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PURPOSE: To compare the success rates of eye drop instillation in the sitting position and supine position among Japanese patients with ocular diseases (cataract, glaucoma, or retinal and vitreous diseases). METHODS: Patients who were hospitalized in Okayama University Hospital for eye surgery were studied. Instillation procedures of each patient in both the sitting and supine positions were recorded using a video camera at the time of instillation. We defined "success" when one drop fell accurately onto the ocular surface at the first attempt. Instillation of two or more drops, drops delivered to a site other than the eye surface, and touching the eyelashes, eyelids, or conjunctiva with the tip of the eye drop bottle were regarded as "failure". We excluded patients with vision below counting finger. RESULTS: One-hundred and two patients (54 males and 58 females, aged 70.2 ± 12.3 years) with ocular disease who were hospitalized for surgery (cataract: 61.8%, glaucoma: 15.7%, retinal and vitreous diseases: 22.5%) were included in this prospective observational study. The mean duration of eye drop use was 3.1 ± 5.2 years. The success rate of eye drop instillation was significantly higher in the supine position than in the sitting position (64.7% vs. 50%, P = 0.0039). The mean age was significantly higher in the failure group than in the success group (74.0 ± 11.5 vs. 67.7 ± 12.4 years, P = 0.0085) for the sitting position, but not significantly different for the supine position (72.3 ± 12.9 vs. 70.1 ± 12.0 years, P = 0.3849). No significant differences in mean duration of drop use, mean corrected VA, and mean spherical equivalent refraction were observed between success and failure groups, for both sitting and supine positions. CONCLUSIONS: In the present study, the success rate of eye drop instillation was significantly higher when applied in the supine position than in the sitting position.
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Soluciones Oftálmicas/administración & dosificación , Sedestación , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Oftalmopatías/tratamiento farmacológico , Femenino , Hospitalización , Humanos , Masculino , Persona de Mediana Edad , Soluciones Oftálmicas/uso terapéutico , Posición Supina , Adulto JovenRESUMEN
In Arabidopsis, a central regulator of copper (Cu) homeostasis is the transcription factor SQUAMOSA promoter binding protein-like7 (SPL7). Under Cu deficiency, SPL7 induces the expression of miR398, which suppresses the expression of the genes CSD1 and CSD2, which encode cytosolic and chloroplastic isoforms of Cu/Zn superoxide dismutase, respectively. Consequently, the limited Cu is preferentially assigned to plastocyanin, which is essential for photosynthetic electron transport. Consistent with this function of miR398 related to photosynthesis, its expression is strongly induced in leaves. In this study, however, we showed that SPL7 was transcribed mainly around the vasculature in roots, where Cu levels were likely sensed. To test the possible long-distance signaling of Cu availability from roots to shoots, we conducted a series of grafting experiments using spl7 mutant and wild-type (WT) plants. Expression of Cu-responsive microRNAs and the resulting suppression of CSD1 and CSD2 mRNAs were observed in leaves only when the aerial part was from WT plants, in which a low level of SPL7 was transcribed also in the vascular tissues. Although local sensing of Cu was disturbed in the spl7 mutant, the Cu level was not affected in the shoots. SPL7 is expressed in specific cell layers in both roots and shoots and locally senses Cu availability, transmitting the information to surrounding cells.
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Proteínas de Arabidopsis/genética , Arabidopsis/genética , Cobre/metabolismo , Regulación de la Expresión Génica de las Plantas , Homeostasis , Superóxido Dismutasa/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Superóxido Dismutasa/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
PURPOSE: To investigate the influence of subretinal injection pressure on the microstructure of the retina in a monkey model. METHODS: After vitrectomy, balanced salt solution was injected subretinally into one eye each of four cynomolgus monkeys while controlling the injection pressure. Initially, a pressure of 2 psi was used, and this was gradually increased to determine the minimum required pressure. Subsequent injections were performed at two pressures: minimum (n = 13) and high (n = 6). To compare the influence of these injection pressures on retinal structure, optical coherence tomography (OCT) was performed before surgery and every week afterwards. The monkeys were euthanized and their eyes were enucleated at 1 or 6 weeks after the injections. The eyes were processed for light microscopy and transmission electron microscopy (TEM) as well as for TdT-mediated dUTP nick end labeling. RESULTS: The minimum pressure required to perform subretinal injection was 6 psi. After injection at this pressure, both OCT and microscopy showed that the retinal structure was well-preserved throughout the experimental period at all injection sites. Conversely, after injection at high pressure (20 psi) OCT images at all injection sites showed disruption of the ellipsoid zone (EZ) after 1 week. Microscopy indicated damage to the photoreceptor outer segment (OS) and stratification of the retinal pigment epithelium (RPE). After 6 weeks, OCT demonstrated that the EZ had become continuous and TEM confirmed that the OS and RPE had recovered. Photoreceptor apoptosis was absent after subretinal injection at both pressures. CONCLUSIONS: The retinal damage caused by subretinal injection increases depending on pressure, indicating that clinicians should perform subretinal injection at pressures as low as possible to ensure safety.
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Inyecciones Intraoculares , Epitelio Pigmentado de la Retina/ultraestructura , Tomografía de Coherencia Óptica , Vitrectomía , Animales , Macaca fascicularis , Masculino , Microscopía Electrónica de Transmisión , PresiónRESUMEN
OBJECTIVE: To examine the usefulness of room temperature vulcanizing (RTV) silicone rubber as a barrier material for cell exclusion zone assays. METHODS: We created barriers using three types of RTV silicone rubber with differing viscosities. We then assessed the adherence of these barriers to culture dishes and their ease of removal from the dishes. We tested the effect of the newly created barriers on the extracellular matrix (ECM) protein fibronectin by attaching and then removing them from fibronectin-coated culture dishes. We also conducted cell exclusion zone assays with MIO-M1 cells using this new barrier in order to measure cell migration. We used real time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining to measure the effect of fibronectin on MIO-M1 cell migration and the effect of migration (with fibronectin coating) on basic fibroblast growth factor (bFGF) expression in MIO-M1 cells. RESULTS: Of the three types of RTV silicon rubber tested, KE-3495-T was the best in terms of adherence to the dish and ease of removal from the dish. When barrier attachment and removal tests were performed, this rubber type did not have an effect on the fibronectin that coated the dish. In the cell exclusion assay, removal of the barrier revealed that a cell-free area with a distinct margin had been created, which allowed us to conduct a quantitative assessment of migration. Fibronectin significantly promoted the migration of MIO-M1 cells (P = 0.02). In addition, both real time RT-PCR and immunohistological staining indicated that bFGF expression in migrating MIO-M1 cells was significantly higher than that in non-migrating cells (P = 0.03). CONCLUSIONS: RTV silicone rubber can be used to create an effective barrier in cell exclusion zone assays and allows simple and low-cost multi-parametric analysis of cell migration.
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Elastómeros de Silicona/química , Temperatura , Línea Celular Transformada , HumanosRESUMEN
Purpose: To investigate the mechanism of macular hole (MH) closure following the inverted internal limiting membrane (ILM) technique. Methods: We performed the inverted ILM flap surgical technique as an experimental MH model in monkeys, and investigated the process of MH closure immunohistochemically. We then investigated the effects of type IV collagen, fibronectin, and laminin, which are constituent proteins of the ILM, on the proliferation and migration of cultivated Müller cells (MIO-M1). We also investigated the expression of neurotrophic factors and basic fibroblast growth factor (bFGF) in human ILM and MIO-M1 cells, and the effect of MIO-M1 migration on the expression of these factors, via immunohistochemical staining and the real-time reverse transcription polymerase chain reaction. Results: Ten days after inverted ILM flap surgery, the MH had closed and proliferating glial fibrillary acidic protein (GFAP)-positive cells surrounded the ILM. Type IV collagen, fibronectin, and laminin all enhanced the proliferation of MIO-M1 cells, and type IV collagen and fibronectin enhanced the migration of MIO-M1 cells. Neurotrophic factors and bFGF were present on the surface of the human ILM, and MIO-M1 cells produced these factors. Neurotrophic factors and bFGF were expressed to a significantly greater extent by migrating MIO-M1 cells than by these cells in their static state. Conclusions: During MH closure, the ILM functioned as a scaffold for the proliferation and migration of Müller cells, and may promote Müller cell activation. Neurotrophic factors and bFGF produced by activated Müller cells and present on the surface of the ILM may contribute to MH closure.
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Membrana Epirretinal/cirugía , Perforaciones de la Retina/cirugía , Colgajos Quirúrgicos , Vitrectomía/métodos , Análisis de Varianza , Animales , Membrana Basal/cirugía , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colágeno Tipo IV/farmacología , Modelos Animales de Enfermedad , Células Ependimogliales/efectos de los fármacos , Células Ependimogliales/metabolismo , Membrana Epirretinal/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Fibronectinas/farmacología , Laminina/farmacología , Macaca fascicularis , Masculino , Factores de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/farmacología , Perforaciones de la Retina/metabolismoRESUMEN
We examined the effectiveness of trabeculectomy in decreasing the slope of mean deviation (MD) in Japanese patients with progressive normal-tension glaucoma (NTG) at low intraocular pressure (IOP) levels. The charts of patients who had undergone initial trabeculectomy with adjunctive mitomycin C for progressive NTG with medically controlled IOP < 15 mmHg in 2010-2013 were retrospectively reviewed. Seventeen eyes of 13 NTG patients who had undergone at least 5 times of visual field (VF) examinations in both of preoperatively and postoperatively with postoperative follow-up of ≥ 2 years were enrolled. Preoperative and postoperative MD slopes were compared to evaluate the effectiveness of trabeculectomy in slowing progression of VF. Mean IOP (8.1 ± 2.9 mmHg) and number of IOP-lowering medications (0.8 ± 1.5) were significantly lower postoperatively than preoperatively (13.9 ± 0.9 mmHg; P < 0.001 and 3.0± 0.4; P < 0.0001). In total, 91.7% of eyes with single-digit IOP postoperatively showed improvement in MD slope, whereas only 20.0% of eyes with IOP ≥ 10 mmHg postoperatively showed the improvement. Three eyes (17.6%) showed a decrease in visual acuity (VA) of ≥ 0.1 unit; this group had a lower mean postoperative IOP (6.0 ± 1.0 vs. 8.6 ± 3.0 mmHg; P = 0.1717) and a higher mean IOP reduction rate (56.2 vs. 38.5%; P = 0.8296) than eyes with a VA decrease of < 0.1 unit or no change. Thus, in this analysis of Japanese NTG patients with medically controlled IOP < 15 mmHg, achieving an IOP < 10 mmHg with trabeculectomy was beneficial for reducing the VF progression rate in progressive NTG at low IOP levels. However, an IOP < 7 mmHg by surgery would be required careful attention to VA decline.
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Presión Intraocular/fisiología , Glaucoma de Baja Tensión/cirugía , Trabeculectomía , Campos Visuales/fisiología , Anciano , Anciano de 80 o más Años , Progresión de la Enfermedad , Femenino , Humanos , Japón , Glaucoma de Baja Tensión/fisiopatología , Masculino , Persona de Mediana Edad , Mitomicina/uso terapéutico , Estudios Retrospectivos , Tonometría Ocular , Resultado del Tratamiento , Agudeza Visual/fisiologíaRESUMEN
For primary open angle glaucoma (POAG), laser treatment or surgery is used when the target intraocular pressure (IOP) cannot be achieved by pharmacological agents, such as prostaglandin (PG) analogs; these drugs also have varied effects. We retrospectively reviewed the medical records of 74 POAG patients (74 eyes) whose IOP was inadequately controlled by PG analogs (bimatoprost [13 eyes], latanoprost [34 eyes], tafluprost [11 eyes], and travoprost [16 eyes]) and underwent primary trabeculectomy. The proportion of patients with no recurrent IOP elevation within 24 months post-trabeculectomy was significantly (P < 0.001) lower in the bimatoprost group (31.3%) than in the latanoprost (83.2%), tafluprost (45.5%), or travoprost groups (65.6%). Deepening of the upper eyelid sulcus (DUES) was observed before trabeculectomy in 18 of 74 eyes (24.3%) treated with bimatoprost (9 eyes; 50.0%), latanoprost (3 eyes; 16.7%), tafluprost (1 eye; 5.5%) and travoprost (5 eyes; 27.8%). The proportion of patients with no recurrent IOP elevation up to 24 months post-trabeculectomy was significantly (P < 0.0001) lower in the DUES(+) group (34.7%) than in the DUES(-) group (74.3%). Multivariate stepwise logistic regression analysis, with no recurrent IOP elevation used as dependent variable, and bimatoprost, latanoprost, travoprost, tafluprost, ß-blocker, carbonic anhydrase inhibitor, brimonidine, gender, age, preoperative IOP, mean deviation, duration of PG analog use before surgery, and the number of ophthalmic solutions used as independent variables, identified only bimatoprost as a significant independent factor (P = 0.0368). Thus, the outcome of trabeculectomy varied depending on the PG analog used preoperatively, and bimatoprost use was associated with a high risk of recurrent IOP elevation up to 2 years post-trabeculectomy. This may indicate that the incidence of DUES differed with the PG analog used. Patients with glaucoma who are treated with bimatoprost should be monitored for DUES, and when these patients undergo trabeculectomy, the postoperative course of IOP should be followed carefully.
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Glaucoma/tratamiento farmacológico , Glaucoma/cirugía , Cuidados Preoperatorios , Prostaglandinas Sintéticas/administración & dosificación , Trabeculectomía , Anciano , Análisis de Varianza , Bimatoprost/administración & dosificación , Femenino , Glaucoma/fisiopatología , Humanos , Presión Intraocular/efectos de los fármacos , Latanoprost , Modelos Logísticos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Prostaglandinas F/administración & dosificación , Prostaglandinas F Sintéticas/administración & dosificación , Recurrencia , Estudios Retrospectivos , Travoprost/administración & dosificación , Resultado del TratamientoRESUMEN
The epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells plays a central role in the development of proliferative vitreoretinopathy (PVR). The purpose of this study was to investigate the effect of AMP-activated protein kinase (AMPK), a key regulator of energy homeostasis, on the EMT in RPE cells. In this study, EMT-associated formation of cellular aggregates was induced by co-stimulation of cultured ARPE-19 cells with tumor necrosis factor (TNF)-α (10 ng/ml) and transforming growth factor (TGF)-ß2 (5 ng/ml). 5-Aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR), a potent activator of AMPK, significantly suppressed TNF-α and TGF-ß2-induced cellular aggregate formation (p < 0.01). Dipyridamole almost completely reversed the suppressive effect of AICAR, whereas 5'-amino-5'-deoxyadenosine restored aggregate formation by approximately 50%. AICAR suppressed the downregulation of E-cadherin and the upregulation of fibronectin and α-smooth muscle actin by TNF-α and TGF-ß2. The levels of matrix metalloproteinase (MMP)-2, MMP-9, interleukin-6, and vascular endothelial growth factor were significantly decreased by AICAR. Activation of the mitogen-activated protein kinase and mammalian target of rapamycin pathways, but not the Smad pathway, was inhibited by AICAR. These findings indicate that AICAR suppresses the EMT in RPE cells at least partially via activation of AMPK. AMPK is a potential target molecule for the prevention and treatment of PVR, so AICAR may be a promising candidate for PVR therapy.
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Proteínas Quinasas Activadas por AMP/metabolismo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Epitelio Pigmentado de la Retina/citología , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Agregación Celular/efectos de los fármacos , Línea Celular , Sinergismo Farmacológico , Activación Enzimática/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Humanos , Interleucina-6/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ribonucleótidos/farmacología , Factor de Crecimiento Transformador beta2/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), a type I transmembrane glycoprotein, is known as one of the most specific lymphatic vessel markers in the skin. In this study, we found that the ectodomain of LYVE-1 undergoes proteolytic cleavage, and this process produces soluble LYVE-1. We further identified the cleavage site for ectodomain shedding and generated an uncleavable mutant of LYVE-1. In lymphatic endothelial cells, ectodomain shedding of LYVE-1 was induced by vascular endothelial growth factor (VEGF)-A, an important factor for angiogenesis and lymphangiogenesis under pathological conditions. VEGF-A-induced LYVE-1 ectodomain shedding was mediated via the extracellular signal-regulated kinase (ERK) and a disintegrin and metalloproteinase (ADAM) 17. Wild-type LYVE-1, but not uncleavable LYVE-1, promoted migration of lymphatic endothelial cells in response to VEGF-A. Immunostaining analyses in human psoriasis skin lesions and VEGF-A transgenic mouse skin suggested that the ectodomain shedding of LYVE-1 occurred in lymphatic vessels undergoing chronic inflammation. These results indicate that the ectodomain shedding of LYVE-1 might be involved in promoting pathological lymphangiogenesis.
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Glicoproteínas/metabolismo , Vasos Linfáticos/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteína ADAM17/genética , Proteína ADAM17/metabolismo , Animales , Línea Celular , Micropartículas Derivadas de Células/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Femenino , Glicoproteínas/genética , Humanos , Linfangiogénesis/fisiología , Sistema de Señalización de MAP Quinasas , Proteínas de Transporte de Membrana , Ratones , Ratones Transgénicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Psoriasis/etiología , Psoriasis/metabolismo , Psoriasis/patología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Proteínas de Transporte Vesicular/genéticaRESUMEN
Poaceae plants release phytosiderophores into the rhizosphere in order to chelate iron (Fe), which often exists in insoluble forms especially under high pH conditions. The impact of phytosiderophore treatment at the physiological and molecular levels in vivo remains largely elusive, although the biosynthesis of phytosiderophores and the transport of phytosiderophore-metal complexes have been well studied. We recently showed that the application of 30 µM of the chemically synthesized phytosiderophore 2'-deoxymugineic acid (DMA) was sufficient for apparent full recovery of otherwise considerably reduced growth of hydroponic rice seedlings at high pH. Moreover, unexpected induction of high-affinity nitrate transporter gene expression as well as nitrate reductase activity indicates that the nitrate response is linked to Fe homeostasis. These data shed light on the biological relevance of DMA not simply as a Fe chelator, but also as a trigger that promotes plant growth by reinforcing nitrate assimilation.
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Ácido Azetidinocarboxílico/análogos & derivados , Hierro/metabolismo , Nitrógeno/metabolismo , Oryza/metabolismo , Plantones/metabolismo , Sideróforos/metabolismo , Ácido Azetidinocarboxílico/metabolismo , Ácido Azetidinocarboxílico/farmacología , Oryza/efectos de los fármacos , Oryza/crecimiento & desarrollo , Plantones/efectos de los fármacos , Plantones/crecimiento & desarrolloRESUMEN
Poaceae plants release 2'-deoxymugineic acid (DMA) and related phytosiderophores to chelate iron (Fe), which often exists as insoluble Fe(III) in the rhizosphere, especially under high pH conditions. Although the molecular mechanisms behind the biosynthesis and secretion of DMA have been studied extensively, little information is known about whether DMA has biological roles other than chelating Fe in vivo. Here, we demonstrate that hydroponic cultures of rice (Oryza sativa) seedlings show almost complete restoration in shoot height and soil-plant analysis development (SPAD) values after treatment with 3-30 µm DMA at high pH (pH 8.0), compared with untreated control seedlings at normal pH (pH 5.8). These changes were accompanied by selective accumulation of Fe over other metals. While this enhanced growth was evident under high pH conditions, DMA application also enhanced seedling growth under normal pH conditions in which Fe was fairly accessible. Microarray and qRT-PCR analyses revealed that exogenous DMA application attenuated the increased expression levels of various genes related to Fe transport and accumulation. Surprisingly, despite the preferential utilization of ammonium over nitrate as a nitrogen source by rice, DMA application also increased nitrate reductase activity and the expression of genes encoding high-affinity nitrate transporters and nitrate reductases, all of which were otherwise considerably lower under high pH conditions. These data suggest that exogenous DMA not only plays an important role in facilitating the uptake of environmental Fe, but also orchestrates Fe and nitrate assimilation for optimal growth under high pH conditions.
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Ácido Azetidinocarboxílico/análogos & derivados , Hierro/metabolismo , Nitratos/metabolismo , Oryza/crecimiento & desarrollo , Oryza/metabolismo , Ácido Azetidinocarboxílico/farmacología , Transporte Biológico/efectos de los fármacos , Concentración de Iones de Hidrógeno , Nitrato-Reductasa/metabolismoRESUMEN
Plants belonging to the Brassicaceae family exhibit species-specific profiles of glucosinolates (GSLs), a class of defence compounds against pathogens and insects. GSLs also exhibit various human health-promoting properties. Among them, glucoraphanin (aliphatic 4-methylsulphinylbutyl GSL) has attracted the most attention because it hydrolyses to form a potent anticancer compound. Increased interest in developing commercial varieties of Brassicaceae crops with desirable GSL profiles has led to attempts to identify genes that are potentially valuable for controlling GSL biosynthesis. However, little attention has been focused on genes of kale (Brassica oleracea var. acephala). In this study, we established full-length kale cDNA libraries containing 59 904 clones, which were used to generate an expressed sequence tag (EST) data set with 119 204 entries. The EST data set clarified genes related to the GSL biosynthesis pathway in kale. We specifically focused on BoMYB29, a homolog of Arabidopsis MYB29/PMG2/HAG3, not only to characterize its function but also to demonstrate its usability as a biological resource. BoMYB29 overexpression in wild-type Arabidopsis enhanced the expression of aliphatic GSL biosynthetic genes and the accumulation of aliphatic GSLs. When expressed in the myb28myb29 mutant, which exhibited no detectable aliphatic GSLs, BoMYB29 restored the expression of biosynthetic genes and aliphatic GSL accumulation. Interestingly, the ratio of methylsulphinyl GSL content, including glucoraphanin, to that of methylthio GSLs was greatly increased, indicating the suitability of BoMYB29 as a regulator for increasing methylsulphinyl GSL content. Our results indicate that these biological resources can facilitate further identification of genes useful for modifications of GSL profiles and accumulation in kale.
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Brassica/genética , Biblioteca de Genes , Glucosinolatos/biosíntesis , Proteínas de Plantas/genética , Factores de Transcripción/genética , Secuencia de Aminoácidos , Arabidopsis/genética , Vías Biosintéticas/genética , Brassica/metabolismo , Clonación Molecular , Etiquetas de Secuencia Expresada , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Prueba de Complementación Genética , Glucosinolatos/genética , Datos de Secuencia Molecular , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Alineación de Secuencia , Análisis de Secuencia de Proteína , Factores de Transcripción/química , Factores de Transcripción/metabolismoRESUMEN
Linum album has been shown to accumulate anti-tumor podophyllotoxin (PTOX) and its related lignans. In the present study, we examined the effects of five fungal extracts on the production of lignans in L. album cell cultures. Fusarium graminearum extract induced the highest increase of PTOX [140µgg(-1) dry weight (DW) of the L. album cell culture] which is seven-fold greater than the untreated control, while Rhizopus stolonifer extract enhanced the accumulation of lariciresinol, instead of PTOX, up to 365µgg(-1) DW, which was 8.8-fold greater than the control. Quantitative PCR analyses showed that expression of the enzyme genes responsible for the PTOX biosynthesis cascade, such as pinoresinol-lariciresinol reductase (PLR), phenylalanine ammonia-lyase (PAL), cinnamoyl-CoA reductase (CCR) and cinnamyl-alcohol dehydrogenase (CAD) genes, were also up-regulated in a fungal extract-selective fashion. These results provide evidence that the fungal extracts used in this study differentially increase the production of PTOX or larisiresinol via the up-regulation of the genes in lignan biosynthesis in L. album cell cultures, and suggest that such selective actions of fungal elicitors on the lignan synthesis will lead to more efficient metabolic engineering-based production of PTOX and other beneficial lignans using L. album cell cultures.
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Lino/genética , Lino/microbiología , Lignanos/biosíntesis , Aldehído Oxidorreductasas/metabolismo , Vías Biosintéticas , Células Cultivadas , Lino/metabolismo , Furanos , Fusarium/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Oxidorreductasas/metabolismo , Fenilanina Amoníaco-Liasa/metabolismo , Podofilotoxina/biosíntesis , Rhizopus/metabolismoRESUMEN
Recent advances in our understanding of how graminaceous plants take up insoluble forms of iron from the rhizosphere and mobilize them in plant tissues are primarily based on the identification of various transporters that are specific to metal-phytosiderophore (PS) complexes containing mugineic acid and deoxymugineic acid. Barley (Hordeum vulgare L.) yellow stripe 1 (HvYS1) is a metal-PS transporter that preferentially transports Fe(III)-PS compared with other metal complexes. Here, we report the cloning and characterization of HvYSL2, a novel metal-PS transporter encoding gene. HvYSL2 is composed of 702 amino acids with 14 transmembrane domains, which are conserved among this class of transporters, and exhibits 67.3% identity to HvYS1. Electrophysiological experiments with Xenopus laevis oocytes revealed that HvYSL2 transports PS complexes with Fe(III), Zn(II), Ni(II), Cu(II), Mn(II) or Co(II); this constitutes a broader range of substrate preference than HvYS1. Real-time PCR analysis revealed that HvYSL2 mRNA is expressed in shoots and also in roots, where it is induced under iron-deficient conditions. Moreover, immunohistochemistry in roots revealed that HvYSL2 is localized to the endodermis, whereas HvYS1 is expressed primarily in the epidermis. These data suggest that HvYSL2 is spatially distinct from HvYS1 and plays a unique role in delivering a broad range of essential metals in barley.
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Hordeum/genética , Proteínas de Transporte de Membrana/metabolismo , Metales/metabolismo , Epidermis de la Planta/metabolismo , Proteínas de Plantas/metabolismo , Sideróforos/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Clonación Molecular , Regulación de la Expresión Génica de las Plantas , Hordeum/metabolismo , Proteínas de Transporte de Membrana/genética , Datos de Secuencia Molecular , Oocitos , Epidermis de la Planta/genética , Proteínas de Plantas/genética , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Especificidad por Sustrato , Xenopus laevisRESUMEN
Nitrate uptake by rice coleoptiles was evaluated using ¹5N-nitrate in relation to the expression of high-affinity nitrate uptake-related genes, OsNRT2s (OsNRT2.1-2.4) and OsNAR2s (OsNAR2.1 and 2.2). Apparent nitrate uptake by coleoptiles was about one-sixth of that by hydroponically cultured seedling roots. Real-time RT-PCR analysis revealed that OsNRT2.1, a root-specific key gene of inducible high-affinity transport system for nitrate, was most strongly induced in coleoptiles following nitrate supply initiation, while other OsNRT2s and OsNAR2s showed modest induction. These results suggest that rice coleoptiles may have high-affinity transport systems for nitrate similar to roots, and can be model organs for nutrient uptake by submerged plant shoots.
Asunto(s)
Regulación de la Expresión Génica de las Plantas , Nitratos/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Transporte de Anión/efectos de los fármacos , Proteínas de Transporte de Anión/genética , Proteínas de Transporte de Anión/metabolismo , Transporte Biológico , Cotiledón/efectos de los fármacos , Cotiledón/genética , Cotiledón/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Transportadores de Nitrato , Oryza/efectos de los fármacos , Oryza/genética , Proteínas de Plantas/efectos de los fármacos , Proteínas de Plantas/genética , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , ARN de Planta/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de TiempoRESUMEN
Understanding plant metabolism as an integrated system is essential for metabolic engineering aimed at the effective production of compounds useful to human life and the global environment. The "omics" approach integrates transcriptome and metabolome data into a single data set and can lead to the identification of unknown genes and their regulatory networks involved in metabolic pathways of interest. One of the intriguing, although poorly described metabolic pathways in plants is the biosynthesis of glucosinolates (GSLs), a group of bioactive secondary products derived from amino acids that are found in the family Brassicaceae. Here we report the discovery of two R2R3-Myb transcription factors that positively control the biosynthesis of GSLs in Arabidopsis thaliana by an integrated omics approach. Combined transcriptome coexpression analysis of publicly available, condition-independent data and the condition-specific (i.e., sulfur-deficiency) data identified Myb28 and Myb29 as candidate transcription factor genes specifically involved in the regulation of aliphatic GSL production. Analysis of a knockout mutant and ectopic expression of the gene demonstrated that Myb28 is a positive regulator for basal-level production of aliphatic GSLs. Myb29 presumably plays an accessory function for methyl jasmonate-mediated induction of a set of aliphatic GSL biosynthetic genes. Overexpression of Myb28 in Arabidopsis-cultured suspension cells, which do not normally synthesize GSLs, resulted in the production of large amounts of GSLs, suggesting the possibility of efficient industrial production of GSLs by manipulation of these transcription factors. A working model for regulation of GSL production involving these genes, renamed Production of Methionine-Derived Glucosinolate (PMG) 1 and 2, are postulated.