Arash Mirzahosseini

Histamine receptor H4 regulates mast cell degranulation and IgE induced FcεRI upregulation in murine bone marrow-derived mast cells

There is increasing evidence that histamine regulates the immune system via histamine H4 receptors, therefore we sought to investigate the functions of the H4 receptor on mast cells. Mast cells were differentiated from murine bone marrow stem cells, and the expression of mast cell surface markers FcεRI and CD117 were measured using flow cytometry. Real-time qRT-PCR was used to determine the expression of mH4R; as a measure of antigen-dependent degranulation, β-hexosaminidase release assay was carried out using IgE sensitized mast cells. We determined that the expression kinetics of FcεRI and mH4R can be described with a function that has one maximum value in the time range of the cultures differentiation. Antigen-dependent degranulation of murine bone marrow-derived mast cells could be inhibited by a selective H4 antagonist/inverse agonist only when it was present during the IgE sensitization phase of degranulation. In addition, flow cytometric analysis revealed that the H4 antagonist/inverse agonist also inhibited IgE induced FcεRI upregulation. The inhibition percentage of H4 antagonist on IgE induced FcεRI upregulation was determined to be dependent upon the maturity of the mast cell cultures, and this time-dependency was consistent with the expression kinetics of both mH4R and FcεRI. These results imply that H4R has regulatory roles in FcεRI expression and FcεRI mediated functions in mast cells. In conclusion the present study shows that H4 receptors potentially play a role in IgE induced FcεRI upregulation and in the sensitization phase but not the effector phase of mast cell degranulation.

Skeletal muscle ATP synthesis and cellular H(+) handling measured by localized (31)P-MRS during exercise and recovery.

31P magnetic resonance spectroscopy (MRS) is widely used for non-invasive investigation of muscle metabolism dynamics. This study aims to extend knowledge on parameters derived from these measurements in detail and comprehensiveness: proton (H+) efflux, buffer capacity and the contributions of glycolytic (L) and oxidative (Q) rates to ATP synthesis were calculated from the evolutions of phosphocreatine (PCr) and pH. Data are reported for two muscles in the human calf, for each subject and over a wide range of exercise intensities. 22 subjects performed plantar flexions in a 7T MR-scanner, leading to PCr changes ranging from barely noticeable to almost complete depletion, depending on exercise protocol and muscle studied by localized MRS. Cytosolic buffer capacity was quantified for the first time non-invasively and individually, as was proton efflux evolution in early recovery. Acidification started once PCr depletion reached 60–75%. Initial and end-exercise L correlated with end-exercise levels of PCr and approximately linear with pH. Q calculated directly from PCr and pH derivatives was plausible, requiring fewer assumptions than the commonly used ADP-model. In conclusion, the evolution of parameters describing cellular energy metabolism was measured over a wide range of exercise intensities, revealing a relatively complete picture of muscle metabolism.

The species- and site-specific acid-base properties of biological thiols and their homodisulfides.

Cysteamine, cysteine, homocysteine, their homodisulfides and 9 related compounds were studied by ¹H NMR-pH titrations and case-tailored evaluation methods. The resulting acid-base properties are quantified in terms of 33 macroscopic and 62 microscopic protonation constants and the concomitant 16 interactivity parameters, providing thus the first complete microspeciation of this vitally important family of biomolecules. The species- and site-specific basicities are interpreted by means of inductive and hydrogen-bonding effects through various intra- and intermolecular comparisons. The pH-dependent distribution of the microspecies is depicted. The thiolate basicities determined this way provide exclusive means for the prediction of thiolate oxidizabilities, a key parameter to understand and influence oxidative stress at the molecular level.

Species-Specific Standard Redox Potential of Thiol-Disulfide Systems: A Key Parameter to Develop Agents against Oxidative Stress.

Microscopic standard redox potential, a new physico-chemical parameter was introduced and determined to quantify thiol-disulfide equilibria of biological significance. The highly composite, codependent acid-base and redox equilibria of thiols could so far be converted into pH-dependent, apparent redox potentials (E’°) only. Since the formation of stable metal-thiolate complexes precludes the direct thiol-disulfide redox potential measurements by usual electrochemical techniques, an indirect method had to be elaborated. In this work, the species-specific, pH-independent standard redox potentials of glutathione were determined primarily by comparing it to 1-methylnicotinamide, the simplest NAD+ analogue. Secondarily, the species-specific standard redox potentials of the two-electron redox transitions of cysteamine, cysteine, homocysteine, penicillamine, and ovothiol were determined using their microscopic redox equilibrium constants with glutathione. The 30 different, microscopic standard redox potential values show close correlation with the respective thiolate basicities and provide sound means for the development of potent agents against oxidative stress.

Species-Specific Thiol-Disulfide Equilibrium Constant: A Tool to Characterize Redox Transitions of Biological Importance

Microscopic redox equilibrium constants, a new species-specific type of physicochemical parameters, were introduced and determined to quantify thiol-disulfide equilibria of biological significance. The thiol-disulfide redox equilibria of glutathione with cysteamine, cysteine, and homocysteine were approached from both sides, and the equilibrium mixtures were analyzed by quantitative NMR methods to characterize the highly composite, co-dependent acid-base and redox equilibria. The directly obtained, pH-dependent, conditional constants were then decomposed by a new evaluation method, resulting in pH-independent, microscopic redox equilibrium constants for the first time. The 80 different, microscopic redox equilibrium constant values show close correlation with the respective thiolate basicities and provide sound means for the development of potent agents against oxidative stress.

The complete microspeciation of ovothiol A, the smallest octafarious antioxidant biomolecule

AbstractOvothiol A, a small biomolecule with highly potent antioxidant capacity, and three newly synthesized derivatives were studied by 1H NMR, 15N NMR, UV-pH titrations, and a customized evaluation method. The omni-interactive imidazole, amino, carboxylate, and thiolate moieties of ovothiol A are quantified in terms of 32 microscopic protonation constants, the relative concentrations of 16 microspecies, 6 pairwise interactivity parameters, and 8 protonation shifts. The highest and lowest imidazole basicities differ by a record-breaking five orders of magnitude, and the predominant thiolate protonation constant is by far the smallest known thiolate logK value. The latter provides an indication as to why ovothiol A occurs naturally under deep-water circumstances only. Since thiolate basicities are in correlation with thiol-disulfide redox potentials, the eight different, fine-tunable thiolate basicities offer versatile and highly specific antioxidant capacities within one single molecular skeleton. This work is the first complete microspeciation of a tetrabasic, nonsymmetrical natural compound. FigureThe protonation microspeciation scheme of ovothiol A. Im, N, O, and S denote the basic moieties; imidazole, amino, carboxylate, and thiolate groups, respectively. Blank and colored fields denote the basic and protonated forms, respectively

Species-specific thiol-disulfide equilibrium constants of ovothiol A and penicillamine with glutathione

The highly composite acid–base and redox equilibria of thiols can so far be converted into pH-dependent, apparent redox equilibrium constants only. In this work, microscopic redox equilibrium constants were determined to quantify the thiol-disulfide equilibria of biologically important substances in intriguing interactions. The thiol-disulfide redox equilibria of glutathione with ovothiol A and penicillamine were analyzed by quantitative NMR methods to characterize the codependent acid–base and redox equilibria. The directly obtained, pH-dependent, conditional constants were then decomposed to pH-independent, microscopic redox equilibrium constants. These equilibria and the 96 different microscopic redox equilibrium constants are highly influenced by the protonation stage of the adjacent basic moieties. Our data show that equilibrium constants referring to redox systems of covalently identical molecular skeletons, but different acid–base status, can differ by orders of magnitude. In fact, this difference can be as many as 14 orders of magnitude in the glutathione–ovothiol system. This enormous diversity is due primarily to the extreme sensitivity of the ovothiol sulfur to the protonation-evoked electron-withdrawing effects of neighboring basic groups. The thiolate oxidizabilities show close correlation with the respective thiolate basicities and provide sound means for the development of potent agents against oxidative stress.

The complete microspeciation of ovothiol A disulfide: A hexabasic symmetric biomolecule

The site-specific acid-base properties of ovothiol A disulfide (OvSSOv), the smallest hexabasic multifunctional biomolecule with complex interdependent moieties, were studied with (1)H NMR-pH and potentiometric titrations. The unprecedented complexity of the protonation microequilibria could be overcome by taking into account the mirror-image molecular symmetry, synthesizing and studying auxiliary model compounds and developing a custom-tailored evaluation method. The amino, imidazole, and carboxylate moieties are quantified in terms of 192 microscopic protonation constants and 64 microspecies, 96 and 36 of which are chemically different ones, respectively. Nine pairwise interactivity parameters also characterize the OvSSOv-proton system at the level of molecular subunits. These data allow understanding and influencing the co-dependent acid-base and redox properties of the highly complex OvSH-OvSSOv and related thiol-disulfide systems, which provide protection against oxidative stress. This work is the first complete microspeciation of a hexabasic molecule.

BODIPY® FL histamine as a new modality for quantitative detection of histamine receptor upregulation upon IgE sensitization in murine bone marrow‐derived mast cells

Flow cytometry is one of the most widely used methods for the qualitative and quantitative analysis of cell surface expressed proteins by making use of fluorescent specific antibodies. Lacking an antibody validated for flow cytometry, an alternative approach for labeling cell surface receptors is the use of fluorescently tagged ligands. In this study, histamine H4 receptor transfected Chinese hamster ovary cells and murine bone marrow‐derived mast cells (mBMMCs) were selected for studying the possibility of staining individual histamine receptors using BODIPY® FL histamine and selective antagonists. Flow cytometric measurements and supporting calculations showed that BODIPY FL histamine is suitable tool for quantitating cell surface histamine receptors. The binding, and competitive inhibition of this fluorescent ligand were characterized, which were in good agreement with a semi‐empirical model constructed from fundamental protein‐binding relationships. Using this method it was shown for the first time that even though mature mBMMCs express H2R and H4R to the same extent, immunoglobulin E sensitization results in H4R upregulation only, while the surface expression of H2R remains unchanged.

Physicochemical Characterization and Cyclodextrin Complexation of the Anticancer Drug Lapatinib

Lapatinib (LAP), the tyrosine kinase inhibitor drug with moderate bioavailability, was characterized in terms of physicochemical properties: acid-base characteristics, lipophilicity, and solubility. The highly lipophilic nature of the drug and its extremely low water solubility (  nM) limit the development of a parenteral formulation. In order to enhance solubility and bioavailability, inclusion complex formation with cyclodextrins (CDs) is a promising method of choice. Therefore, LAP-CD interactions were also studied by a multianalytical approach. The stability constants of LAP with native cyclodextrins, determined by UV spectroscopy, identified the seven-membered β-CD as the most suitable host. Continuous variation method (Job’s plot) by 1H NMR showed a 1 : 1 stoichiometry for the complexes. The geometry of the complex was elucidated by 2D ROESY NMR measurements and molecular modeling, indicating that the partial molecular encapsulation includes the fluorophenyl ring of LAP. Phase-solubility studies with four CDs, β-CD, (2-hydroxypropyl)-β-cyclodextrin (HP-β-CD), randomly methylated-β- (RAMEB-) cyclodextrin, and sulfobutylether-β-cyclodextrin (SBE-β-CD), show an type diagram and highly increased solubility via CD complexation. The results are especially promising with SBE-β-CD, exerting more than 600-fold gain in solubility. The equilibrium and structural information presented herein can offer the molecular basis for an improved drug formulation with enhanced bioavailability.