RESUMEN
Dynamically controlled posttranslational modifications of nucleosomal histones alter chromatin condensation to regulate transcriptional activation. We report that a nuclear tandem kinase, JIL-1, controls gene expression by activating poly(ADP-ribose) polymerase-1 (PARP-1). JIL-1 phosphorylates the C terminus of the H2Av histone variant, which stimulates PARP-1 enzymatic activity in the surrounding chromatin, leading to further modification of histones and chromatin loosening. The H2Av nucleosome has a higher surface representation of PARP-1 binding patch, consisting of H3 and H4 epitopes. Phosphorylation of H2Av by JIL-1 restructures this surface patch, leading to activation of PARP-1. Exposure of Val61 and Leu23 of the H4 histone is critical for PARP-1 binding on nucleosome and PARP-1 activation following H2Av phosphorylation. We propose that chromatin loosening and associated initiation of gene expression is activated by phosphorylation of H2Av in a nucleosome positioned in promoter regions of PARP-1-dependent genes.
Asunto(s)
Proteínas de Drosophila/química , Histonas/química , Nucleosomas/química , Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/química , Animales , Cromatina/química , ADN/química , Drosophila/genética , Proteínas de Drosophila/metabolismo , Epítopos/química , Inmunohistoquímica , Nucleasa Microcócica/metabolismo , Modelos Moleculares , Conformación Molecular , Sistemas de Lectura Abierta , Fosforilación , Poli(ADP-Ribosa) Polimerasa-1 , Poli Adenosina Difosfato Ribosa/genética , Regiones Promotoras Genéticas , Conformación Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
We report the measurement of the binding constants (Ka) for cucurbit[n]uril (n = 7, 8) toward four series of guests based on 2,6-disubstituted adamantanes, 4,9-disubstituted diamantanes, 1,6-disubstituted diamantanes, and 1-substituted adamantane ammonium ions by direct and competitive 1H NMR spectroscopy. Compared to the affinity of CB[7]·Diam(NMe3)2, the adamantane diammonium ion complexes (e.g., CB[7]·2,6-Ad(NH3)2 and CB[7]·2,6-Ad(NMe3)2) are less effective at realizing the potential 1000-fold enhancement in affinity due to ion-dipole interactions at the second ureidyl CâO portal. Comparative crystallographic investigation of CB[7]·Diam(NMe3)2, CB[7]·DiamNMe3, and CB[7]·1-AdNMe3 revealed that the preferred geometry positions the +NMe3 groups ≈0.32 Å above the CâO portal; the observed 0.80 Å spacing observed for CB[7]·Diam(NMe3)2 reflects the simultaneous geometrical constraints of CH2···OâC close contacts at both portals. Remarkably, the CB[8]·IsoDiam(NHMe2)2 complex displays femtomolar binding affinity, placing it firmly alongside the CB[7]·Diam(NMe3)2 complex. Primary or quaternary ammonium ion looping strategies lead to larger increases in binding affinity for CB[8] than for CB[7], which we attribute to the larger size of the carbonyl portals of CB[8]; this suggests routes to develop CB[8] as the tightest binding host in the CB[n] family. We report that alkyl group fluorination (e.g., CB[7]·1-AdNH2Et versus CB[7]·1-AdNH2CH2CF3) does not result in the expected increase in Ka value. Finally, we discuss the role of solvation in nonempirical quantum mechanical computational methodology, which is used to estimate the relative changes in Gibbs binding free energies.
RESUMEN
A training set of eleven X-ray structures determined for biomimetic complexes between cucurbit[n]uril (CB[7 or 8]) hosts and adamantane-/diamantane ammonium/aminium guests were studied with DFT-D3 quantum mechanical computational methods to afford ΔGcalcd binding energies. A novel feature of this work is that the fidelity of the BLYP-D3/def2-TZVPP choice of DFT functional was proven by comparison with more accurate methods. For the first time, the CB[n]â guest complex binding energy subcomponents [for example, ΔEdispersion , ΔEelectrostatic , ΔGsolvation , binding entropy (-TΔS), and induced fit Edeformation(host) , Edeformation(guest) ] were calculated. Only a few weeks of computation time per complex were required by using this protocol. The deformation (stiffness) and solvation properties (with emphasis on cavity desolvation) of cucurbit[n]uril (n=5, 6, 7, 8) isolated host molecules were also explored by means of the DFT-D3 method. A high ρ2 =0.84 correlation coefficient between ΔGexptl and ΔGcalcd was achieved without any scaling of the calculated terms (at 298â K). This linear dependence was utilized for ΔGcalcd predictions of new complexes. The nature of binding, including the role of high energy water molecules, was also studied. The utility of introduction of tethered [-(CH2 )n NH3 ]+ amino loops attached to N,N-dimethyl-adamantane-1-amine and N,N,N',N'-tetramethyl diamantane-4,9-diamine skeletons (both from an experimental and a theoretical perspective) is presented here as a promising tool for the achievement of new ultra-high binding guests to CB[7] hosts. Predictions of not yet measured equilibrium constants are presented herein.
RESUMEN
We report the binding constants of CB[7] toward a series of naphthalene diammonium and 4,4'-dipiperidinium derivatives and compare the results with those obtained previously for CB[7]·3b by (1)H NMR and X-ray crystallography. The nature of binding in the host·guest complexes was investigated using quantum mechanical tools.
RESUMEN
The endosymbiotic bacteria Wolbachia pipientis Hertig infects a wide variety of insect species and can increase viral resistance in its host. Wolbachia naturally infects Culex quinquefasciatus Say and Culex pipiens L. mosquitoes, both vectors of West Nile virus (WNV). We recently demonstrated that Wolbachia infection of Cx. quinquefasciatus laboratory strain Ben95 increases host resistance to WNV infection, reducing vector competence. This observation raised the possibility that Wolbachia could impact vector competence in other populations of Cx. quinquefasciatus or Cx. pipiens. To investigate this possibility, Wolbachia densities were measured in Ben95 Cx. quinquefasciatus and compared with densities in a newly established colony of Cx. quinquefasciatus, and in field-collected and colonized Cx. pipiens. Wolbachia densities in somatic tissues of Ben95 Cx. quinquefasciatus were significantly higher than densities in the other mosquito populations tested. There was also no significant spatiotemporal variation in Wolbachia density in the field-collected Cx. pipiens, although significant familial differences were observed. Correlating Wolbachia densities and vector competence in individual colonized Cx. pipiens indicated that the densities of somatic Wolbachia observed in the mosquitoes other than Ben95 Cx. quinquefasciatus were too low to inhibit WNV infection and reduce vector competence. These results suggest that the high Wolbachia densities capable of inducing resistance to WNV in Ben95 Cx. quinquefasciatus are not a general characteristic of Cx. quinquefasciatus or Cx. pipiens mosquitoes and that the impact of Wolbachia on vector competence in field populations of Cx. quinquefasciatus and Cx. pipiens, if any, is likely to be limited to specific populations.
Asunto(s)
Culex/microbiología , Insectos Vectores/microbiología , Virus del Nilo Occidental , Wolbachia/fisiología , Animales , Culex/inmunología , Femenino , Insectos Vectores/inmunologíaRESUMEN
Comparative genomics of nucleosome positions provides a powerful means for understanding how the organization of chromatin and the transcription machinery co-evolve. Here we produce a high-resolution reference map of H2A.Z and bulk nucleosome locations across the genome of the fly Drosophila melanogaster and compare it to that from the yeast Saccharomyces cerevisiae. Like Saccharomyces, Drosophila nucleosomes are organized around active transcription start sites in a canonical -1, nucleosome-free region, +1 arrangement. However, Drosophila does not incorporate H2A.Z into the -1 nucleosome and does not bury its transcriptional start site in the +1 nucleosome. At thousands of genes, RNA polymerase II engages the +1 nucleosome and pauses. How the transcription initiation machinery contends with the +1 nucleosome seems to be fundamentally different across major eukaryotic lines.
Asunto(s)
Drosophila melanogaster/genética , Genoma de los Insectos/genética , Nucleosomas/genética , Nucleosomas/metabolismo , Animales , Secuencia Conservada/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/enzimología , Regulación de la Expresión Génica/genética , Genes de Insecto/genética , Histonas/metabolismo , Regiones Promotoras Genéticas/genética , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/genética , Sitio de Iniciación de la Transcripción , Transcripción Genética/genéticaRESUMEN
Hostâ guest complexes between cucurbit[7] (CB[7]) or CB[8] and diamantane diammonium ion guests 3 or 6 were studied by (1) Hâ NMR spectroscopy and X-ray crystallography. (1) Hâ NMR competition experiments revealed that CB[7]â 6 is among the tightest monovalent non-covalent complexes ever reported in water with Ka =7.2×10(17) M(-1) in pure D2 O and 1.9×10(15) M(-1) in D2 O buffered with NaO2 CCD3 (50â mM). The crystal structure of CB[7]â 6 allowed us to identify some of the structural features responsible for the ultratight binding, including the distance between the NMe3 (+) groups of 6 (7.78â Å), which allows it to establish 14 optimal ion-dipole interactions with CB[7], the complementarity of the convex van der Waals surface contours of 6 with the corresponding concave surfaces of CB[7], desolvation of the CO portals within the CB[7]â 6 complex, and the co-linearity of the C7 â axis of CB[7] with the N(+) â â â N(+) line in 6. This work further blurs the lines of distinction between natural and synthetic receptors.
Asunto(s)
Hidrocarburos Aromáticos con Puentes/química , Imidazoles/química , Hidrocarburos Aromáticos con Puentes/síntesis química , Cristalografía por Rayos X , Imidazoles/síntesis química , Modelos Moleculares , Estructura MolecularRESUMEN
Alkyl analogues of methylphenidate (Ritalin) salts are slow onset, long duration dopamine reuptake inhibitors with a potential use as a cocaine abuse pharmacotherapy. X-ray crystallographic studies and nuclear magnetic resonance (NMR) investigations strongly suggest that avoidance of sterically unfavorable gauche(-)gauche(+) orientations effectively influences both the C(α)-alkyl side chain conformation and the formation of a predominant rotamer about the CH-CH bond ligating piperidine and C(Ar)R moieties. The favored CH-CH rotamer in D(2)O and in CD(2)Cl(2) of the pharmacologically interesting i-Bu and CH(2)-cyc-Pnt (RS,RS)-salts has the same antiperiplanar arrangement that was found in the crystal structures, although there clearly is a fast equilibrium involving smaller amounts of synclinal partners. While the rotamer in the (RS,SR)-i-Bu HCl crystal structure exhibits a synclinal orientation for the vicinal pair of adjacent methine protons, the weighted time-averaged arrangement for these protons becomes almost completely antiperiplanar when the crystals are dissolved in D(2)O. Increased steric congestion around the CH-CH bond in the analogous N-methyl tertiary ammonium salts seems to augment the quantity of the preferred rotamer within the mixture. The stereochemistry of the species observed via NMR seems to arise from specific combinations of N-methyl orientation and avoidance of sterically unfavorable gauche(-)gauche(+) arrangements.
Asunto(s)
Alcanos/química , Metilfenidato/química , Sales (Química)/química , Cristalografía por Rayos X , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Conformación Molecular , Estructura Molecular , Soluciones , EstereoisomerismoRESUMEN
FACT (facilitates chromatin transcription) is an evolutionarily conserved histone chaperone that was initially identified as an activity capable of promoting RNA polymerase II (Pol II) transcription through nucleosomes in vitro. In this report, we describe a global analysis of FACT function in Pol II transcription in Drosophila. We present evidence that loss of FACT has a dramatic impact on Pol II elongation-coupled processes including histone H3 lysine 4 (H3K4) and H3K36 methylation, consistent with a role for FACT in coordinating histone modification and chromatin architecture during Pol II transcription. Importantly, we identify a role for FACT in the maintenance of promoter-proximal Pol II pausing, a key step in transcription activation in higher eukaryotes. These findings bring to light a broader role for FACT in the regulation of Pol II transcription.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , ARN Polimerasa II/metabolismo , Elongación de la Transcripción Genética , Animales , Proteínas Portadoras/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Histonas/genética , ARN Polimerasa II/genéticaRESUMEN
A rapid, sensitive, and quantitative novel immunoassay [FluoroChrome ImmunoAssay, FCIA] technique was developed which auspiciously combines both the high sensitivity of fluorescence measurements with the high specificity of an antibody. As opposed to existing immunoassays, FCIA is performed without separation of antibody-bound haptens from those that are free, and utilizes fluorescence measurements from widely available standard commercial fluorimeters. FCIA is based on the hypothesis that an appropriately designed stilbene-antigen analogue probe will suffer considerable steric hindrance to trans-cis photoisomerization when bound within the combined constraints of both an antibody binding site and a second globular protein. Specifically, an appropriately designed 2,4-dinitrophenyl-hapten derivative of fluorescent trans-4,4'-diaminostilbene (DAS), was squeezed between two large globular proteins: lysozyme (Lys) from one side, and anti-2,4,6-trinitrophenyl antibody (antiTNP) from the other side, in order to provide the desired constricted environment to restrict trans/cis-stibene isomerization within the antiTNP-DNP-DAS-Lys adduct. As was theoretically predicted and then experimentally verified, the trans-cis photoisomerization rate for the bound probe was found to be markedly inhibited, compared to that expected for the free probe in solution. The fluorescence-photochrome labeled probe was competitively displaced from the antiTNP binding site in the presence of the picric acid hapten, and photoisomerization then commenced to produce the fluorescence-silent cis-stilbene diastereomer. The process of association and dissociation of a hapten-antibody complex was readily monitored by the fluorescence technique in the presence of both antibody-bound and free haptens.
Asunto(s)
Compuestos de Cloro/análisis , Inmunoensayo/métodos , Muramidasa/metabolismo , Fármacos Fotosensibilizantes/análisis , Pliegue de Proteína , Espectrometría de Fluorescencia/métodos , Reactivos de Enlaces Cruzados/química , Estructura Molecular , Picratos , EstilbenosRESUMEN
Hexatertiary butyl-substituted D(3)-symmetrical cage ligands composed of octahedral bidentate complexes of Ni(II) or Cu(II) ligated on either side to triskelion arrangements of three salicyl rings bound to a central nitrogen hub atom exhibit triple-helical conformations. Maximization of intermolecular aromatic-aromatic interactions between these complexes promote extended tongue and groove interleaving and affords a supramolecular array of diastereomeric intermeshed C(3)-symmetrical triple-helices perpetuating sideways throughout alternating enantiomeric layer stacks in the entire P-3 space group crystal lattice. Chiral recognition within these supramolecular ensembles is based upon the principles of helical sense and phase. A discussion of the symmetry, mechanical and chemical factors, and constraints influencing the formation of these self-assembled crystalline supramolecular ensembles will be presented.
RESUMEN
Bacillus coagulans is an interesting facultative anaerobic microorganism for biotechnological production of lactic acid that arouses interest. To determine the efficiency of biotechnological production of lactic acid from lignocellulosic feedstock hydrolysates, five Bacillus coagulans strains were grown in lignocellulose organosolv hydrolysate from ethanol/water-pulped beechwood. Parameter estimation based on a Monod-type model was used to derive the basic key parameters for a performance evaluation of the batch process. Three of the Bacillus coagulans strains, including DSM No. 2314, were able to produce lactate, primarily via uptake of glucose and xylose. Two other strains were identified as having the ability of utilizing cellobiose to a high degree, but they also had a lower affinity to xylose. The lactate yield concentration varied from 79.4⯱â¯2.1â¯g/L to 93.7⯱â¯1.4â¯g/L (85.4⯱â¯4.7 % of consumed carbohydrates) from the diluted organosolv hydrolysate.
RESUMEN
Human neuronal ceroid lipofuscinoses (NCLs) are a group of genetic neurodegenerative diseases characterized by progressive death of neurons in the central nervous system (CNS) and accumulation of abnormal lysosomal storage material. Infantile NCL (INCL), the most severe form of NCL, is caused by mutations in the Ppt1 gene, which encodes the lysosomal enzyme palmitoyl-protein thioesterase 1 (Ppt1). We generated mutations in the Ppt1 ortholog of Drosophila melanogaster to characterize phenotypes caused by Ppt1 deficiency in flies. Ppt1-deficient flies accumulate abnormal autofluorescent storage material predominantly in the adult CNS and have a life span 30% shorter than wild type, phenotypes that generally recapitulate disease-associated phenotypes common to all forms of NCL. In contrast, some phenotypes of Ppt1-deficient flies differed from those observed in human INCL. Storage material in flies appeared as highly laminar spherical deposits in cells of the brain and as curvilinear profiles in cells of the thoracic ganglion. This contrasts with the granular deposits characteristic of human INCL. In addition, the reduced life span of Ppt1-deficient flies is not caused by progressive death of CNS neurons. No changes in brain morphology or increases in apoptotic cell death of CNS neurons were detected in Ppt1-deficient flies, even at advanced ages. Thus, Ppt1-deficient flies accumulate abnormal storage material and have a shortened life span without evidence of concomitant neurodegeneration.
Asunto(s)
Drosophila melanogaster/genética , Tioléster Hidrolasas/genética , Tioléster Hidrolasas/fisiología , Animales , Apoptosis , Modelos Animales de Enfermedad , Drosophila melanogaster/fisiología , Femenino , Masculino , Microscopía Electrónica , Microscopía Fluorescente , Mutación , Enfermedades Neurodegenerativas/metabolismo , Lipofuscinosis Ceroideas Neuronales/metabolismo , Neuronas/metabolismo , Fenotipo , Interferencia de ARNRESUMEN
The data presented in this article are related to the research article entitled "Model-based characterization of growth performance and l-lactic acid production with high optical purity by thermophilic Bacillus coagulans in a lignin-supplemented mixed substrate medium (R. Glaser and J. Venus, 2016) [1]". This data survey provides the information on characterization of three Bacillus coagulans strains. Information on cofermentation of lignocellulose-related sugars in lignin-containing media is given. Basic characterization data are supported by optical-density high-throughput screening and parameter adjustment to logistic growth models. Lab scale fermentation procedures are examined by model adjustment of a Monod kinetics-based growth model. Lignin consumption is analyzed using the data on decolorization of a lignin-supplemented minimal medium.
RESUMEN
Three Bacillus coagulans strains were characterised in terms of their ability to grow in lignin-containing fermentation media and to consume the lignocellulose-related sugars glucose, xylose, and arabinose. An optical-density high-throughput screening was used for precharacterisation by means of different mathematical models for comparison (Logistic, Gompertz, Baranyi, Richards & Stannard, and Schnute). The growth response was characterised by the maximum growth rate and lag time. For a comparison of the screening and fermentation results, an unstructured mathematical model was proposed to characterise the lactate production, bacterial growth and substrate consumption. The growth model was then applied to fermentation procedures using wheat straw hydrolysates. The results indicated that the unstructured growth model can be used to evaluate lactate producing fermentation. Under the experimental fermentation conditions, one strain showed the ability to tolerate a high lignin concentration (2.5g/L) but lacked the capacity for sufficient pentose uptake. The lactate yield of the strains that were able to consume all sugar fractions of glucose, xylose and arabinose was â¼83.4%. A photometric measurement at 280nm revealed a dynamic change in alkali-lignin concentrations during lactate producing fermentation. A test of decolourisation of vanillin, ferulic acid, and alkali-lignin samples also showed the decolourisation performance of the B. coagulans strains under study.
Asunto(s)
Bacillus coagulans/metabolismo , Medios de Cultivo/metabolismo , Ácido Láctico/biosíntesis , Lignina/metabolismo , Modelos Biológicos , Temperatura , Medios de Cultivo/química , Suplementos Dietéticos , Fermentación , Lignina/administración & dosificación , Lignina/química , Fenómenos ÓpticosRESUMEN
Wolbachia pipientis, a bacterial symbiont infecting arthropods and nematodes, is vertically transmitted through the female germline and manipulates its host's reproduction to favor infected females. Wolbachia also infects somatic tissues where it can cause nonreproductive phenotypes in its host, including resistance to viral pathogens. Wolbachia-mediated phenotypes are strongly associated with the density of Wolbachia in host tissues. Little is known, however, about how Wolbachia density is regulated in native or heterologous hosts. Here, we measure the broad-sense heritability of Wolbachia density among families in field populations of the mosquito Culex pipiens, and show that densities in ovary and nongonadal tissues of females in the same family are not correlated, suggesting that Wolbachia density is determined by distinct mechanisms in the two tissues. Using introgression analysis between two different strains of the closely related species C. quinquefasciatus, we show that Wolbachia densities in ovary tissues are determined primarily by cytoplasmic genotype, while densities in nongonadal tissues are determined by both cytoplasmic and nuclear genotypes and their epistatic interactions. Quantitative-trait-locus mapping identified two major-effect quantitative-trait loci in the C. quinquefasciatus genome explaining a combined 23% of variance in Wolbachia density, specifically in nongonadal tissues. A better understanding of how Wolbachia density is regulated will provide insights into how Wolbachia density can vary spatiotemporally in insect populations, leading to changes in Wolbachia-mediated phenotypes such as viral pathogen resistance.
Asunto(s)
Culex/microbiología , Epistasis Genética , Gónadas/microbiología , Simbiosis/genética , Wolbachia/crecimiento & desarrollo , Wolbachia/genética , Animales , Mapeo Cromosómico , Femenino , Patrón de Herencia/genética , Masculino , Sitios de Carácter Cuantitativo/genéticaRESUMEN
In nature, proteins have evolved sophisticated cavities tailored for capturing target guests selectively among competitors of similar size, shape, and charge. The fundamental principles guiding the molecular recognition, such as self-assembly and complementarity, have inspired the development of biomimetic receptors. In the current work, we report a self-assembled triple anion helicate (host 2) featuring a cavity resembling that of the choline-binding protein ChoX, as revealed by crystal and density functional theory (DFT)-optimized structures, which binds choline in a unique dual-site-binding mode. This similarity in structure leads to a similarly high selectivity of host 2 for choline over its derivatives, as demonstrated by the NMR and fluorescence competition experiments. Furthermore, host 2 is able to act as a fluorescence displacement sensor for discriminating choline, acetylcholine, L-carnitine, and glycine betaine effectively.The choline-binding protein ChoX exhibits a synergistic dual-site binding mode that allows it to discriminate choline over structural analogues. Here, the authors design a biomimetic triple anion helicate receptor whose selectivity for choline arises from a similar binding mechanism.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Portadoras/química , Colina/química , Fosfatos/química , Dominios Proteicos , Sinorhizobium meliloti/metabolismo , Acetilcolina/química , Acetilcolina/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Unión Competitiva , Proteínas Portadoras/metabolismo , Colina/metabolismo , Cristalografía por Rayos X , Cinética , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Modelos Moleculares , Fosfatos/metabolismo , Unión Proteica , Espectroscopía de Protones por Resonancia MagnéticaRESUMEN
The response of eukaryotic cells to the formation of a double-strand break (DSB) in chromosomal DNA is highly conserved. One of the earliest responses to DSB formation is phosphorylation of the C-terminal tail of H2A histones located in nucleosomes near the break. Histone variant H2AX and core histone H2A are phosphorylated in mammals and budding yeast, respectively. We demonstrate the DSB-induced phosphorylation of histone variant H2Av in Drosophila melanogaster. H2Av is a member of the H2AZ family of histone variants. Ser137 within an SQ motif located near the C- terminus of H2Av was phosphorylated in response to gamma-irradiation in both tissue culture cells and larvae. Phosphorylation was detected within 1 min of irradiation and detectable after only 0.3 Gy of radiation exposure. Photochemically induced DSBs, but not general oxidative damage or UV-induced nicking of DNA, caused H2Av phosphorylation, suggesting that phosphorylation is DSB specific. Imaginal disc cells from Drosophila expressing a mutant allele of H2Av with its C-terminal tail deleted, and therefore unable to be phosphorylated, were more sensitive to radiation-induced apoptosis than were wildtype controls, suggesting that phosphorylation of H2Av is important for repair of radiation-induced DSBs. These observations suggest that in addition to providing the function of an H2AZ histone, H2Av is also the functional homolog in Drosophila of H2AX.
Asunto(s)
Apoptosis/efectos de la radiación , Daño del ADN , Drosophila/genética , Histonas/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis/genética , Western Blotting , Línea Celular , ADN/genética , ADN/metabolismo , Reparación del ADN , Drosophila/metabolismo , Drosophila/efectos de la radiación , Genotipo , Histonas/genética , Larva/citología , Larva/genética , Larva/efectos de la radiación , Datos de Secuencia Molecular , Mutación , Fosforilación , Homología de Secuencia de AminoácidoRESUMEN
A fluorophore-nitroxide free radical dual-functional probe (FN) was utilized to study the kinetics of ascorbate (AH(-)) binding to Bovine Serum Albumin (BSA). Since the free radical fragment in the FN probe intramolecularly quenches fluorescence, ascorbate reduction of the nitroxide function is accompanied by a concomitant fluorescence intensity increase from the fluorophore. Thus, both fluorescence and the EPR techniques could be utilized to measure the reaction rate. In the presence of BSA protein, the observed rate of the overall process is the sum of that from at least two reactions: the reaction between free ascorbate and free probe, and the reaction between bound ascorbate and bound probe. Our findings show that the observed rate is strongly dependent on the ionic strength of the medium. A corollary of this observation is the indication of a purely electrostatic interaction between ascorbate and the BSA protein. This conclusion was further corroborated by 1H NMR measurement of the transverse relaxation time, T(2), of ascorbate protons in BSA solutions. Ascorbate ion was released from the ascorbate/BSA ensemble in the presence of increasing concentrations of NaCl. Binding constants of AH(-) to BSA were calculated at different ionic strengths at pH 7.4. Furthermore, an increase in ionic strength did not affect the ability of albumin to protect ascorbate against autoxidation. This suggests that the protein's protective antioxidant effect may be attributed to BSA binding of trace quantities of transition-metal cations (rather than ascorbate binding to BSA). This conclusion is supported by ascorbate UV-absorption measurements in the presence of albumin and Cu(2+) ions as a function of ionic strength.
Asunto(s)
Ácido Ascórbico/química , Albúmina Sérica Bovina/química , Ácido Ascórbico/metabolismo , Relación Dosis-Respuesta a Droga , Espectroscopía de Resonancia por Spin del Electrón , Colorantes Fluorescentes/química , Cinética , Espectroscopía de Resonancia Magnética , Concentración Osmolar , Oxidación-Reducción , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Albúmina Sérica Bovina/metabolismo , Cloruro de Sodio/química , Cloruro de Sodio/farmacología , Espectrometría de Fluorescencia , Espectrofotometría UltravioletaRESUMEN
The data presented here supports the informational background of enzyme-based lignocellulose hydrolyzation, cellulase characterization, and sugar yield prediction for the work "Enzyme-based lignocellulose hydrolyzation - Sauter mean diameter of raw materials as a basis for cellulase performance characterization and yield prediction" by Glaser [1]. Glucose yields from the enzymatic hydrolysis of the raw materials were shown as a function of cellulase enzyme loading as well as of particle size with different solid loading. The data for the proposed methods of the determination of enzyme activity in inhomogeneous samples of lignocellulosic raw materials are presented. The data of the empirical model that was developed for the prediction of hydrolysis yields for different enzyme concentrations, substrate specific particle size, and solid loadings, are given. Data are also given in relation of terms of scale-up opportunities.