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In this study, resistive random-access memory (ReRAM) devices with ZnO nanoparticles (NPs) are suggested to enhance performance and reduce variation in device switching parameters. The ZnO NPs are formed by annealing ZnO prepared via atomic layer deposition on HfO2, which is verified using transmission electron microscopy, x-ray diffraction pattern, and atomic force microscopy. The depth profile analysis of x-ray photoelectron spectroscopy shows that oxygen diffuses from HfO2to ZnO NPs during annealing. This can be explained by the calculation results using density functional theory (DFT) where the formation energy of oxygen vacancies is reduced at the interface of ZnO NPs and HfO2compared to single HfO2. The fabricated ZnO NPs ReRAM demonstrates reduced forming voltage, stable resistive switching behavior, and improved cycle-to-cycle uniformity in a high-resistance state.
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Nanopartículas , Óxido de Zinc , Microscopía de Fuerza Atómica , Microscopía Electrónica de Transmisión , OxígenoRESUMEN
The nucleosome remodelling factor (NURF) is an ISWI-class ATP-dependent chromatin remodeling enzyme required both for gene expression and higher order chromatin organisation. NURF binds to histone modifications that decorate the Drosophila polytene male X chromosome and is required to maintain correct organisation of this chromosome. NURF mutants exhibit distorted and decondensed polytene male X chromosomes dependent on the presence of the male-specific lethal (MSL) complex. Here we tested whether mitotic chromosomes similarly require NURF to maintain correct morphology. Surprisingly, although the MSL complex remains associated with mitotic male X chromosomes, NURF is not required to maintain morphology. While the ISWI subunit of NURF is known to remain associated with mitotic chromosomes we show that the NURF specificity subunit Nurf301/BPTF dissociates from chromatin during both Drosophila and human mitosis, further illuminating that NURF is dispensable for mitotic chromosome organisation.
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Retinal pigment epithelium (RPE) cell dysfunction caused by excessive oxidative damage is partly involved in age-related macular degeneration, which is among the leading causes of visual impairment in elderly people. Here, we investigated the protective role of chrysoeriol against hydrogen peroxide (H2O2)-induced oxidative stress in RPE cells. The cellular viability, reactive oxygen species (ROS) generation, and mitochondrial function of retinal ARPE-19 cells were monitored under oxidative stress or pre-treatment with chrysoeriol. The expression levels of mitochondrial-related genes and associated transcription factors were assessed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Moreover, the protein expression of antioxidant signal molecules was characterized by Western blot analysis. Chrysoeriol significantly increased cell viability, reduced ROS generation, and increased the occurrence of antioxidant molecules in H2O2-treated ARPE-19 cells. Additionally, mitochondrial dysfunction caused by H2O2-induced oxidative stress was also considerably diminished by chrysoeriol treatment, which reduced the mitochondrial membrane potential (MMP) and upregulated mitochondrial-associated genes and proteins. Chrysoeriol also markedly enhanced key transcription factors (Nrf2) and antioxidant-associated genes (particularly HO-1 and NQO-1). Therefore, our study confirms the protective effect of chrysoeriol against H2O2-induced oxidative stress in RPE cells, thus confirming that it may prevent mitochondrial dysfunction by upregulating antioxidant-related molecules.
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Antioxidantes/farmacología , Flavonas/farmacología , Mitocondrias/efectos de los fármacos , Factor 2 Relacionado con NF-E2/antagonistas & inhibidores , Antioxidantes/química , Antioxidantes/aislamiento & purificación , Línea Celular , Flavonas/química , Flavonas/aislamiento & purificación , Humanos , Peróxido de Hidrógeno/antagonistas & inhibidores , Peróxido de Hidrógeno/farmacología , Mitocondrias/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Estrés Oxidativo/efectos de los fármacos , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
BACKGROUND: The present study aimed to compare the volumetric changes of three calcium silicate cements after retrofilling and placing under different pH conditions via micro-computed tomography (micro-CT) scan. METHODS: Forty-two extracted human single-rooted teeth were randomly assigned to three groups according to the retrofilling materials used (Biodentine, Endocem MTA, and ProRoot MTA). Each group was divided into two subgroups according to the setting condition. The teeth in one group were immersed in normal saline for 5 days at room temperature, and the teeth in the other group were immersed in butyric acid (pH = 5.4) for 5 days at room temperature. The volume ratios of the retrofilling material were calculated via micro-CT imaging. RESULTS: The volume ratios of the Biodentine and Endocem MTA groups were significantly different between the two setting environment, and these groups had significantly lower filled volume ratio (Vf, %) in the acidic environment than in the saline environment (pH = 5.4). Meanwhile, the volume ratio of the ProRoot MTA group did not significantly differ between the two setting environments. All materials under the acidic setting condition had relative radiolucency in the area in contact with the acidic solution. CONCLUSION: The Vf ratio of the Biodentine and Endocem MTA cements was significantly lower in the acidic environment than in the saline environment. No statistically significant difference was observed in the Vf ratio of ProRoot MTA between the two setting environments.
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Compuestos de Calcio , Materiales de Obturación del Conducto Radicular , Compuestos de Aluminio , Combinación de Medicamentos , Humanos , Concentración de Iones de Hidrógeno , Ensayo de Materiales , Óxidos , Silicatos , Microtomografía por Rayos XRESUMEN
Obesity-induced inflammation, or meta-inflammation, plays key roles in metabolic syndrome and is a significant risk factor in diabetes and cardiovascular disease. To investigate causal links between obesity, meta-inflammation, and insulin signaling we established a Drosophila model to determine how elevated dietary fat and changes in the levels and balance of saturated fatty acids (SFAs) and polyunsaturated fatty acids (PUFAs) influence inflammation. We observe negligible effect of saturated fatty acid on inflammation but marked enhancement or suppression by omega-6 and omega-3 PUFAs, respectively. Using combined lipidomic and genetic analysis, we show omega-6 PUFA enhances meta-inflammation by producing linoleic acid-derived lipid mediator 9-hydroxy-octadecadienoic acid (9-HODE). Transcriptome analysis reveals 9-HODE functions by regulating FOXO family transcription factors. We show 9-HODE activates JNK, triggering FOXO nuclear localisation and chromatin binding. FOXO TFs are important transducers of the insulin signaling pathway that are normally down-regulated by insulin. By activating FOXO, 9-HODE could antagonise insulin signaling providing a molecular conduit linking changes in dietary fatty acid balance, meta-inflammation, and insulin resistance.
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Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Proteína Forkhead Box O3/metabolismo , Factores de Transcripción Forkhead/metabolismo , Ácido Linoleico/metabolismo , Obesidad/metabolismo , Animales , Animales Modificados Genéticamente , Núcleo Celular/metabolismo , Cromatina/metabolismo , Grasas de la Dieta/efectos adversos , Modelos Animales de Enfermedad , Drosophila/genética , Proteínas de Drosophila/genética , Ácidos Grasos Omega-3/metabolismo , Ácidos Grasos Omega-3/farmacología , Proteína Forkhead Box O3/genética , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Inflamación/inducido químicamente , Inflamación/metabolismo , Insulina/metabolismo , Ácido Linoleico/farmacología , Ácidos Linoleicos Conjugados/genética , Ácidos Linoleicos Conjugados/metabolismo , Ácidos Linoleicos Conjugados/farmacología , Obesidad/inducido químicamente , Unión Proteica , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transcriptoma , TransfecciónRESUMEN
A new species, Gammarus baengnyeongensis sp. nov., belonging to the family Gammaridae Leach, 1814 was collected from the Baengnyeongdo and Daecheongdo islands in South Korea. This new species is characterized by the flagellum of antenna 2 with calceoli, and the length ratio of outer ramus to inner ramus of uropod 3. Descriptions of diagnostic characteristics of the species are provided in the text. We performed statistical analysis to confirm the significance of morphological differences of interspecies. Additionally, we compare the new species to related species by the partial sequence of the mitochondrial cytochrome c oxidase subunit 1 (CO1) gene. Genetic distances between the new species and closely related species rated from 29.1-40.8%, which highly supports Gammarus baengnyeongensis sp. nov. as a valid species. In this study, we add one new Gammarus species in Korean waters.
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Anfípodos , Anfípodos/genética , Animales , Islas , República de CoreaRESUMEN
Myometrial relaxation of mouse via expression of two-pore domain acid sensitive (TASK) channels was studied. In our previous report, we suggested that two-pore domain acid-sensing K(+) channels (TASK-2) might be one of the candidates for the regulation of uterine circular smooth muscles in mice. In this study, we tried to show the mechanisms of relaxation via TASK-2 channels in marine myometrium. Isometric contraction measurements and patch clamp technique were used to verify TASK conductance in murine myometrium. Western blot and immunehistochemical study under confocal microscopy were used to investigate molecular identity of TASK channel. In this study, we showed that TEA and 4-AP insensitive non-inactivating outward K(+) current (NIOK) may be responsible for the quiescence of murine pregnant longitudinal myometrium. The characteristics of NIOK coincided with two-pore domain acid-sensing K(+) channels (TASK-2). NIOK in the presence of K(+) channel blockers was inhibited further by TASK inhibitors such as quinidine, bupivacaine, lidocaine, and extracellular acidosis. Furthermore, oxytocin and estrogen inhibited NIOK in pregnant myometrium. When compared to non-pregnant myometrium, pregnant myometrium showed stronger inhibition of NIOK by quinidine and increased immunohistochemical expression of TASK-2. Finally, TASK-2 inhibitors induced strong myometrial contraction even in the presence of L-methionine, a known inhibitor of stretch-activated channels in the longitudinal myometrium of mouse. Activation of TASK-2 channels seems to play an essential role for relaxing uterus during pregnancy and it might be one of the alternatives for preventing preterm delivery.
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NURF is a conserved higher eukaryotic ISWI-containing chromatin remodeling complex that catalyzes ATP-dependent nucleosome sliding. By sliding nucleosomes, NURF is able to alter chromatin dynamics to control transcription and genome organization. Previous biochemical and genetic analysis of the specificity-subunit of Drosophila NURF (Nurf301/Enhancer of Bithorax (E(bx)) has defined NURF as a critical regulator of homeotic, heat-shock and steroid-responsive gene transcription. It has been speculated that NURF controls pathway specific transcription by co-operating with sequence-specific transcription factors to remodel chromatin at dedicated enhancers. However, conclusive in vivo demonstration of this is lacking and precise regulatory elements targeted by NURF are poorly defined. To address this, we have generated a comprehensive map of in vivo NURF activity, using MNase-sequencing to determine at base pair resolution NURF target nucleosomes, and ChIP-sequencing to define sites of NURF recruitment. Our data show that, besides anticipated roles at enhancers, NURF interacts physically and functionally with the TRF2/DREF basal transcription factor to organize nucleosomes downstream of active promoters. Moreover, we detect NURF remodeling and recruitment at distal insulator sites, where NURF functionally interacts with and co-localizes with DREF and insulator proteins including CP190 to establish nucleosome-depleted domains. This insulator function of NURF is most apparent at subclasses of insulators that mark the boundaries of chromatin domains, where multiple insulator proteins co-associate. By visualizing the complete repertoire of in vivo NURF chromatin targets, our data provide new insights into how chromatin remodeling can control genome organization and regulatory interactions.
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Ensamble y Desensamble de Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulación de la Expresión Génica/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Nucleosomas/genética , Animales , Secuencia de Bases , Línea Celular , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Mapeo Cromosómico , Drosophila , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Análisis de Secuencia de ADN , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/genéticaRESUMEN
Galbonolide (GAL) A and B are antifungal macrolactone polyketides produced by Streptomyces galbus. During their polyketide chain assembly, GAL-A and -B incorporate methoxymalonate and methylmalonate, respectively, in the fourth chain extension step. The methoxymalonyl-acyl carrier protein biosynthesis locus (galG to K) is specifically involved in GAL-A biosynthesis, and this locus is neighbored by a gene cluster composed of galA-E. GalA-C constitute a single module, highly reducing type I polyketide synthase (PKS). GalD and GalE are cytochrome P450 and Rieske domain protein, respectively. Gene knock-out experiments verified that galB, -C, and -D are essential for GAL biosynthesis. A galD mutant accumulated a GAL-C that lacked two hydroxyl groups and a double bond when compared with GAL-B. A [U-(13)C]propionate feeding experiment indicated that no rare precursor other than methoxymalonate was incorporated during GAL biogenesis. A search of the S. galbus genome for a modular type I PKS system, the type that was expected to direct GAL biosynthesis, resulted in the identification of only one modular type I PKS gene cluster. Homology analysis indicated that this PKS gene cluster is the locus for vicenistatin biosynthesis. This cluster was previously reported in Streptomyces halstedii. A gene deletion of the vinP2 ortholog clearly demonstrated that this modular type I PKS system is not involved in GAL biosynthesis. Therefore, we propose that GalA-C direct macrolactone polyketide formation for GAL. Our studies provide a glimpse into a novel biochemical strategy used for polyketide synthesis; that is, the iterative assembly of propionates with highly programmed ß-keto group modifications.
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Sintasas Poliquetidas/metabolismo , Streptomyces/enzimología , Silenciador del Gen , Lactonas/metabolismo , Familia de Multigenes/genética , Sintasas Poliquetidas/química , Sintasas Poliquetidas/deficiencia , Sintasas Poliquetidas/genética , Propionatos/metabolismo , Estructura Terciaria de Proteína , Streptomyces/genética , Streptomyces/metabolismoAsunto(s)
Regulación Neoplásica de la Expresión Génica , Leucemia Mieloide Aguda/genética , Proteínas del Grupo Polycomb/genética , Receptores de Factores de Crecimiento/genética , Cromosomas Humanos Par 21 , Cromosomas Humanos Par 8 , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Mutación , Factor de Transcripción PAX5/genética , Factor de Transcripción PAX5/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Factores de Crecimiento/metabolismo , Transducción de Señal , Translocación GenéticaRESUMEN
The activation of B-cell-specific genes, such as CD19 and PAX5, is a hallmark of t(8;21) acute myeloid leukemia (AML) which expresses the translocation product RUNX1/ETO. PAX5 is an important regulator of B-lymphoid development and blocks myeloid differentiation when ectopically expressed. To understand the molecular mechanism of PAX5 deregulation, we examined its chromatin structure and regulation in t(8;21) AML cells, non-t(8;21) myeloid precursor control cells, and pre-B cells. In non-t(8;21) myeloid precursors, PAX5 is poised for transcription, but is repressed by polycomb complexes. In t(8;21) AML, PAX5 is not directly activated by RUNX1/ETO, but expression requires constitutive mitogen-activated protein (MAP) kinase signaling. Using a model of t(8;21) carrying an activating KIT mutation, we demonstrate that deregulated MAP kinase signaling in t(8;21) AML abrogates the association of polycomb complexes to PAX5 and leads to aberrant gene activation. Our findings therefore suggest a novel role of activating tyrosine kinase mutations in lineage-inappropriate gene expression in AML.
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Linaje de la Célula/genética , Leucemia Mieloide Aguda/genética , Factor de Transcripción PAX5/genética , Proteínas del Grupo Polycomb/fisiología , Línea Celular Tumoral , Cromosomas Humanos Par 21/genética , Cromosomas Humanos Par 8/genética , Regulación hacia Abajo/fisiología , Regulación Leucémica de la Expresión Génica , Células HL-60 , Células HeLa , Humanos , Leucemia Mieloide Aguda/patología , Sistema de Señalización de MAP Quinasas/fisiología , Modelos Biológicos , Factor de Transcripción PAX5/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Unión Proteica , Transducción de Señal/genética , Transducción de Señal/fisiología , Translocación GenéticaRESUMEN
dTDP-L-rhamnose (dTDP-Rha)-synthesizing dTDP-6-deoxy-L-lyxo-4-hexulose reductase (4-KR) and dTDP-Rha 4-epimerase were characterized from Burkholderia thailandensis E264 by utilizing rmlD(Bth) (BTH_I1472) and wbiB(Bth) (BTH_I1476), respectively. Incubation of the recombinant WbiB(Bth) with RmlA/RmlB/RmlC/Tal, which has previously been shown to generate dTDP-6-deoxy-L-talose (dTDP-6dTal) from α-D-glucose-1-phosphate, dTTP, and NADPH, produced dTDP-Rha. (1)H NMR measurements confirmed that both RmlA/RmlB/RmlC/Tal/WbiB(Bth) and RmlA/RmlB/RmlC/RmlD produced dTDP-Rha. WbiB(Bth) alone produced dTDP-Rha when incubated with dTDP-6dTal. This is the first report to demonstrate epimerase activity interconverting between dTDP-Rha and dTDP-6dTal.
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Burkholderia/enzimología , Carbohidrato Epimerasas/química , Desoxiazúcares/química , Hexosas/química , Azúcares de Nucleósido Difosfato/química , Nucleótidos de Timina/química , Burkholderia/genética , Cromatografía Líquida de Alta Presión , Estructura Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
The filipin biosynthetic gene cluster of Streptomyces avermitilis contains pteB, a homolog of crotonyl-CoA carboxylase/reductase. PteB was predicted to be 2-octenoyl-CoA carboxylase/reductase, supplying hexylmalonyl-CoA to filipin biosynthesis. Recombinant PteB displayed selective reductase activity toward 2-octenoyl-CoA while generating a broad range of alkylmalonyl-CoAs in the presence of bicarbonate.
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Acilcoenzima A/metabolismo , Acil-CoA Deshidrogenasas/metabolismo , Antibacterianos/química , Filipina , Streptomyces/enzimología , Acil-CoA Deshidrogenasas/química , Acil-CoA Deshidrogenasas/genética , Cromatografía Líquida de Alta Presión , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Filipina/biosíntesis , Familia de Multigenes , Plásmidos , Homología de Secuencia de Aminoácido , Espectrofotometría , Streptomyces/química , Streptomyces/genética , Transformación BacterianaRESUMEN
The Elongator complex has been implicated in several cellular processes, including gene expression and tRNA modification. We investigated the biological importance of the Elp3 gene in Drosophila melanogaster. Deletion of Elp3 results in larval lethality at the pupal stage. During early development, larval growth is dramatically impaired, with progression to the third instar delayed for â¼24 hr, and pupariation occurring only at day 14 after egg laying. Melanotic nodules appear after 4 days. Microarray analysis shows that stress response genes are induced and ecdysone-induced transcription factors are severely repressed in the mutant. Interestingly, the phenotypes of Elp3 flies are similar to those of flies lacking the domino gene, encoding a SWI/SNF-like ATP-dependent chromatin-remodeling enzyme. Indeed, the gene expression profiles of these mutants are also remarkably similar. Together, these data demonstrate that Drosophila Elp3 is essential for viability, normal development, and hematopoiesis and suggest a functional overlap with the chromatin remodeler Domino.
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Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/genética , Genes Letales , Histona Acetiltransferasas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Cromatina/genética , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Proteínas de Drosophila/genética , Ecdisona/fisiología , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Histona Acetiltransferasas/genética , Larva/genética , Larva/metabolismo , Modelos Lineales , Proteínas del Tejido Nervioso/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Pupa/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Leukocyte-like cells called hemocytes have key functions in Drosophila innate immunity. Three hemocyte types occur: plasmatocytes, crystal cells, and lamellocytes. In the absence of qimmune challenge, plasmatocytes are the predominant hemocyte type detected, while crystal cells and lamellocytes are rare. However, upon infestation by parasitic wasps, or in melanotic mutant strains, large numbers of lamellocytes differentiate and encapsulate material recognized as "non-self". Current models speculate that lamellocytes, plasmatocytes and crystal cells are distinct lineages that arise from a common prohemocyte progenitor. We show here that over-expression of the CoREST-interacting transcription factor Chn in plasmatocytes induces lamellocyte differentiation, both in circulation and in lymph glands. Lamellocyte increases are accompanied by the extinction of plasmatocyte markers suggesting that plasmatocytes are transformed into lamellocytes. Consistent with this, timed induction of Chn over-expression induces rapid lamellocyte differentiation within 18 hours. We detect double-positive intermediates between plasmatocytes and lamellocytes, and show that isolated plasmatocytes can be triggered to differentiate into lamellocytes in vitro, either in response to Chn over-expression, or following activation of the JAK/STAT pathway. Finally, we have marked plasmatocytes and show by lineage tracing that these differentiate into lamellocytes in response to the Drosophila parasite model Leptopilina boulardi. Taken together, our data suggest that lamellocytes arise from plasmatocytes and that plasmatocytes may be inherently plastic, possessing the ability to differentiate further into lamellocytes upon appropriate challenge.
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Linaje de la Célula , Drosophila melanogaster/genética , Hemocitos/metabolismo , Macrófagos/metabolismo , Animales , Diferenciación Celular/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Drosophila melanogaster/parasitología , Femenino , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hemocitos/citología , Interacciones Huésped-Parásitos , Quinasas Janus/genética , Quinasas Janus/metabolismo , Larva/genética , Larva/metabolismo , Larva/parasitología , Macrófagos/citología , Masculino , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción STAT/genética , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Avispas/fisiologíaRESUMEN
Kitasatospora kifunensis, the talosin producer, was used as a source for the dTDP-6-deoxy-l-talose (dTDP-6dTal) biosynthetic gene cluster, serving as a template for four recombinant proteins of RmlA(Kkf), RmlB(Kkf), RmlC(Kkf), and Tal, which complete the biosynthesis of dTDP-6dTal from dTTP, alpha-d-glucose-1-phosphate, and NAD(P)H. The identity of dTDP-6dTal was validated using (1)H and (13)C NMR spectroscopy. K. kifunensistal and tll, the known dTDP-6dTal synthase gene of Actinobacillus actinomycetemcomitans origin, have low sequence similarity and are distantly related within the NDP-6-deoxy-4-ketohexose reductase family, providing an example of the genetic diversity within the dTDP-6dTal biosynthetic pathway.
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Lactonas/metabolismo , Azúcares de Nucleósido Difosfato/metabolismo , Streptomycetaceae/genética , Streptomycetaceae/metabolismo , Deshidrogenasas del Alcohol de Azúcar/genética , Deshidrogenasas del Alcohol de Azúcar/metabolismo , Clonación Molecular , Variación Genética , Datos de Secuencia Molecular , Streptomycetaceae/enzimología , Deshidrogenasas del Alcohol de Azúcar/aislamiento & purificaciónRESUMEN
Galbonolides A and B are antifungal compounds, which are produced by Streptomyces galbus. A multimodular polyketide synthase (PKS) was predicted to catalyze their biosynthesis, and a methoxymalonyl-acyl carrier protein (methoxymalonyl-ACP) was expected to be involved in the biosynthesis of galbonolide A. Cloning of a methoxymalonyl-ACP biosynthesis locus (galGHIJK) and the flanking regions has revealed that the locus is colocalized with beta-ketoacyl synthase (KAS)-related genes (orf3, 4, and 5), but separated from any multimodular PKS gene cluster in S. galbus. A galI-disruption mutant (SK-galI-5) is unable to produce galbonolide A, but can synthesize galbonolide B, indicating that galGHIJK is involved in the biosynthesis of galbonolide A. A disruption mutant of orf4 is severely impaired in the production of both galbonolides A and B. These results indicate that galGHIJK and the KAS genes are involved in the biosynthesis of galbonolides, although they are not colocalized with a multimodular PKS gene cluster. We further propose that a single galbonolide PKS generates two discrete structures, galbonolides A and B, by alternatively incorporating methoxymalonate and methylmalonate, respectively.
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Proteína Transportadora de Acilo/genética , Vías Biosintéticas/genética , Familia de Multigenes , Sintasas Poliquetidas/genética , Streptomyces/enzimología , Antifúngicos/metabolismo , Clonación Molecular , Eliminación de Gen , Orden Génico , Lactonas/metabolismo , Modelos Biológicos , Mutagénesis Insercional , Streptomyces/genética , Streptomyces/metabolismoRESUMEN
Drosophila NURF is an ISWI-containing chromatin remodeling complex that catalyzes ATP-dependent nucleosome sliding. By sliding nucleosomes, NURF can alter chromatin structure and regulate transcription. NURF301/BPTF is the only NURF-specific subunit of NURF and is instrumental in recruiting the complex to target genes. Here we demonstrate that three NURF301 isoforms are expressed and that these encode functionally distinct NURF chromatin remodeling complexes. Full-length NURF301 contains a C-terminal bromodomain and juxtaposed PHD finger that bind histone H3 trimethylated at Lys4 (H3K4me3) and histone H4 acetylated at Lys16 (H4K16Ac) respectively. However, a NURF301 isoform that lacks these C-terminal domains is also detected. This truncated NURF301 isoform assembles a complex containing ISWI, NURF55, and NURF38, indicating that a second class of NURF remodeling complex, deficient in H3K4me3 and H4K16Ac recognition, exists. By comparing microarray expression profiles and phenotypes of null Nurf301 mutants with mutants that remove the C-terminal PHD fingers and bromodomain, we show that full-length NURF301 is not essential for correct expression of the majority of NURF gene targets in larvae. However, full-length NURF301 is required for spermatogenesis. Mutants that lack full-length NURF exhibit a spermatocyte arrest phenotype and fail to express a subset of spermatid differentiation genes. Our data reveal that variants of the NURF ATP-dependent chromatin remodeling complex that recognize post-translational histone modifications are important regulators of primary spermatocyte differentiation in Drosophila.
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Empalme Alternativo , Ensamble y Desensamble de Cromatina , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila/genética , Animales , Ciclina B/metabolismo , Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Perfilación de la Expresión Génica , Histonas/metabolismo , Mutación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas de Unión al ARN/metabolismo , EspermatogénesisRESUMEN
In Drosophila, defense against foreign pathogens is mediated by an effective innate immune system, the cellular arm of which is composed of circulating hemocytes that engulf bacteria and encapsulate larger foreign particles. Three hemocyte types occur: plasmatocytes, crystal cells, and lamellocytes. The most abundant larval hemocyte type is the plasmatocyte, which is responsible for phagocytosis and is present either in circulation or in adherent sessile domains under the larval cuticle. The mechanisms controlling differentiation of plasmatocytes and their migration toward these sessile compartments are unclear. To address these questions we have conducted a misexpression screen using the plasmatocyte-expressed GAL4 driver Peroxidasin-GAL4 (Pxn-GAL4) and existing enhancer-promoter (EP) and EP yellow (EY) transposon libraries to systematically misexpress approximately 20% of Drosophila genes in larval hemocytes. The Pxn-GAL4 strain also contains a UAS-GFP reporter enabling hemocyte phenotypes to be visualized in the semitransparent larvae. Among 3412 insertions screened we uncovered 101 candidate hemocyte regulators. Some of these are known to control hemocyte development, but the majority either have no characterized function or are proteins of known function not previously implicated in hemocyte development. We have further analyzed three candidate genes for changes in hemocyte morphology, cell-cell adhesion properties, phagocytosis activity, and melanotic tumor formation.
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Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Técnicas Genéticas , Hemocitos/metabolismo , Larva/metabolismo , Animales , Cruzamientos Genéticos , Proteínas de Drosophila/genética , Femenino , Hemocitos/citología , Masculino , Modelos Genéticos , Fagocitosis , Fenotipo , Regiones Promotoras Genéticas , Transducción de SeñalRESUMEN
The Drosophila nucleosome remodeling factor (NURF) is an ISWI-containing chromatin remodeling complex that catalyzes ATP-dependent nucleosome sliding. By sliding nucleosomes, NURF has the ability to alter chromatin structure and regulate transcription. Previous studies have shown that mutation of Drosophila NURF induces melanotic tumors, implicating NURF in innate immune function. Here, we show that NURF mutants exhibit identical innate immune responses to gain-of-function mutants in the Drosophila JAK/STAT pathway. Using microarrays, we identify a common set of target genes that are activated in both mutants. In silico analysis of promoter sequences of these defines a consensus regulatory element comprising a STAT-binding sequence overlapped by a binding-site for the transcriptional repressor Ken. NURF interacts physically and genetically with Ken. Chromatin immunoprecipitation (ChIP) localizes NURF to Ken-binding sites in hemocytes, suggesting that Ken recruits NURF to repress STAT responders. Loss of NURF leads to precocious activation of STAT target genes.