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1.
Sci Rep ; 7(1): 2198, 2017 05 19.
Artículo en Inglés | MEDLINE | ID: mdl-28526864

RESUMEN

Previous studies of the oligoacyllysyl (OAK) series acyl-lysyl-lysyl-aminoacyl-lysine-amide, suggested their utility towards generating robust linear lipopeptide-like alternatives to antibiotics, although to date, none exhibited potent broad-spectrum bactericidal activity. To follow up on this premise, we produced a new analog (C14KKc12K) and investigated its properties in various media. Mechanistic studies suggest that C14KKc12K uses a non-specific membrane-disruptive mode of action for rapidly reducing viability of Gram-negative bacteria (GNB) similarly to polymyxin B (PMB), a cyclic lipopeptide used as last resort antibiotic. Indeed, C14KKc12K displayed similar affinity for lipopolysaccharides and induced cell permeabilization associated with rapid massive membrane depolarization. Unlike PMB however, C14KKc12K was also bactericidal to Gram-positive bacteria (GPB) at or near the minimal inhibitory concentration (MIC), as assessed against a multispecies panel of >50 strains, displaying MIC50 at 3 and 6 µM, respectively for GPB and GNB. C14KKc12K retained activity in human saliva, reducing the viability of cultivable oral microflora by >99% within two minutes of exposure, albeit at higher concentrations, which, nonetheless, were similar to the commercial gold standard, chlorhexidine. This equipotent bactericidal activity was also observed in pre-formed biofilms of Streptococcus mutans, a major periodontal pathogen. Such compounds therefore, may be useful for eradication of challenging poly-microbial infections.


Asunto(s)
Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Lipopéptidos/farmacología , Antibacterianos/química , Relación Dosis-Respuesta a Droga , Lipopéptidos/química , Estructura Molecular , Streptococcus mutans/efectos de los fármacos
2.
Phys Chem Chem Phys ; 18(32): 22516-25, 2016 Aug 10.
Artículo en Inglés | MEDLINE | ID: mdl-27468431

RESUMEN

Kappa-casein (κCN) and beta-casein (ßCN) are disordered proteins present in mammalian milk. In vitro, ßCN self-assembles into core-shell micelles. κCN self assembles into similar micelles, as well as into amyloid-like fibrils. Recent studies indicate that fibrillization can be suppressed by mixing ßCN and κCN, but the mechanism of fibril inhibition has not been identified. Examining the interactions of native and reduced kappa-caseins (N-κCN and R-κCN) with ßCN, we expose a competition between two different self-assembly processes: micellization and fibrillization. Quite surprisingly, however, we find significant qualitative and quantitative differences in the self-assembly between the native and reduced κCN forms. Specifically, thermodynamic analysis reveals exothermic demicellization for ßCN and its mixtures with R-κCN, as opposed to endothermic demicellization of N-κCN and its mixtures with ßCN at the same temperature. Furthermore, with time, R-κCN/ßCN mixtures undergo phase separation into pure ßCN micelles and R-κCN fibrils, while in the N-κCN/ßCN mixtures fibril formation is considerably delayed and mixed micelles persist for longer periods of time. Fibrils formed in N-κCN/ßCN mixtures are shorter and more flexible than those formed in R-κCN/ßCN systems. Interestingly, in the N-κCN/ßCN mixtures, the sugar moieties of N-κCN oligomers seem to organize on the mixed micelles surface in a manner similar to the organization of κCN in milk casein micelles.


Asunto(s)
Caseínas/química , Micelas , Leche/química , Amiloide/metabolismo , Animales , Caseínas/metabolismo , Temperatura , Termodinámica
3.
Chirality ; 24(7): 500-5, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22570171

RESUMEN

Supramolecular chiral assemblies of R(-) and S(+) 2-butanol, in their neat form or when dissolved in their nonchiral isomer isobutanol, were evaluated by isothermal titration calorimetry (ITC) ensuing mixing. Dilution of 0.5 M solution of R(-) 2-butanol in isobutanol into the latter liberated heat of several calories per mole, which was approximately double than that obtained in parallel dilutions of S(+) 2-butanol in isobutanol. The ITC dilution profiles indicated an estimate of about 100 isobutanol solvent molecules surrounding each of the 2-butanol enantiomers, presumably arranged in chiral configurations, with different adopted order between the isomers. Mixings of neat R and S 2-butanol were followed by endothermic ITC profiles, indicating that, in racemic 2-butanol, both the supramolecular order and the intermolecular binding energies are lower than in each of the neat chiral isomers. The diversion from symmetrical ITC patterns in these mixings indicated again a subtle difference in molecular organization between the neat enantiomers. It should be noted that the presence of impurities, α-pinene and teterhydrofuran, at a level totaling 0.5%, did not influence the ITC heat flow profiles. The findings of this study demonstrate for the first time that chiral solutes in organic solvents are expected to acquire asymmetric solvent envelopes that may be different between the enantiomers, thus broadening this phenomenon beyond the previously demonstrated cases in aqueous solutions.


Asunto(s)
Butanoles/química , Calorimetría , Calor , Estereoisomerismo
4.
Phys Chem Chem Phys ; 13(8): 3153-60, 2011 Feb 28.
Artículo en Inglés | MEDLINE | ID: mdl-21079858

RESUMEN

Amphiphilic block copolymers and mixtures of amphiphiles find broad applications in numerous technologies, including pharma, food, cosmetic and detergency. Here we report on the interactions between a biological charged diblock copolymer, ß-casein, and a synthetic uncharged triblock copolymer, Lutrol F-127 (EO(101)PO(56)EO(101)), on their mixed micellization characteristics and the micelles' structure and morphology. Isothermal titration calorimetry (ITC) experiments indicate that mixed micelles form when Lutrol is added to monomeric as well as to assembled ß-casein. The main driving force for the mixed micellization is the hydrophobic interactions. Above ß-casein CMC, strong perturbations caused by penetration of the hydrophobic oxypropylene sections of Lutrol into the protein micellar core lead to disintegration of the micelles and reformation of mixed Lutrol/ß-casein micelles. The negative enthalpy of micelle formation (ΔH) and cooperativity increase with raising ß-casein concentration in solution. ζ-potential measurements show that Lutrol interacts with the protein micelles to form mixed micelles even below its critical micellization temperature (CMT). They further indicate that Lutrol effectively masks the protein charges, probably by forming a coating layer of the ethyleneoxide rich chains. Small-angle X-ray scattering (SAXS) and cryogenic-transmission electron microscopy (cryo-TEM) indicate relatively small changes in the oblate micellar shape, but do show swelling along the small axis of ß-casein micelles in the presence of Lutrol, thereby confirming the formation of mixed micelles.


Asunto(s)
Caseínas/química , Micelas , Polietilenos/química , Polímeros/química , Polipropilenos/química , Calorimetría , Microscopía Electrónica de Transmisión , Unión Proteica , Dispersión del Ángulo Pequeño , Temperatura , Termodinámica , Difracción de Rayos X
5.
J Agric Food Chem ; 56(6): 2192-8, 2008 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-18303844

RESUMEN

Beta-casein is an intrinsically unstructured amphiphilic protein that self-assembles into micelles at neutral pH. This paper reports that beta-casein self-organizes into micelles also under acidic conditions. The protein association behavior and micelle characteristics at pH 2.6, well below the p I, are presented. The pH was found to strongly affect the micelle shape and dimensions. Cryogenic transmission electron microscopy (cryo-TEM) experiments revealed disk-like micelles of 20-25 nm in length and approximately 3.5 nm in height in acidic conditions. An aggregation number of 6 was determined by sedimentation equilibrium under these conditions. Isothermal titration calorimetry experiments verified the association below the p I and allowed determination of the micellization enthalpy, the critical micellar concentration, and the micellization relative cooperativity (MR). Small-angle X-ray scattering results at concentrations below the critical micellization concentration (CMC) suggest that the monomeric protein is likely in a premolten globule state at low pH. Calculations of the protein charge at acidic and neutral pH reveal a similar high net charge but considerable differences in the charge distribution along the protein backbone. Overall the results show that beta-casein is amphiphilic at low pH, but the distribution of charge along the protein chain creates packing constraints that affect the micelle organization, leading at concentrations above the CMC to the formation of disk micelles.


Asunto(s)
Caseínas/química , Micelas , Animales , Bovinos , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Dispersión de Radiación , Termodinámica , Rayos X
6.
Chem Biol ; 14(1): 75-85, 2007 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17254954

RESUMEN

Truncation and acylation were combined to investigate the broad-spectrum bactericidal and hemolytic peptide S4(1-15). Substitution of up to seven residues with dodecanoic acid (C(12)) gradually led to specific antipseudomonal activity: out of 40 bacterial strains tested in vitro, C(12)-S4(8-15) displayed similar minimal inhibitory concentrations (MICs) as S4(1-15) against Pseudomonas aeruginosa sp. (identical MIC(90)) but was practically inactive against most other bacteria or erythrocytes. Surface plasmon resonance and isothermal titration calorimetry experiments revealed the binding properties of S4(1-15) to be consistent with its nonselective activities, while discriminative activities of C(12)-S4(8-15) correlated with high binding affinity to a membrane containing pseudomonal lipopolysaccharides and with lower affinities to membranes containing nonpseudomonal lipopolysaccharides or cholesterol. Various mechanistic studies failed to detect significant differences in secondary structure, bactericidal kinetics, or ability to perturb the cytoplasmic membrane, pointing to a similar mode of action.


Asunto(s)
Proteínas Anfibias/química , Proteínas Anfibias/farmacología , Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Pseudomonas/efectos de los fármacos , Acilación , Secuencia de Aminoácidos , Animales , Antibacterianos/farmacología , Membrana Celular/metabolismo , Hemólisis/efectos de los fármacos , Humanos , Lipopolisacáridos/metabolismo , Pruebas de Sensibilidad Microbiana , Pseudomonas/ultraestructura , Relación Estructura-Actividad
7.
Evid Based Complement Alternat Med ; 3(3): 329-38, 2006 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-16951717

RESUMEN

Teucrium polium L. (Lamiaceae) (RDC 1117) is a medicinal plant whose species have been used for over 2000 years in traditional medicine due to its diuretic, diaphoretic, tonic, antipyretic, antispasmodic and cholagogic properties. The therapeutic benefit of medicinal plants is often attributed to their antioxidant properties. We previously reported that an aqueous extract of the leaves and stems of this plant could inhibit iron-induced lipid peroxidation in rat liver homogenate at concentrations that were not toxic to cultured hepatic cells. Others have reported that organic extracts of the aerial components of this plant could inhibit oxidative processes. Against this background, we felt further investigation on the antioxidant action of the extract of T. polium prepared according to traditional Arab medicine was warranted. Accordingly, we assessed (i) its ability to inhibit (a) oxidation of beta-carotene, (b) 2,2'-azobis(2-amidinopropan) dihydrochloride (AAPH)-induced plasma oxidation and (c) iron-induced lipid peroxidation in rat liver homogenates; (ii) to scavenge the superoxide (O2*-) radical and the hydroxyl radical (OH(*)); (iii) its effects on the enzyme xanthine oxidase activity; (iv) its capacity to bind iron; and (v) its effect on cell glutathione (GSH) homeostasis in cultured Hep G2 cells. We found that the extract (i) inhibited (a) oxidation of beta-carotene, (b) AAPH-induced plasma oxidation (c) Fe(2+)-induced lipid peroxidation in rat liver homogenates (IC(50) = 7 +/- 2 mug ml(-1)); (ii) scavenged O2*-(IC(50) = 12 +/- 3 mug ml(-1)) and OH(*) (IC(50) = 66 +/- 20 mug ml(-1)); (iii) binds iron (IC(50) = 79 +/- 17 mug ml(-1)); and (iv) tended to increase intracellular GSH levels resulting in a decrease in the GSSG/GSH ratio. These results demonstrate that the extract prepared from the T. polium possesses antioxidant activity in vitro. Further investigations are needed to verify whether this antioxidant effect occurs in vivo.

8.
J Agric Food Chem ; 54(15): 5555-61, 2006 Jul 26.
Artículo en Inglés | MEDLINE | ID: mdl-16848545

RESUMEN

The association behavior, critical micellization concentration (CMC), and enthalpy of demicellization (DeltaHdemic) of bovine beta-casein were studied, for the first time by isothermal titration calorimetry, in a pH 7.0 phosphate buffer with 0.1 ionic strength and in pure water. In the buffer solutions, the CMC decreased asymptotically from 0.15 to 0.006 mM as the temperature was raised from 16 to 45 degrees C. DeltaHdemic decreased with increasing temperature between 16 and 28 degrees C but increased from 28 to 45 degrees C. Thermodynamic analysis below 30 degrees C is consistent with the Kegeles shell model, which suggests a stepwise association process. At higher temperatures, this model exhibits limitations, and the micellization becomes much more cooperative. The CMC values in water, measured between 17 and 28 degrees C, decreased with increasing temperature and, expectedly, were higher than those found in the buffer solutions. beta-Casein micelles were visualized and characterized, for the first time in their hydrated state, using advanced digital-imaging cryogenic transmission electron microscopy. The images revealed small, oblate micelles, about approximately 13 nm in diameter. The micelles shape and dimensions remained nearly constant in the temperature range of 24-35 degrees C.


Asunto(s)
Calorimetría/métodos , Caseínas/química , Microscopía por Crioelectrón , Micelas , Termodinámica
9.
Phys Chem Chem Phys ; 8(3): 333-9, 2006 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-16482275

RESUMEN

Mirror-image asymmetric molecules, i.e., chiral isomers or enantiomers, are classically considered as chemically identical. Recent studies, however, have indicated that parity violation by the nuclear weak force induces a tiny energy difference between chiral isomers. Upon combination with a massive amplification process, expansion of this difference to a detectable macroscopic level may be achieved. Yet, experimental tests of this possibility, where one enantiomer is compared to the other in solution, are hampered by the possible presence of undetectable impurities. In this study we have overcome this problem by comparing structural and dynamic features of synthetic D- and L-polyglutamic acid and polylysine molecules each of 24 identical residues. In these water-soluble polypeptides helix formation is an intramolecular autocatalytic process amplified by each turn, which is actually unaffected by low level of putative impurities in the solvent. The helix and random coil configurations and their transition were determined in this study by circular dichroism (CD) and isothermal titration calorimetry (ITC) in water and deuterium oxide. Distinct differences in structure and transition energies between the enantiomeric polypeptides were detected by both CD and ITC when dissolved in water. Intriguingly, these differences were by and large abolished in deuterium oxide. Our findings suggest that deviation from physical invariance between the D- and L-polyamino acids is induced in part by different hydration in water which is eliminated in deuterium oxide. Based on the recent findings by Tikhonov and Volkov (V. I. Tikhonov and A. A. Volkov, Science 2002, 296, 2363) we suggest that ortho-H(2)O, which constitutes 75% of bulk H(2)O, has a preferential affinity to L-enantiomers. Differential hydration of enantiomers may have played a role in the selection of L-amino acids by early forms of life.


Asunto(s)
Ácido Poliglutámico/química , Polilisina/química , Agua/química , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Conformación Molecular , Solubilidad , Estereoisomerismo
10.
Evid Based Complement Alternat Med ; 2(4): 549-56, 2005 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-16322813

RESUMEN

In response to increased popularity and greater demand for medicinal plants, a number of conservation groups are recommending that wild medicinal plants be brought into cultivation systems. We collected four medicinal herbs Cichorium pumilum, Eryngium creticum, Pistacia palaestina and Teucrium polium used in traditional Arab medicine for greenhouse cultivation to assess the effects of different fertilization regimes on their growth and antioxidant activity. Wild seedlings were collected and fertilized with either 100% Hoagland solution, 50% Hoagland solution, 20% Hoagland solution or irrigated with tap water. Plant height was measured and the number of green leaves and branches counted weekly. Thereafter, the aboveground parts of plants were harvested for preparing a water-soluble powder extracts of which antioxidant activity was measured by their ability to suppress the oxidation of beta-carotene. Of the fertilization regimes, we found either 20 or 50% Hoagland solution produced the most consistent response of the plant growth parameters. All powders prepared from the four wild growing plants inhibited oxidation of beta-carotene. Increasing the amount of fertilizer caused a significant concentration-dependent increase in antioxidant activity of the cultivated T. polium compared with the wild type. In contrast, increasing the amount of fertilizer caused a significant concentration-dependent reduction in the antioxidant activity of powders prepared from the cultivated E. creticum when compared with wild plants. Our results showed that cultivation success should not rely solely on parameters of growth but should incorporate assessment related to indices of therapeutic potential.

11.
J Ethnopharmacol ; 100(1-2): 198-204, 2005 Aug 22.
Artículo en Inglés | MEDLINE | ID: mdl-16054533

RESUMEN

In a previous study, we identified Pistacia lentiscus was worthy for further laboratory evaluation because an aqueous extract of the plant suppressed iron-induced lipid peroxidation in rat liver homogenates without affecting mitochondrial respiration in cultured HepG2 and PC12 cells. The present study was undertaken to evaluate the efficacy of an aqueous extract prepared from the dried leaves of Pistacia lentiscus in a rat model of hepatic injury caused by the hepatotoxin, thioacetamide. We assessed the impact of daily dosing on biochemical and morphological indices and the extent of oxidative stress in the livers of healthy and thioacetamide-treated rats. In healthy rats, long-term administration of the extract induced hepatic fibrosis and an inflammatory response, mild cholestasis and depletion of reduced glutathione associated with an increase in its oxidized form. In thioacetamide-treated rats, long-term administration of extract aggravated the inflammatory and fibrotic and glutathione depleting responses without affecting the extent of lipid peroxidation. Although our previous in vitro study established that extracts prepared from the leaves of Pistacia lentiscus had antioxidant activity, this in vivo study establishes these extracts also contains hepatotoxins whose identity may be quite different from those compounds with antioxidant properties. The results of this study suggest complementing in vitro experiments with those involving animals are essential steps in establishing the safety of medicinal plants. Furthermore, these data confirm that complete reliance on data obtained using in vitro methodologies may lead to erroneous conclusions pertaining to the safety of phytopharmaceuticals.


Asunto(s)
Hepatopatías/tratamiento farmacológico , Pistacia/química , Hojas de la Planta/química , Animales , Peso Corporal/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas , Glutatión/sangre , Peroxidación de Lípido/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/patología , Masculino , Tamaño de los Órganos/efectos de los fármacos , Extractos Vegetales/química , Extractos Vegetales/farmacología , Ratas , Ratas Sprague-Dawley , Tioacetamida
12.
J Ethnopharmacol ; 101(1-3): 153-61, 2005 Oct 03.
Artículo en Inglés | MEDLINE | ID: mdl-15970411

RESUMEN

BACKGROUND: The medicinal use of extracts prepared from plant parts of the genus Crataegus dates back to ancient times. Furthermore, it has been proposed that its antioxidant constituents account for its beneficial therapeutic effects. A decoction of leaves and unripe fruits from Crataegus aronia syn. azarolus (L) (Rosaceae), the indigenous Israeli hawthorn, is used to treat cardiovascular diseases, cancer, diabetes and sexual weakness in Arab traditional medicine. PURPOSE: Because laboratory data on the bioactivity of extracts prepared from the indigenous Israeli hawthorn is lacking, we evaluated the antioxidant and cytotoxic potentials of an extract prepared from leaves and unripe fruits in a variety of cell and cell-free in vitro assays. METHODS: The antioxidant assays measured: (a) its ability to inhibit (i) oxidation of beta-carotene, (ii) 2,2'-azobis(2-amidino-propan) dihydrochloride (AAPH)-induced plasma oxidation and (iii) iron-induced lipid peroxidation in rat liver homogenates; (b) its ability to scavenge the superoxide (O2-) radical; (c) its effects on the enzyme xanthine oxidase (XO) activity; (d) its effect on the redox state of glutathione (GSH) in cultured Hep G2 cells. In addition, we also evaluated the effects of the extract on cell membrane integrity and mitochondrial respiration in cultured Hep G2 cells. RESULTS AND CONCLUSIONS: Water-soluble extracts inhibited (1) oxidation of beta-carotene, (2) AAPH-induced plasma oxidation and (3) Fe(2+)-induced lipid peroxidation in rat liver homogenates. In addition, the extract (4) is an efficient scavenger of the O2- (5) increases intracellular GSH levels and (6) is not cytotoxic. Accordingly, we propose that the therapeutic benefit of Crataegus aronia can be, at least in part, attributed to its effective inhibition of oxidative processes, efficient scavenging of O2- and possible increasing GSH biosynthesis.


Asunto(s)
Antioxidantes/farmacología , Fitoterapia , Extractos Vegetales/farmacología , Animales , Crataegus , Glutatión/metabolismo , Israel , Peroxidación de Lípido/efectos de los fármacos , Masculino , Medicina Tradicional , Oxidación-Reducción , Ratas , Ratas Sprague-Dawley , Superóxidos/metabolismo , beta Caroteno/metabolismo
13.
J Ethnopharmacol ; 99(1): 43-7, 2005 May 13.
Artículo en Inglés | MEDLINE | ID: mdl-15848018

RESUMEN

Ethnopharmacological surveys conducted among herbal practitioners of traditional Arab medicine in Israel and the Palestinian area have revealed a large number of indigenous plant species are used as sources of their herbal therapies. Some of these herbal therapies are used to treat liver disease, jaundice or diabetes, conditions in which oxidative stress is prominent. No laboratory data on the bioactivity of herbal medicines in these settings exist in traditional Arab medicine. We hypothesized that the beneficial effect of these plants might be due to their antioxidant properties. Accordingly, we selected eight plants used to treat these two conditions and assessed their antioxidant potential by measuring their ability to suppress the extent of iron-induced lipid peroxidation in rat liver homogenates and their potential toxicity by evaluating their effects on mitochondrial respiration and cell membrane integrity in cultured PC12 and HepG2 cells. We found that all the extracts can suppress iron-induced lipid peroxidation and are not toxic. Of these extracts, those prepared from Teucrium polium and Pistacia lentiscus were the most effective in suppressing iron-induced lipid peroxidation. Further investigations are now needed to establish the exact mechanism of action and identify the active bio-ingredient(s) of each extract in order to explain their therapeutic efficacy.


Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Antioxidantes/farmacología , Animales , Arabia , Membrana Celular/efectos de los fármacos , Respiración de la Célula/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Hierro/antagonistas & inhibidores , Israel , L-Lactato Deshidrogenasa/metabolismo , Peroxidación de Lípido/efectos de los fármacos , Medicina Tradicional , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Células PC12 , Extractos Vegetales/farmacología , Ratas , Sales de Tetrazolio , Tiazoles , Células Tumorales Cultivadas
14.
J Allergy Clin Immunol ; 114(5): 1151-8, 2004 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-15536424

RESUMEN

BACKGROUND: The increased consumption of foods containing sesame seeds is paralleled by an increase in reported sesame-induced allergic reactions. OBJECTIVE: This study aimed at identifying and characterizing the linear B-cell epitopes of the 14-kd beta-globulin, the major allergen of sesame seed. METHODS: A peptide containing 71 amino acids (peptide B) was previously identified by us as the IgE-binding region on beta-globulin. To determine the amino acid sequence of the IgE-binding sites on peptide B, we synthesized overlapping peptides 20 and 10 amino acid residues long that span the entire length of peptide B, which were offset from each other by 10 and 2 amino acid residues, respectively. Sera from 20 subjects given diagnoses of allergy to sesame beta-globulin served to identify the epitopes by using the dot-blot test. RESULTS: At least 9 different IgE-recognition sites were identified on peptide B. Three of them, numbers 2, 3, and 13 (corresponding to amino acids 46-55, 48-57, and 76-86, respectively, in the beta-globulin sequence), appeared to be immunodominant IgE-binding epitopes. Also, these peptides were best recognized in terms of intensity of response. There was no obvious sequence motif shared by the 9 different IgE-binding epitopes of beta-globulin. However, approximately 60% of the amino acids represented in the epitopes are hydrophobic residues. CONCLUSION: Identification of the IgE-binding epitopes might provide a better understanding of the functional role the allergens play in the disease and might have implications for immunodiagnosis and probably immunotherapy.


Asunto(s)
Alérgenos/inmunología , beta-Globulinas/inmunología , Epítopos de Linfocito B , Semillas/inmunología , Sesamum/inmunología , Adolescente , Adulto , Secuencia de Aminoácidos , Niño , Preescolar , Humanos , Inmunoglobulina E/inmunología , Lactante , Datos de Secuencia Molecular
15.
Proc Natl Acad Sci U S A ; 101(31): 11275-80, 2004 Aug 03.
Artículo en Inglés | MEDLINE | ID: mdl-15277671

RESUMEN

Relating thermodynamic parameters to structural and biochemical data allows a better understanding of substrate binding and its contribution to catalysis. The analysis of the binding of carbohydrates to proteins or enzymes is a special challenge because of the multiple interactions and forces involved. Isothermal titration calorimetry (ITC) provides a direct measure of binding enthalpy (DeltaHa) and allows the determination of the binding constant (free energy), entropy, and stoichiometry. In this study, we used ITC to elucidate the binding thermodynamics of xylosaccharides for two xylanases of family 10 isolated from Geobacillus stearothermophilus T-6. The change in the heat capacity of binding (DeltaCp = DeltaH/DeltaT) for xylosaccharides differing in one sugar unit was determined by using ITC measurements at different temperatures. Because hydrophobic stacking interactions are associated with negative DeltaCp, the data allow us to predict the substrate binding preference in the binding subsites based on the crystal structure of the enzyme. The proposed positional binding preference was consistent with mutants lacking aromatic binding residues at different subsites and was also supported by tryptophan fluorescence analysis.


Asunto(s)
Endo-1,4-beta Xilanasas/metabolismo , Geobacillus stearothermophilus/enzimología , Sitios de Unión/fisiología , Calorimetría , Endo-1,4-beta Xilanasas/química , Endo-1,4-beta Xilanasas/genética , Entropía , Mutagénesis Sitio-Dirigida , Espectrometría de Fluorescencia , Especificidad por Sustrato , Volumetría , Xilosa/metabolismo
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