Single-Step Protocol for Isolating the Recombinant Extracellular Domain of the Luteinizing Hormone Receptor from the Ovis aries Testis.

The luteinizing hormone receptor (LHR) is a glycoprotein member of the G protein-coupled receptors superfamily. It participates in corpus luteum formation and ovulation in females and acts in testosterone synthesis and spermatogenesis in males. In this study, we extracted RNA from sheep testicles and synthetized the cDNA to amplify the gene lhr-bed. This gene consists of 762 bp and encodes 273 amino acids of the extracellular domain of LHR. The lhr-bed was cloned into pJET1.2/blunt, then subcloned into pCOLD II, and finally, transformed in E. coli BL21 (DE3) cells. Because the induced rLHR-Bed protein was found in the insoluble fraction, we followed a modified purification protocol involving induction at 25 °C, subjection to denaturing conditions, and on-column refolding to increase solubility. We confirmed rLHR-Bed expression by means of Western blot and mass spectrometry analysis. It is currently known that the structure stem-loop 5'UTR on pCOLD II vector is stable at 15 °C. We predicted and obtained RNAfold stability at 25 °C. We successfully obtained the recombinant LHR extracellular domain, with protein yields of 0.2 mg/L, and purity levels of approximately 90%, by means of a single chromatographic purification step. The method described here may be used to obtain large quantities of rLHR-Bed in the future.

Atomic and molecular analysis highlights the biophysics of unprotonated and protonated retinal in UV and scotopic vision.

During the photoreaction of rhodopsin, retinal isomerizes, rotating the C11[double bond, length as m-dash]C12 π-bond from cis to an all-trans configuration. Unprotonated (UR) or protonated (PR) retinal in the Schiff's base (SB) is related to UV and light vision. Because the UR and PR have important differences in their physicochemical reactivities, we compared the atomic and molecular properties of these molecules using DFT calculations. The C10-C11[double bond, length as m-dash]C12-C13 dihedral angle was rotated from 0° to 180° in 45° steps, giving five conformers, and the following were calculated from them: atomic orbital (AO) contributions to the HOMO and LUMO, atomic charges, bond length, bond order, HOMO, LUMO, hardness, electronegativity, polarizability, electrostatic potential, UV-vis spectra and dipole moment (DM). Similarly, the following were analyzed: the energy profile, hybridization, pyramidalization and the hydrogen-out-of-plane (HOOP) wagging from the H11-C11[double bond, length as m-dash]C12-H12 dihedral angle. In addition, retinal with a water H-bond (HR) in the SB was included for comparison. Interestingly, in the PR, C11 and C12 are totally the LUMO and the HOMO, respectively, and have a large electronegativity difference, which predicts an electron jump in these atoms during photoexcitation. At the same time, the PR showed a longer bond length and lower bond order, with a larger DM, lower HOMO-LUMO gap, lower hardness and higher electronegativity. In addition, the AOs of -45° and -90° conformers changed significantly, from pz to py, during the rotation concomitantly with marked hybridization, smooth pyramidalization and lower HOOP activity. Clearly, the atomic and molecular differences between the UR and PR are overwhelming, including the rotational energy profile and light absorption spectra, which indicates that light absorption of UR and PR is already determined by the retinal characteristics of the SB protonation. The HR-model compared with UR shows a lower energy barrier and a discreet bathochromic effect in the UV region.

Molecular biosensor based on a coordinated iron complex.

A sensor model based on the porphyrin nucleus of the soluble guanylate cyclase enzyme is modeled and tested with nitric oxide and carbon monoxide. Molecular oxygen is tested as a possible interferer. Geometries and electronic structures of the model are assessed by density functional theory. Vibrational circular dichroism (VCD), infrared, and Raman spectra are obtained for the iron complexes uncoordinated and coordinated with the gas moieties. The sensor is capable of detecting the ligands to different extents. Carbon monoxide is less detectable than nitric oxide due to the adopted position of the molecule in the sensor; carbon oxide is aligned with the iron atom, while nitric oxide and molecular oxygens bend with an angle detectable by the VCD. It is suggested that pollutants may be detected and measured with the proposed biosensors.

Ion trap MS/MS of intact testosterone and epitestosterone conjugates--adducts, fragile ions and the advantages of derivatisation.

In ion trap mass spectrometry, fragile ions may fragment under the application of resonance ejection during precursor mass isolation, reducing MS/MS spectral intensity. In this study the steroidal epimers testosterone glucuronide (TG) and epitestosterone glucuronide (EG) have been chosen as a model for exploring whether compound structure is linked to ion trap fragility. Both compounds form multiple adducts by ESI-MS, namely protonation, ammonium and sodium, however, the mass spectrum of EG displays a more intense ammonium adduct peak than TG. [TG+NH(4)](+), [EG+NH(4)](+) and [EG+H](+) were found to be fragile ions. To explain the differences in adduct formation and fragility, molecular modelling was employed. Ammonium adduction was localised to the glucuronide ring oxygens and while EG has eight possible adduction sites, only seven were located for TG explaining the increased ammonium adduct abundance with EG. In EG the bond between the steroid and the glucuronide was slightly longer and the oxygen in this bond was more basic than TG. This shows that the EG bond is weaker which may contribute to the fact that [EG+H](+) but not [TG+H](+) is fragile. To investigate whether stability could be restored by chemical means, EG was derivatised with tris(trimethoxyphenyl)phosphonium chloride or methylated on the carboxylic acid and Girard P or methoxylamine on the 3-keto group. Derivatisation of the steroid rather than the glucuronide eliminated fragility and using a charged derivative eliminated adduct formation. This work demonstrates the importance of carefully considering the nature of the derivative and the site of derivatisation.

Insights on the mechanism of action of immunostimulants in relation to their pharmacological potency. The effects of imidazoquinolines on TLR8.

Imidazoquinolines are powerful immunostimulants (IMMS) that function through Toll-like receptors, particularly TLR7 and TLR8. In addition to enhancing the immune response, IMMS also function as antineoplastic drugs and vaccine adjuvants. These small compounds display almost the same molecular structure, except in some cases in which atom in position 1 varies and changes the imidazole characteristics. A variable acyclic side chain is also always attached at atom in position 2, while another chain may be attached at atom in position 1. These structural differences alter immune responses, such as the production of interferon regulatory factor and nuclear factor-κB (IRF-NFκB). In this work, quantum mechanics theory and computational chemistry methods were applied to study the physicochemical properties of the crystal binding site of TLR8 complexed with the following six IMMS molecules: Hybrid-2, XG1-236, DS802, CL075, CL097 and R848 (resiquimod). The PDB IDs of the crystals were: 4R6A, 4QC0, 4QBZ, 3W3K, 3W3J, and 3W3N respectively. Thus, were calculated, the total energy, solvation energy, interaction energy (instead of free energy) of the system and interaction energy of the polar region of the IMMS. Additionally, the dipole moment, electrostatic potential, polar surface, atomic charges, hydrogen bonds, and polar and hydrophobic interactions, among others, were assessed. Together, these properties revealed important differences among the six TLR8-immunostimulant complexes, reflected as different interaction energies and therefore different electrostatic environments and binding energies. Remarkably, the interaction energy of a defined polar region composed of the highly polarized N3, N5 atoms and the N11 amino group, acted as a polar pharmacophore that correlates directly with the reported immunopharmacological potency of the six complexed molecules. Based on these results, it was concluded that accurate physicochemical analysis of the crystal binding site could reveal the binding energy (measured as interaction energy) and associated molecular mechanism of action between IMMS and TLR8. These findings may facilitate the development and design of improved small molecules with IMMS properties that are targeted to the TLR system and have enhanced pharmacological effectiveness and reduced toxicity.

A theoretical model of the catalytic mechanism of the Delta5-3-ketosteroid isomerase reaction.

The present paper describes a theoretical approach to the catalytic reaction mechanism involved in the conversion of 5-androstene-3,17-dione to 4-androstene-3,17-dione. The model incorporates the side chains of the residues tyrosine (Tyr(14)), aspartate (Asp(38)) and aspartic acid (Asp(99)) of the enzyme Delta(5)-3-ketosteroid isomerase (KSI; EC 5.3.3.1). The reaction involves two steps: first, Asp(38) acts as a base, abstracting the 4beta-H atom (proton) from C-4 of the steroid to form a dienolate as the intermediate; next, the intermediate is reketonized by proton transfer to the 6beta-position. Each step goes through its own transition state. Functional groups of the Tyr(14) and Asp(99) side chains act as hydrogen bond donors to the O1 atom of the steroid, providing stability along the reaction coordinate. Calculations were assessed at high level Hartree-Fock theory, using the 6-31G(*) basis set and the most important physicochemical properties involved in each step of the reaction, such as total energy, hardness, and dipole moment. Likewise, to explain the mechanism of reaction, highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), atomic orbital contributions to frontier orbitals formation, encoded electrostatic potentials, and atomic charges were used. Energy minima and transition state geometries were confirmed by vibrational frequency analysis. The mechanism described herein accounts for all of the properties, as well as the flow of atomic charges, explaining both catalytic mechanism and proficiency of KSI.

Reversing gastric mucosal alterations during ethanol-induced chronic gastritis in rats by oral administration of Opuntia ficus-indica mucilage.

AIM: To study the effect of mucilage obtained from cladodes of Opuntia ficus-indica (Cactaceae) on the healing of ethanol-induced gastritis in rats. METHODS: Chronic gastric mucosa injury was treated with mucilage (5 mg/kg per day) after it was induced by ethanol. Lipid composition, activity of 5'-nucleotidase (a membrane-associated ectoenzyme) and cytosolic activities of lactate and alcohol dehydrogenases in the plasma membrane of gastric mucosa were determined. Histological studies of gastric samples from the experimental groups were included. RESULTS: Ethanol elicited the histological profile of gastritis characterized by loss of the surface epithelium and infiltration of polymorphonuclear leukocytes. Phosphatidylcholine (PC) decreased and cholesterol content increased in plasma membranes of the gastric mucosa. In addition, cytosolic activity increased while the activity of alcohol dehydrogenases decreased. The administration of mucilage promptly corrected these enzymatic changes. In fact, mucilage readily accelerated restoration of the ethanol-induced histological alterations and the disturbances in plasma membranes of gastric mucosa, showing a univocal anti-inflammatory effect. The activity of 5'-nucleotidase correlated with the changes in lipid composition and the fluidity of gastric mucosal plasma membranes. CONCLUSION: The beneficial action of mucilage seems correlated with stabilization of plasma membranes of damaged gastric mucosa. Molecular interactions between mucilage monosaccharides and membrane phospholipids, mainly PC and phosphatidylethanolamine (PE), may be the relevant features responsible for changing activities of membrane-attached proteins during the healing process after chronic gastric mucosal damage.

The importance of the chemical structure of pregnanes in the concurrent inhibition of estrous behavior in the female rat.

An investigation of aspects ranging from behavior to molecular electronic structure and physicochemical properties was performed to explore the role of 5α-pregnanedione (5α-DHP), 5ß-pregnanedione (5ß-DHP) and their precursor progesterone (P) on the concurrent inhibition of the sexual lordosis response in female rats. The concurrent inhibition of lordosis behavior occurs when ovariectomized rodents are primed simultaneously with estradiol (E2) and P. Thus, a second administration of P 40h later fails to induce the expected sexual response that takes place when E2 and P are administered sequentially 40h apart. In this study, it is hypothesized that the modulation of the sexual behavior display depends to some extent on the molecular structure and associated physicochemical properties of steroid hormones such as P and its metabolites. Therefore, these molecules must be studied chemically and structurally to explain their role in sexual behavior, including the concurrent inhibition effect. Analysis of the electronic structure and physicochemical properties demonstrated striking differences in the A-ring region of P, 5α-DHP and 5ß-DHP, particularly in atomic charges, dipole moment (DM) and electrostatic potentials. Similarly, the structural differences between the trans (5α-DHP) and cis (5ß-DHP) configurations were remarkable. 5α-DHP most significantly promoted the concurrent inhibition of the lordosis behavior, followed by P and 5ß-DHP. These data indicate that variations in pregnane structure are related to the extent of the concurrent inhibition effect and also suggest that P may act as a prehormone in certain functions of the central nervous system.

Ab initio electronic structure of the progestogen norethisterone and its 5 alpha-derivatives.

The steroid 17 alpha-ethynyl-19-nor-4-androsten-17 beta-ol, 3-one (Norethisterone; NET) and its 5 alpha-dihydro (5 alpha-NET), 3 alpha- and 3 beta-tetrahydro derivatives (3 alpha,5 alpha- and 3 beta,5 alpha-NET), were comparatively studied by the ab initio quantum mechanics theory. Additionally, 5 alpha-androstan-3 beta,17 beta-diol (ADIOL) was also studied. The Hartree-Fock method and the 6-31G(*) basis set were used to obtain the lowest energy conformation, geometries, electronic structure and physicochemical properties of the steroids. The results showed bond distances and valence angles similar among all steroids, but some differences in dihedral angles in the A-B-ring system were observed. The electronic structure analysis showed that NET has both frontier orbitals that is, the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) located at the C4-C5 pi-bond. In A-ring reduced derivatives, the HOMO was found at the 17 beta-OH and ethynyl groups. In the case of 5 alpha-NET, the LUMO was confined to the A-ring and its C3 carbonyl group while the two NET tetrahydro-reduced derivatives showed the LUMO at the 17 beta-OH and ethynyl groups. The energy changes of the rotational barrier of the 17 beta-OH group suggest that its movement is somewhat restricted by the 17 alpha-ethynyl group. Interestingly both groups at C17 form a single electrostatic potential with high electronic density. On the other side, the 19-nor condition increases the A-ring mobility. However, the 3 beta-OH group of 3 beta,5 alpha-NET may rotate without significant energy differences as compared to the same group in ADIOL. The electronic structure of NET and its A-ring reduced derivatives explains in some extent their interaction with androgen and progesterone receptors as well as their selectivity for the estrogen alpha-receptor.

Computational design of a CNT carrier for a high affinity bispecific anti-HER2 antibody based on trastuzumab and pertuzumab Fabs.

This is a preliminary cross multidisciplinary theoretical-computational approach for the design of a drug delivery system based on immunoconjugated carbon nanotube against HER2- overexpressing cancer cells. This drug delivery system allows the release of an encapsulated cytotoxic cocktail in a controlled manner under pulsed radio frequency (RF) irradiation. Our effort is focused on the computational aided design of a high affinity bispecific anti-HER2 antibody and an opening mechanism of the carbon nanotube (CNT) based cytotoxic carrier for controlling multiple drug release. We study the main interactions between the antibody and the antigen by a computational scanning mutagenesis approach of trastuzumab and pertuzumab fragment antigen binding (Fab) structures in order to enhance their binding affinity. Then, each Fab fragments is joined by a polypeptide linker which should be stable enough to avoid the "open form" of antibody. On the other hand, we also conjugate the engineered antibody to functionalized CNTs (f-CNTs), which encapsulate the inhibitors of the HER2/PI3K/Akt/mTOR signaling pathway. We take advantage of the fact that f-CNT converts the RF radiation absorption into heat release. A pulsed laser at 13.45 MHz increments the temperature around 40 °C for triggering the nano-caps destabilization, which allows the switching of the opening mechanism of the drug carrier. Nano-caps will be a dual pH/temperature responsive in order to take advantage of lysosome characteristic (acidic pH) and heat release from the carrier. Nano-caps are functionalized with organic amide moieties, which hydrolyze quickly at an acidic pH into primary amines, and protonated amines generate repulsion interactions with other charged species, which trigger the cytotoxics release.

Bimodal binding and free energy of the progesterone receptor in the induction of female sexual receptivity by progesterone and synthetic progestins.

Synthetic progestins (SPs) are used for regulation of fertility, contraception and hormone replacement therapy. The acetylated medroxyprogesterone (MPA), megestrol (MGA) and chlormadinone (CLA) are related to progesterone (P). Other SPs are 19-nortestosterone derivatives such as: norethisterone (NET), norethynodrel (NED) or the 13-ethyl gonane, levonorgestrel (LNG). We studied MPA, NET, NED and LNG in a dose-response manner to induce sexual receptivity in rats. Results showed that MPA, NET and NED act as partial agonists, with similar or lower potency than P. However, LNG is a full agonist. Additionally, the molecules of MPA, MGA, CLA, NET, NED, LNG, and P, were submitted to computer calculations at ab initio quantum mechanics theory, to obtain their electronic structure and molecular properties. The aim was to correlate their behavioral effect with their physicochemical properties. In addition, the crystals of P, NET and LNG bound to the progesterone receptor (PR) were studied. The PR crystallizes as a dimer forming two monomers (mA and mB), in which Gln725 interacts in either of two possible ways with the C3-carbonyl pharmacophore of progestins. P binds differentially to both PR monomers, while NET binds exclusively as mA and LNG binds only as mB in both monomers with no difference. Energetically, binding of LNG and P to mB, is more favorable than that of NET and P to mA. Consequently, this bimodal mechanism increases the action possibilities of SPs on biological systems. Interestingly, progestin potency depends mostly on local molecular structure and electronic features, prevailing over total molecular properties.

Amyloid-ß peptide binds to cytochrome C oxidase subunit 1.

Extracellular and intraneuronal accumulation of amyloid-beta aggregates has been demonstrated to be involved in the pathogenesis of Alzheimer's disease (AD). However, the precise mechanism of amyloid-beta neurotoxicity is not completely understood. Previous studies suggest that binding of amyloid-beta to a number of macromolecules has deleterious effects on cellular functions. Mitochondria were found to be the target for amyloid-beta, and mitochondrial dysfunction is well documented in AD. In the present study we have shown for the first time that Aß 1-42 bound to a peptide comprising the amino-terminal region of cytochrome c oxidase subunit 1. Phage clone, selected after screening of a human brain cDNA library expressed on M13 phage and bearing a 61 amino acid fragment of cytochrome c oxidase subunit 1, bound to Aß 1-42 in ELISA as well as to Aß aggregates present in AD brain. Aß 1-42 and cytochrome c oxidase subunit 1 co-immunoprecipitated from mitochondrial fraction of differentiated human neuroblastoma cells. Likewise, molecular dynamics simulation of the cytochrome c oxidase subunit 1 and the Aß 1-42 peptide complex resulted in a reliable helix-helix interaction, supporting the experimental results. The interaction between Aß 1-42 and cytochrome c oxidase subunit 1 may explain, in part, the diminished enzymatic activity of respiratory chain complex IV and subsequent neuronal metabolic dysfunction observed in AD.

Light activation of the isomerization and deprotonation of the protonated Schiff base retinal.

We perform an ab initio analysis of the photoisomerization of the protonated Schiff base of retinal (PSB-retinal) from 11-cis to 11-trans rotating the C10-C11=C12-C13 dihedral angle from 0° (cis) to -180° (trans). We find that the retinal molecule shows the lowest rotational barrier (0.22 eV) when its charge state is zero as compared to the barrier for the protonated molecule which is ∼0.89 eV. We conclude that rotation most likely takes place in the excited state of the deprotonated retinal. The addition of a proton creates a much larger barrier implying a switching behavior of retinal that might be useful for several applications in molecular electronics. All conformations of the retinal compound absorb in the green region with small shifts following the dihedral angle rotation; however, the Schiff base of retinal (SB-retinal) at trans-conformation absorbs in the violet region. The rotation of the dihedral angle around the C11=C12 π-bond affects the absorption energy of the retinal and the binding energy of the SB-retinal with the proton at the N-Schiff; the binding energy is slightly lower at the trans-SB-retinal than at other conformations of the retinal.

Theoretical mechanism of the formation of cholesteryl chloride from cholesterol and thionyl chloride.

This work describes a theoretical approach to the substitution reaction mechanism involving the conversion of cholesterol to cholesteryl chloride. Two chlorosulfite ester molecules were formed as intermediates. An iso-steroid was found as the transition state. The final product was cholesteryl chloride and the side products were HCl and SO2. Calculations were carried out at high level Hartree-Fock theory, using the 6-31G* basis set. From the electronic structure of the reactants, the most important physicochemical properties involved in the reaction pathway were used. Thus, to determine the participation of each molecule and to explain the mechanism of reaction; the total energy, HOMO and LUMO, atomic orbital contribution to frontier orbitals formation, electrostatic potentials, atomic charges, hardness and dipole moment were used. Characterization of intermediates and transition state was supported by their respective energy minima, fundamental frequencies and equilibrium geometry.

Hypotalamic monoamine oxidase activity in ovariectomized rats after sexual behavior restoration

The effect of estradiol benzoate, progesterone and a sequential treatment with both on the activity of the enzyme monoamine-oxidase (MAO) was assessed in mitochondria from hypothalami of ovariectomized rats. A differential effect on the subtypes A and B MAO was found according to the type of treatment. Estradiol benzoate administration decreases MAO activity, mainly that of MAO-A. Progesterone alone had no effect, and sequential treatment with estradiol benzoate plus progesterone restored sexual behavior and produced a significant increase of MAO-A activity, whitout changes in total MAO activity. Since MAO-A is an isoform of MAO that preferentially uses norepinephrine and serotonin as substrates and MAO-B acts on phenylethylamine and benzylamine as substrates, our findings suggest that the restoration of sexual behabior after the treatment with estradiol benzoate followed by progesterone may be associated with the differential effect exerted by the hormones on MAO subtypes, rather than to the simple decrease in hypothalamic monoamine concentrations as reported in the literature

Relaxant action of androgens, progestins and corticosteroids on the isolated ileum of the guinea pig

La acción de tres diferentes clases de derivados de la progesterona fueron probados sobre las contracciones espontáneas del íleon aislado de cobayo. Los resultados mostraron que este tejido es muy sensible a la acción de los esteroides. Se observó una marcada relajación que fue dependiente de la dosis y diferente para cada compuesto. Esta diferencia fue asociada a la estructura molecular del esteroide. Así, las progestinas 5ß-reducidas fueron las más potentes, seguidas de los andrógenos 5 alfa y 5ß-reducidos. Los compuestos 4-en, 17 alfa-OH-progesterona y los corticosteroides, fueron los más bajos en potencia. La 5alfa-pregnandiona y los pregnandioles fueron prácticamente inefectivos. La gran sensibilidad del músculo liso del íleon permite postular a este órgano como blanco de esteroides. Es posible que en algunas circunstancias fisiológicas, como podría ser el embarazo, los trastornos de motilidad intestinal observados en este estado estén asociados al incremento notable de esteroides circulantes

Cambios electroencefalográficos y de la conducta producidos por derivados 5 Beta de la progesterona y su antagonismo por la 4-aminopiridina / Electroencephalographic and behavioral changes produced by 5 beta progestins and its antagonism by 4-aminopyridine

Se estudiaron en ratas en libre movimiento los cambios de la conducta y el EEG producidos por pregnandiona, pregnanolona y epipregnanolona, y su antagonismo por la 4-aminopiridina (4-AP). Las progestinas produjeron pérdida del reflejo de enderezamiento, ataxia, sedación, estado anestésico y aparición de husos en el EEG. La 4-AP produjo temblor y contracciones musculares, e incrementó la amplitud del EEG. La epipregnanolona fue más potente que la pregnandiona y la pregnanolona para producir los cambios en el EEG y la anestesia. El estado anestésico experimentó inversión por acción de la 4-AP en menos de tres minutos. Los animales despertaron temblorosos y, en algunos casos, con convulsiones. Sin embargo, la actividad del EEG permaneció sincronizada. La potencialización de la 4-AP por los derivados 5 ß de la progesterona refuerza la idea de un efecto bifásico de los últimos que podrían excitar más que deprimir algunas áreas cerebrales. Se concluye que la inversión por la 4-AP de la anestesia producida por los derivados de la 5 ß progesterona podría incluir efectos en la membrana neuronal, liberación de neurotransmisores y movimientos iónicos