RESUMEN
We investigated whether the abundance of bitter receptor mRNA expression from human taste papillae is related to an individual's perceptual ratings of bitter intensity and habitual intake of bitter drinks. Ratings of the bitterness of caffeine and quinine and three other bitter stimuli (urea, propylthiouracil, and denatonium benzoate) were compared with relative taste papilla mRNA abundance of bitter receptors that respond to the corresponding bitter stimuli in cell-based assays ( TAS2R4, TAS2R10, TAS2R38, TAS2R43, and TAS2R46). We calculated caffeine and quinine intake from a food frequency questionnaire. The bitterness of caffeine was related to the abundance of the combined mRNA expression of these known receptors, r = 0.47, p = .05, and self-reported daily caffeine intake, t(18) = 2.78, p = .012. The results of linear modeling indicated that 47% of the variance among subjects in the rating of caffeine bitterness was accounted for by these two factors (habitual caffeine intake and taste receptor mRNA abundance). We observed no such relationships for quinine but consumption of its primary dietary form (tonic water) was uncommon. Overall, diet and TAS2R gene expression in taste papillae are related to individual differences in caffeine perception.
Asunto(s)
Cafeína/farmacología , Quinina/farmacología , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/fisiología , Percepción del Gusto/efectos de los fármacos , Percepción del Gusto/fisiología , Adulto , Variaciones en el Número de Copia de ADN , Conducta Alimentaria , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Masculino , Propiltiouracilo/farmacología , Compuestos de Amonio Cuaternario/farmacología , Papilas Gustativas/metabolismo , Urea/farmacología , Adulto JovenRESUMEN
Fundamental concepts in biochemistry important for drug design often lack connection to the macroscopic world and can be difficult for students to grasp, particularly those in introductory science courses at the high school and college level. Educational research has shown that multisensory teaching facilitates learning, but teaching at the high school and college level is almost exclusively limited to the visual and auditory senses. This approach neglects the lifetime of experience our students bring to the classroom in the form of taste perception and makes our teaching less supportive of those with sensory impairment. In this article, we outline a novel guided-inquiry activity that utilizes taste perception for a series of natural and artificial sweetener solutions to introduce the concepts of substrate affinity and selectivity in the context of drug design. The findings from this study demonstrate clear gains in student knowledge, as well as an increase in enthusiasm for the fields of biochemistry and drug design. © 2016 by The International Union of Biochemistry and Molecular Biology, 44(6):550-554, 2016.
Asunto(s)
Bioquímica/educación , Investigación Biomédica/educación , Diseño de Fármacos , Aprendizaje Basado en Problemas/métodos , Estudiantes/psicología , Edulcorantes/análisis , Curriculum , Evaluación Educacional , Humanos , Laboratorios/normas , Modelos Biológicos , Gusto/fisiologíaRESUMEN
BACKGROUND: The present study aimed to determine if salty and sweet taste preferences in children are related to each other, to markers of growth, and to genetic differences. METHODS: We conducted a 2-day, single-blind experimental study using the Monell two-series, forced-choice, paired-comparison tracking method to determine taste preferences. The volunteer sample consisted of a racially/ethnically diverse group of children, 5-10 years of age (nâ=â108), and their mothers (nâ=â83). After excluding those mothers who did not meet eligibility and children who did not understand or comply with study procedures, the final sample was 101 children and 76 adults. The main outcome measures were most preferred concentration of salt in broth and crackers; most preferred concentration of sucrose in water and jelly; reported dietary intake of salty and sweet foods; levels of a bone growth marker; anthropometric measurements such as height, weight, and percent body fat; and TAS1R3 (sweet taste receptor) genotype. RESULTS: Children preferred higher concentrations of salt in broth and sucrose in water than did adults, and for both groups, salty and sweet taste preferences were significantly and positively correlated. In children, preference measures were related to reported intake of sodium but not of added sugars. Children who were tall for their age preferred sweeter solutions than did those that were shorter and percent body fat was correlated with salt preference. In mothers but not in children, sweet preference correlated with TAS1R3 genotype. CONCLUSIONS AND RELEVANCE: For children, sweet and salty taste preferences were positively correlated and related to some aspects of real-world food intake. Complying with recommendations to reduce added sugars and salt may be more difficult for some children, which emphasizes the need for new strategies to improve children's diets.
Asunto(s)
Preferencias Alimentarias , Cloruro de Sodio Dietético/farmacología , Sacarosa/farmacología , Gusto , Adiposidad , Adulto , Factores de Edad , Antropometría , Biomarcadores/orina , Estatura , Resorción Ósea/orina , Niño , Preescolar , Colágeno Tipo I/orina , Dieta , Femenino , Humanos , Masculino , Análisis Multivariante , Péptidos/orina , Receptores Acoplados a Proteínas G/genética , Análisis y Desempeño de TareasRESUMEN
BACKGROUND: Alleles of the receptor gene TAS2R38 are responsible in part for the variation in bitter taste perception of 6-n-propylthiouracil (PROP) and structurally similar compounds (eg, glucosinolates in cruciferous vegetables). At low concentrations, people with the PAV ("taster" amino acid sequence) form of TAS2R38 perceive these bitter compounds, whereas most with the AVI ("nontaster" amino acid sequence) form do not; heterozygotes (PAV/AVI) show the widest range of bitter perception. OBJECTIVES: The objectives were to examine individual differences in expression of PAV-TAS2R38 messenger RNA (mRNA) among heterozygotes, to test the hypotheses that the abundance of allele-specific gene expression accounts for the variation in human bitter taste perception, and to relate to dietary intake of bitter-tasting beverages and foods. DESIGN: Heterozygous individuals (n = 22) provided psychophysical evaluation of the bitterness of PROP, glucosinolate-containing broccoli juice, non-glucosinolate-containing carrot juice, and several bitter non-TAS2R38 ligands as well as dietary recalls. Fungiform taste papillae were examined for allele-specific TAS2R38 expression by using quantitative polymerase chain reaction. RESULTS: PAV-TAS2R38 mRNA expression was measured in 18 of 22 heterozygous subjects. Relative expression varied widely and positively correlated with ratings of bitterness intensity of PROP (P = 0.007) and broccoli juice (P = 0.004) but not of the control solutions carrot juice (P = 0.26), NaCl (P = 0.68), caffeine (P = 0.24), or urea (P = 0.47). Expression amounts were related to self-reported recent and habitual caffeine intake (P = 0.060, P = 0.005); vegetable intake was too low to analyze. CONCLUSIONS: We provide evidence that PAV-TAS2R38 expression amount correlates with individual differences in bitter sensory perception and diet. The nature of this correlation calls for additional research on the molecular mechanisms associated with some individual differences in taste perception and food intake. The trial was registered at clinicaltrials.gov as NCT01399944.
Asunto(s)
ARN Mensajero/genética , Receptores Acoplados a Proteínas G/genética , Papilas Gustativas/química , Percepción del Gusto/genética , Percepción del Gusto/fisiología , Gusto/fisiología , Alelos , Bebidas/análisis , Daucus carota , Dieta , Conducta Alimentaria , Expresión Génica , Glucosinolatos , Heterocigoto , Humanos , Reacción en Cadena de la Polimerasa , Propiltiouracilo , ARN Mensajero/análisis , Receptores Acoplados a Proteínas G/fisiologíaRESUMEN
BACKGROUND: Children often refuse to take medication in liquid formulation because of its unpleasant taste. Recent advances in taste genetics have provided some insight into individual differences in taste among children: due to their genotype, some prefer more intense sweetness and are more sensitive to bitter tastes and thus may have different needs for medication formulation. OBJECTIVE: The aims of this study were to: (1) test the a priori hypothesis that children with the bitter-sensitive genotypes are more likely to have experienced solid medication formulations than those with the bitter-insensitive genotype; and (2) conduct post hoc analyses to examine the relationship between genotype and most preferred level of sucrose in water because sweeteners are a common component of liquid formulations. METHODS: The following data were obtained from healthy, 3- to 10-year-old children who participated in 1 of 5 taste-research studies: taste genotype for alleles of the bitter-taste receptor TAS2R38; data from retrospective reports of solid medication formulation usage and favorite beverage; and most preferred level of sucrose in water, as determined psychophysically. RESULTS: Data from 448 children were included (245 girls, 203 boys; mean age, 7.8 years). Children with ≥1 bitter-sensitive allele (TAS2R38 PP or AP genotype) were more likely to have taken medication in solid formulation than were bitter-insensitive (AA genotype) children. Children with the PP genotype preferred higher concentrations of sucrose in water, and their favorite beverage contained more grams of sugar compared with children with the AA genotype. CONCLUSIONS: Taste genotype was associated with experience with solid medication formulations and preference for more intense sweetness. This finding suggests that taste genetics might be an important factor in formulation choice and compliance in the pediatric population.
Asunto(s)
Receptores Acoplados a Proteínas G/genética , Comprimidos/administración & dosificación , Gusto/genética , Administración Oral , Alelos , Niño , Preescolar , Deglución , Femenino , Preferencias Alimentarias , Genotipo , Humanos , Masculino , Estudios Retrospectivos , SacarosaRESUMEN
The cyclic dinucleotide c-di-GMP regulates lifestyle transitions in many bacteria, such as the change from a free motile state to a biofilm-forming community. Riboswitches that bind this second messenger are important downstream targets in this bacterial signaling pathway. The breakdown of c-di-GMP in the cell is accomplished enzymatically and results in the linear dinucleotide pGpG. The c-di-GMP-binding riboswitches must be able to discriminate between their cognate cyclic ligand and linear dinucleotides in order to be selective biological switches. It has been reported that the c-di-GMP-I riboswitch binds c-di-GMP 5 orders of magnitude better than the linear pGpG, but the cause of this large energetic difference in binding is unknown. Here we report binding data and crystal structures of several linear c-di-GMP analogues in complex with the c-di-GMP-I riboswitch. These data reveal the parameters for phosphate recognition and the structural basis of linear dinucleotide binding to the riboswitch. Additionally, the pH dependence of binding shows that exclusion of pGpG is not due to the additional negative charge on the ligand. These data reveal principles that, along with published work, will contribute to the design of c-di-GMP analogues with properties desirable for use as chemical tools and potential therapeutics.
Asunto(s)
Aptámeros de Nucleótidos/química , Proteínas Bacterianas/química , GMP Cíclico/química , ARN Bacteriano/química , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Regulación Bacteriana de la Expresión Génica , Nucleótidos de Guanina/química , Nucleótidos de Guanina/genética , Guanosina Monofosfato/química , Humanos , Concentración de Iones de Hidrógeno , Ligandos , Conformación de Ácido Nucleico , ARN Bacteriano/genética , Riboswitch/genética , Sistemas de Mensajero Secundario/genéticaRESUMEN
This article reviews the development of the senses of taste and smell, which provide information on the flavor of foods, and discusses how innate predispositions interact with early-life feeding experiences to form children's dietary preferences and habits. A basic understanding of the development and functioning of the chemical senses during early childhood may assist in forming evidence-based strategies to improve children's diets, especially for those who experience a discontinuity or disruption in early flavor experiences.
Asunto(s)
Desarrollo Infantil/fisiología , Conducta Alimentaria/fisiología , Olfato/fisiología , Gusto/fisiología , Humanos , Recién Nacido , Factores de RiesgoRESUMEN
The bacterial second messenger c-di-GMP is used in many species to control essential processes that allow the organism to adapt to its environment. The c-di-GMP riboswitch (GEMM) is an important downstream target in this signaling pathway and alters gene expression in response to changing concentrations of c-di-GMP. The riboswitch selectively recognizes its second messenger ligand primarily through contacts with two critical nucleotides. However, these two nucleotides are not the most highly conserved residues within the riboswitch sequence. Instead, nucleotides that stack with c-di-GMP and that form tertiary RNA contacts are the most invariant. Biochemical and structural evidence reveals that the most common natural variants are able to make alternative pairing interactions with both guanine bases of the ligand. Additionally, a high-resolution (2.3 A) crystal structure of the native complex reveals that a single metal coordinates the c-di-GMP backbone. Evidence is also provided that after transcription of the first nucleotide on the 3'-side of the P1 helix, which is predicted to be the molecular switch, the aptamer is functional for ligand binding. Although large energetic effects occur when several residues in the RNA are altered, mutations at the most conserved positions, rather than at positions that base pair with c-di-GMP, have the most detrimental effects on binding. Many mutants retain sufficient c-di-GMP affinity for the RNA to remain biologically relevant, which suggests that this motif is quite resilient to mutation.
Asunto(s)
Guanosina Monofosfato/metabolismo , Emparejamiento Base , Secuencia de Bases , GMP Cíclico/análogos & derivados , Guanina/química , Guanina/metabolismo , Ligandos , Mutación , Estructura Terciaria de Proteína , Sistemas de Mensajero Secundario/fisiologíaRESUMEN
The kink turn (K-turn) is an RNA structural motif found in many biologically significant RNAs. While most examples of the K-turn have a similar fold, the crystal structure of the Azoarcus group I intron revealed a novel RNA conformation, a reverse kink turn bent in the direction opposite that of a consensus K-turn. The reverse K-turn is bent toward the major grooves rather than the minor grooves of the flanking helices, yet the sequence differs from the K-turn consensus by only a single nucleotide. Here we demonstrate that the reverse bend direction is not solely defined by internal sequence elements, but is instead affected by structural elements external to the K-turn. It bends toward the major groove under the direction of a tetraloop-tetraloop receptor. The ability of one sequence to form two distinct structures demonstrates the inherent plasticity of the K-turn sequence. Such plasticity suggests that the K-turn is not a primary element in RNA folding, but instead is shaped by other structural elements within the RNA or ribonucleoprotein assembly.
Asunto(s)
ARN Bacteriano/química , ARN/química , Azoarcus/genética , Azoarcus/metabolismo , Secuencia de Bases , Cristalografía por Rayos X , Intrones , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación de Ácido NucleicoRESUMEN
The second messenger signaling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) regulates many processes in bacteria, including motility, pathogenesis and biofilm formation. c-di-GMP-binding riboswitches are important downstream targets in this signaling pathway. Here we report the crystal structure, at 2.7 A resolution, of a c-di-GMP riboswitch aptamer from Vibrio cholerae bound to c-di-GMP, showing that the ligand binds within a three-helix junction that involves base-pairing and extensive base-stacking. The symmetric c-di-GMP is recognized asymmetrically with respect to both the bases and the backbone. A mutant aptamer was engineered that preferentially binds the candidate signaling molecule c-di-AMP over c-di-GMP. Kinetic and structural data suggest that genetic regulation by the c-di-GMP riboswitch is kinetically controlled and that gene expression is modulated through the stabilization of a previously unidentified P1 helix, illustrating a direct mechanism for c-di-GMP signaling.
Asunto(s)
GMP Cíclico/análogos & derivados , Regulación Bacteriana de la Expresión Génica , ARN Bacteriano/química , ARN Bacteriano/metabolismo , Sistemas de Mensajero Secundario/fisiología , Vibrio cholerae/fisiología , Emparejamiento Base , Cristalografía por Rayos X , GMP Cíclico/química , GMP Cíclico/metabolismo , Fosfatos de Dinucleósidos/metabolismo , Sustancias Intercalantes , Cinética , Modelos Moleculares , Conformación de Ácido Nucleico , ARN Bacteriano/genética , Dispersión del Ángulo Pequeño , Vibrio cholerae/químicaRESUMEN
The glmS ribozyme is the first naturally occurring catalytic RNA that relies on an exogenous, nonnucleotide cofactor for reactivity. From a biochemical perspective, the glmS ribozyme derived from Bacillus anthracis is the best characterized. However, much of the structural work to date has been done on a variant glmS ribozyme, derived from Thermoanaerobacter tengcongensis. Here we present structures of the B. anthracis glmS ribozyme in states before the activating sugar, glucosamine 6-phosphate (GlcN6P), has bound and after the reaction has occurred. These structures show an active site preorganized to bind GlcN6P that retains some affinity for the sugar even after cleavage of the RNA backbone. A structure of an inactive glmS ribozyme with a mutation distal from the ligand-binding pocket highlights a nucleotide critical to the reaction that does not affect GlcN6P binding. Structures of the glmS ribozyme bound to a naturally occurring inhibitor, glucose 6-phosphate (Glc6P), and a nonnatural activating sugar, mannosamine 6-phosphate (MaN6P), reveal a binding mode similar to that of GlcN6P. Kinetic analyses show a pH dependence of ligand binding that is consistent with titration of the cofactor's phosphate group and support a model in which the major determinant of activity is the sugar amine independent of its stereochemical presentation.
Asunto(s)
Proteínas Bacterianas/química , Glucosamina/análogos & derivados , Glucosa-6-Fosfato/análogos & derivados , ARN Catalítico/química , Bacillus anthracis , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Unión Competitiva/genética , Catálisis , Cristalografía por Rayos X , Activación Enzimática/genética , Glucosamina/química , Glucosamina/metabolismo , Glucosa-6-Fosfato/química , Glucosa-6-Fosfato/metabolismo , Cinética , Ligandos , Mutación , Conformación de Ácido Nucleico , Unión Proteica/genética , ARN Catalítico/genética , ARN Catalítico/metabolismo , Relación Estructura-ActividadRESUMEN
During RNA maturation, the group I intron promotes two sequential phosphorotransfer reactions resulting in exon ligation and intron release. Here, we report the crystal structure of the intron in complex with spliced exons and two additional structures that examine the role of active-site metal ions during the second step of RNA splicing. These structures reveal a relaxed active site, in which direct metal coordination by the exons is lost after ligation, while other tertiary interactions are retained between the exon and the intron. Consistent with these structural observations, kinetic and thermodynamic measurements show that the scissile phosphate makes direct contact with metals in the ground state before exon ligation and in the transition state, but not after exon ligation. Despite no direct exonic interactions and even in the absence of the scissile phosphate, two metal ions remain bound within the active site. Together, these data suggest that release of the ligated exons from the intron is preceded by a change in substrate-metal coordination before tertiary hydrogen bonding contacts to the exons are broken.
Asunto(s)
Polinucleótido Ligasas/metabolismo , ARN/biosíntesis , Sitios de Unión , Cristalografía por Rayos X , Exones , Enlace de Hidrógeno , Intrones , Cinética , Metales , ARN/química , Empalme del ARN , TermodinámicaRESUMEN
The GlmS riboswitch is located in the 5'-untranslated region of the gene encoding glucosamine-6-phosphate (GlcN6P) synthetase. The GlmS riboswitch is a ribozyme with activity triggered by binding of the metabolite GlcN6P. Presented here is the structure of the GlmS ribozyme (2.5 A resolution) with GlcN6P bound in the active site. The GlmS ribozyme adopts a compact double pseudoknot tertiary structure, with two closely packed helical stacks. Recognition of GlcN6P is achieved through coordination of the phosphate moiety by two hydrated magnesium ions as well as specific nucleobase contacts to the GlcN6P sugar ring. Comparison of this activator bound and the previously published apoenzyme complex supports a model in which GlcN6P does not induce a conformational change in the RNA, as is typical of other riboswitches, but instead functions as a catalytic cofactor for the reaction. This demonstrates that RNA, like protein enzymes, can employ the chemical diversity of small molecules to promote catalytic activity.