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Elevated environmental temperatures can induce heat stress which could reduce fertility and early embryonic development. Fatty acids can initiate an endergonic reaction that absorbs cellular heat and decreases intracellular temperature. This study's objective was to minimize heat stress-induced damage to in vitro matured oocytes by supplementing maturation media with either 50 μM linoleic or linolenic acid or both (25 or 50 μM) during maturation at either 38.5 or 41.5°C. Oocytes were evaluated for intracellular antioxidative pathways, fertilization characteristics, or early embryonic development. Elevated maturation temperatures increased (p < 0.05) reactive oxygen species (ROS) formation and supplementation with linoleic or linolenic acid decreased (p < 0.05) ROS in oocytes matured at 41.5°C. Maturation temperature had an effect (p < 0.05) on the intracellular antioxidative pathways of the oocyte except for glutathione peroxidase activity. Regardless of maturation temperature, supplementation with linoleic or linolenic acid increased (p < 0.05) the enzyme activities and glutathione concentrations in the oocytes compared to no fatty acid supplementation. Supplementation of both linoleic and linolenic acid decreased (p < 0.05) polyspermic fertilization rates. Supplementing either 25 or 50 μM linoleic and linolenic acid to maturing oocytes at 41.5°C increased (p < 0.05) cleavage rates by 48 h after IVF and the blastocyst formation rates by 144 h after IVF compared to other treatments. Oocytes matured at 38.5°C had greater (p < 0.05) embryonic development than those matured at 41.5°C except for those supplemented with 50 μM linoleic and linolenic acid. Supplementing 50 μM linoleic and linolenic acid to the maturation medium of pig oocytes reduces the effects of heat stress-induced damage., (© 2022. The Society for In Vitro Biology.)

The objective of this study was to reduce the negative effects of oxidative stress by decreasing the levels of reactive oxygen species (ROS) through supplementation of the major antioxidants present in elderberries: kuromanin and cyanidin. Oocytes (n = 1150) were supplemented with 100 or 200 μM of kuromanin or cyanidin during maturation, and then evaluated for ROS levels or fertilized and evaluated for penetration, polyspermic penetration, male pronucleus formation, and embryonic development. The ROS levels and incidence of polyspermic penetration were lower (P < 0.05) in oocytes supplemented with 100 μM cyanidin when compared with other treatments. Supplementation of 100 μM cyanidin increased (P < 0.05) MPN and blastocyst formation compared with other treatments. However, supplementation of 100 μM kuromanin did not have significant effects on the criteria evaluated, and supplementation of 200 μM kuromanin had significant (P < 0.05) detrimental effects for each criterion. Additional oocytes (n = 1438) were supplemented with 100 μM cyanidin during maturation and evaluated for glutathione, glutathione peroxidase, catalase, and superoxide dismutase activity. Supplementation of 100 μM cyanidin increased (P < 0.05) catalase activity and intracellular GSH levels compared with no supplementation of cyanidin. These results indicate that supplementing cyanidin during maturation reduces oxidative stress by reducing ROS levels and increasing GSH concentrations within the oocyte.

The cellular prion protein (PrP(C)) is a Cu(2+) binding protein connected to the outer cell membrane. The molecular features of the Cu(2+) binding sites have been investigated and characterized by spectroscopic experiments on PrP(C)-derived peptides and the recombinant human full-length PrP(C )(hPrP-[23-231]). The hPrP-[23-231] was loaded with (63)Cu under slightly acidic (pH 6.0) or neutral conditions. The PrP(C)/Cu(2+)-complexes were investigated by extended X-ray absorption fine structure (EXAFS), electron paramagnetic resonance (EPR), and electron nuclear double resonance (ENDOR). For comparison, peptides from the copper-binding octarepeat domain were investigated in different environments. Molecular mechanics computations were used to select sterically possible peptide/Cu(2+) structures. The simulated EPR, ENDOR, and EXAFS spectra of these structures were compared with our experimental data. For a stoichiometry of two octarepeats per copper the resulting model has a square planar four nitrogen Cu(2+) coordination. Two nitrogens belong to imidazole rings of histidine residues. Further ligands are two deprotonated backbone amide nitrogens of the adjacent glycine residues and an axial oxygen of a water molecule. Our complex model differs significantly from those previously obtained for shorter peptides. Sequence context, buffer conditions and stoichiometry of copper show marked influence on the configuration of copper binding to PrP(C).

Since high-intensity synchrotron radiation is available, "extended X-ray absorption fine structure" spectroscopy (EXAFS) is used for detailed structural analysis of metal ion environments in proteins. However, the information acquired is often insufficient to obtain an unambiguous picture. ENDOR spectroscopy allows the determination of hydrogen positions around a metal ion. However, again the structural information is limited. In the present study, a method is proposed which combines computations with spectroscopic data from EXAFS, EPR, electron nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM). From EXAFS a first picture of the nearest coordination shell is derived which has to be compatible with EPR data. Computations are used to select sterically possible structures, from which in turn structures with correct H and N positions are selected by ENDOR and ESEEM measurements. Finally, EXAFS spectra are re-calculated and compared with the experimental data. This procedure was successfully applied for structure determination of the Cu(2+) complex of the octapeptide repeat of the human prion protein. The structure of this octarepeat complex is rather similar to a pentapeptide complex which was determined by X-ray structure analysis. However, the tryptophan residue has a different orientation: the axial water is on the other side of the Cu.

In the physiological form, the prion protein is a glycoprotein tethered to the cell surface via a C-terminal glycosylphosphatidylinositol anchor, consisting of a largely alpha-helical globular C-terminal domain and an unstructured N-terminal portion. This unstructured part of the protein contains four successive octapeptide repeats, which were shown to bind up to four Cu(2+) ions in a cooperative manner. To mimic the location of the protein on the cell membrane and to analyze possible structuring effects of the lipid/water interface, the conformational preferences of a single octapeptide repeat and its tetrameric form, as well of the fragment 92-113, proposed as an additional copper binding site, were comparatively analyzed in aqueous and dodecylphosphocholine micellar solution as a membrane mimetic. While for the downstream fragment 92-113 no conformational effects were detectable in the presence of DPC micelles by CD and NMR, both the single octapeptide repeat and, in an even more pronounced manner, its tetrameric form are restricted into well-defined conformations. Because of the repetitive character of the rigid structural subdomain in the tetrarepeat molecule, the spatial arrangement of these identical motifs could not be resolved by NMR analysis. However, the polyvalent nature of the repetitive subunits leads to a remarkably enhanced interaction with the micelles, which is not detectably affected by copper complexation. These results strongly suggest interactions of the cellular form of PrP (PrP(c)) N-terminal tail with the cell membrane surface at least in the octapeptide repeat region with preorganization of these sequence portions for copper complexation. There are sufficient experimental facts known that support a physiological role of copper complexation by the octapeptide repeat region of PrP(c) such as a copper-buffering role of the PrP(c) protein on the extracellular surface., (Copyright 2004 Wiley Periodicals, Inc.)

The binding of vanadate (V) to human serum albumin (HSA) in infusion solutions, to human fresh frozen plasma (FFP), and to human transferrin (TF) was investigated over a wide concentration range. Free V concentrations were obtained by ultrafiltration. Total and free V concentrations were determined using electrothermal atomic absorption spectrometry (ETAAS). Binding parameters were obtained by non-linear regression. V only bound appreciably to HSA at low concentrations (<1 microM). The binding capacity of HSA was about 1000-fold lower than that of FFP and TF per mole of protein. Binding to FFP and TF in the concentration range investigated could be described by a combination of saturable and additional non-saturable binding. The respective maximal binding capacities (B(max), microM), dissociation constants (k(D), microM), and proportionality constants (C) for the non-saturable, linear binding were B(max)=27, k(D)=2.5, C=0.19 for FFP and B(max)=47, k(D)=0.47, C=0.38 for TF. The results suggest that V is predominantly bound to transferrin in FFP. It is concluded that HSA in infusion solutions represents a reservoir of readily accessible V. Nevertheless, given the high binding capacity of transferrin in plasma, the amount of vanadate delivered via the brief administration of HSA solutions is unlikely to be of major importance.

Mössbauer, 57Fe ENDOR, CW and pulsed EPR experiments were performed on the reduced and the oxidized high-potential iron proteins (HiPIPs) of the wild type (WT) and the C77S mutant from Chromatium vinosum. The EPR spectra of the oxidized WT and mutant show three species respectively having nearly the same g-values but strongly changed spectral contributions. Relaxation times were estimated for oxidized WT and mutant at T = 5 K with pulsed EPR. A-tensor components of both iron pairs were obtained by 57Fe ENDOR, proving a similar magnetic structure for the WT and the mutant. Electronic relaxation has to be taken into account at T = 5 K in native and mutated oxidized HiPIPs to achieve agreement between Mössbauer and 57Fe ENDOR spectroscopies. The Mössbauer spectroscopy shows that the oxidized cluster contains a pure ferric and a mixed-valence iron pair coupled antiparallel. While all cluster irons from reduced C. vinosum WT are indistinguishable in the Mössbauer spectrum, the reduced C77S mutant shows a non-equivalence between the serine-bound and the three cysteine-ligated iron ions. The Mössbauer parameters confirm a loss of the covalent character of the iron bond when S is replaced by O and indicate a shift of the cluster's electron cloud towards the serine. Mössbauer spectra of the oxidized mutant can be simulated with two models: model I introduces a single electronic isomer with the serine always ligated to a ferric iron. Model II assumes two equally populated electronic isomers with the serine ligated to a ferric iron and a mixed-valence iron, respectively. The latter model is in better agreement with EPR and NMR.

Mössbauer spectra of the oxidized [Fe4S4]3+ and the reduced [Fe4S4]2+ clusters in the high-potential iron protein I from Ectothiorhodospira halophila were measured in a temperature range from 5 K to 240 K. EPR measurements and 57Fe electron-nuclear double resonance (ENDOR) experiments were carried out with the oxidized protein. In the oxidized state the cluster has a net spin S = 1/2 and is paramagnetic. As common in [Fe4S4]3+ clusters, the Mössbauer spectrum was simulated with two species contributing equally to the absorption area: two Fe3+ atoms couple to the "ferric-ferric" pair, and one Fe2+ and one Fe3+ atom give the "ferric-ferrous pair". For the simulation of the Mössbauer spectrum, g-values were taken from EPR measurements. A-tensor components were determined by 57Fe ENDOR experiments that turned out to be a necessary source of estimating parameters independently. In order to obtain a detailed agreement of Mössbauer and ENDOR data, electronic relaxation has to be taken into account. Relaxing the symmetry condition in a way that the electric field gradient tensor does not coincide with g- and A-tensors yielded an even better agreement of experimental and theoretical Mössbauer spectra. Spin-spin and spinlattice relaxation times were estimated by pulsed EPR; the former turned out to be the dominating mechanism at T = 5 K. Relaxation times measured by pulsed EPR and obtained from the Mössbauer fit were compared and yield nearly identical values. The reduced cluster has one additional electron and has a diamagnetic (S = 0) ground state. All the four irons are indistinguishable in the Mössbauer spectrum, indicating a mixed-valence state of Fe2.5+ for each.