epsilon of the distal histidine E7 which is observed in methemoglobin in solution cannot be detected in single crystals. Our results suggest that crystallization of hemoglobin has little influence on small-scale structural fluctuations which are necessary for ligands to get to the binding sites and large-scale structural motions are suppressed.

Abstract: The refolding kinetics of horse cyanometmyoglobin induced by concentration jump of urea was investigated by five optical probe stopped-flow methods: absorption at 422 nm, tryptophyl fluorescence at around 340 nm, circular dichroism (CD) at 222 nm, CD at 260 nm, and CD at 422 nm. In the refolding process, we detected three phases with rate constants of > 1 x 10(2) s-1, (4.5-9.3) s-1, and (2-5) x 10(-3) s-1. In the fastest phase, a substantial amount of secondary structure (approximately 40%) is formed within the dead time of the CD stopped-flow apparatus (10.7 ms). The kinetic intermediate populated in the fastest phase is shown to capture a hemindicyanide, suggesting that a “heme pocket precursor” recognized by hemindicyanide must be constructed within the dead time. In the middle phase, most of secondary and tertiary structures, especially around the captured hemindicyanide, have been constructed. In the slowest phase, we detected a minor structural rearrangement accompanying the ligand-exchange reaction in the fifth coordination of ferric iron. We present a possible model for the refolding process of myoglobin in the presence of the heme group.

Abstract: Regulation of local overpopulations of free-roaming feral equids is in demand worldwide for ecological balance and habitat preservation. Contraceptive vaccines have proven effective in feral horses, which breed seasonally, but no data are available for equids such as the burro, which is reproductively active all year round. In the present study, 27 individually identified female feral burros (Equus asinus) roaming free in Virgin Islands National Park (St John, US Virgin Islands; Lesser Antilles) were remotely treated with pig zonae pellucidae (PZP) vaccine. Between January and May, 16 burros were darted with a 1 ml emulsion of PZP plus Freund's adjuvant. Ten to twelve months later each treated burro was given a single booster injection of PZP plus adjuvant to maintain contraception through a second year. Eleven adult untreated jennies served as controls. Beginning one year after initial vaccination, these burros were monitored for pregnancy and foal production. Collection of data to determine treatment effect was not begun until 12 months after initial treatment to ensure that pregnancies existing before vaccination were not included. Pregnancy was assessed using previously validated methods for steroid metabolite measurement in fresh faecal samples. None of the PZP-treated burros produced foals between 0 and 12 months after the last inoculation. One PZP-treated burro tested positive for pregnancy at 10 months after the final inoculation. During this same period, six of 11 untreated burros tested pregnancy-positive, and four were observed with foals. There was no difference in pregnancy rates among treated, control and randomly sampled jennies between 12 and 24 months after the last inoculation. The results demonstrate that, in free-roaming feral burros that are reproductively active all year round: (1) burros can be accessed for remotely delivered PZP vaccination; (2) PZP contraception is effective; (3) PZP contraception is reversible; and (4) pregnancy can be reliably detected by faecal steroid analysis.