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Background: Adequate nutrient intake during the first few months of life plays a critical role on brain structure and function development. Objectives: To analyze the long-term effects of an experimental infant formula (EF) on neurocognitive function and brain structure in healthy children aged 6 years compared to those fed with a standard infant formula or breastfed. Methods: The current study involved 108 healthy children aged 6 years and participating in the COGNIS Study. At 0-2 months, infants were randomized to receive up to 18 months of life a standard infant formula (SF) or EF enriched with milk fat globule membrane (MFGM), long-chain polyunsaturated fatty acids (LC-PUFAs) and synbiotics. Furthermore, a reference group of breastfed (BF) infants were also recruited. Children were assessed using neurocognitive tests and structural Magnetic Resonance Imaging (MRI) at 6 years old. Results: Experimental infant formula (EF) children showed greater volumes in the left orbital cortex, higher vocabulary scores and IQ, and better performance in an attention task than BF children. EF children also presented greater volumes in parietal regions than SF kids. Additionally, greater cortical thickness in the insular, parietal, and temporal areas were found in children from the EF group than those fed with SF or BF groups. Further correlation analyses suggest that higher volumes and cortical thickness of different parietal and frontal regions are associated with better cognitive development in terms of language (verbal comprehension) and executive function (working memory). Finally, arachidonic acid (ARA), adrenic acid (AdA), docosahexaenoic acid (DHA) levels in cheek cell glycerophospholipids, ARA/DHA ratio, and protein, fatty acid, and mineral intake during the first 18 months of life seem to be associated with changes in the brain structures at 6 years old. Conclusions: Supplemented infant formula with MFGM components, LC-PUFAs, and synbiotics seems to be associated to long-term effects on neurocognitive development and brain structure in children at 6 years old. Clinical Trial Registration: https://www.clinicaltrials.gov/, identifier: NCT02094547.
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Nutrition during early life is essential for brain development and establishes the basis for cognitive and language skills development. It is well established that breastfeeding, compared to formula feeding, has been traditionally associated with increased neurodevelopmental scores up to early adulthood. We analyzed the long-term effects of a new infant formula enriched with bioactive compounds on healthy children's language development at four years old. In a randomized double-blind COGNIS study, 122 children attended the follow-up call at four years. From them, 89 children were fed a standard infant formula (SF, n = 46) or an experimental infant formula enriched with functional nutrients (EF, n = 43) during their first 18 months of life. As a reference group, 33 exclusively breastfed (BF) were included. Language development was assessed using the Oral Language Task of Navarra-Revised (PLON-R). ANCOVA, chi-square test, and logistic regression models were performed. EF children seemed to show higher scores in use of language and oral spontaneous expression than SF children, and both SF and EF groups did not differ from the BF group. Moreover, it seems that SF children were more frequently categorized into "need to improve and delayed" in the use of language than EF children, and might more frequently present "need to improve and delayed" in the PLON-R total score than BF children. Finally, the results suggest that SF children presented a higher risk of suffering language development than BF children. Secondary analysis also showed a slight trend between low socioeconomic status and poorer language skills. The functional compound-enriched infant formula seems to be associated with beneficial long-term effects in the development of child's language at four years old in a similar way to breastfed infants.
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Desarrollo Infantil/fisiología , Lenguaje Infantil , Fórmulas Infantiles , Fenómenos Fisiológicos Nutricionales del Lactante/fisiología , Nutrientes/administración & dosificación , Preescolar , Suplementos Dietéticos , Método Doble Ciego , Femenino , Alimentos Fortificados , Humanos , Lactante , Recién Nacido , Masculino , Estudios ProspectivosRESUMEN
Postnatal nutrition is essential for growth and neurodevelopment. We analyzed the influence of a new enriched-infant formula with bioactive compounds on growth, neurodevelopment, and visual function (VF) in healthy infants during their first 18 months of life. A total of 170 infants were randomized in the COGNIS randomized clinical trial (RCT) to receive a standard infant formula (SF = 85) or a new experimental infant formula supplemented with functional nutrients (EF = 85). As a control, 50 breastfed infants (BF) were enrolled. Growth patterns were evaluated up to 18 months of life; neurodevelopment was assessed by general movements at 2, 3, and 4 months; VF was measured by cortical visual evoked potentials at 3 and 12 months. No differences in growth and neurodevelopment were found between groups. Regarding VF, SF and EF infants presented prolonged latencies and lower amplitudes in the P100 wave than BF infants. In the EF group, a higher percentage of infants presented response at 7½'of arc at 12 months compared to 3 months of age; a similar proportion of BF and EF infants presented responses at 7½'of arc at 12 months of age. Early nutritional intervention with bioactive compounds could narrow the gap in growth and neurodevelopment between breastfed and formula-fed infants.
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Desarrollo Infantil/efectos de los fármacos , Potenciales Evocados Visuales/efectos de los fármacos , Fórmulas Infantiles/análisis , Fitoquímicos/administración & dosificación , Adulto , Lactancia Materna , Humanos , Lactante , Fenómenos Fisiológicos Nutricionales del Lactante , PadresRESUMEN
We aimed to investigate the effects of fish oil (FO) supplementation to pregnant women on the maternal and fetal fatty acid profile in plasma and erythrocyte phospholipids (PL) and to identify the best compartment for the assessment of fatty acid status. A multi-centre, double-blind, controlled trial was conducted. Healthy pregnant women from three European centres were randomly assigned to receive from week 20 of gestation until delivery a daily dietary supplement with either FO (500 mg DHA+150 mg EPA), 400 µg 5-methyltetrahydrofolate, both or placebo. Fatty acids in plasma and erythrocyte PL were determined in maternal blood (week 20, week 30 of pregnancy and delivery) and in cord blood (delivery). FO supplementation increased DHA levels in maternal and cord plasma and erythrocyte PL. Higher percentage changes were observed in erythrocyte PL than in plasma PL. There were significant correlations between plasma and erythrocyte fatty acid levels in maternal and cord blood. Significant correlations between maternal and cord fatty acid levels at delivery in plasma and erythrocytes were also observed; however, correlation coefficients were higher for erythrocyte phophatidylethanolamine. FO supplementation increases maternal and fetal DHA status. Both plasma and erythrocytes appear to be suitable to evaluate the fatty acid status of mothers but erythrocytes seem to be a more reliable marker in neonates.
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Membrana Eritrocítica/química , Ácidos Grasos/análisis , Aceites de Pescado/administración & dosificación , Fosfolípidos/sangre , Adulto , Método Doble Ciego , Femenino , Humanos , Recién Nacido , Fosfolípidos/química , EmbarazoRESUMEN
Several conditions have been used in the coupling reaction of stepwise SPPS at elevated temperature (SPPS-ET), but we have elected the following as our first choice: 2.5-fold molar excess of 0.04-0.08 M Boc or Fmoc-amino acid derivative, equimolar amount of DIC/HOBt (1:1) or TBTU/DIPEA (1:3), 25% DMSO/toluene, 60 degrees C, conventional heating. In this study, aimed to further examine enantiomerization under such condition and study the applicability of our protocols to microwave-SPPS, peptides containing L-Ser, L-His, L-Cys and/or L-Met were manually synthesized traditionally, at 60 degrees C using conventional heating and at 60 degrees C using microwave heating. Detailed assessment of all crude peptides (in their intact and/or fully hydrolyzed forms) revealed that, except for the microwave-assisted coupling of L-Cys, all other reactions occurred with low levels of amino acid enantiomerization (<2%). Therefore, herein we (i) provide new evidences that our protocols for SPPS at 60 degrees C using conventional heating are suitable for routine use, (ii) demonstrate their appropriateness for microwave-assisted SPPS by Boc and Fmoc chemistries, (iii) disclose advantages and limitations of the three synthetic approaches employed. Thus, this study complements our past research on SPPS-ET and suggests alternative conditions for microwave-assisted SPPS.
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Microondas , Péptidos/química , Péptidos/síntesis química , Secuencia de Aminoácidos , Aminoácidos/química , Colecistoquinina/química , Colecistoquinina/genética , Cromatografía Liquida , Electroforesis Capilar , Cromatografía de Gases y Espectrometría de Masas , Calefacción , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/aislamiento & purificación , Estereoisomerismo , Espectrometría de Masas en Tándem , TemperaturaRESUMEN
This paper describes the development and application of an RP HPLC method using a C(18) monolithic stationary phase for the separation and quantification of extra- and intracellular amino acids in a batch cultivation of the marine alga Tetraselmis gracilis. Fluorimetric detection was made after separation of the o-phthaldialdehyde 2-mercaptoethanol (OPA-2MCE) derivatives using a binary gradient elution. Separation of 19 amino acids was achieved with resolution >1.5 in about 39 min at a flow rate of 1.5 mL/min. RSD of analyses in seawater medium ranged from 0.36% for Orn (0.50 micromol/L) to 12% for Ile (0.10 micromol/L). The main constituents of the intracellular dissolved free amino acids (DFAAs) in the exponential growth phase were arginine (Arg), asparagine (Asn), alanine (Ala), aspartic acid (Asp), glutamic acid (Glu), serine (Ser), glycine (Gly), glutamine (Gln), and leucine (Leu). The major amino acids excreted to the media were valine (Val), Ala, Ser, and Gly. The monolithic phase facilitates the analysis by shortening the separation time and saving solvents and instrumentation costs (indeed conventional HPLC instrumentation can be used, running at lower pressures than those ones used with packed particle columns).
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Aminoácidos/análisis , Chlorophyta , Fluorometría/métodos , Resinas de Intercambio Aniónico/química , Chlorophyta/química , Chlorophyta/genética , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Fluorometría/instrumentación , Agua de MarAsunto(s)
Calor , Péptidos/síntesis química , Animales , Bovinos , Cromatografía Líquida de Alta Presión , Electroforesis Capilar , HumanosRESUMEN
Glycine-rich proteins (GRPs) serve a variety of biological functions. Acanthoscurrin is an antimicrobial GRP isolated from hemocytes of the Brazilian spider Acanthoscurria gomesiana. Aiming to contribute to the knowledge of the secondary structure and stepwise solid-phase synthesis of GRPs' glycine-rich domains, we attempted to prepare G(101)GGLGGGRGGGYG(113)GGGGYGGGYG(123) GGY(126)GGGKYK(132)-NH(2), acanthoscurrin C-terminal amidated fragment. Although a theoretical prediction did not indicate high aggregation potential for this peptide, repetitive incomplete aminoacylations were observed after incorporating Tyr(126) to the growing peptide-MBHA resin (Boc chemistry) at 60 degrees C. The problem was not solved by varying the coupling reagents or solvents, adding chaotropic salts to the reaction media or changing the resin/chemistry (Rink amide resin/Fmoc chemistry). Some improvement was made when CLEAR amide resin (Fmoc chemistry) was used, as it allowed for obtaining fragment G(113)-K(132). NIR-FT-Raman spectra collected for samples of the growing peptide-MBHA, -Rink amide resin and -CLEAR amide resin revealed the presence of beta-sheet structures. Only the combination of CLEAR-amide resin, 60 degrees C, Fmoc-(Fmoc-Hmb)Gly-OH and LiCl (the last two used alternately) was able to inhibit the phenomenon, as proven by NIR-FT-Raman analysis of the growing peptide-resin, allowing the total synthesis of desired fragment Gly(101)-K(132). In summary, this work describes a new difficult sequence, contributes to understanding stepwise solid-phase synthesis of this type of peptide and shows that, at least while protected and linked to a resin, this GRP's glycine-rich motif presents an early tendency to assume beta-sheet structures.
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Antibacterianos/síntesis química , Proteínas de Insectos/síntesis química , Fragmentos de Péptidos/síntesis química , Temperatura , Secuencia de Aminoácidos , Animales , Antibacterianos/química , Compuestos de Bencidrilo , Cromatografía Líquida de Alta Presión , Proteínas de Insectos/química , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Espectrometría Raman , ArañasRESUMEN
The aim of this study was to analyse whether a tactical training program based on a constructivist model canimprove decision making related to keeping control of the ball for a men's senior basketball team composed of ten players. Thedependent variables were: player distribution around the ball, and achieving support on both sides of the ball at an effectivepassing distance. Data collection was made through observational analysis utilizing a previously validated tool. A pretest-posttestdesign without a control group was used. Results demonstrated an improvement in decision making after the posttest for boththe number of support players near the player with the ball, as it increased from 85% in the pretest to 100% in the posttest, andthe number of collective or team actions around the player with the ball (from 5% to 76.5%) with highly significant differences.The primary conclusion is that a training program for teaching team tactics based on a constructivist model has a positiveinfluence on players capability to facilitate the pass to their teammates(AU)
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Humanos , Masculino , Adulto , Baloncesto/educación , Baloncesto/estadística & datos numéricos , Baloncesto/normas , Enseñanza/métodos , Enseñanza/estadística & datos numéricos , Enseñanza/normas , Toma de Decisiones , Formulación de Políticas , Deportes/educación , Deportes/estadística & datos numéricos , Equipo Deportivo/éticaRESUMEN
The concept of sequential injection chromatography (SIC) was exploited to automate the fluorimetric determination of amino acids after pre-column derivatization with o-phthaldialdehyde (OPA) in presence of 2-mercaptoethanol (2MCE) using a reverse phase monolithic C(18) stationary phase. The method is low-priced and based on five steps of isocratic elutions. The first step employs the mixture methanol: tetrahydrofuran: 10 mmol L(-1) phosphate buffer (pH 7.2) at the volumetric ratio of 8:1:91; the other steps use methanol: 10 mmol L(-1) phosphate buffer (pH 7.2) at volumetric ratios of 20:80, 35:65, 50:50 and 65:35. At a flow rate of 10 microL s(-1) a 25 mm long-column was able to separate aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), serine (Ser), glutamine (Gln), glycine (Gly), threonine (Thr), citruline (Ctr), arginine (Arg), alanine (Ala), tyrosine (Tyr), phenylalanine (Phe), ornithine (Orn) and lysine (Lys) with resolution >1.2 as well as methionine (Met) and valine (Val) with resolution of 0.6. Under these conditions isoleucine (Ile) and leucine (Leu) co-eluted. The entire cycle of amino acids derivatization, chromatographic separation and column conditioning at the end of separation lasted 25 min. At a flow rate of 40 microL s(-1) such time was reduced to 10 min at the cost of resolution worsening for the pairs Ctr/Arg and Orn/Lys. The detection limits varied from 0.092 micromol L(-1) for Tyr to 0.51 micromol L(-1) for Orn. The method was successfully applied to the determination of intracellular free amino acids in the green alga Tetraselmis gracilis during a period of seven days of cultivation. Samples spiked with known amounts of amino acids resulted in recoveries between 94 and 112%.
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Aminoácidos/aislamiento & purificación , Chlorophyta/química , Cromatografía Liquida/métodos , Análisis de Inyección de Flujo/métodos , Solventes/química , Mercaptoetanol/química , Sensibilidad y Especificidad , Espectrometría de Fluorescencia/métodosRESUMEN
The preparation of small-sized protected peptide acids related to cholecystokinin and gomesin was attempted using peptide-Kaiser oxime resins (KOR) as starting materials. For comparison, peptide-2-Cl-trityl resin (CLTR) was also employed. While peptide detachment from KOR was achieved through the oxime ester bond hydrolysis mediated by DBU, hydroxide ion or Ca(+2) ion, peptide release from CLTR was accomplished through acid-catalysed hydrolysis of the peptide-resin ester linkage. Amino acid analysis of the peptide-resins before and after peptide release allowed the calculation of the reaction yields. RP-HPLC and LC/ESI-MS of the resulting crude peptides allowed estimation of their quality. The data collected indicated that: (i) among the procedures used for peptide displacement from KOR, the one employing DBU was the most efficient since it furnished all model protected peptide acids with the highest quality in a very short time; (ii) although slow, Ca(+2)-assisted peptide detachment from KOR was selective and was suitable for generating high-quality protected peptide acids containing up to five residues; (iii) even though the protocols employed for peptide release from CLTR have shown to be appropriate, the one employing 1% TFA/DCM was the most productive; (iv) in terms of product quality, DBU-catalysed peptide detachment from KOR was similar to TFA-catalysed peptide release from CLTR although the latter was more productive. These findings are relevant to peptide chemists in general, but especially to those interested in preparing acyl donors for convergent peptide syntheses by the t-Boc chemistry.
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Ácidos/química , Péptidos/química , Catálisis , Cromatografía Líquida de Alta Presión , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
Peptides derived from endogenous hemoglobin play important biological roles in a variety of living systems. In previous works we showed that the fragment 33-61 of bovine alpha-hemoglobin (Hb33-61) and its C-terminus amidated analogue (Hb33-61a) exhibit antimicrobial activity and we determined the 3D structure of Hb33-61a bound to sodium dodecyl sulfate micelles. Here we report that Hb33-61a is lethal to Candida albicans at 6.25 microM probably through disruption of its plasma membrane. In addition, we show that, even when used at 50 microM, Hb33- 61a produces low hemolysis (16% +/- 3.0%). Recognizing that one of the key steps to study new compounds with potential pharmaceutical application is to identify the structural elements essential to express biological activity, we also investigated the anticandidal activity of Hb33- 61a fragments. The results indicated that Hb40-61a exhibits the same minimal inhibitory concentration as Hb33-61a, whereas Hb33-52a and Hb48-61a are significantly less active. Noteworthy, for all the peptides tested, we observed that C-terminus amidation produces a potentiation of their anticandidal activity and we associate that increased biological activity to a preferred structural and spatial organization of the C-terminal region favored by amidation. Finally, the data show that the most active peptides (Hb33-61a and Hb40-61a) are characterized by a central hinge joining the C-terminal region that presents, containing a beta-turn, followed by and a helical element, to the N-terminal region that presents only a beta-turn. We hypothesize that these two structured regions, by fluctuating independently in the lipid environment, may act in a coordinated fashion disrupting the yeast plasma membrane.
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Hemoglobinas/química , Hemoglobinas/farmacología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Secuencia de Aminoácidos , Animales , Antifúngicos/síntesis química , Antifúngicos/química , Antifúngicos/farmacología , Candida albicans/efectos de los fármacos , Bovinos , Diseño de Fármacos , Hemoglobinas/genética , Hemólisis/efectos de los fármacos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Técnicas In Vitro , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/toxicidad , Estructura Secundaria de Proteína , Electricidad EstáticaRESUMEN
Gomesin (Gm) is a potent antimicrobial peptide isolated from the spider Acanthoscurria gomesiana. The two disulfide bridges Cys(2,15) and Cys(6,11) facilitate the folding of the molecule in a beta-hairpin structure, conferring on the peptide a high stability in human plasma. We report herein biological and structural features of new linear Gm analogues, obtained by combining the removal of both disulfide bridges and the incorporation of a D- or L-proline. Regarding their biological properties, two analogues, namely, [D-Thr(2,6,11,15), Pro(9)]-D-Gm and [Thr(2,6,11,15), D-Pro(9)]-Gm, are as potent as Gm against Candida albicans and only fourfold less against Staphylococcus aureus and Escherichia coli. In addition, at 100 microM they are approximately threefold less hemolytic than Gm. The best therapeutic indices were found for [D-Thr(2,6,11,15), Pro(9)]-D-Gm and for [(Des-pGlu(1), -Thr(2), -Arg(3)), Thr(6,11,15), D-Pro(9)]-Gm with a 32-fold increase of their activity against bacteria, and from 128- to 512-fold against yeast when compared with Gm. Regarding the stability, [D-Thr(2,6,11,15), Pro(9)]-D-Gm appeared to be the most resistant in human serum, along with [D-Thr(2,6,11,15), Pro(8)]-D-Gm and [Thr(2,6,11,15), D-Arg(4,16), D-Pro(9)]-Gm. When evaluating their conformation by CD spectroscopy in sodium dodecyl sulfate (SDS), most linear analogues display beta-conformation characteristics. Moreover, considering its high therapeutic index and stability in serum, [D-Thr(2,6,11,15), Pro(9)]-D-Gm was further analyzed by NMR spectroscopy. (1)H NMR experiments in SDS micelles demonstrated that [D-Thr(2,6,11,15), Pro(9)]-D-Gm presents a conformation very similar to that of Gm. In our search for Gm analogues with enhanced potential for drug development, we demonstrated that designing cysteine-free analogues can improve the therapeutic index of Gm derivatives.
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Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Secuencia de Aminoácidos , Péptidos Catiónicos Antimicrobianos/síntesis química , Péptidos Catiónicos Antimicrobianos/toxicidad , Candida albicans/efectos de los fármacos , Dicroismo Circular , Disulfuros/química , Diseño de Fármacos , Estabilidad de Medicamentos , Escherichia coli/efectos de los fármacos , Hemólisis/efectos de los fármacos , Humanos , Técnicas In Vitro , Micelas , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Prolina/química , Conformación Proteica , Staphylococcus aureus/efectos de los fármacos , Electricidad EstáticaRESUMEN
The aim of this work was to examine the bioactivity and the conformational behavior of some gomesin (Gm) analogues in different environments that mimic the biological membrane/water interface. Thus, manual peptide synthesis was performed by the solid-phase method, antimicrobial activity was evaluated by a liquid growth inhibition assay, and conformational studies were performed making use of several spectroscopic techniques: CD, fluorescence and EPR. [TOAC(1)]-Gm; [TOAC(1), Ser(2,6,11,15)]-Gm; [Trp(7)]-Gm; [Ser(2,6,11,15), Trp(7)]-Gm; [Trp(9)]-Gm; and [Ser(2,6,11,15), Trp(9)]-Gm were synthesized and tested. The results indicated that incorporation of TOAC or Trp caused no significant reduction of antimicrobial activity; the cyclic analogues presented a beta-hairpin conformation similar to that of Gm. All analogues interacted with negatively charged SDS both above and below the detergent's critical micellar concentration (cmc). In contrast, while Gm and [TOAC(1)]-Gm required higher LPC concentrations to bind to micelles of this zwitterionic detergent, the cyclic Trp derivatives and the linear derivatives did not seem to interact with this membrane-mimetic system. These data corroborate previous results that suggest that electrostatic interactions with the lipid bilayer of microorganisms play an important role in the mechanism of action of gomesin. Moreover, the results show that hydrophobic interactions also contribute to membrane binding of this antimicrobial peptide.
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Antiinfecciosos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Membrana Celular/efectos de los fármacos , Dicroismo Circular , Espectroscopía de Resonancia por Spin del Electrón , Espectrometría de Fluorescencia , Antiinfecciosos/síntesis química , Antiinfecciosos/química , Péptidos Catiónicos Antimicrobianos/síntesis química , Péptidos Catiónicos Antimicrobianos/química , Bacillus megaterium/efectos de los fármacos , Bacillus megaterium/crecimiento & desarrollo , Candida albicans/efectos de los fármacos , Candida albicans/crecimiento & desarrollo , Membrana Celular/química , Óxidos N-Cíclicos , Escherichia coli/efectos de los fármacos , Escherichia coli/crecimiento & desarrollo , Interacciones Hidrofóbicas e Hidrofílicas , Membrana Dobles de Lípidos/química , Lípidos de la Membrana/química , Micelas , Pruebas de Sensibilidad Microbiana , Conformación Proteica , Marcadores de Spin , Relación Estructura-Actividad , Triptófano/química , Agua/químicaRESUMEN
We have isolated a 417Da antibacterial molecule, named mygalin, from the hemocytes of the spider Acanthoscurria gomesiana. The structure of mygalin was elucidated by tandem mass spectrometry (MS/MS) and by two spectroscopic techniques, nuclear magnetic resonance (NMR) and ultraviolet (UV) spectroscopy. Mygalin was identified as bis-acylpolyamine N1,N8-bis(2,5-dihydroxybenzoyl)spermidine, in which the primary amino groups of the spermidine are acylated with the carboxyl group of the 2,5-dihydroxybenzoic acid. Mygalin was active against Escherichia coli at 85muM, being this activity inhibited completely by catalase. Therefore, the antibacterial activity of mygalin was attributed to its production of hydrogen peroxide (H(2)O(2)). The putative mechanisms of formation of H(2)O(2) from mygalin are discussed. To our knowledge this is the first report of one bis-acylpolyamine with antibacterial activity purified from animal source.
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Antibacterianos/química , Antibacterianos/farmacología , Hemocitos/química , Espermidina/análogos & derivados , Arañas/química , Animales , Antibacterianos/aislamiento & purificación , Candida albicans/efectos de los fármacos , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Escherichia coli/efectos de los fármacos , Isomerismo , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Micrococcus luteus/efectos de los fármacos , Estructura Molecular , Espermidina/química , Espermidina/aislamiento & purificación , Espermidina/farmacologíaRESUMEN
Gomesin is an antimicrobial peptide isolated from hemocytes of the Brazilian spider Acanthoscurria gomesiana that contains two disulfide bridges Cys(2-15)/Cys(6-11) and presents a beta-hairpin structure. To investigate the role of the disulfide bridges on gomesin conformation, bioactivities, and serum stability, structure-activity relationship (SAR) studies were conducted. Initially, gomesin and variants lacking one or both disulfide bridges were synthesized. CD studies showed that the gomesin structure is very rigid independently of the solvent environment. On the other hand, the linearized analogues adopted secondary structures according to the environment, while the monocyclic disulfide-bridged peptides had a tendency to adopt a turn structure. The absence of one or both bridges resulted in a decrease in the antimicrobial and hemolytic activities. In addition, serum stability studies revealed that, contrasting to gomesin that was stable even after 48 h of incubation, the linearized analogues were rapidly degraded. The replacement of the disulfide bounds by lactam bridges led to monocyclic and bicyclic compounds. SAR studies indicated that the monocyclic lactam-bridged analogues tend to assume a alpha-helical structure being less potent, hemolytic, and serum stable than the wild-type gomesin. On the other hand, the bicyclic lactam/disulfide-bridged analogues displayed a similar conformation and degradation kinetics identical to gomesin. However, the antimicrobial activity appeared to be dependent on the lactam bridge position and size. These findings indicated that (i) the secondary structure plays a pivotal role for the full activity of gomesin; (ii) the antimicrobial and hemolytic activities of gomesin are correlated events; (iii) while at least one of the disulfide bridges is needed for the maintenance of a significant antimicrobial activity of gomesin, both bridges are required for high serum stability and optimal conformation; and finally (iv) the best analogue obtained was the bicyclo (2-15,6-11)[Glu2, Cys(6,11), Lys15]-Gm since it is as stable and potent as gomesin.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/metabolismo , Disulfuros/química , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos/síntesis química , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/farmacología , Proteínas Sanguíneas/química , Candida albicans/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Cisteína/química , Escherichia coli/efectos de los fármacos , Hemólisis/efectos de los fármacos , Humanos , Cinética , Lactamas/química , Peso Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Soluciones , Espectrometría de Masa por Ionización de Electrospray , Arañas/química , Staphylococcus aureus/efectos de los fármacos , Relación Estructura-Actividad , Agua/químicaRESUMEN
Hemoglobin is known to be a source of peptides involved in several functions. The peptide FLSFPTTKTYFPHFDLSHGSAQVKGHGAK (Hb33-61) is a proteolytic product of the bovine hemoglobin alpha-chain found in the gut content of the cattle tick, Boophilus microplus, and it possesses antimicrobial activity. Since in the past we showed that the amidated form of Hb33-61, Hb33-61a, is active against a few Gram-positive bacteria and fungi strains at micromolar concentration [Fogaca et al. (1999) J. Biol. Chem. 274, 25330-25334], we have been prompted to shed more light on its functional and structural features. Here we show that the peptide is able to disrupt the bacterial membrane ofMicrococcus luteus A270. As for its structure, it has a random conformation in water, and it does not interact with zwitterionic micelles. On the other hand, it binds to negatively charged micelles acquiring a finite structural organization. The 3D structure of Hb33-61a bound to SDS micelles exhibits a nonconventional conformation for an antimicrobial peptide. The backbone is characterized by the presence of a beta-turn in the N-terminus and by a beta-turn followed by a alpha-helical stretch in the C-terminus. A hinge, whose spatial organization is stabilized by side-chain-side-chain interactions, joins these two regions. Interestingly, it preserves structural features present in the corresponding segment of the bovine hemoglobin alpha-chain. Hb33-61a does not possess a well-defined amphipathic nature, and H/D exchange experiments show that while the C-terminal region is embedded in the SDS micelle, one face of the N-terminal half is partly exposed to the solvent.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Hemoglobina A/química , Ixodidae/química , Micelas , Fragmentos de Péptidos/química , Subunidades de Proteína/química , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/farmacología , Sitios de Unión , Bovinos , Dicroismo Circular/métodos , Medición de Intercambio de Deuterio , Hemoglobina A/aislamiento & purificación , Hemoglobina A/farmacología , Espectroscopía de Resonancia Magnética/métodos , Micrococcus luteus/efectos de los fármacos , Micrococcus luteus/crecimiento & desarrollo , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/farmacología , Estructura Secundaria de Proteína , Subunidades de Proteína/aislamiento & purificación , Subunidades de Proteína/farmacología , Relación Estructura-ActividadRESUMEN
Leptin presents an important role in energy balance and neuroendocrine control in mammals. In an attempt to identify regions of the leptin molecule responsible for its bioactivity, we have synthesized six peptides based on the protein three-dimensional structure. Fragments were synthesized by the solid-phase methodology, purified by reverse-phase high-performance liquid chromatography (RP-HPLC), and characterized by liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS). They were injected intravenously and their ability to induce Fos immunoreactivity (Fos-ir) in rat hypothalamus was compared with that of the recombinant human leptin and saline. Fragment Ac-[Ser117]Lep116-140-NH2 (V) induced Fos-ir in hypothalamic nuclei that express leptin receptor long form. No similar ability was observed for the other five fragments. To investigate whether Fos-ir was induced in the same neuronal group activated by leptin, we proceeded with a dual-label immunohistochemistry for cocaine- and amphetamine-regulated transcript (CART), a neuropeptide related to leptin action in rat hypothalamus. We found that Ac-[Ser117]Lep116-140-NH2 (V) differentially activates CART neurons through the rostrocaudal extension of the arcuate nucleus. These results suggest that this fragment acts in the same group of neurons that mediate leptin response. This approach may offer the basis for the development of leptin-related compounds, having potential application in human or veterinary medicine.
Asunto(s)
Hipotálamo/efectos de los fármacos , Leptina/farmacología , Fragmentos de Péptidos/farmacología , Proteínas Proto-Oncogénicas c-fos/metabolismo , Animales , Humanos , Hipotálamo/citología , Hipotálamo/metabolismo , Inmunohistoquímica , Leptina/administración & dosificación , Leptina/química , Masculino , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Neuronas/metabolismo , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Ratas , Ratas WistarRESUMEN
The synthesis of mutual prodrugs of nitrofurazone with primaquine, using specific and nonspecific spacer groups, has been previously attempted seeking selective antichagasic agents. The intermediate reaction product, hydroxymethylnitrofurazone (NFOH-121), was isolated and tested in LLC-MK(2) culture cells infected with trypomastigotes forms of Trypanosoma cruzi showing higher trypanocidal activity than nitrofurazone and benznidazol in all stages. The mutagenicity tests showed that the prodrug was less toxic than the parent drug. Degradation assays were carried out in pH 1.2 and 7.4.