- [Show abstract] [Hide abstract] ABSTRACT: Telomerase is a prototype-shared tumor Ag and represents an attractive target for anticancer immunotherapy. We have previously described promiscuous and immunogenic HLA-DR-restricted peptides derived from human telomerase reverse transcriptase (hTERT) and referred as universal cancer peptide (UCP). In nonsmall cell lung cancer, the presence of spontaneous UCP-specific CD4 T cell responses increases the survival of chemotherapy-responding patients. However, the precise mechanisms of hTERT's uptake, processing, and presentation on MHC-II molecules to stimulate CD4 T cells are poorly understood. In this work, by using well-characterized UCP-specific CD4 T cell clones, we showed that hTERT processing and presentation on MHC-II involve both classical endolysosomal and nonclassical cytosolic pathways. Furthermore, to our knowledge, we demonstrated for the first time that hTERT's internalization by dendritic cells requires its interaction with surface heparan sulfate proteoglycans. Altogether, our findings provide a novel mechanism of tumor-specific CD4 T cell activation and will be useful for the development of novel cancer immunotherapies that harness CD4 T cells.
- [Show abstract] [Hide abstract] ABSTRACT: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis of cancer cells when bound to its cognate receptors, TRAIL-R1 and TRAIL-R2 (DR4 and DR5), without being toxic to healthy cells. Nanovectorized TRAIL (abbreviated as NPT) is 10 to 20 times more efficient than one of the most potent soluble TRAIL used in preclinical studies (His-TRAIL). To determine whether differences in affinity may account for NPT superiority, a thermodynamic study was undertaken to evaluate NPT versus TRAIL binding affinity to DR5. Docking calculations showed that TRAIL in homotrimer configuration was more stable than in heterotrimer, because of the presence of one Zn ion in its structure. Indeed, TRAIL trimers can have head-to-tail orientations when Zn is missing. Altogether these data suggest that TRAIL homotrimer structures are predominant in solution and then are grafted on NPT. When docked to DR5, NPT carrying TRAIL homotrimer leads to a more stable complex than TRAIL monomer-based NPT. To comfort these observations, the extracellular domain of DR5 was immobilized on a chromatographic support using an "in situ" immobilization technique. The determination of the thermodynamic data (enthalpy ∆H° and entropy ∆S°*) of TRAIL and NPT binding to DR5 showed that the binding mechanism was pH dependent. The affinity of NPT to DR5 increased with pH, and the ionized energy was more important for NPT than for soluble TRAIL. Moreover, because of negative values of ∆H° and ∆S°* quantities, we demonstrated that van der Waals and hydrogen bonds governed the strong NPT-DR5 association for pH > 7.4 (as for TRAIL alone). Copyright © 2016 John Wiley & Sons, Ltd.
- [Show abstract] [Hide abstract] ABSTRACT: A carbon nanotube (CNT) stationary phase was used for the first time to study the β-cyclodextrin (β-CD) solute complexation mechanism using high performance liquid chromatography (HPLC). For this, the β-CD was added at various concentrations in the mobile phase and the effect of column temperature was studied on both the retention of a series of aniline and benzoic acid derivatives with the CNT stationary phase and their complexation mechanism with β-CD. A decrease in the solute retention factor was observed for all the studied molecules without change in the retention order. The apparent formation constant KF of the inclusion complex β-CD/solute was determined at various temperatures. Our results showed that the interaction of β-CD with both the mobile phase and the stationary phase interfered in the complex formation. The enthalpy and entropy of the complex formation (ΔHF and ΔSF) between the solute molecule and CD were determined using a thermodynamic approach. Negative enthalpies and entropies indicated that the inclusion process of the studied molecule in the CD cavity was enthalpically driven and that the hydrogen bonds between carboxylic or aniline groups and the functional groups on the β-CD rim play an important role in the complex formation.
- [Show abstract] [Hide abstract] ABSTRACT: In a previous paper Guillaume's group demonstrated that magnesium (Mg2+ concentration range 0.00–2.60 mm) increased the passive diffusion of statins and thus played a role in their potential toxicity. In order to confirm an increase in this passive diffusion by divalent salt cations, the role of calcium chloride (CaCl2) on the statin–immobilized artificial membrane (IAM) association was studied. It was demonstrated that calcium supplementation (Ca2+ concentration range 0.00–3.25 mm) increases the statin passive diffusion. In addition, it was shown that the Ca2+ effect on the statin–IAM association is higher than that of Mg2+. These results show that Ca2+ enhances the passive diffusion of drugs into biological membranes and thus their potential toxicity. Also, addition of H2O2 to the medium showed a hyperbolic response for the statin passive diffusion and this effect was enhanced for the highest Ca2+ or Mg2+ concentrations in the medium. H2O2 is likely to interact with the polar head groups of the IAM through dipole–dipole interactions. The conformational changes in H2O2–IAM result in a higher degree of exposure of hydrophobic areas, thus explaining why the binding of pravastatin, which showed the lowest logP value, was less affected by H2O2. This result shows the significant contribution of H2O2 and thus the oxidative stress on the statin passive diffusion. Much of the sensitivity derives from the action of Ca2+ or Mg2+, in turn supported the idea that H2O2 may serve a Ca2+ or Mg2+ sensing function in statin passive diffusion Copyright © 2015 John Wiley & Sons, Ltd.
- [Show abstract] [Hide abstract] ABSTRACT: A novel HPLC stationary phase based on fluorinated boron nitride nanotubes (F-BNNTs) incorporated into a monolithic polymeric material was developed. This F-BNNT stationary phase was synthesized to combine the analytical performance of boron nitride nanotubes and the fluorine-based unique selectivity for polar compounds. This F-BNNT column appeared to work well when fluorinated or halogenated compounds were encountered.
- [Show abstract] [Hide abstract] ABSTRACT: The arginase enzyme was bound to porous silica using a reactive polymer where two types of nanomaterials were entrapped, i.e., carbon nanotubes (CNTs) and boron nitride nanotubes (BNNTs). For the first time, it was shown that BNNTs were highly efficient for increasing the performance of a particulate bioactive support. Also, we demonstrated that BNNTs enhanced more strongly this effect in comparison with CNTs. In addition, with this novel bioactive support, the relative IC50 values of the well-known arginase inhibitors were found to be in agreement with those derived by the conventional spectrometric method. It was shown the ethylacetate extract of the roots of Spirotropis longifolia (SL) and of the ethanol extract of sunflower (Helianthus annuus) seed (SS) and Lonicera japonica Thunb, i.e., honeysuckle (H) on the arginase activity inhibited the enzyme activity.
- [Show abstract] [Hide abstract] ABSTRACT: A novel column based on silica-containing immobilized fluorinated double-wall carbon nanotubes (F-DWCNTs) was developed. This F-DWCNT stationary phase was synthesized to combine the analytical performance of carbon nanotubes and the fluorine-based unique selectivity for polar compounds. First, the chromatographic support was coated with DWCNTs in a noncovalent way to preserve the sp(2) internal nanotube structure. Second, the DWCNT silica particles were functionalized with fluorine atoms via a solution of Br-2 and BrF3 at room temperature. This F-DWCNT stationary phase was applied for a variety of separations. The solute retention behaviour was particularly studied under isocratic conditions with a high fraction of ACN in the ACN/water (v/v) mobile phase. The retention factors of the solute molecule do not depend linearly on the ACN fraction, but follow a quadratic relationship. This fluorinated stationary phase separated compounds based upon a combination of hydrophobic and polar selective stationary phase interactions. This F-DWCNT appeared to work best when fluorinated or halogenated compounds were encountered. They have longer retention time, better selectivity and work well with high fraction of organic modifiers. This novel stationary phase could thus be a good choice for LC-MS experiments.
- [Show abstract] [Hide abstract] ABSTRACT: The tetramer destabilization of transthyretin into monomers and its fibrillation are phenomena leading to amyloid deposition. Heparan sulfate proteoglycan (HSPG) has been found in all amyloid deposits. A chromatographic approach was developed to compare binding parameters between wild-type transthyretin (wtTTR) and an amyloidogenic transthyretin (sTTR). Results showed a greater affinity of sTTR for HSPG at pH 7.4 compared with wtTTR owing to the monomeric form of sTTR. Analysis of the thermodynamic parameters showed that van der Waals interactions were involved at the complex interface for both transthyretin forms. For sTTR, results from the plot representing the number of protons exchanged vs pH showed that the binding mechanism was pH-dependent with a critical value at a pH 6.5. This observation was due to the protonation of a histidine residue as an imidazolium cation, which was not accessible when TTR was in its tetrameric structure. At pH >6.5, dehydration at the binding interface and several contacts between nonpolar groups of sTTR and HSPG were also coupled to binding for an optimal hydrogen-bond network. At pH <6.5, the protonation of the His residue from sTTR monomer when pH decreased broke the hydrogen-bond network, leading to its destabilization and thus producing slight conformational changes in the sTTR monomer structure. Copyright © 2014 John Wiley & Sons, Ltd.
- [Show abstract] [Hide abstract] ABSTRACT: Patients suffering of Alzheimer's disease (AD) are characterized by a low transthyretin (TTR) level in the brain. The effect of pH and TTR concentration in the medium on the β-amyloid protein (Aβ)/heparan sulfate proteoglycan (HSPG) association mechanism were studied using a biochromatographic approach. For this purpose, HSPG was immobilized via amino groups onto the amino propyl silica pre-packed column, activated with glutaraldehyde, by using the Schiff base method. Using an equilibrium perturbation method, it was clearly shown that Aβ can be bound with HSPG. This approach allowed the determination of the thermodynamic data of this binding mechanism. The role of the pH was also analyzed. Results from enthalpy-entropy compensation and the plot of the number of protons exchanged versus pH showed that the binding mechanism was dependent on pH with a critical value at pH=6.5. This value agreed with a histidine protonation as an imidazolium cation. Moreover, the corresponding thermodynamical data showed that at pH>6.5, van der Waals and hydrogen bonds due to aromatic amino acids as tyrosine or phenylalanine present in the N-terminal (NT) part governed the Aβ/HSPG association. Aβ remained in its physiological structure in a random coil form (i.e. the non-amyloidogenic structure) because van der Waals interactions and hydrogen bonds were preponderant. At acidic pH (pH<6.5), ionic and hydrophobic interactions, created by histidine protonation and hydrophobic amino acids, appeared in the Aβ/HSPG binding. These hydrophobic and ionic interactions led to the conversion of the random coil form of Aβ into a β-sheet structure which was the amyloidogenic folding. When TTR was incubated with Aβ, the Aβ/HSPG association mechanism was enthalpy driven at all pH values. The affinity of Aβ for HSPG decreased when TTR concentration increased due to the complexation of Aβ with TTR. Also, the decrease of the peak area with the increase of TTR concentration demonstrated that this Aβ/TTR association led to the cleavage of Aβ full length to a smaller fragment. For acidic pH (pH<6.5), it was shown that the importance of the hydrophobic and ionic interactions decreased when TTR concentration increased. This result confirmed that Aβ was cleaved by TTR in a part containing only the NT part. Our results demonstrated clearly that TTR reversed the effect of acidic pH and thus played a protective role in AD.
Dataset: Supplementary Material
- [Show abstract] [Hide abstract] ABSTRACT: A new carbon nanotube porous silica poroshell stationary phase was developed. The chromatographic support was coated with ultrashort single-wall carbon nanotubes (SWCNTs) in a noncovalent way. It was demonstrated that the porous amino silica surface of the 300 NH2 poroshell column stabilized with 1-methyl-2-pyrrolidinone efficiently and stably adsorbed SWCNTs onto the chromatographic support. It was shown that this novel poroshell carbon nanotube (CNT) stationary phase was very useful for the HPLC separation of a series of monoclonal antibodies (mAbs) in a short analysis time (<3 min). The high-performance liquid chromatography (HPLC) method was validated and was successfully tested for the fast quantitative and qualitative control of chemotherapeutic bags fabricated in a hospital pharmacy.
- [Show abstract] [Hide abstract] ABSTRACT: This work deals with the development of a carbon nanotube (CNT) monolithic column coated with a pyrenyl derivative as chiral selector. For this, a solution of pyrenyl neomycine A was pumping through a monolithic CNT column previously developed by our group. This coating was stable against the desorption for months when aqueous mobile phases were used. This column was applied to the chiral separation of underivatized amino acids. As well, ultra fast separations in the range of seconds were achieved using high flow-rates.
- [Show abstract] [Hide abstract] ABSTRACT: A biochromatographic system was used to study the direct effect of carbon nanoparticles (CNPs) on the acetylcholinesterase (AChE) activity. The AChE enzyme was covalently immobilized on a monolithic CIM-disk via its NH2 residues. Our results showed an increase in the AChE activity in presence of CNPs. The catalytic constant (kcat) was increased while the Michaelis constant (Km) was slightly decreased. This indicated an increase in the enzyme efficiency with increase of the substrate affinity to the active site. The thermodynamic data of the activation mechanism of the enzyme, i.e. ΔH* and ΔS*, showed no change in the substrate interaction mechanism with the anionic binding site. The increase of the enthalpy (ΔH*) and the entropy (ΔS*) with decrease in the free energy of activation (Ea) was related to structural conformation change in the active site gorge. This affected the stability of water molecules in the active site gorge and facilitated water displacement by substrate for entering to the active site of the enzyme.
- [Show abstract] [Hide abstract] ABSTRACT: Acetylcholinesterase (AChE) is a serine protease that hydrolyzes the neurotransmitter acetylcholine. Here, the effects of hydroxyl radical (OH˙) and nitric oxide (NO) on AChE activity were studied using a biochromatographic process. The enzyme was immobilized on an ethylenediamine (EDA) monolithic convective interaction media (CIM) disk. The AChE enzymatic mechanism was demonstrated from the chromatographic peak shape. A decrease in AChE activity was observed for each concentration of NO, while OH˙ radical formation led to an increase in the rate of enzymatic catalysis. Michaelis-Menten and Lineweaver-Burk plots were obtained in the presence or absence of the free radicals and their effects on Km and Vmax were evaluated. Our results indicated classical deactivation/activation kinetics without significant influence on the rate of substrate binding. The variation in transition state energies (ΔΔGES) induced by the free radicals indicated that a conformational change was occurring in the active site, while changes in the binding site were negligible. These results clearly demonstrate the direct role of OH˙ and NO on AChE activity and confirm the role they may play in Alzheimer’s disease.
- [Show abstract] [Hide abstract] ABSTRACT: The influence of the mobile phase composition and column temperature on the chromatographic separation of five buckminsterfullerenes (C60, C70, C76, C78, C84) on a stationary phase based on silica gel with chemically bonded humic acid (Bonded humic acid column (BHAC)) was studied. The retention behavior of the fullerenes was measured under isocratic conditions with different mobile phase compositions, ranging from 0.05–0.70 (v/v) of toluene in cyclohexane. The column temperature was analysed in the range 35–75°C. The retention factors of the five fullerenes do not depend linearly on the toluene fraction but follow a quadratic relationship. The best chromatographic conditions for baseline separation of the five fullerenes were selected. The retention of the fullerenes on the HA stationary phase was strongly affected by temperature. Positive values of thermodynamic parameters (changes of enthalpy and entropy) were due to the abnormal solubility behaviour of fullerenes in toluene in the temperature range 35–75°C. The information obtained in this work makes this BHAC very simple to prepare and low cost, useful for fullerene research applications.
- [Show abstract] [Hide abstract] ABSTRACT: In this paper, an effective and simple method was used for the immobilization of single wall carbon nanotubes (SWCNTs) on a monolithic HPLC material containing 2μm macropore sizes and 13nm mesopore sizes. The chromatographic support was coated with ultra short SWCNTs in a noncovalent way to preserve the sp(2) nanotube structure and thus their physico-chemical properties. It was demonstrated that the amino-surface of the monolith stabilized with 1-methyl-2-pyrrolidinone efficiently and stably adsorbed SWCNTs onto the chromatographic support. It was shown that this novel stationary phase was very useful for the HPLC isocratic mode separation of a series of small aromatic compounds in a very short analysis time. The comparison with a classical equivalent C18 monolithic column showed that the SWCNT column presented the best efficiency in similar chromatographic conditions.
- [Show abstract] [Hide abstract] ABSTRACT: In this paper, a new and effective method was described for attaching gold nanoparticles (Au-NPs) on to the surface of thiol-terminated Boron Nitride Nanotubes (BNNT) functionalized with quinuclidine-3-thiol, acting as a bridging agent. The quinuclidine-3-thiol was first grafted onto the surface of the BNNTs via strong interactions between the electron pair from the nitrogen atom of the quinuclidine structure and the electronic gap from the boron atom of the BNNT. The bare surface of Au-NPs facilitates to attach on the thiol group of the thiol-terminated BNNTs. These two nanomaterials (pristine BNNTs and Au-BNNTs) were then incorporated into a monolithic polymer. The obtained monolithic BNNT and AuBNNT stationary phases were very useful columns for the HPLC isocratic mode separation of a series of benzene and naphtalene derivatives. The retention on these two stationary phases was due to the different intermolecular interactions including the dispersion interaction (area of the delocalized π bond), the dipole-dipole interactions, and the electrostatic repulsion. The presence of Au-NPs on the BNNT surface improved significantly the retention and column efficiency for compounds with thiol groups in their structure. As well, it was shown that both retention and column efficiency linearly increased with the nanotube (NT) amount in the polymerization mixture. This manuscript thus established for the first time the fact that BNNT was a very useful nanomaterial for the development of novel HPLC stationary phases and increased the performance of classical equivalent C18 monolithic columns.
- [Show abstract] [Hide abstract] ABSTRACT: The interaction of a series of steroid hormones (DHEA, progesterone, testosterone, estradiol) with serum albumin immobilized on porous silica particles and the effect of reactive oxygen species and nitric oxide on this interaction were studied using a biochromatographic approach. The determination of enthalpy and entropy changes of this binding indicated that van der Waals interactions and hydrogen bonds predominated the hormone association with albumin. Reactive oxygen species (H(2)O(2) and OH*) increased the hormone binding affinity to albumin. On the other hand, this binding was decreased with the presence of NO*. This variation was due to conformational changes in the binding region explained by the oxidation of some residues such as free thiol and arginine. The thermodynamic analysis showed that free radical affects the van der Waals forces and/or a hydrogen bond of the hormone binding with albumin. These results explained the role of reactive oxygen species and nitric oxide in the hormone free fraction level in the blood.
- [Show abstract] [Hide abstract] ABSTRACT: β-Amyloid (Aβ) is a major component of the senile plaques characteristic of Alzheimer disease (AD). Chondroitin sulfate (CS) and glycoaminoglycan (GAG) are also localized throughout the senile plaques in AD. In previous studies, the interaction of the Aβ protein with CS immobilized on a chromatographic support and the role of aluminum and copper cations was studied using a molecular chromatographic approach [1, 2]. Here, we demonstrated the direct implication of OH· radical formation on this binding via a novel analytical procedure. The binding of Aβ amyloid on CS was accompanied by an OH· radical uptake. The Aβ–CS complex was stabilized by the OH· radical via the creation of about one to two hydrogen bonds. The addition in the medium of a radical scavenger allowed decreasing the Aβ/CS association and thus confirmed the positive role of these compounds in amyloidosis.
University of Franche-ComtéBecoinson, Franche-Comté, France
Laboratoire des Sciences du Climat et l'EnvironnementGif, Île-de-France, France