Histamine uptake by leukocytes in vitro

Human leukocytes at the concentration of 10(6) cells/ml were incubated for 1h at 37.5 degrees C in a tris-albumin buffer, containing 0, 6.25, 12.5, 25, 50, and 100 micrograms/ml of histamine. Histamine uptake was measured by a photofluorometric method. We found that the leukocytes take up exogenous histamine and attain respective intracellular concentration of 307%, 423%, 438%, 1000% and 645% above controls. It was found that neutrophils and eosinophils possess the greatest capacity to take up exogenous histamine whereas, contrary to the expectation, basophils display poor capacity.     

Acetylation of (R,S)-propranolol catalyzed by Candida antarctica lipase B: An experimental and computational study

The chemo- and enantioselectivity of the Candida antarctica lipase B (CalB)-catalyzed acetylation reaction of (R,S)-propranolol using vinyl acetate as acyl donor and toluene as organic solvent was studied. Because of the poor solubility of propranolol in toluene small quantities of methanol were added as cosolvent. The effects of the propranolol/vinyl acetate ratio, the enzyme purification procedure and the methanol concentration on the reaction were investigated. The reactions occurring in the system were quantitatively investigated using ¹H and ¹³C NMR spectroscopy. The major reactions were the hydrolysis and alcoholysis of vinyl acetate, as a consequence of the presence of residual water and methanol in the reaction medium. Furthermore, the NMR analysis confirmed that O-acetyl-propranolol was formed exclusively. The reaction was also found to be enantioselective favoring the faster transformation of the R-propranolol. In addition to the experiments, molecular modeling was used to study the formation of the reactive Michaelis complexes between propranolol and acetylated CalB, using a combined molecular docking and molecular dynamics (MD) procedure. Only for the O-acetylation we found binding modes of the substrate leading to formation of the product, which explains the experimentally observed chemoselectivity of CalB.     

Implementation of an experimental and computational tool set to study protein-mAb interactions

This work focused on the development of a combined experimental and computational tool set to study protein-mAb interactions. A model protein library was first screened using cross interaction chromatography to identify proteins with the strongest retention. Fluorescence polarization was then employed to study the interactions and thermodynamics of the selected proteins—lactoferrin, pyruvate kinase, and ribonuclease B with the mAb. Binding affinities of lactoferrin and pyruvate kinase to the mAb were seen to be relatively salt insensitive in the range examined. Further, a strong entropic contribution was observed, suggesting the importance of hydrophobic interactions. On the other hand, ribonuclease B-mAb binding was seen to be enthalpically driven and salt sensitive, indicating the importance of electrostatic interactions. Protein–protein docking was then carried out and the results identified the CDR region on the mAb as an important binding site for all three proteins. The binding interfaces identified for the mAb-lactoferrin and mAb-pyruvate kinase systems were found to contain complementary hydrophobic and oppositely charged clusters on the interacting regions which were indicative of both hydrophobic and electrostatic interactions. On the other hand, the binding site on ribonuclease B was predominantly positively charged with minimal hydrophobicity. This resulted in an alignment with negatively charged clusters on the mAb, supporting the contention that these interactions were primarily electrostatic in nature. Importantly, these computational results were found to be consistent with the fluorescence polarization data and this combined approach may have utility in examining mAb-HCP interactions which can often complicate the downstream processing of biologics. © 2019 American Institute of Chemical Engineers     

Histamine improves antigen uptake and cross-presentation by dendritic cells

Previous studies have shown that histamine is able to modulate the function of dendritic cells (DCs). Histamine seems to be required for the normal differentiation of DCs. Moreover, it is capable of stimulating the chemotaxis of immature DCs and of promoting the differentiation of T CD4+ cells into a Th2 profile. In this study, we analyzed whether histamine was able to modulate endocytosis and cross-presentation mediated by immature DCs. Our results show that both functions are stimulated by histamine. Endocytosis of soluble HRP and FITC-OVA and cross-presentation of soluble OVA were markedly increased by histamine. Interestingly, stimulation of endocytosis and cross-presentation appeared to be mediated through different histamine receptors. In fact, the enhancement of endocytosis was prevented by the histamine2 receptor (H2R) antagonist cimetidine, whereas the stimulation of cross-presentation was prevented by the H3R/H4R antagonist thioperamide. Of note, contrasting with the observations made with soluble Ags, we found that histamine did not increase either the uptake of OVA-attached to latex beads, or the cross-presentation of OVA immobilized on latex beads. This suggests that the ability of histamine to increase endocytosis and cross-presentation is dependent on the Ag form and/or the mechanisms through which the Ag is internalized by DCs. Our results support that histamine may favor cross-presentation of soluble allergens by DCs enabling the activation of allergen-specific T CD8+ cells, which appears to play an important role in the development of allergic responses in the airway.     

The pH-dependent unfolding mechanism of P2 myelin protein: an experimental and computational study

The P2 protein is a small, extrinsic protein of the myelin membrane in the peripheral nervous system that structurally belongs to the fatty acid binding proteins (FABPs) family, sharing with them a 10 strands beta-barrel structure. FABPs appear to be involved in cellular fatty acid transport, but very little is known about the role of P2 in the metabolism of peripheral myelin lipids. Study of protein conformation at different pHs is a useful tool for the characterization of the unfolding mechanisms and the intrinsic conformational properties of the protein, and may give insight into factors that guide protein folding pathways. In particular, low pH conditions have been shown to induce partially folded states in several proteins. In this paper, the acidic unfolding of purified P2 protein was studied with both spectroscopic techniques and molecular dynamics simulation. Both experimental and computational results indicate the presence of a partly folded state at low pH, which shows structural changes mainly involving the lid that is formed by the helix-turn-helix domain. The opening of the lid, together with a barrel relaxation, could regulate the ligand exchanges near the cell membrane, supporting the hypothesis that the P2 protein may transport fatty acids between Schwann cells and peripheral myelin.     

Histamine uptake by human normal neutrophils in vitro

The uptake of different concentrations of (2.5-3H) histamine by normal human neutrophils at 37.5 degrees C in a tris-albumin buffer was determined at 20, 60 and 90 min of incubation. Uptake was correlated with the concentration of amine and incubation time with a saturation-like curve. On the basis of these results, it is suggested that neutrophils play a role in the regulation of histamine plasma levels.     

Study of the interaction of Co(III) polypyridyl complexes with DNA: an experimental and computational approach

Three new cobalt(III) polypyridyl complexes, [Co(L - L)₂IIP]³⁺ where IIP = 2-(2H-isoindol-1-yl)-2H-imidazo[4,5-f][1, 10]phenanthroline, L?=?1) phen (1,10-phenanthroline), 2) bpy (2,2’bipyridyl), 3) dmb (4, 4-dimethyl 2, 2’-bipyridine) have been synthesized, characterized (UV –VIS, IR, ¹HNMR and ¹³C NMR spectroscopy) and screened for their in vitro antibacterial activity against E.coli, Staphylococcus aureus and Bacillus subtilis. The binding of these complexes with calf-thymus DNA (CT-DNA) has been investigated by absorption and fluorescence spectroscopy, viscosity measurements. The experimental studies indicate that complexes bind to CT-DNA by means of intercalation, but with different binding affinities due to differences in the planarity of the ancillary ligand. The complexes promote photocleavage of plasmid DNA from super coiled form I to the open circular form II. The antibacterial activities suggest that the metal complexes are more active as compared to the prepared un-complexed IIP ligand.

In addition, a conformational search was carried out by Molecular Dynamics Simulations, and docking revealed that complexes intercalate between base pairs of DNA. The experimental and computational approaches reveal that the length of the intercalator and the nature of ancillary ligand are highly important factors for DNA binding.     

[3H]Histamine uptake and release by astrocytes from rat brain: Effects of sodium deprivation,high potassium,and potassium channel blockers

Histamine transport has been characterized in cultured astroglial cells of rat brain. The kinetics of [³H]-histamine uptake yielded a K_m of 0.19±0.03 M and a V_max of 3.12±0.75 pmol×mg protein^–1×min^–1. Transport system revealed high affinity for histamine and an approximately ten times higher capacity than that shown in cultured glial cells of chick embryonic brain. Ouabain which interferes with utilization of ATP to generate ion gradients, and the replacement of Na⁺ with choline inhibited the initial rate of uptake showing a strong Na⁺-dependency and suggesting the presence of a tightly coupled sodium/histamine symporter. Dissipation of K⁺-gradient (in>out) by high K⁺ or by K⁺-channel blockers, BaCl₂, (100 M), quinine (100 M) or Sparteine (20 M) produced also remarkable inhibitions in the uptake of [³H]-histamine. Impromidine, a structural histamine-analogue could inhibit the uptake non-competitively in a range of concentrations of 1 to 10 M with a K_i value of 2.8 M, indicating the specificity of the uptake. [³H]histamine uptake measurements carried out by using a suspension of dissociated hypothalamic cells, of rat brain showed a strong gliotoxin-sensitivity and yielded a Km of 0.33±0.08 M; and a V_max of 2.65±0.35 pmoles×mg protein^–1×min^–1. The uptake could be reversed by incubating the cells in histamine-free Krebs medium. The [³H]histamine efflux was sensitive to Na⁺ omission, ouabain treatment and high K⁺ or K⁺ channel blockers, resulting in marked elevations in the efflux. Data indicate that glial uptake of histamine is a high affinity, Na⁺-dependent and electrogenic, driven by an inward-oriented sodium ion gradient and an outward-oriented potassium ion gradient and functions as part of histamine inactivation, at least in a shunt mechanism.Abbreviations used HA histamine - [³H]HA [2.5-³H]-histamine - dl--aAA dl-alpha-aminoadipic acid - (Na⁺+K⁺) ATP-ase sodium and potassium activated adenosine triphosphatase - SAH S-Adenosyl-d-Homocysteine - HNMT histamine-N-methyltransferase     

Deoxyglucose uptake by mouse astrocytes: Effects of temperature and retrovirus infection

Deoxyglucose uptake by FVB/N mouse astrocytes was studied before and after infecton by tsl retrovirus which causes a neurodegenerative disease in mice similar to HIV-1 encephalopathy in man. The Michaelis-Menten kinetic parameters, Km and Vmax, of 2-deoxy-D-glucose uptake by brain and cerebellar astrocytes were measured following culture at 34°C where tsl retrovirus replicates optimally, and at 37°C. Compared to astrocytes cultured at 37°C, astrocytes cultured at 34°C had increased Km and decreased deoxyglucose uptake despite increased or unchanged Vmax. Following ts1 retrovirus infection, brain astrocyte deoxyglucose uptake doubled [132%] associated with decreased Km but unchanged Vmax, whereas cerebellar astrocyte deoxyglucose uptake doubled [102%] associated with increased Vmax but unchanged Km. These observations of altered deoxyglucose uptake kinetic parameters following retrovirus infection indicate different neurochemical mechanisms for the regional variation in deoxyglucose uptake observed following retrovirus infection of the CNS in vivo.     

Polyamine uptake in cultured astrocytes: characterization and modulation by protein kinases

The properties and regulation of the polyamine transport system in brain are still poorly understood. The present study shows, for the first time, the existence of a polyamine transport system in cerebellar astrocytes and suggests that polyamine uptake is mediated by a single and saturable high-affinity transport system for putrescine, spermine, and spermidine (K:(m) = 3.2, 1.2, and 1.8 microM:, respectively). Although substitution of NaCl by choline chloride produced a decrease in the putrescine, spermine, and spermidine uptake, it seems that polyamine transport in cerebellar astrocytes is not mediated by an Na(+) cotransport as in the presence of Na(+) and cholinium, polyamine uptake was much lower than when measured in a sucrose-based medium. On the other hand, ouabain, gramicidin (a Na(+) ionophore), and ionomycin (a Ca(2+) ionophore) produced a strong inhibition of polyamine uptake, suggesting that membrane potential could have an important role in the functioning of the astroglial polyamine uptake system. Moreover, protein kinase C inhibition produced an enhancement of polyamine uptake, whereas stimulation of protein kinase C with phorbol esters inhibited polyamine uptake. Alternatively, the tyrosine kinase inhibitor genistein caused a marked reduction in the uptake. No effects on polyamine uptake were observed with inhibitors and activators of cyclic AMP-dependent protein kinase or when Ca(2+)/calmodulin-dependent protein kinase II was inhibited with KN-62. These results suggest that the polyamine uptake system in cerebellar astrocytes could be modulated by protein kinase C and tyrosine kinase activities.     

Mechanobiological modeling of endochondral ossification: an experimental and computational analysis

Long bone formation starts early during embryonic development through a process known as endochondral ossification. This is a highly regulated mechanism that involves several mechanical and biochemical factors. Because long bone development is an extremely complex process, it is unclear how biochemical regulation is affected when dynamic loads are applied, and also how the combination of mechanical and biochemical factors affect the shape acquired by the bone during early development. In this study, we develop a mechanobiological model combining: (1) a reaction–diffusion system to describe the biochemical process and (2) a poroelastic model to determine the stresses and fluid flow due to loading. We simulate endochondral ossification and the change in long bone shapes during embryonic stages. The mathematical model is based on a multiscale framework, which consisted in computing the evolution of the negative feedback loop between Ihh/PTHrP and the diffusion of VEGF molecule (on the order of days) and dynamic loading (on the order of seconds). We compare our morphological predictions with the femurs of embryonic mice. The results obtained from the model demonstrate that pattern formation of Ihh, PTHrP and VEGF predict the development of the main structures within long bones such as the primary ossification center, the bone collar, the growth fronts and the cartilaginous epiphysis. Additionally, our results suggest high load pressures and frequencies alter biochemical diffusion and cartilage formation. Our model incorporates the biochemical and mechanical stimuli and their interaction that influence endochondral ossification during embryonic growth. The mechanobiochemical framework allows us to probe the effects of molecular events and mechanical loading on development of bone.     

Substrate diffusion and oxidation in GMC oxidoreductases: an experimental and computational study on fungal aryl-alcohol oxidase

AAO (aryl-alcohol oxidase) provides H?O? in fungal degradation of lignin, a process of high biotechnological interest. The crystal structure of AAO does not show open access to the active site, where different aromatic alcohols are oxidized. In the present study we investigated substrate diffusion and oxidation in AAO compared with the structurally related CHO (choline oxidase). Cavity finder and ligand diffusion simulations indicate the substrate-entrance channel, requiring side-chain displacements and involving a stacking interaction with Tyr?2. Mixed QM (quantum mechanics)/MM (molecular mechanics) studies combined with site-directed mutagenesis showed two active-site catalytic histidine residues, whose substitution strongly decreased both catalytic and transient-state reduction constants for p-anisyl alcohol in the H502A (over 1800-fold) and H546A (over 35-fold) variants. Combination of QM/MM energy profiles, protonation predictors, molecular dynamics, mutagenesis and pH profiles provide a robust answer regarding the nature of the catalytic base. The histidine residue in front of the FAD ring, AAO His??2 (and CHO His???), acts as a base. For the two substrates assayed, it was shown that proton transfer preceded hydride transfer, although both processes are highly coupled. No stable intermediate was observed in the energy profiles, in contrast with that observed for CHO. QM/MM, together with solvent KIE (kinetic isotope effect) results, suggest a non-synchronous concerted mechanism for alcohol oxidation by AAO.     

FACE-selective fluorogenic cycloaddition reaction between coumarin azides and sugar terminal alkynes: an experimental and computational study

Copper catalyzed azide–alkyne cycloaddition reaction (CuAAC) of non-fluorescent coumarin azides and sugar terminal alkynes afforded intense fluorescent 1,2,3-triazoles in 75–85% of yield. The photophysical properties of coumarin–sugar triazoles influenced greatly, upon introducing different substituents at 6th and 7th positions. The experimental observations were further supported by TD-DFT computational studies.     

Biophysical probing of the binding properties of a Cu(II) complex with G‐quadruplex DNA: an experimental and computational study

Telomerase inhibition through G‐quadruplex stabilization by small molecules is of great interest as a novel anticancer therapeutic strategy. Here, we show that newly synthesized Cu‐complex binds to G‐quadruplex DNA and induces changes in its stability. This biophysical interaction was investigated in vitro using spectroscopic, voltammetric and computational techniques. The binding constant for this complex to G‐quadruplex using spectroscopic and electrochemical methods is in the order of 10⁵. The binding stoichiometry was investigated using spectroscopic techniques and corresponded to a ratio of 1: 1. Fluorescence titration results reveal that Cu‐complex is quenched in the presence of G‐quadruplex DNA. Analysis of the fluorescence emission at different temperatures shows that ΔH° > 0, ΔS° > 0 and ΔG° < 0, and indicates that hydrophobic interactions played a major role in the binding processes. MD simulation results suggested that this ligand could stabilize the G‐quadruplex structure. An optimized docked model of the G‐quadruplex–ligand mixture confirmed the experimental results. Based on the results, we conclude that Cu‐complex as an anticancer candidate can bind and stabilize the G‐quadruplex DNA structure. Copyright © 2015 John Wiley & Sons, Ltd.     

Methamphetamine inhibits the glucose uptake by human neurons and astrocytes: stabilization by acetyl-L-carnitine

Methamphetamine (METH), an addictive psycho-stimulant drug exerts euphoric effects on users and abusers. It is also known to cause cognitive impairment and neurotoxicity. Here, we hypothesized that METH exposure impairs the glucose uptake and metabolism in human neurons and astrocytes. Deprivation of glucose is expected to cause neurotoxicity and neuronal degeneration due to depletion of energy. We found that METH exposure inhibited the glucose uptake by neurons and astrocytes, in which neurons were more sensitive to METH than astrocytes in primary culture. Adaptability of these cells to fatty acid oxidation as an alternative source of energy during glucose limitation appeared to regulate this differential sensitivity. Decrease in neuronal glucose uptake by METH was associated with reduction of glucose transporter protein-3 (GLUT3). Surprisingly, METH exposure showed biphasic effects on astrocytic glucose uptake, in which 20 μM increased the uptake while 200 μM inhibited glucose uptake. Dual effects of METH on glucose uptake were paralleled to changes in the expression of astrocytic glucose transporter protein-1 (GLUT1). The adaptive nature of astrocyte to mitochondrial β-oxidation of fatty acid appeared to contribute the survival of astrocytes during METH-induced glucose deprivation. This differential adaptive nature of neurons and astrocytes also governed the differential sensitivity to the toxicity of METH in these brain cells. The effect of acetyl-L-carnitine for enhanced production of ATP from fatty oxidation in glucose-free culture condition validated the adaptive nature of neurons and astrocytes. These findings suggest that deprivation of glucose-derived energy may contribute to neurotoxicity of METH abusers.     

Orchestrated neuronal migration and cortical folding: A computational and experimental study

Brain development involves precisely orchestrated genetic, biochemical, and mechanical events. At the cellular level, neuronal proliferation in the innermost zone of the brain followed by migration towards the outermost layer results in a rapid increase in brain surface area, outpacing the volumetric growth of the brain, and forming the highly folded cortex. This work aims to provide mechanistic insights into the process of brain development and cortical folding using a biomechanical model that couples cell division and migration with volumetric growth. Unlike phenomenological growth models, our model tracks the spatio-temporal development of cohorts of neurons born at different times, with each cohort modeled separately as an advection-diffusion process and the total cell density determining the extent of volume growth. We numerically implement our model in Abaqus/Standard (2020) by writing user-defined element (UEL) subroutines. For model calibration, we apply in utero electroporation (IUE) to ferret brains to visualize and track cohorts of neurons born at different stages of embryonic development. Our calibrated simulations of cortical folding align qualitatively with the ferret experiments. We have made our experimental data and finite-element implementation available online to offer other researchers a modeling platform for future study of neurological disorders associated with atypical neurodevelopment and cortical malformations.     

Inhibition of cathepsin B by Au(I) complexes: a kinetic and computational study

Gold(I) compounds have been used in the treatment of rheumatoid arthritis for over 80 years, but the biological targets and the structure–activity relationships of these drugs are not well understood. Of particular interest is the molecular mechanism behind the antiarthritic activity of the orally available drug triethylphosphine(2,3,4,6-tetra-O-acetyl-β-1-d-thiopyranosato-S) gold(I) (auranofin, Ridaura). The cathepsin family of lysosomal, cysteine-dependent enzymes is an attractive biological target of Au(I) and is inhibited by auranofin and auranofin analogs with reasonable potency. Here we employ a combination of experimental and computational investigations into the effect of changes in the phosphine ligand of auranofin on its in vitro inhibition of cathepsin B. Sequential replacement of the ethyl substituents of triethylphosphine by phenyl groups leads to increasing potency in the resultant Au(I) complexes, due in large part to favorable interactions of the more sterically bulky Au(I)–PR₃ fragments with the enzyme active site.