RESUMEN
Defensive responses to visually threatening stimuli represent an essential fear-related survival instinct, widely detected across species. The neural circuitry mediating visually triggered defensive responses has been delineated in the midbrain. However, the molecular mechanisms regulating the development and function of these circuits remain unresolved. Here, we show that midbrain-specific deletion of the transcription factor Brn3b causes a loss of neurons projecting to the lateral posterior nucleus of the thalamus. Brn3b deletion also down-regulates the expression of the neuropeptide tachykinin 2 (Tac2). Furthermore, Brn3b mutant mice display impaired defensive freezing responses to visual threat precipitated by social isolation. This behavioral phenotype could be ameliorated by overexpressing Tac2, suggesting that Tac2 acts downstream of Brn3b in regulating defensive responses to threat. Together, our experiments identify specific genetic components critical for the functional organization of midbrain fear-related visual circuits. Similar mechanisms may contribute to the development and function of additional long-range brain circuits underlying fear-associated behavior.
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Miedo , Mesencéfalo , Animales , Ratones , Miedo/fisiología , Mesencéfalo/fisiología , Neuronas/fisiología , TálamoRESUMEN
Developmental control of long-range neuronal connections in the mammalian midbrain remains unclear. We explored the mechanisms regulating target selection of the developing superior colliculus (SC). The SC is a midbrain center that directs orienting behaviors and defense responses. We discovered that a transcription factor, Rorß, controls establishment of axonal projections from the SC to two thalamic nuclei: the dorsal lateral geniculate nucleus (dLGN) and the lateral posterior nucleus (LP). A genetic strategy used to visualize SC circuits revealed that in control animals Rorß+ neurons abundantly innervate the dLGN but barely innervate the LP. The opposite phenotype was observed in global and conditional Rorb mutants: projections to the dLGN were strongly decreased, and projections to the LP were increased. Furthermore, overexpression of Rorb in the wild type showed increased projections to the dLGN and decreased projections to the LP. In summary, we identified Rorß as a key developmental mediator of colliculo-thalamic innervation. Such regulation could represent a general mechanism orchestrating long-range neuronal connections in the mammalian brain.
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Axones/fisiología , Mesencéfalo/embriología , Mesencéfalo/crecimiento & desarrollo , Neuronas/metabolismo , Miembro 2 del Grupo F de la Subfamilia 1 de Receptores Nucleares/fisiología , Vías Visuales/metabolismo , Animales , Animales Recién Nacidos , Embrión de Mamíferos , Femenino , Cuerpos Geniculados/fisiología , Células HEK293 , Humanos , Masculino , Mesencéfalo/citología , Ratones , Ratones Transgénicos , Miembro 2 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Miembro 2 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Embarazo , Colículos Superiores/fisiologíaRESUMEN
Fatty aldehydes (FALs) can be derived from fatty acids (FAs) and related compounds and are frequently used as flavors and fragrances. Although chemical methods have been conventionally used, their selective biotechnological production aiming at more efficient and eco-friendly synthetic routes is in demand. α-Dioxygenases (α-DOXs) are heme-dependent oxidative enzymes biologically involved in the initial step of plant FA α-oxidation during which molecular oxygen is incorporated into the Cα -position of a FA (Cn ) to generate the intermediate FA hydroperoxide, which is subsequently converted into the shortened corresponding FAL (Cn-1 ). α-DOXs are promising biocatalysts for the flavor and fragrance industries, they do not require NAD(P)H as cofactors or redox partner proteins, and they have a broad substrate scope. Here, we highlight recent advances in the biocatalytic utilization of α-DOXs with emphasis on newly discovered cyanobacterial α-DOXs as well as analytical methods to measure α-DOX activity inâ vitro and inâ vivo.
Asunto(s)
Dioxigenasas , Dioxigenasas/metabolismo , Odorantes , Oxidación-ReducciónRESUMEN
α-Dioxygenases (α-DOXs) are known as plant enzymes involved in the α-oxidation of fatty acids through which fatty aldehydes, with a high commercial value as flavor and fragrance compounds, are synthesized as products. Currently, little is known about α-DOXs from non-plant organisms. The phylogenic analysis reported here identified a substantial number of α-DOX enzymes across various taxa. Here, we report the functional characterization and Escherichia coli whole-cell application of two novel α-DOXs identified from cyanobacteria: CalDOX from Calothrix parietina and LepDOX from Leptolyngbya sp. The catalytic behavior of the recombinantly expressed CalDOX and LepDOX revealed that they are heme-dependent like plant α-DOXs but exhibit activities toward medium carbon fatty acids ranging from C10 to C14 unlike plant α-DOXs. The in-depth molecular investigation of cyanobacterial α-DOXs and their application in an E. coli whole system employed in this study is useful not only for the understanding of the molecular function of α-DOXs, but also for their industrial utilization in fatty aldehyde biosynthesis.Key points⢠Two novel α-dioxygenases from Cyanobacteria are reported⢠Both enzymes prefer medium-chain fatty acids⢠Both enzymes are useful for fatty aldehyde biosynthesis.
Asunto(s)
Cianobacterias , Dioxigenasas , Aldehídos , Escherichia coli/genética , Ácidos GrasosRESUMEN
Sensitive and accurate detection of specific metal ions is important for sensor development and can advance analytical science and support environmental and human medical examinations. Fluorescent proteins (FPs) can be quenched by specific metal ions and spectroscopically show a unique fluorescence-quenching sensitivity, suggesting their potential application as FP-based metal biosensors. Since the characteristics of the fluorescence quenching are difficult to predict, spectroscopic analysis of new FPs is important for the development of FP-based biosensors. Here we reported the spectroscopic and structural analysis of metal-induced fluorescence quenching of the photoconvertible fluorescent protein DendFP. The spectroscopic analysis showed that Fe2+, Fe3+, and Cu2+ significantly reduced the fluorescence emission of DendFP. The metal titration experiments showed that the dissociation constants (Kd) of Fe2+, Fe3+, and Cu2+ for DendFP were 24.59, 41.66, and 137.18 µM, respectively. The tetrameric interface of DendFP, which the metal ions cannot bind to, was analyzed. Structural comparison of the metal-binding sites of DendFP with those of iq-mEmerald and Dronpa suggested that quenchable DendFP has a unique metal-binding site on the ß-barrel that does not utilize the histidine pair for metal binding.
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Técnicas Biosensibles , Metales , Fluorescencia , Colorantes Fluorescentes/química , Histidina , Humanos , Iones/química , Proteínas Luminiscentes/química , Metales/químicaRESUMEN
ß-Glucosidases (Bgls) convert cellobiose and other soluble cello-oligomers into glucose and play important roles in fundamental biological processes, providing energy sources in living organisms. Bgls are essential terminal enzymes of cellulose degradation systems and attractive targets for lignocellulose-based biotechnological applications. Characterization of novel Bgls is important for broadening our knowledge of this enzyme class and can provide insights into its further applications. In this study, we report the biochemical and structural analysis of a Bgl from the hemicellulose-degrading thermophilic anaerobe Thermoanaerobacterium saccharolyticum (TsaBgl). TsaBgl exhibited its maximum hydrolase activity on p-nitrophenyl-ß-d-glucopyranoside at pH 6.0 and 55 °C. The crystal structure of TsaBgl showed a single (ß/α)8 TIM-barrel fold, and a ß8-α14 loop, which is located around the substrate-binding pocket entrance, showing a unique conformation compared with other structurally known Bgls. A Tris molecule inhibited enzyme activity and was bound to the active site of TsaBgl coordinated by the catalytic residues Glu163 (proton donor) and Glu351 (nucleophile). Titration experiments showed that TsaBgl belongs to the glucose-tolerant Bgl family. The gatekeeper site of TsaBgl is similar to those of other glucose-tolerant Bgls, whereas Trp323 and Leu170, which are involved in glucose tolerance, show a unique configuration. Our results therefore improve our knowledge about the Tris-mediated inhibition and glucose tolerance of Bgl family members, which is essential for their industrial application.
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Thermoanaerobacterium/enzimología , beta-Glucosidasa/química , Secuencia de Aminoácidos , Biodegradación Ambiental , Fenómenos Químicos , Glucosa/metabolismo , Modelos Moleculares , Estructura Molecular , Polisacáridos/química , Polisacáridos/metabolismo , Conformación Proteica , Relación Estructura-Actividad , Thermoanaerobacterium/metabolismo , beta-Glucosidasa/metabolismoRESUMEN
The fluorescence of fluorescent proteins (FPs) is quenched when they are exposed to certain transition metals, which makes them promising receptor materials for metal biosensors. In this study, we report the spectroscopic analysis of metal-induced fluorescence quenching of the fluorescent protein ZsGreen from Zoanthus sp. The fluorescence of ZsGreen was reduced to 2%, 1%, and 20% of its original intensity by Fe2+, Fe3+, and Cu2+, respectively. Metal titration experiments indicated that the dissociation constants of Fe2+, Fe3+, and Cu2+ for ZsGreen were 11.5, 16.3, and 68.2 µM, respectively. The maximum binding capacities of ZsGreen for Fe2+, Fe3+, and Cu2+ were 103.3, 102.2, and 82.9, respectively. Reversibility experiments indicated that the fluorescence of ZsGreen, quenched by Fe2+ and Fe3+, could be recovered, but only to about 15% of its original intensity, even at a 50-fold molar excess of EDTA. In contrast, the fluorescence quenched by Cu2+ could be recovered up to 89.47% of its original intensity at a Cu2+: EDTA ratio of 1:5. The homology model of ZsGreen revealed that the protein does not share any metal-binding sites with previously reported FPs, suggesting that ZsGreen contains unprecedented binding sites for fluorescence quenching metal ions.
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Antozoos/química , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/análisis , Compuestos de Hierro/química , Animales , Cobre/química , Iones/química , Espectrometría de FluorescenciaRESUMEN
Understanding how much solvents influence the structures and dynamics of proteins is important to understand functional mechanisms of solvated proteins. We propose a solvated potential model that approximates the potential energy of a solvated protein by projecting the solvent information into the protein structure. Using the model, we derive three properties of the solvent. First, the influence of the solvent on protein structure and dynamics, mostly by the bulk solvent, decays drastically (near-exponentially) as the distances of the solvent from the protein increase. Using this decay pattern, we suggest the economical size of solvent boxes in molecular dynamics simulations. Second, the hydration shell regulates the protein dynamics by effecting extra interactions within the protein structure. Lastly, the lowest frequency modes are determined mostly by protein structures.
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Simulación de Dinámica Molecular , Proteínas/química , Cinética , Conformación Proteica , Solventes/químicaRESUMEN
Iron is an essential requirement for the survival and virulence of most bacteria. The bacterial ferrous iron transporter protein FeoB functions as a major reduced iron transporter in prokaryotes, but its biochemical mechanism has not been fully elucidated. In the present study, we compared enzymatic properties of the cytosolic portions of pathogenic bacterial FeoBs to elucidate each bacterial strain-specific characteristic of the Feo system. We show that bacterial FeoBs are classified into two distinct groups that possess either a sole GTPase or an NTPase with a substrate promiscuity. This difference in nucleotide preference alters cellular requirements for monovalent and divalent cations. While the hydrolytic activity of the GTP-dependent FeoBs was stimulated by potassium, the action of the NTP-dependent FeoBs was not significantly affected by the presence of monovalent cations. Mutation of Asn11, having a role in potassium-dependent GTP hydrolysis, changed nucleotide specificity of the NTP-dependent FeoB, resulting in loss of ATPase activity. Sequence analysis suggested a possible association of alanine in the G5 motif for the NTP-dependent activity in FeoBs. This demonstration of the distinct enzymatic properties of bacterial FeoBs provides important insights into mechanistic details of Feo iron transport processes, as well as offers a promising species-specific anti-virulence target.
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Proteínas Bacterianas/química , Proteínas de Transporte de Catión/química , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/química , Secuencia de Aminoácidos , Bacterias/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/metabolismo , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/metabolismo , Guanosina Trifosfato/química , Hidrólisis , Mutagénesis Sitio-Dirigida , Mutación , Nucleósido-Trifosfatasa/química , Nucleósido-Trifosfatasa/metabolismo , Potasio/metabolismo , Unión Proteica , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Inhibitory interneurons sculpt the outputs of excitatory circuits to expand the dynamic range of information processing. In mammalian retina, >30 types of amacrine cells provide lateral inhibition to vertical, excitatory bipolar cell circuits, but functional roles for only a few amacrine cells are well established. Here, we elucidate the function of corticotropin-releasing hormone (CRH)-expressing amacrine cells labeled in Cre-transgenic mice of either sex. CRH cells costratify with the ON alpha ganglion cell, a neuron highly sensitive to positive contrast. Electrophysiological and optogenetic analyses demonstrate that two CRH types (CRH-1 and CRH-3) make GABAergic synapses with ON alpha cells. CRH-1 cells signal via graded membrane potential changes, whereas CRH-3 cells fire action potentials. Both types show sustained ON-type responses to positive contrast over a range of stimulus conditions. Optogenetic control of transmission at CRH-1 synapses demonstrates that these synapses are tuned to low temporal frequencies, maintaining GABA release during fast hyperpolarizations during brief periods of negative contrast. CRH amacrine cell output is suppressed by prolonged negative contrast, when ON alpha ganglion cells continue to receive inhibitory input from converging OFF-pathway amacrine cells; the converging ON- and OFF-pathway inhibition balances tonic excitatory drive to ON alpha cells. Previously, it was demonstrated that CRH-1 cells inhibit firing by suppressed-by-contrast (SbC) ganglion cells during positive contrast. Therefore, divergent outputs of CRH-1 cells inhibit two ganglion cell types with opposite responses to positive contrast. The opposing responses of ON alpha and SbC ganglion cells are explained by differing excitation/inhibition balance in the two circuits.SIGNIFICANCE STATEMENT A goal of neuroscience research is to explain the function of neural circuits at the level of specific cell types. Here, we studied the function of specific types of inhibitory interneurons, corticotropin-releasing hormone (CRH) amacrine cells, in the mouse retina. Genetic tools were used to identify and manipulate CRH cells, which make GABAergic synapses with a well studied ganglion cell type, the ON alpha cell. CRH cells converge with other types of amacrine cells to tonically inhibit ON alpha cells and balance their high level of excitation. CRH cells diverge to different types of ganglion cell, the unique properties of which depend on their balance of excitation and inhibition.
Asunto(s)
Células Amacrinas/fisiología , Hormona Liberadora de Corticotropina/metabolismo , Vías Visuales/citología , Potenciales de Acción , Células Amacrinas/metabolismo , Animales , Femenino , Neuronas GABAérgicas/metabolismo , Neuronas GABAérgicas/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/fisiología , Potenciales Sinápticos , Vías Visuales/fisiologíaRESUMEN
Substrate-binding proteins (SBPs) bind to specific ligands and are associated with membrane protein complexes for transport or signal transduction. Most SBPs recognize substrates by the hinge motion between two distinct α/ß domains. However, short SBP motifs are often observed in protein databases, which are located around methyl-accepting chemotaxis protein genes, but structural and functional studies have yet to be performed. Here, we report the crystal structure of an unusually small SBP from Rhodothermus marinus (named as RmSBP) at 1.9â¯Å. This protein is composed of a single α/ß-domain, unlike general SBPs that have two distinct domains. RmSBP exhibits a high structural similarity to the C-terminal domain of the previously reported amino acid bound SBPs, while it does not contain an N-terminal domain for substrate recognition. As a result of the structural comparison analysis, RmSBP has a putative SBP that is different from the previously reported SBP. Our results provide insight into a new class of substrate recognition mechanism by the mini SBP protein.
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Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Proteínas Portadoras/química , Proteínas Portadoras/ultraestructura , Modelos Químicos , Modelos Moleculares , Rhodothermus/química , Secuencia de Aminoácidos , Sitios de Unión , Simulación por Computador , Cristalización , Unión Proteica , Conformación Proteica , Dominios Proteicos , Análisis de Secuencia de Proteína , Especificidad por SustratoRESUMEN
BACKGROUND: The relationship between anemia and sudden cardiac arrest (SCA) is unclear in the general population, so we assessed it in a nationwide cohort.MethodsâandâResults: We studied 494,948 subjects (mean age, 47.8 years; 245,333 men [49.6%]) with national health check-up data from the Korean National Health Insurance Database Cohort. During a mean follow-up period of 5.4 years, SCA occurred in 616 participants (396 men, 220 women). The incidence rates of SCA increased across the 4 anemia groups in both men (0.3, 1.5, 5.3, and 4.5 per 1,000 person-years) and women (0.2, 0.5, 0.5, and 1.2 per 1,000 person-years). The SCA risk per 1-unit decrease in hemoglobin (Hb) increased by 21% and 24%, respectively, in multivariable models adjusted for cardiovascular factors, in men (95% confidence interval [CI], 13-29%; P<0.001) and women (95% CI, 13-37%; P<0.001). A negative correlation between QTc interval and Hb level was observed in men, and a trend was observed in women. CONCLUSIONS: Anemia was associated with an increased risk of SCA even after accounting for concomitant conditions in a South Korean nationwide cohort. The correlation between anemia and SCA might be explained by an increase in arrhythmic risks, such as QTc prolongation.
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Anemia/mortalidad , Muerte Súbita Cardíaca/etiología , Adulto , Anciano , Femenino , Estudios de Seguimiento , Humanos , Incidencia , Masculino , Persona de Mediana Edad , República de Corea/epidemiología , Factores de Riesgo , Factores SexualesRESUMEN
Glucose isomerase (GI) is an intramolecular oxidoreductase that interconverts aldoses and ketoses. These characteristics are widely used in the food, detergent, and pharmaceutical industries. In order to obtain an efficient GI, identification of novel GI genes and substrate binding/inhibition have been studied. Xylitol is a well-known inhibitor of GI. In Streptomyces rubiginosus, two crystal structures have been reported for GI in complex with xylitol inhibitor. However, a structural comparison showed that xylitol can have variable conformation at the substrate binding site, e.g., a nonspecific binding mode. In this study, we report the crystal structure of S. rubiginosus GI in a complex with xylitol and glycerol. Our crystal structure showed one metal binding mode in GI, which we presumed to represent the inactive form of the GI. The metal ion was found only at the M1 site, which was involved in substrate binding, and was not present at the M2 site, which was involved in catalytic function. The O2 and O4 atoms of xylitol molecules contributed to the stable octahedral coordination of the metal in M1. Although there was no metal at the M2 site, no large conformational change was observed for the conserved residues coordinating M2. Our structural analysis showed that the metal at the M2 site was not important when a xylitol inhibitor was bound to the M1 site in GI. Thus, these findings provided important information for elucidation or engineering of GI functions.
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Isomerasas Aldosa-Cetosa/antagonistas & inhibidores , Isomerasas Aldosa-Cetosa/ultraestructura , Magnesio/química , Modelos Químicos , Modelos Moleculares , Xilitol/química , Isomerasas Aldosa-Cetosa/clasificación , Proteínas Bacterianas/química , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/ultraestructura , Sitios de Unión , Simulación por Computador , Cristalografía por Rayos X , Activación Enzimática , Inhibidores Enzimáticos/química , Unión Proteica , Conformación Proteica , Relación Estructura-ActividadRESUMEN
Many fluorescent proteins (FPs) exhibit fluorescence quenching at a low pH. This pH-induced non-fluorescent state of an FP serves as a useful indicator of the cellular pH. ZsYellow is widely used as an optical marker in molecular biology, but its pH-induced non-fluorescent state has not been characterized. Here, we report the pH-dependent spectral properties of ZsYellow, which exhibited the pH-induced non-fluorescence state at a pH below 4.0. We determined the crystal structures of ZsYellow at pH 3.5 (non-fluorescence state) and 8.0 (fluorescence state), which revealed the cis-configuration of the chromophore without pH-induced isomerization. In the non-fluorescence state, Arg95, which is involved in stabilization of the exited state of the chromophore, was found to more loosely interact with the carbonyl oxygen atom of the chromophore when compared to the interaction at pH 8.0. In the fluorescence state, Glu221, which is involved in the hydrogen bonding network around the chromophore, stably interacted with Gln42 and His202. By contrast, in the non-fluorescence state, the protonated conserved Glu221 residue exhibited a large conformational change and was separated from His202 by 5.46 Å, resulting in breakdown of the hydrogen bond network. Our results provide insight into the critical role of the conserved Glu221 residue for generating the pH-induced non-fluorescent state.
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Ácido Glutámico/química , Concentración de Iones de Hidrógeno , Hidrógeno/química , Proteínas Luminiscentes/química , Proteínas Luminiscentes/ultraestructura , Espectrometría de Fluorescencia/métodos , Sitios de Unión , Simulación por Computador , Enlace de Hidrógeno , Proteínas Luminiscentes/análisis , Modelos Químicos , Modelos Moleculares , Unión Proteica , ProtonesRESUMEN
Processivity is a typical characteristic of cellobiohydrolases (CBHs); it enables the enzyme to successively hydrolyze the ends of cellulose chains and to produce cellobiose as the major product. Some microbes, which do not have CBHs, utilize endoglucanases (EGs) that exhibit processivity, commonly referred to as processive EGs. A processive EG identified from Hahella chejuensis, HcCel5, has a catalytic domain (CD) belonging to the glycoside hydrolase family 5 (GH5) and two carbohydrate-binding modules (CBM6s). In this study, we compared HcCel5-CD with the CD of Saccharophagus degradans Cel5H (SdCel5H-CD), which is a processive EG reported previously. Our results showed that in comparison to SdCel5H-CD, HcCel5-CD has more suitable characteristics for cellulose hydrolysis, such as higher hydrolytic activity, thermostability (40-80 °C), and processivity. Noticeably, HcCel5-CD is capable of hydrolyzing cellotriose, unlike HcCel5. These features of HcCel5-CD for cellulose hydrolysis could be employed for efficient saccharification of lignocellulose to produce cellobiose and glucose, which may be used to produce renewable fuels and chemicals.
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Proteínas Bacterianas/química , Celulosa 1,4-beta-Celobiosidasa/química , Gammaproteobacteria/enzimología , Calor , Lignina/química , Estabilidad de Enzimas , HidrólisisRESUMEN
Visual processing in the retina depends on coordinated signaling by interneurons. Photoreceptor signals are relayed to â¼20 ganglion cell types through a dozen excitatory bipolar interneurons, each responsive to light increments (ON) or decrements (OFF). ON and OFF bipolar cell pathways become tuned through specific connections with inhibitory interneurons: horizontal and amacrine cells. A major obstacle for understanding retinal circuitry is the unknown function of most of the â¼30-40 amacrine cell types, each of which synapses onto a subset of bipolar cell terminals, ganglion cell dendrites, and other amacrine cells. Here, we used a transgenic mouse line in which vasoactive intestinal polypeptide-expressing (VIP+) GABAergic interneurons express Cre recombinase. Targeted whole-cell recordings of fluorescently labeled VIP+ cells revealed three predominant types: wide-field bistratified and narrow-field monostratified cells with somas in the inner nuclear layer (INL) and medium-field monostratified cells with somas in the ganglion cell layer (GCL). Bistratified INL cells integrated excitation and inhibition driven by both ON and OFF pathways with little spatial tuning. Narrow-field INL cells integrated excitation driven by the ON pathway and inhibition driven by both pathways, with pronounced hyperpolarizations at light offset. Monostratified GCL cells integrated excitation and inhibition driven by the ON pathway and showed center-surround spatial tuning. Optogenetic experiments showed that, collectively, VIP+ cells made strong connections with OFF δ, ON-OFF direction-selective, and W3 ganglion cells but weak, inconsistent connections with ON and OFF α cells. Revealing VIP+ cell morphologies, receptive fields and synaptic connections advances our understanding of their role in visual processing. SIGNIFICANCE STATEMENT: The retina is a model system for understanding nervous system function. At the first stage, rod and cone photoreceptors encode light and communicate with a complex network of interneurons. These interneurons drive the responses of ganglion cells, which form the optic nerve and transmit visual information to the brain. Presently, we lack information about many of the retina's inhibitory amacrine interneurons. In this study, we used genetically modified mice to study the light responses and intercellular connections of specific amacrine cell types. The results show diversity in the shape and function of the studied amacrine cells and elucidate their connections with specific types of ganglion cell. The findings advance our understanding of the cellular basis for retinal function.
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Interneuronas/citología , Interneuronas/fisiología , Retina/citología , Retina/fisiología , Vías Visuales/citología , Vías Visuales/fisiología , Animales , Células Cultivadas , Femenino , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Confocal , Optogenética , Técnicas de Placa-Clamp , Péptido Intestinal Vasoactivo/biosíntesisRESUMEN
The objective of this study was to validate fork test which is a simple tool to assess the consistency of food. The consistencies of 27 water and thickener mixtures were measured with a viscometer. These measures were then compared to those obtained with fork test to evaluate the validity of fork test. The inter-observer and intra-observer reliabilities of the fork test were assessed with an intra-class correlation coefficient. The viscometer was used to obtain reference values for three categories (0-300 cP, 300-10,000 cP, and >10,000 cP) in order to categorize water and thickener mixtures into grade 1, grade 2, or grade 3 according to the results of fork test. Our results revealed that the fork test showed excellent validity (r = -0.889, p < 0.05), intra-observer reliability, and inter-observer reliability. Therefore, fork test may be used as a practical tool to assess food consistency.
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Trastornos de Deglución/dietoterapia , Dieta/métodos , Viscosidad , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Variaciones Dependientes del Observador , Reproducibilidad de los ResultadosRESUMEN
Direction selectivity represents a fundamental visual computation. In mammalian retina, On-Off direction-selective ganglion cells (DSGCs) respond strongly to motion in a preferred direction and weakly to motion in the opposite, null direction. Electrical recordings suggested three direction-selective (DS) synaptic mechanisms: DS GABA release during null-direction motion from starburst amacrine cells (SACs) and DS acetylcholine and glutamate release during preferred direction motion from SACs and bipolar cells. However, evidence for DS acetylcholine and glutamate release has been inconsistent and at least one bipolar cell type that contacts another DSGC (On-type) lacks DS release. Here, whole-cell recordings in mouse retina showed that cholinergic input to On-Off DSGCs lacked DS, whereas the remaining (glutamatergic) input showed apparent DS. Fluorescence measurements with the glutamate biosensor intensity-based glutamate-sensing fluorescent reporter (iGluSnFR) conditionally expressed in On-Off DSGCs showed that glutamate release in both On- and Off-layer dendrites lacked DS, whereas simultaneously recorded excitatory currents showed apparent DS. With GABA-A receptors blocked, both iGluSnFR signals and excitatory currents lacked DS. Our measurements rule out DS release from bipolar cells onto On-Off DSGCs and support a theoretical model suggesting that apparent DS excitation in voltage-clamp recordings results from inadequate voltage control of DSGC dendrites during null-direction inhibition. SAC GABA release is the apparent sole source of DS input onto On-Off DSGCs.
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Potenciales de Acción/fisiología , Ácido Glutámico/metabolismo , Células Bipolares de la Retina/fisiología , Células Ganglionares de la Retina/fisiología , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Acetilcolina/metabolismo , Células Amacrinas/fisiología , Animales , Dendritas/fisiología , Femenino , Colorantes Fluorescentes , Masculino , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Modelos Neurológicos , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Auxiliary activity family 9 (AA9, formerly known as glycoside hydrolase family 61 or polysaccharide monooxygenase) is a group of fungal proteins that were recently found to have a significant synergism with cellulase in cellulose hydrolysis via the oxidative cleavage of glycosidic bonds of cellulose chains. In this study, we report the active expression of a recombinant fungal AA9 from Chaetomium globosum (CgAA9) in a bacterial host, Escherichia coli, and the optimization of its synergistic activity in cellulose hydrolysis by using cellulase. The recombinant CgAA9 (0.9 mg/g cellulose) exhibited 1.7-fold synergism in the hydrolysis of Avicel when incubated with 0.9 filter paper units of Celluclast 1.5 L/g cellulose. The first study of the active expression of AA9 using a bacterial host and its synergistic optimization could be useful for the industrial application of AA9 for the saccharification of lignocellulose.
Asunto(s)
Celulosa/metabolismo , Chaetomium/enzimología , Escherichia coli/genética , Escherichia coli/metabolismo , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/metabolismo , Chaetomium/genética , Hidrólisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The retina reports the visual scene to the brain through many parallel channels, each carried by a distinct population of retinal ganglion cells. Among these, the population with the smallest and densest receptive fields encodes the neural image with highest resolution. In human retina, and those of cat and macaque, these high-resolution ganglion cells act as generic pixel encoders: They serve to represent many different visual inputs and convey a neural image of the scene downstream for further processing. Here we identify and analyze high-resolution ganglion cells in the mouse retina, using a transgenic line in which these cells, called "W3", are labeled fluorescently. Counter to the expectation, these ganglion cells do not participate in encoding generic visual scenes, but remain silent during most common visual stimuli. A detailed study of their response properties showed that W3 cells pool rectified excitation from both On and Off bipolar cells, which makes them sensitive to local motion. However, they also receive unusually strong lateral inhibition, both pre- and postsynaptically, triggered by distant motion. As a result, the W3 cell can detect small moving objects down to the receptive field size of bipolar cells, but only if the background is featureless or stationary--an unusual condition. A survey of naturalistic stimuli shows that W3 cells may serve as alarm neurons for overhead predators.