Delta opioid receptor on equine sperm cells: subcellular localization and involvement in sperm motility analyzed by computer assisted sperm analyzer (CASA)

<p>Abstract</p> <p>Background</p> <p>Opioid receptors and endogenous opioid peptides act not only in the control of nociceptive pathways, indeed several reports demonstrate the effects of opiates on sperm cell motility and morphology suggesting the importance of these receptors in the modulation of reproduction in mammals. In this study we investigated the expression of delta opioid receptors on equine spermatozoa by western blot/indirect immunofluorescence and its relationship with sperm cell physiology.</p> <p>Methods</p> <p>We analyzed viability, motility, capacitation, acrosome reaction and mitochondrial activity in the presence of naltrindole and DPDPE by means of a computer assisted sperm analyzer and a fluorescent confocal microscope. The evaluation of viability, capacitation and acrosome reaction was carried out by the double CTC/Hoechst staining, whereas mitochondrial activity was assessed by means of MitoTracker Orange dye.</p> <p>Results</p> <p>We showed that in equine sperm cells, delta opioid receptor is expressed as a doublet of 65 and 50 kDa molecular mass and is localized in the mid piece of tail; we also demonstrated that naltrindole, a delta opioid receptor antagonist, could be utilized in modulating several physiological parameters of the equine spermatozoon in a dose-dependent way. We also found that low concentrations of the antagonist increase sperm motility whereas high concentrations show the opposite effect. Moreover low concentrations hamper capacitation, acrosome reaction and viability even if the percentage of cells with active mitochondria seems to be increased; the opposite effect is exerted at high concentrations. We have also observed that the delta opioid receptor agonist DPDPE is scarcely involved in affecting the same parameters at the employed concentrations.</p> <p>Conclusions</p> <p>The results described in this paper add new important details in the comprehension of the mammalian sperm physiology and suggest new insights for improving reproduction and for optimizing equine breeding.</p

Decoding of grasping information from neural signals recorded using peripheral intrafascicular interfaces

The restoration of complex hand functions by creating a novel bidirectional link between the nervous system and a dexterous hand prosthesis is currently pursued by several research groups. This connection must be fast, intuitive, with a high success rate and quite natural to allow an effective bidirectional flow of information between the user's nervous system and the smart artificial device. This goal can be achieved with several approaches and among them, the use of implantable interfaces connected with the peripheral nervous system, namely intrafascicular electrodes, is considered particularly interesting

Modulation of Antioxidant Defense in Farmed Rainbow Trout (Oncorhynchus mykiss) Fed with a Diet Supplemented by the Waste Derived from the Supercritical Fluid Extraction of Basil (Ocimum basilicum)

Phytotherapy is based on the use of plants to prevent or treat human and animal diseases. Recently, the use of essential oils and polyphenol-enriched extracts is also rapidly increasing in the aquaculture sector as a means of greater industrial and environmental sustainability. Previous studies assessed the antibacterial and antiparasitic effects of these bioactive compounds on fish. However, studies on the modulation of oxidative stress biomarkers are still scant to date. Thus, in this study, the modulation of antioxidant defense against oxidative stress exerted by fish diets supplemented with a basil supercritical extract (F1-BEO) was assessed in rainbow trout Oncorhynchus mykiss. The F1-BEO extracted with supercritical fluid extraction was added to the commercial feed flour (0.5, 1, 2, 3% w/w) and mixed with fish oil to obtain a suitable compound for pellet preparation. Fish were fed for 30 days. The levels of stress biomarkers such as superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, glutathione reductase, glyoxalase I, glyoxalase II, lactate dehydrogenase, glutathione and malondialdehyde showed a boost in the antioxidant pathway in fish fed with a 0.5% F1-BEO-supplemented diet. Higher F1-BEO supplementation led to a failure of activity of several enzymes and the depletion of glutathione levels. Malondialdehyde concentration suggests a sufficient oxidative stress defense against lipid peroxidation in all experimental groups, except for a 3% F1-BEO-supplemented diet (liver 168.87 ± 38.79 nmol/mg prot; kidney 146.86 ± 23.28 nmol/mg prot), compared to control (liver 127.76 ± 18.15 nmol/mg prot; kidney 98.68 ± 15.65 nmol/mg prot). Our results suggest supplementing F1-BEO in fish diets up to 0.5% to avoid potential oxidative pressure in farmed trout.This research was funded by Italian Ministry of Health, Ricerca Finalizzata, grant number GR-2013-02355796.Peer reviewe

Determination of the hyperfine coupling constant and zero-field splitting in the ESR spectrum of Mn²⁺ in calcite

The hyperfine coupling constant (A in units of energy or A in units of magnetic field) and zero-field splitting (D in units of energy or D in units of magnetic field) in the ESR spectrum of Mn2+ in calcite were determined. The spreading of the non-central allowed transitions |3/2, m&gt; → |5/2, m&gt;, |1/2, m&gt; → |3/2, m&gt;, |-3/2, m&gt; → |-1/2, m&gt; and |-5/2, m&gt; → |-3/2, m&gt; was analysed and the experimental transitions were attributed. Particular relevance was given to the difference between the two types of resonances, shoulder or divergence, and to their origin (M, m). The analysis explains the presence of a weak shoulder between each of the five central doublets |-1/2, m&gt; → |1/2, m&gt;. Six independent methods for calculating the hyperfine coupling constant and five methods for calculating the zero-field splitting, based on the analysis of the allowed and forbidden transitions, were provided. The values of the hyperfine coupling constant range from -93.9 to -94.6 G and those of the zero-field splitting range from -79.5 to -80.5 G. A critical evaluation of the advantages and drawbacks of the 11 methods is included: the best value for A is -94.30 G and that for D is -79.95 G

Application of DFT methods in the study of V^IVO²⁺–peptide interactions

VIVO2+ complexes formed by histidylglycylglycine (HisGlyGlyH), glycylglycylhistidine (GlyGlyHisH), glycylglycylcysteine (GlyGlyCysH2), N-glycyl-bis(imidazol-2-yl)methylamine (Gly–BIMA), N-glycyl-bis(pyridin-2-yl)methylamine (Gly–BPMA), salicylglycyl-L-alanine (SalGly-L-AlaH2) and 1,2-bis(2-hydroxybenzamido)benzene (H2hybeb) in their fully deprotonated form were studied by density functional theory (DFT) methods. They are characterised by different total electric charges, total equatorial charges and number of V–N–amide bonds. DFT calculations enable structural features, like V–donor bond lengths, and spectroscopic features, like electron paramagnetic resonance (EPR) and electronic absorption parameters, to be calculated. The results suggest that an amide group coordinates vanadium in the“amide-” rather than the “imine-like” form with the nitrogen atom negatively charged and with a double bond between the carbon and oxygen atoms of the carbonyl group, and that the equatorial charge is delocalised among all the donors bound to vanadium (Ooxido included). The analysis of the molecular orbital composition reveals that the dxy orbital is the vanadium orbital at lower energy, that it can participate in a π bond with the nitrogen pz orbital of the amide groups, that the vanadium dxz and dyz orbitals are involved in a large π interaction with the oxido px and py orbitals and that differences in the donor strengths of the ligands and deviations from the ideal square-pyramidal symmetry can result in the separation of the energies of the vanadium dxz and dyz orbitals

Is the spin-orbit coupling important in the prediction of the ⁵¹V hyperfine coupling constants of V^IVO²⁺ species? ORCA versus Gaussian performance and biological applications

Density functional theory calculations of the 51V hyperfine coupling (HFC) tensor A, have been completed for eighteen VIVO2+ complexes with different donor set, electric charge and coordination geometry. A tensor was calculated with ORCA software with several functionals and basis sets taking into account the spin-orbit coupling contribution. The results were compared with those obtained with Gaussian 03 software using the half-and-half functional BHandHLYP and 6-311g(d,p) basis set. The order of accuracy of the functionals in the prediction of Aiso, Az and dipolar term Az,anis is BHandHLYP &gt; PBE0 &gt;&gt; B3PW &gt; TPSSh &gt;&gt; B3LYP &gt;&gt; BP86 &gt; VWN5 (for Aiso), BHandHLYP &gt; PBE0 &gt;&gt; B3PW &gt; TPSSh &gt; B3LYP &gt;&gt;; BP86 &gt; VWN5 (for Az), B3LYP &gt; PBE0 ∼ B3PW ∼ BHandHLYP &gt;&gt; TPSSh &gt; BP86 ∼ VWN5 (for Az,anis). The good agreement in the prediction of Az with BHandHLYP is due to a compensation between the overestimation of Aiso and underestimation of Az,anis (Az = Aiso +Az,anis), whereas among the hybrid functionals PBE0 performs better than the other ones. BHandHLYP functional and Gaussian software are recommended when the VIVO2+ species contains only V-O and/or V-N bonds, whereas PBE0 functional and ORCA software for VIVO2+ complexes with one or more VAS bonds. Finally, the application of these methods to the coordination environment of VIVO2+ ion in V-proteins, like vanadylsubstituted insulin, carbonic anhydrase, collagen and S-adenosylmethionine synthetase, was discussed

The Determination of the geometry of Cu(II) complexes: an EPR spectroscopy experiment

An instrumental experiment is presented in which eleven Cu(II) complexes are studied with electron paramagnetic resonance (EPR) spectroscopy. The EPR spectroscopy allows the characterization of the geometry and electronic structure of the copper complexes. Three common ligands, ethylenediamine (en), 1,10-phenanthroline (phen), and 2,2′-bipyridine (bpy), were used. Examination of the EPR spectra of the solid-state compounds demonstrates that [Cu(en)2(ClO4)2], [Cu(en)2(BF4)2], and [Cu(en)2(NO3)2] have a dx2-y2 ground state and an elongated octahedral geometry; that [Cu(phen)2(H2O)](NO3)2, [Cu(phen)2(H2O)](BF4)2, and [Cu(bpy)2Cl]ClO4 are characterized by a geometry intermediate between the square pyramid and the trigonal bipyramid and a ground state corresponding to the linear combination of the dx2-y2 and dz2 orbitals; and that [Cu(phen)2Cl]ClO4, [Cu(phen)2Br]ClO4, [Cu(bpy)2Br]ClO4, [Cu(phen)2]ClO4, and [Cu(bpy)2I]ClO show a geometry close to the trigonal bipyramid and a ground state of dz2. A theoretical explanation of the results is presented

Interaction of VO²⁺ ion with human serum transferrin and albumin

The complexation of VO2+ ion with the high molecular mass components of the blood serum, human serum transferrin (hTf) and albumin (HSA), has been re-examined using EPR spectroscopy. In the case of transferrin, the results confirm those previously obtained, showing that VO2+ ion occupies three different binding sites, A, B1 and B2, distinguishable in the X-band anisotropic spectrum recorded in D2 O. With albumin the results show that a dinuclear complex (VO)1dHSA is formed in equimolar aqueous solutions or with an excess of protein; in the presence of an excess of VO2+, the multinuclear complex (VO)xmHSA is the prevalent species, where x = 5–6 indicates the equivalents of metal ion coordinated by HSA. The structure of the dinuclear species is discussed and the donor atoms involved in the metal coordination are proposed on the basis of the measured EPR parameters. Two different binding modes of albumin can be distinguished varying the pH, with only one species being present at the physiological value. The results show that the previously named “strong” site is not the N-terminal copper binding site, and some hypothesis on the metal coordination is discussed, with the 51V Az values for the proposed donor sets obtained by DFT (density functional theory) calculations. Finally, preliminary results obtained in the ternary system VO2+/hTf/HSA are shown in order to determine the different binding strength of the two proteins. Due to the low VO2+ concentration used, the recording of the EPR spectra through the repeated acquisition of the weak signals is essential to obtain a good signal to noise ratio in these systems

Monomeric versus dimeric structures in ternary complexes of manganese(II) with derivatives of benzoic acid and nitrogenous bases: structural details and spectral properties

Adducts formed by [Mn(2,6-dmb)2(H2O)3]n · nH2O, 2,6-dmb=2,6-dimethoxybenzoate(1–), Mn(2,4-dhb)2 · 8H2O, Mn(2,5-dhb)2 · 4H2O or Mn(2,6-dhb)2 · 8H2O, dhb=dihydroxybenzoate(1–), and 2,2′-bipyridine (bpy), 4,4′-dimethyl-2,2′-bipyridine (Me2bpy) or 4,7-dimethyl-1,10-phenanthroline (Me2phen) were isolated in the solid state and characterised by IR, EPR and thermogravimetry. Two of them, [Mn(2,6-dhb)2(bpy)2] (1) and [Mn2(2,6-dmb)4(Me2Phen)2(H2O)2] · 2EtOH (2), were studied by single crystal X-ray diffraction. The adduct 1 is mononuclear and consists of hexa-co-ordinate manganese(II) ions bound to two bipyridine and two 2,6-dihydroxybenzoate ligands in a cis-octahedral arrangement. The complex 2 exhibits a dinuclear structure in which two manganese(II) ions share two carboxylate groups adopting a rather uncommon single-atom bridging mode. The results allow us to conclude that weak, e.g., hydrogen bonding and stacking interactions govern the type of structure, monomeric or dimeric. The spectral features of the complexes are discussed. In particular, the solid-state EPR features of the complexes are interpreted in terms of D, E and Hmax, the high-field resonance. For the monomeric species, the higher is the D value, the higher is Hmax. A series of adducts formed by Mn2+ ion with derivatives of benzoic acid and aromatic nitrogenous bases is described. The formation of monomeric or dimeric complexes is influenced by weak interactions, like hydrogen bonding and stacking interactions

The Equilibrium between the octahedral and square pyramidal form and the influence of an axial ligand on the molecular properties of V^IVO complexes: a spectroscopic and DFT study

The previously unreported equilibrium in aqueous solution between the VIVO square pyramidal and trans octahedral form with an axial water molecule for a number of bidentate ligands with (N,O) and (O,O) donor sets (6-methylpicolinic (6-mepicH) and 6-methyl-2,3-pyridinedicarboxylic (6-me-2,3-pdcH2) acids, dipyridin-2-ylmethanol (Hdpmo) and 1,2-dimethyl-3-hydroxy-4(1H)-pyridinone (Hdhp)) has been demonstrated by the combined application of EPR spectroscopy and DFT methods. The EPR spectra suggest that, with increasing ionic strength, the equilibrium is shifted towards the formation of the pentacoordinated species and values of K≈4.0 and 7.0 for the systems containing 6-methyl-2,3-pyridinedicarboxylic acid and dipyridin-2-ylmethanol were measured. DFT calculations performed with Gaussian 03 and ORCA software predict the 51V anisotropic hyperfine coupling constant along the z axis (Az), which can be used to demonstrate the presence of an axially bound ligand trans to the V=O bond. The results suggest that an axial donor (charged or not) can lower |Az|, in contrast to what was previously believed on the basis of the “additivity rule”, and this explains the anomalous behaviour of the VIVO complexes formed by N-{2-[(2-pyridylmethylene)amino]phenyl}-pyridine-2-carboxamide (Hcapca) and several amidrazone derivatives. The decrease in |Az| for the axial binding of a solvent molecule is mainly a result of the reduction of |Aiso| and this was also observed when the solid [VO(6-methylpicolinato)2] was dissolved in DMSO or DMF. The variations in the structural (V=O, V-O and V-N distances, O-V-O and N-V-N angles, and the trigonality index t) and spectroscopic (|Az|, |Aiso| and v(V=O)) properties as a function of the axial VOH2 distance (R) are also presented. Finally, the electronic structures of the pentaand hexacoordinated complexes are discussed