RESUMEN
Phosphorylation of alcohols is a fundamentally important reaction in both life science and physical science. Product phosphate monoesters play key roles in living organisms, natural products, pharmaceuticals, and organic materials. Most of the chemical methods to date for synthesizing phosphate monoesters, however, require multistep sequences or are limited to specific types of substrates possibly due to harsh conditions. An alternative way to enable the simple production of phosphate monoesters from highly functionalized precursor alcohols is, thus, highly desired. We report herein a catalytic phosphorylation of alcohols with high functional group tolerance using tetrabutylammonium hydrogen sulfate (TBAHS) and phosphoenolpyruvic acid monopotassium salt (PEP-K) as the catalyst and phosphoryl donor, respectively. This method enables the direct introduction of a nonprotected phosphate group to the hydroxy group of a diverse menu of alcohol substrates, including functionalized small molecules, carbohydrates, and unprotected peptides. Nuclear magnetic resonance, mass spectrometric, and density functional theory analyses suggest that an unprecedented mixed anhydride species, generated from PEP-K and TBAHS, acts as an active phosphoryl donor in this reaction. This operationally simple and chemoselective catalytic phosphorylation allows for the efficient production of densely functionalized O-phosphorylated compounds, which are useful in diverse fields including biology and medicine.
RESUMEN
Ingestion of Bacillus subtilis C-3102 spores (C-3102) has relieved the symptoms of diarrhoea in piglets and changed the composition of gut microbiota in humans. Recently, it was suggested that the composition of the human gut microbiota affects stool consistency. In this study, a double-blind, randomised, placebo-controlled trial was conducted to assess the preventive effects of chronic diarrhoea in healthy volunteers with loose stools by ingestion of C-3102. The results showed that oral doses of C-3102 tablets significantly decreased the Bristol Stool Scale score and stool frequency, and also significantly improved abdominal sounds. With regard to gut microbiota, the relative abundance of Lachnospira, Actinomyces and SMB53 were significantly changed. This study shows that C-3102 could be effective for treating loose stools (Trial registration: UMIN000022583, http://tinyurl.com/ya4refqn ).
Asunto(s)
Antidiarreicos/administración & dosificación , Bacillus subtilis/crecimiento & desarrollo , Voluntarios Sanos , Probióticos/administración & dosificación , Administración Oral , Método Doble Ciego , Microbioma Gastrointestinal , Humanos , Placebos/administración & dosificación , Comprimidos/administración & dosificaciónRESUMEN
BACKGROUND: Renal fibrosis (RF) is a well-known marker for chronic kidney disease (CKD) progression, including chronic renal injury after renal transplantation. However, invasive biopsy is an available examination for evaluation of RF. Diffusion MRI was once recognized as a promising option for RF. However, it is now controversial for RF evaluation in a unilateral ureteral obstruction (UUO) model. METHODS: To seek an optimal imaging method applicable for RF in UUO model kidneys, we attempted a series of MRI methods, including proton density-weighted imaging, T1-weighted imaging, T2-weighted imaging, T2*-weighted imaging, diffusion-weighted imaging, and diffusion tensor imaging (DTI). RESULTS: We identified DTI MRI by spin-echo sequence plus a special kidney attachment as the best option for evaluation of renal UUO fibrosis, compared with normal kidney on the opposite side. To confirm these results, we applied this technique to a rat UUO therapeutic model with the anti-fibrotic reagent Fasudil. Fractional anisotropy values calculated from DTI MRI showed statistically significant linear correlation with the RF area measured by use of Sirius red or Masson trichrome staining of the positive area [cortex (r = 0.6397, P = .0283) and outer stripe of the outer medulla (r = 0.7810, P = .0039)]. CONCLUSIONS: By use of the DTI MRI with spin-echo sequence, it may be possible to accurately evaluate RF in CKD.
Asunto(s)
Imagen de Difusión Tensora/métodos , Enfermedades Renales/patología , Imagen por Resonancia Magnética/métodos , Animales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Fibrosis/patología , Masculino , RatasRESUMEN
BACKGROUND: On the basis of a comparison of the hemolytic complement titer in pigs with that in humans, the complement system of pigs was investigated. The response of innate immunity, such as the natural antibodies, against humans was also examined. METHODS: Hemolytic complement activity of pig serum was measured with the use of a microtitration technique. CH50 was determined according to the method of Mayer. ACH50 was assayed according to the methods of Platts-Milles and Ishizaka. Hemolytic activities of C1, C4, C2, C3, C5, C8, and C9 were estimated through the use of intermediate cells and reagents, as described previously. In addition, the pig natural anti-human antibody was studied with the use of human peripheral blood mononuclear cells (PBMCs). Human PBMCs were stained with 5% pig serum, followed by staining with fluorescein isothiocyanate-labeled goat anti-pig IgG and IgM. The resulting stained cells were quantified by use of a FACScalibur system. The alternative pathway of pig complement was also measured with the use of human erythrocytes and normal pooled pig serum with or without Mg(++)EGTA. RESULTS: Both the CH50 and ACH50 titers were lower than those of humans. Concerning the components, except for C3, each component, that is, C1, C4, C2, C5, C8, and C9, was also lower than that of humans, based on measured values for human complement components. Pig serum clearly contains natural antibodies, IgG and IgM, to human PBMCs. The alternative pathway of pig complement reacted with human erythrocytes. CONCLUSIONS: As a whole, pig innate immunity, the complement system and natural antibody, recognizes the surfaces of human cells.
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Proteínas del Sistema Complemento/inmunología , Hemólisis/inmunología , Inmunidad Innata/inmunología , Animales , Anticuerpos Antiidiotipos/metabolismo , Activación de Complemento/inmunología , Ensayo de Actividad Hemolítica de Complemento , Proteínas del Sistema Complemento/metabolismo , Eritrocitos/inmunología , Fibronectinas/metabolismo , Humanos , Leucocitos Mononucleares/inmunología , Proteínas Recombinantes/metabolismo , Sus scrofa , PorcinosRESUMEN
Emerging data illustrate a pivotal role for activation of ß-cell endoplasmic reticulum (ER) stress pathways in diabetes pathophysiology. The purpose of this review is to appraise the evidence for ß-cell ER stress in human type 1 and 2 diabetes, review the molecular signalling pathways involved in the unfolded protein response and ER stress signalling, and to provide data from polyribosome profiling to illustrate the impact of ER stress on the mRNA translation response. Finally, we will discuss existing and novel therapeutic strategies that target ß-cell ER stress and discuss their use in rodent and human type 1 and 2 diabetes.
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Diabetes Mellitus/etiología , Diabetes Mellitus/fisiopatología , Estrés del Retículo Endoplásmico/fisiología , Células Secretoras de Insulina/fisiología , Biosíntesis de Proteínas/fisiología , Respuesta de Proteína Desplegada , Animales , Humanos , ARN Mensajero/metabolismo , Respuesta de Proteína Desplegada/genéticaRESUMEN
Survival and germination rate of Bacillus subtilis C-3102 spores were investigated in a stomach and small intestine model (TIM-1), while the impact of C-3102 cells that had passed through TIM-1 on human colon microbiota was evaluated in a model of the large intestine (TIM-2). The survival of C-3102 spores in TIM-1 was 99%; 8% of the spores had germinated. Effluent of TIM-1 was subsequently introduced into TIM-2 and a micro-array platform was employed to assess changes in the microbiota composition. The effluent, which contained germinated C-3102 cells, increased some Bifidobacterium species and decreased some Clostridium groups. These changes were greater compared to those obtained by adding C-3102 spores directly to TIM-2. The present study suggests that oral doses of B. subtilis C-3102 spores have the potential to modulate the human colon microbiota. This effect may be caused by germination of the spores in the gastrointestinal tract.
Asunto(s)
Bacillus subtilis/crecimiento & desarrollo , Tracto Gastrointestinal/microbiología , Metagenoma , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Línea Celular , Humanos , Viabilidad Microbiana , Modelos Biológicos , Esporas Bacterianas/crecimiento & desarrolloRESUMEN
Many immunedeficiency syndromes are associated with autoimmune disorders. We here report on a girl with a systemic lupus erythematosus-like disease who suffered from both hyperimmunoglobulin M syndrome (HIGMS) and C1q deficiency. Despite severe central nervous system-lupus like disease, probably due to C1q deficiency, kidney function was relatively spared. IgM autoantibody might play a protective role against lupus-glomerulonephritis.
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Complemento C1q/deficiencia , Síndrome de Inmunodeficiencia con Hiper-IgM/complicaciones , Lupus Eritematoso Sistémico/etiología , Niño , Femenino , Humanos , Síndrome de Inmunodeficiencia con Hiper-IgM/diagnóstico , Inmunoglobulina M/fisiología , Lupus Eritematoso Sistémico/complicaciones , Lupus Eritematoso Sistémico/diagnóstico , Nefritis Lúpica/fisiopatología , Nefritis Lúpica/prevención & controlRESUMEN
AIMS/HYPOTHESIS: The WFS1 gene encodes an endoplasmic reticulum (ER) membrane-embedded protein called Wolfram syndrome 1 protein, homozygous mutations of which cause selective beta cell loss in humans. The function(s) of this protein and the mechanism by which the mutations of this gene cause beta cell death are still not fully understood. We hypothesised that increased insulin demand as a result of obesity/insulin resistance causes ER stress in pancreatic beta cells, thereby promoting beta cell death. METHODS: We studied the effect of breeding Wfs1 ( -/- ) mice on a C57BL/6J background with mild obesity and insulin resistance, by introducing the agouti lethal yellow mutation (A ( y ) /a). We also treated the mice with pioglitazone. RESULTS: Wfs1 ( -/- ) mice bred on a C57BL/6J background rarely develop overt diabetes by 24 weeks of age, showing only mild beta cell loss. However, Wfs1 ( -/- ) A ( y ) /a mice developed selective beta cell loss and severe insulin-deficient diabetes as early as 8 weeks. This beta cell loss was due to apoptosis. In Wfs1 ( +/+ ) A ( y ) /a islets, levels of ER chaperone immunoglobulin-binding protein (BiP)/78 kDa glucose-regulated protein (GRP78) and phosphorylation of eukaryotic translation initiation factor 2, subunit alpha (eIF2alpha) apparently increased. Levels of both were further increased in Wfs1 ( -/- ) A ( y ) /a murine islets. Electron micrography revealed markedly dilated ERs in Wfs1 (-/-) A ( y ) /a murine beta cells. Interestingly, pioglitazone treatment protected beta cells from apoptosis and almost completely prevented diabetes development. CONCLUSIONS/INTERPRETATION: Wfs1-deficient beta cells are susceptible to ER stress. Increased insulin demand prompts apoptosis in such cells in vivo. Pioglitazone, remarkably, suppresses this process and prevents diabetes. As common WFS1 gene variants have recently been shown to confer a risk of type 2 diabetes, our findings may be relevant to the gradual but progressive loss of beta cells in type 2 diabetes.
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Células Secretoras de Insulina/fisiología , Insulina/deficiencia , Insulina/farmacología , Proteínas de la Membrana/deficiencia , Tiazolidinedionas/farmacología , Envejecimiento , Animales , Apoptosis , Peso Corporal , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/fisiología , Chaperón BiP del Retículo Endoplásmico , Variación Genética , Prueba de Tolerancia a la Glucosa , Humanos , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/patología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , PioglitazonaAsunto(s)
Trasplante de Médula Ósea , Enfermedad Injerto contra Huésped/tratamiento farmacológico , Neutropenia/terapia , Acondicionamiento Pretrasplante , Trasplante de Médula Ósea/mortalidad , Preescolar , Femenino , Enfermedad Injerto contra Huésped/sangre , Enfermedad Injerto contra Huésped/etiología , Enfermedad Injerto contra Huésped/mortalidad , Prueba de Histocompatibilidad , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Lactante , Masculino , Neutropenia/sangre , Neutropenia/historia , Neutropenia/mortalidad , Síndrome , Acondicionamiento Pretrasplante/métodosRESUMEN
We report the observations of a new type of changing process in the Burgers vector of dislocations by in situ transmission electron microscopy. Small interstitial-type perfect dislocation loops in bcc iron with diameters less than approximately 50 nm are transformed from a 1/2<111> loop to another 1/2<111> one or an energetically unfavorable <100> one; furthermore, a <100> loop is transformed to a 1/2<111> one. These transformations occurred on high-energy electron irradiation or simple heating without contact with external dislocations. The origin of these phenomena is discussed.
RESUMEN
We report here the cDNA cloning and functional analysis of Xenopus DNase gamma (xDNase gamma). Two forms of cDNAs are isolated from adult spleen: one composing a 933 bp open reading frame for the enzymatically active xDNase gamma protein, and the other encoding an inactive short alternative form. Northern blot analysis revealed that the xDNase gamma mRNA is expressed in spleen, liver, testis, and ovary. xDNase gamma expression is scarcely detected in the tail muscle of tadpoles; however, it increases during metamorphosis and reaches a maximum during the late metamorphic climax. The ectopic expression of xDNase gamma results in the appearance of extensive DNA fragmentation in C2C12 myoblasts after the induction of apoptosis. In contrast, Xenopus DNase I fails to induce apoptotic DNA ladder formation under the same conditions. Our results suggest a possible involvement of xDNase gamma in apoptosis during amphibian metamorphosis.
Asunto(s)
Apoptosis , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , ADN Complementario/química , ADN Complementario/aislamiento & purificación , Endodesoxirribonucleasas/química , Larva/anatomía & histología , Larva/crecimiento & desarrollo , Metamorfosis Biológica , Ratones , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Proteínas de Xenopus/química , Xenopus laevis/genética , Xenopus laevis/crecimiento & desarrolloRESUMEN
AIM: To compare the results of the water drinking test between glaucomatous eyes with and without visual field progression. METHODS: Retrospective analysis of 76 eyes of 76 open angle glaucoma patients followed for a mean period of 26.0 (SD 13.8) months. Patients were submitted to the water drinking test at the beginning of the follow up period. Reliable achromatic automated perimetry tests performed during the studied period were used to characterise visual field progression. All subjects were under clinical therapy and had an intraocular pressure (IOP) lower than 17 mm Hg monitored by isolated measurements during the follow up period. The results of the water drinking test were compared between glaucomatous eyes with and without visual field progression. RESULTS: Twenty eight eyes reached definite visual field progression. There were no significant differences in the mean age, sex, race, basal IOP, number of antiglaucomatous drugs, initial mean deviation (MD), and corrected pattern standard deviation (CPSD) between eyes that showed visual field progression and the ones who did not progress. A significant difference of 1.9 (SD 0.6) mm Hg (p = 0.001, analysis of covariance; 95% CI 0.8 to 3.0) was observed between glaucomatous eyes that showed visual field deterioration and glaucomatous eyes that did not progress. A significant difference of 16.8% (SD 4.6%) in the mean percentage of IOP variation was also observed between the two groups (p<0.001, analysis of covariance; 95% CI 7.7 to 26.0). CONCLUSIONS: Mean IOP peak and percentage of IOP variation during water drinking test were significantly higher in patients with visual field progression compared with patients who did not progress.
Asunto(s)
Ingestión de Líquidos , Glaucoma de Ángulo Abierto/fisiopatología , Campos Visuales , Adulto , Anciano , Anciano de 80 o más Años , Antihipertensivos/uso terapéutico , Ritmo Circadiano , Técnicas de Diagnóstico Oftalmológico , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Glaucoma de Ángulo Abierto/tratamiento farmacológico , Humanos , Presión Intraocular , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Pruebas del Campo VisualRESUMEN
AIM: To assess the intraocular pressure (IOP) variability in patients with primary open angle glaucoma (POAG) under clinical treatment who reached an established target pressure based on isolated office readings. METHODS: Retrospective analysis of 65 eyes from 65 POAG patients under clinical therapy who submitted to modified diurnal tension curve (mDTC) (measurements at every 3 hours between 8 am and 5 pm) followed by a water drinking test (WDT). All subjects had established target IOP < or =15 mm Hg at 11 am or 2 pm. IOP variability during mDTC or WDT was evaluated. RESULTS: mDTC revealed IOP measurements > or =17 mm Hg in 16 of 65 eyes (24.6%). Nine eyes (13.8%) presented values > or =18 mm Hg. The highest IOP detected by mDTC was 20 mm Hg in one patient (1.5%). WDT demonstrated IOP values > or =17 mm Hg in 32 of 65 eyes (49.2%). 22 eyes (33.8%) presented values > or =18 mm Hg after water ingestion. Moreover, IOP levels > or =20 mm Hg were observed in 14 eyes (21.5%). CONCLUSION: A great percentage of POAG patients undergoing clinical treatment and with IOP control based on single office measurement present significantly higher IOP measurements when performing mDTC and, especially, the WDT.
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Glaucoma de Ángulo Abierto/fisiopatología , Presión Intraocular/fisiología , Adulto , Anciano , Anciano de 80 o más Años , Ritmo Circadiano , Ingestión de Líquidos , Femenino , Glaucoma de Ángulo Abierto/terapia , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , AguaRESUMEN
Listeria monocytogenes contamination of ready-to-eat seafood products commercially available in Osaka was examined between 1999 and 2000. L. monocytogenes was isolated from 12 (13%) of the 95 products tested. All positive samples were from cold-smoked fish with 9 being obtained during the summer. Thirteen isolates of L. monocytogenes were typed by pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction (PCR)-based typing methods. Isolates of the same serotype originating from the same manufacturer gave similar DNA profiles, irrespective of the type of sample or date of isolation. The finding suggest that persistent strains in each manufacturing facility proliferate during the summer and contaminate products during manufacturing processes.
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Productos Pesqueros/microbiología , Contaminación de Alimentos/análisis , Industria de Procesamiento de Alimentos , Listeria monocytogenes/aislamiento & purificación , Animales , Seguridad de Productos para el Consumidor , ADN Bacteriano/análisis , Electroforesis en Gel de Campo Pulsado , Contaminación de Equipos , Japón , Reacción en Cadena de la Polimerasa , Prevalencia , Estaciones del AñoRESUMEN
Human immunodeficiency virus type 1 (HIV-1) Tat repressed the p53-dependent gene expression through its C-terminal domain of Tat (amino acid residues 73-86) independent of the involvement of NF-kappaB and coactivator CBP/p300. Although Tat did not directly bind to p53, this repression required the N-terminal domain of p53. In contrast, Tat and p53 cooperated in the activation of HIV-1 gene expression. Thus, the cross-talk between Tat and p53 may be linked with cellular transformation by HIV-1 infection or activation of HIV-1 replication.
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Productos del Gen tat/farmacología , VIH-1/química , Proteína p53 Supresora de Tumor/metabolismo , Animales , Células COS , Expresión Génica/efectos de los fármacos , Expresión Génica/fisiología , Productos del Gen tat/química , Silenciador del Gen , Duplicado del Terminal Largo de VIH/fisiología , VIH-1/fisiología , FN-kappa B/metabolismo , Estructura Terciaria de Proteína , Activación Transcripcional/efectos de los fármacos , Proteína p53 Supresora de Tumor/genética , Replicación Viral , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaRESUMEN
To study the inhibitory effects of calcium phosphate-associated proteins on calcium oxalate crystallization and urinary concentrations of proteins in people who form stones and healthy controls. From 60 L of urine from healthy men, calcium phosphate-associated proteins (alpha-2-HS-glycoprotein, prothrombin fragment 1 and osteopontin) were obtained. The effects of the proteins on calcium oxalate (CaOx) crystallization were studied with a mixed suspension mixed product removal system. To examine urinary concentrations of the proteins, urine samples were collected from 17 healthy subjects and 15 stone formers and analyzed using anion-exchange chromatography and an enzyme immunoassay. Prothrombin fragment 1 (PTF1) and osteopontin (OPN) had strong inhibitory effects on CaOx crystallization, while alpha-2-HS-glycoprotein had a mild inhibitory effect. Urinary concentrations of PTF1 and OPN were lower in stone formers than in healthy controls. Low urinary concentrations of PTF1 and OPN might be one of the reasons for stone formation.
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Proteínas Sanguíneas/orina , Oxalato de Calcio/química , Fragmentos de Péptidos/orina , Fosfoproteínas/orina , Precursores de Proteínas/orina , Protrombina/orina , Sialoglicoproteínas/orina , Cálculos Urinarios/orina , Adulto , Cristalización , Femenino , Humanos , Masculino , Osteopontina , alfa-2-Glicoproteína-HSRESUMEN
Replication protein A (RPA), which is comprised of three subunits, is an important factor involved in DNA replication, repair, and transcription. We isolated and characterized 70 and 32 kDa subunits of RPA from rice (Oryza sativa cv. Nipponbare) termed OsRPA70a and OsRPA32. OsRPA70a shows a low level of homology with OsRPA1 which was isolated from deepwater rice (Oryza sativa cv. Pin Gaew 56), previously. We also succeeded to isolate OsRPA70b which is homologue to OsRPA1 from Oryza sativa cv. Nipponbare. OsRPA70a shows only 33.8% sequence identity with OsRPA70b, indicating that two different types of 70 kDa RPA subunits are present in Oryza sativa cv. Nipponbare. These subunits showed differences in their expression patterns among tissues. The transcripts of OsRPA70a and OsRPA32 were expressed strongly in proliferating tissues such as root tips and young leaves that contain root apical meristem and marginal meristem, respectively, and weakly in the mature leaves which have no proliferating tissues. On the other hand, OsRPA70b was expressed mostly in the proliferating tissues. The roles of these molecules in plant DNA replication and DNA repair are discussed.
Asunto(s)
Proteínas de Unión al ADN/genética , Oryza/genética , Secuencia de Aminoácidos , Northern Blotting , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Proteínas de Unión al ADN/química , Expresión Génica , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Datos de Secuencia Molecular , Peso Molecular , Oryza/citología , Oryza/efectos de los fármacos , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Subunidades de Proteína , ARN de Planta/efectos de los fármacos , ARN de Planta/genética , ARN de Planta/metabolismo , Proteína de Replicación A , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Sacarosa/farmacología , Distribución TisularRESUMEN
PURPOSE: To compare scanning laser polarimeter (GDx, Laser Diagnostic Technologies, San Diego, Calif) measurements of the peripapillar retinal nerve fiber layer in amblyopic and normal eyes. METHODS: Scanning laser polarimetry was performed on 21 patients with unilateral strabismic amblyopia who had an absence of neurologic diseases or glaucoma and a minimum age of 7 years. A mean retardation map was calculated from separate scans or was considered to be the best scan obtained for each eye. Polarimetric indices were analyzed comparing amblyopic and contralateral normal eyes. RESULTS: The mean age was 15 +/- 9 years (7-35 years) and the male:female ratio was 13:8. There were 6 right and 15 left amblyopic eyes, with the amblyopic group having a mean visual acuity of 0.3 +/- 0.1. The mean (+/- SD) indices did not differ significantly between normal and amblyopic eyes, except the number that summates information from the individual parameters, which was higher in normal (20.71 +/- 11.98) than in amblyopic (15.14 +/- 6.81) eyes, P =.02. CONCLUSION: There was no statistical difference in thickness of the nerve fiber layer between amblyopic and normal eyes. A previous study found similar results in adults with strabismic amblyopia.
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Ambliopía/patología , Fibras Nerviosas/patología , Células Ganglionares de la Retina/patología , Adolescente , Adulto , Niño , Femenino , Humanos , Rayos Láser , Masculino , Agudeza VisualRESUMEN
Spore germination, a transition from the quiescent G0 phase to the proliferation cycle, is triggered by glucose in Schizosaccharomyces pombe. The role of cAMP/protein kinase A (PKA) signalling in germination is investigated. Gene disruption of cyr1+, pka1+ and gpa2+ encoding adenylate cyclase, PKA and the alpha-subunit of a trimeric GTP-binding protein, respectively, reduced the colony-forming efficiency of spores in minimal medium. Isolated spores of these null mutants did not germinate in minimal medium for up to 12 h, at which time wild-type spores had completed germination and formed germ projections. In wild-type spores, cortical actin patches randomly distributed in the early stage of outgrowth and then localized to one side of spores before the formation of projections. In contrast, the mutant spores exhibited no actin patches, but the cell surface was predominantly stained, like ungerminated spores of wild-type. Flow fluorocytometric analysis of propidium iodide-stained spores revealed a distinct 1C DNA peak after germination was completed. The fluorescent profile of the mutant spores, however, did not change during 12 h incubation in the minimal medium. These observations indicate that spores harbouring either cyr1Delta, pka1Delta or gpa2Delta are hardly triggered to germination. When wild-type spores were exposed to glucose, the intracellular cAMP level transiently increased in a few minutes, but gpa2Delta spores did not respond to glucose. We conclude that S. pombe spores initiate germination in response to glucose through the cyclic AMP-PKA pathway.