RESUMEN
Svx proteins are virulence factors secreted by phytopathogenic bacteria of the Pectobacterium genus into the host plant cell wall. Svx-encoding genes are present in almost all species of the soft rot Pectobacteriaceae (Pectobacterium and Dickeya genera). The Svx of P. atrosepticum (Pba) has been shown to be a gluzincin metallopeptidase that presumably targets plant extensins, proteins that contribute to plant cell wall rigidity and participate in cell signaling. However, the particular "output" of the Pba Svx action in terms of plant-pathogen interactions and plant immune responses remained unknown. The Svx proteins are largely unexplored in Dickeya species, even though some of them have genes encoding two Svx homologs. Therefore, our study aims to compare the structural and catalytic properties of the Svx proteins of Pba and D. solani (Dso) and to test the phytoimmune properties of these proteins. Two assayed Dso Svx proteins, similar to Pba Svx, were gluzincin metallopeptidases with conservative tertiary structures. The two domains of the Svx proteins form electronegative clefts where the active centers of the peptidase domains are located. All three assayed Svx proteins possessed phytoimmunosuppressory properties and induced ethylene-mediated plant susceptible responses that play a decisive role in Pba-caused disease.
Asunto(s)
Bacterias , Péptido Hidrolasas , Bioensayo , Transporte Biológico , Catálisis , DickeyaRESUMEN
Amyloid fibroproliferation leads to organ damage and is associated with a number of neurodegenerative diseases affecting populations worldwide. There are several ways to protect against fibril formation, including inhibition. A variety of organic compounds based on molecular recognition of amino acids within the protein have been proposed for the design of such inhibitors. However, the role of macrocyclic compounds, i.e., thiacalix[4]arenes, in inhibiting fibrillation is still almost unknown. In the present work, the use of water-soluble thiacalix[4]arene derivatives for the inhibition of hen egg-white lysozyme (HEWL) amyloid fibrillation is proposed for the first time. The binding of HEWL by the synthesized thiacalix[4]arenes (logKa = 5.05-5.13, 1:1 stoichiometry) leads to the formation of stable supramolecular systems capable of stabilizing the protein structure and protecting against fibrillation by 29-45%. The macrocycle conformation has little effect on protein binding strength, and the native HEWL secondary structure does not change via interaction. The synthesized compounds are non-toxic to the A549 cell line in the range of 0.5-250 µg/mL. The results obtained may be useful for further investigation of the anti-amyloidogenic role of thiacalix[4]arenes, and also open up future prospects for the creation of new ways to prevent neurodegenerative diseases.
Asunto(s)
Ácidos Carboxílicos , Muramidasa , Muramidasa/química , Humanos , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacología , Animales , Células A549 , Amiloide/química , Amiloide/metabolismo , Amiloide/antagonistas & inhibidores , Unión Proteica , Fenoles/química , Fenoles/farmacología , Calixarenos/química , Calixarenos/farmacología , SulfurosRESUMEN
1,3,5-Tris-(α-naphthyl)benzene is an organic non-electrolyte with notable stability of an amorphous phase. Its glassy and supercooled liquid states were previously studied by spectroscopic and calorimetric methods. Despite the continuing interest in its amorphous state and, particularly, vapor-deposited glasses, the thermodynamic parameters of the vaporization of 1,3,5-tris-(α-naphthyl)benzene have not been obtained yet. Likewise, the reliable evaluation of the thermodynamic parameters of fusion below the melting point, required to establish the thermodynamic state of its glass, is still an unsolved problem. In this work, the heat capacities of crystalline and liquid phases, the temperature dependence of the saturated vapor pressures, fusion and vaporization enthalpies were determined using differential and fast scanning calorimetry and were verified using the estimates based on solution calorimetry. The structural features of 1,3,5-tris-(α-naphthyl)benzene are discussed based on the computations performed and the data on the molecular refractivity. The consistency between the values obtained by independent techniques was demonstrated.
RESUMEN
The application of drugs in the amorphous state is one way to improve their bioavailability. As such, the determination of the optimal conditions for production and the assessment of the stability of the amorphous system are actively researched topics of present-day pharmaceutical science. In the present work, we have studied the kinetic stability and glass-forming ability of the thermally labile quinolone antibiotics using fast scanning calorimetry. The critical cooling rates for avoiding crystallization of the melts of oxolinic and pipemidic acids and sparfloxacin were determined to be 10â¯000, 40, and 80 K·s-1, respectively. The studied antibiotics were found to be "strong" glass formers. Based on a combination of nonisothermal and isothermal kinetic approaches, the Nakamura model was suitable for describing the crystallization process of the amorphous forms of the quinolone antibiotics.
Asunto(s)
Antibacterianos , Quinolonas , Rastreo Diferencial de Calorimetría , Cristalización , Calorimetría , CinéticaRESUMEN
Amorphous polymers currently have a wide range of applications, including the production of amorphous solid dispersions in the pharmaceutical industry. This application requires knowledge of the kinetic parameters of the glass transition process, which are the key to the formation of the end product. In the present work, we have thoroughly investigated the glass transition in the biocompatible polymer polyvinylpyrrolidone as a function of the polymer molecular mass, using differential scanning calorimetry, fast scanning calorimetry, and broadband dielectric spectroscopy. We have determined the dependence of the difference between the isobaric specific heat capacities of the liquid and the glass on the dynamic glass transition temperature, volume, and number of particles included in the cooperatively rearranging regions. A linear dependence between the shift factor from the Frenkel-Kobeko-Reiner equation and the molecular mass of polyvinylpyrrolidone was established. The results of the present work help in choosing the optimal excipient for the development of solid dispersions based on amorphous polymers.
RESUMEN
The specific features of crystal nucleation widely determine the morphology of the evolving crystalline material. Crystal nucleation is, as a rule, not accessible by direct observation of the nuclei, which develop with time. This limitation is caused by the small size (nanometer scale) of the critical nuclei and the stochastic nature of their formation. We describe an experimental approach to the determination of specific features of the cluster size distribution employing fast scanning calorimetry at scanning rates up to 10 000 K s-1. The surviving cluster fraction is determined by selectively melting/dissolving clusters smaller than the critical size corresponding to the highest temperature of a short spike positioned between the nucleation and the development stage in Tammann's two-stage method. This approach allows for estimating the time evolution of the radius of the largest detectable clusters in the distribution. Knowing this radius as a function of nucleation time allows for determining a radial growth rate. In the example of poly(l-lactic acid) (PLLA), the order of magnitude estimate of radial growth rates of clusters of about 2-5 nm yields values between 10-5 and 10-3 nm s-1. The radial growth rate of micrometer-sized spherulites is available from optical microscopy. The corresponding values are about three orders of magnitude higher than the values for the nanometer-sized clusters. This difference is explainable by stochastic effects, transient features, and the size dependence of the growth processes on the nanometer scale. The experimental and (order of magnitude) classical nucleation theory estimates agree well.
RESUMEN
It is known that four peptide fragments of predominant protein in human semen Semenogelin 1 (SEM1) (SEM1(86-107), SEM1(68-107), SEM1(49-107) and SEM1(45-107)) are involved in fertilization and amyloid formation processes. In this work, the structure and dynamic behavior of SEM1(45-107) and SEM1(49-107) peptides and their N-domains were described. According to ThT fluorescence spectroscopy data, it was shown that the amyloid formation of SEM1(45-107) starts immediately after purification, which is not observed for SEM1(49-107). Seeing that the peptide amino acid sequence of SEM1(45-107) differs from SEM1(49-107) only by the presence of four additional amino acid residues in the N domain, these domains of both peptides were obtained via solid-phase synthesis and the difference in their dynamics and structure was investigated. SEM1(45-67) and SEM1(49-67) showed no principal difference in dynamic behavior in water solution. Furthermore, we obtained mostly disordered structures of SEM1(45-67) and SEM1(49-67). However, SEM1(45-67) contains a helix (E58-K60) and helix-like (S49-Q51) fragments. These helical fragments may rearrange into ß-strands during amyloid formation process. Thus, the difference in full-length peptides' (SEM1(45-107) and SEM1(49-107)) amyloid-forming behavior may be explained by the presence of a structured helix at the SEM1(45-107) N-terminus, which contributes to an increased rate of amyloid formation.
Asunto(s)
Amiloide , Péptidos , Humanos , Secuencia de Aminoácidos , Péptidos/química , Amiloide/química , Fragmentos de Péptidos/química , Proteínas Amiloidogénicas , Dicroismo Circular , Pliegue de Proteína , Péptidos beta-Amiloides/químicaRESUMEN
In this work, we present a comprehensive study of the thermodynamic properties of 3-and 4-ethoxyacetanilides. The heat capacities in crystalline, liquid, and supercooled liquid states from 80 to 475 K were obtained using adiabatic, differential scanning (DSC), and fast scanning (FSC) calorimetries. The fusion enthalpies at Tm were combined from DSC measurement results and the literature data. The fusion enthalpies at 298.15 K were evaluated in two independent ways: adjusted according to Kirchhoff's law of thermochemistry, and using Hess' law. For the latter approach, the enthalpies of the solution in DMF in crystalline and supercooled liquid states were derived. The values obtained by the two methods are consistent with each other. The standard thermodynamic functions (entropy, enthalpy, and Gibbs energy) between 80 and 470 K were calculated.
RESUMEN
SEM1(68-107) is a peptide corresponding to the region of semenogelin 1 protein from 68 to 107 amino acid position. SEM1(68-107) is an abundant component of semen, which participates in HIV infection enhanced by amyloid fibrils forming. To understand the causes influencing amyloid fibril formation, it is necessary to determine the spatial structure of SEM1(68-107). It was shown that the determination of SEM1(68-107) structure is complicated by the non-informative NMR spectra due to the high intramolecular mobility of peptides. The complementary approach based on the geometric restrictions of individual peptide fragments and molecular modeling was used for the determination of the spatial structure of SEM1(68-107). The N- (SEM1(68-85)) and C-terminuses (SEM1(86-107)) of SEM1(68-107) were chosen as two individual peptide fragments. SEM1(68-85) and SEM1(86-107) structures were established with NMR and circular dichroism CD spectroscopies. These regions were used as geometric restraints for the SEM1(68-107) structure modeling. Even though most of the SEM1(68-107) peptide is unstructured, our detailed analysis revealed the following structured elements: N-terminus (70His-84Gln) forms an α-helix, (86Asp-94Thr) and (101Gly-103Ser) regions fold into 310-helixes. The absence of a SEM1(68-107) rigid conformation leads to instability of these secondary structure regions. The calculated SEM1(68-107) structure is in good agreement with experimental values of hydrodynamic radius and dihedral angles obtained by NMR spectroscopy. This testifies the adequacy of a combined approach based on the use of peptide fragment structures for the molecular modeling formation of full-size peptide spatial structure.
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Amiloide , Infecciones por VIH , Humanos , Espectroscopía de Resonancia Magnética , Péptidos , Fragmentos de PéptidosRESUMEN
Chiral crystals remain one of the probable sources of first minute chiral symmetry breaking, a trigger that potentially causes an as-yet unknown type of asymmetric autocatalysis during the formation of chiral biopolymers under the conditions of the Archean Earth. Therefore, studying adsorption processes on the surface of such crystals may help improve the understanding of the nature of the initial chiral shift. The adsorptive activity of non-porous crystals with respect to the majority of organic molecules essentially depends on the ability of a crystal surface to engage in specific intermolecular interactions. In this work, the enantioselectivity provided by hippuric acid and phloroglucinol crystals, obtained under Viedma ripening conditions, was studied by the adsorption of menthol enantiomers from solutions and the adsorption of limonene and α-pinene enantiomers from vapors. To establish the reliability of chiral recognition, the experimental adsorption isotherms on chiral crystals were compared with the isotherms on achiral (racemic mixtures) crystals, obtained under similar conditions. The data obtained were confirmed using CD spectra, XRD patterns and SEM images. A t-test was used to assess the statistical significance of differences in adsorption. From the adsorption isotherms of vapors at different temperatures, the isosteric heats of adsorption and the differential entropies of adsorption were calculated. It was determined that the chiral recognition ability depends not only on the difference between enantiomers in the thermodynamic functions of adsorption, but also on the isosteric heats of adsorption at low coverages and the heat of liquefaction ratio. If intermolecular interactions between the enantiomer and the surface are too weak, then enantiomer layer formation becomes difficult. This reduces the enantioselectivity or even makes chiral recognition impossible. The physicochemical regularities revealed in this present work made it possible to formulate the requirements that enantiomorphous crystals must meet for satisfactory chiral recognition of molecules of different polarities.
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Adsorción , Reproducibilidad de los Resultados , Estereoisomerismo , TermodinámicaRESUMEN
The folding of lysozyme in glycerol was monitored by the fast scanning calorimetry technique. Application of a temperature-time profile with an isothermal segment for refolding allowed assessment of the state of the non-equilibrium protein ensemble and gave information on the kinetics of folding. We found that the non-equilibrium protein ensemble mainly contains a mixture of unfolded and folded protein forms and partially folded intermediates, and enthalpic barriers control the kinetics of the process. Lysozyme folding in glycerol follows the same or similar triangular mechanism described in the literature for folding in water. The unfolding enthalpy of the intermediate must be no lower than 70% of the folded form, while the activation barrier for the unfolding of the intermediate (ca. 140 kJ/mol) is about 100 kJ/mol lower than that of the folded form (ca. 240-260 kJ/mol).
Asunto(s)
Glicerol , Muramidasa , Calorimetría , Concentración de Iones de Hidrógeno , Cinética , Muramidasa/química , Desnaturalización Proteica , Pliegue de Proteína , Proteínas/metabolismo , TermodinámicaRESUMEN
The Svx proteins are virulence factors of phytopathogenic bacteria of the Pectobacterium genus. The specific functions of these proteins are unknown. Here we show that most of the phytopathogenic species of Pectobacterium, Dickeya, and Xanthomonas genera have genes encoding Svx proteins, as well as some plant-non-associated species of different bacterial genera. As such, the Svx-like proteins of phytopathogenic species form a distinct clade, pointing to the directed evolution of these proteins to provide effective interactions with plants. To get a better insight into the structure and functions of the Svx proteins, we analyzed the Svx of Pectobacterium atrosepticum (Pba)-an extracellular virulence factor secreted into the host plant cell wall (PCW). Using in silico analyses and by obtaining and analyzing the recombinant Pba Svx and its mutant forms, we showed that this protein was a gluzincin metallopeptidase. The 3D structure model of the Pba Svx was built and benchmarked against the experimental overall secondary structure content. Structure-based substrate specificity analysis using molecular docking revealed that the Pba Svx substrate-binding pocket might accept α-glycosylated proteins represented in the PCW by extensins-proteins that strengthen the PCW. Thus, these results elucidate the way in which the Pba Svx may contribute to the Pba virulence.
Asunto(s)
Pectobacterium , Factores de Virulencia , Simulación del Acoplamiento Molecular , Pectobacterium/metabolismo , Enfermedades de las Plantas/microbiología , Plantas , Virulencia/genética , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
The first evidence of native cyclodextrins fusion was registered using fast scanning calorimetry (FSC) with heating rates up to 40,000 K s-1. The endothermal effects, detected at low heating rates, correspond to the decomposition processes. Upon the increase of the heating rate the onset of these effects shifts to higher temperatures, reaching a limiting value at high heating rates. The limiting temperatures were identified as the melting points of α-, ß- and γ-cyclodextrins, as the decomposition processes are suppressed at high heating rates. For γ-cyclodextrin the fusion enthalpy was measured. The activation energies of thermal decomposition of cyclodextrins were determined by dependence of the observed thermal effects on heating rates from 4 K min-1 in conventional differential scanning calorimetry to 40,000 K s-1 in FSC. The lower thermal stability and activation energy of decomposition of ß-cyclodextrin than for the other two cyclodextrins were found, which may be explained by preliminary phase transition and chemical reaction without mass loss. The obtained values of fusion parameters of cyclodextrins are needed in theoretical models widely used for prediction of solubility and solution rates and in preparation of cyclodextrin inclusion compounds involving heating.
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Ciclodextrinas , Ciclodextrinas/química , Rastreo Diferencial de Calorimetría , Calorimetría , Termodinámica , SolubilidadRESUMEN
The therapeutic application of serum albumin is determined by the relative content of the monomeric form compared to dimers, tetramers, hexamers, etc. In this paper, we propose and develop an approach to synthesize the cone stereoisomer of p-tert-butylthiacalix[4]arene with sulfobetaine fragments stabilization of monomeric bovine serum albumin and preventing aggregation. Spectral methods (UV-vis, CD, fluorescent spectroscopy, and dynamic light scattering) established the influence of the synthesized compounds on the content of monomeric and aggregated forms of BSA even without the formation of stable thiacalixarene/protein associates. The effect of thiacalixarenes on the efficiency of protein binding with the antibiotic ciprofloxacin was shown by fluorescence spectroscopy. The binding constant increases in the presence of the macrocycles, likely due to the stabilization of monomeric forms of BSA. Our study clearly shows the potential of this macrocycle design as a platform for the development of the fundamentally new approaches for preventing aggregation.
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Ciprofloxacina , Nanopartículas , Ciprofloxacina/química , Unión Proteica , Albúmina Sérica Bovina/química , Espectrometría de FluorescenciaRESUMEN
Artificial gene delivery systems are in great demand from both scientific and practical biomedical points of view. In this paper, we present the synthesis of a new click chemistry calix[4]arene precursor with free lower rim and new water-soluble calixarene triazoles with 12 amino-groups on the upper rim (one with free phenol hydroxyl groups and two another containing four butyl or tetradecyl fragments). Aggregation in the series of amino-triazole calixarenes of different lipophilicity (calixarene with free phenol hydroxyl groups or butyl and tetradecyl fragments on the lower rim) was studied using dynamic light scattering and fluorescent pyrene probe. It was found that calix[4]arene with a free lower rim, like alkyl-substituted butyl calix[4]arene, forms stable submicron aggregates 150-200 nm in size, while the more lipophilic tetradecyl -substituted calix[4]arene forms micellar aggregates19 nm in size. Using UV-Vis spectroscopy, fluorimetry and CD, it was shown that amino-triazole calix[4]arenes bind to calf thymus DNA by classical intercalation. According to DLS and TEM data, all studied macrocycles cause significant DNA compaction, forming stable nanoparticles 50-20 nm in size. Among all studied calix[4]arenes the most lipophilic tetradecyl one proved to be the best for both binding and compaction of DNA.
Asunto(s)
Calixarenos , Triazoles , Poliaminas , Fenol , Calixarenos/química , ADNRESUMEN
As a result of bright complexation properties, easy functionalization and the ability to self-organize in an aqueous solution, amphiphilic supramolecular macrocycles are being actively studied for their application in nanomedicine (drug delivery systems, therapeutic and theranostic agents, and others). In this regard, it is important to study their potential toxic effects. Here, the synthesis of amphiphilic calix[4]resorcinarene carboxybetaines and their esters and the study of a number of their microbiological properties are presented: cytotoxic effect on normal and tumor cells and effect on cellular and non-cellular components of blood (hemotoxicity, anti-platelet effect, and anticoagulant activity). Additionally, the interaction of macrocycles with bovine serum albumin as a model plasma protein is estimated by various methods (fluorescence spectroscopy, synchronous fluorescence spectroscopy, circular dichroic spectroscopy, and dynamic light scattering). The results demonstrate the low toxicity of the macrocycles, their anti-platelet effects at the level of acetylsalicylic acid, and weak anticoagulant activity. The study of BSA-macrocycle interactions demonstrates the dependence on macrocycle hydrophilic/hydrophobic group structure; in the case of carboxybetaines, the formation of complexes prevents self-aggregation of BSA molecules in solution. The present study demonstrates new data on potential drug delivery nanosystems based on amphiphilic calix[4]resorcinarenes for their cytotoxicity and effects on blood components.
Asunto(s)
Ésteres , Albúmina Sérica Bovina , Ésteres/farmacología , Albúmina Sérica Bovina/química , Espectrometría de Fluorescencia , Interacciones Hidrofóbicas e Hidrofílicas , Agua/químicaRESUMEN
Pillar[5]arenes containing sulfonate fragments have been shown to form supramolecular complexes with therapeutic proteins to facilitate targeted transport with an increased duration of action and enhanced bioavailability. Regioselective synthesis was used to obtain a water-soluble pillar[5]arene containing the fluorescent label FITC and nine sulfoethoxy fragments. The pillar[5]arene formed complexes with the therapeutic proteins binase, bleomycin, and lysozyme in a 1:2 ratio as demonstrated by UV-vis and fluorescence spectroscopy. The formation of stable spherical nanosized macrocycle/binase complexes with an average particle size of 200 nm was established by dynamic light scattering and transmission electron microscopy. Flow cytometry demonstrated the ability of macrocycle/binase complexes to penetrate into tumor cells where they exhibited significant cytotoxicity towards A549 cells at 10-5-10-6 M while maintaining the enzymatic activity of binase.
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Calixarenos/química , Excipientes/química , Proteínas/química , Compuestos de Amonio Cuaternario/química , Células A549 , Bleomicina/química , Bleomicina/farmacología , Endorribonucleasas/química , Endorribonucleasas/farmacología , Humanos , Muramidasa/química , Muramidasa/farmacología , Estabilidad Proteica/efectos de los fármacos , Proteínas/farmacología , Solubilidad , Agua/químicaRESUMEN
A convenient method for the synthesis of the first generation PAMAM dendrimers based on the thiacalix[4]arene has been developed for the first time. Three new PAMAM-calix-dendrimers with the macrocyclic core in cone, partial cone, and 1,3-alternate conformations were obtained with high yields. The interaction of the obtained compounds with salmon sperm DNA resulted in the formation of the associates of the size up to 200 nm, as shown by the UV-Vis spectroscopy, DLS, and TEM. It was demonstrated by the CD method that the structure of the DNA did not undergo significant changes upon binding. The PAMAM-calix-dendrimer based on the macrocycle in cone conformation stabilized DNA and prevented its degradation.
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ADN/química , ADN/metabolismo , Dendrímeros/química , Fenoles/química , Sulfuros/química , Animales , Masculino , Conformación Molecular , Salmón , Espermatozoides/metabolismoRESUMEN
In the present work, the thermochemistry of solution, solvation, and hydrogen bonding of cyclic amides in proton acceptor (B) and proton donor (RXH) solvents were studied. The infinite dilution solution enthalpies of δ-valerolactam, N-methylvalerolactam, ε-caprolactam, and N-methylcaprolactam were measured at 298.15 K. The solvation enthalpies of cyclic amides were calculated based on the measured solution enthalpies and sublimation/vaporization enthalpies from literature. The enthalpies of hydrogen bonding between cyclic amides and proton acceptor and donor solvents were then calculated as a difference between the total solvation enthalpy and the non-specific contribution. The latter was estimated via two different approaches in proton donor and proton accepting solvents. The effect of the cycle size on the strength of hydrogen bonding of the cyclic amides in solution is discussed.
Asunto(s)
Amidas/química , Lactamas/química , Solventes/química , Calorimetría , Caprolactama/química , Enlace de Hidrógeno , Piperidonas/química , ProtonesRESUMEN
Novel water-soluble multifunctional pillar[5]arenes containing amide-ammonium-amino acid moiety were synthesized. The compounds demonstrated a superior ability to bind (1S)-(+)-10-camphorsulfonic acid (S-CSA) and methyl orange dye depending on the nature of the substituent, resulting in the formation one-to-one complexes with both guests. The formation of host-guest complexes was confirmed by ultraviolet (UV), circular dichroism (CD) and 1H NMR spectroscopy. This work demonstrates the first case of using S-CSA as a chiral template for the non-covalent self-assembly of architectures based on pillar[5]arenes. It was shown that pillar[5]arenes with glycine or L-alanine fragments formed aggregates with average hydrodynamic diameters (d) of 165 and 238 nm, respectively. It was established that the addition of S-CSA to the L-alanine-containing derivative led to the formation of micron-sized aggregates with d of 713 nm. This study may advance the design novel stereoselective catalysts and transmembrane amino acid channels.