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PURPOSE: To assess variability in the coefficient of variation (COV) in cell area estimates when using different numbers of cells for endothelial morphometry. METHODS: Using non-contact specular microscopy images of the corneal endothelium, 4 sets of 20 cases were selected that included 200 cells and had overall (global) COV values of less than 30 (group 1), 31-40 (group 2), 41-50 (group 3) and over 50% (group 4). Subjects could be normal, or had ophthalmic disease (such as diabetes), a history of rigid or soft contact lens wear or were assessed after cataract surgery. A step-wise analysis was undertaken, 20 cells at a time, of the variability in cell area estimates when using different numbers of cells for the calculations. RESULTS: Variability in the average cell area values was higher if only 20-60 cells were used in the calculations and then tended to decrease. The standard deviation values on these average cell area values and the calculated COV showed the same overall trends and were more than twice as large for endothelia with marked polymegethism. Using more than 100 cells/image in markedly polymegethous endothelia only increased the variability in the calculations. CONCLUSIONS: These analyses indicate that substantial region variability in cell area values can be expected in polymegethous endothelia. The analysis further confirm that using only small numbers of cells (e.g. less than 50/image) in such cases is likely to yield far less reliable estimates of COV.
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Recuento de Células/métodos , Enfermedades de la Córnea/diagnóstico , Técnicas de Diagnóstico Oftalmológico/normas , Endotelio Corneal/citología , Adulto , Humanos , Microscopía/métodosRESUMEN
Genetic analysis in mice has most commonly employed two general strategies: phenotypic screens for spontaneous or induced mutations and genotypic analysis using homologous recombination or gene trapping to produce deletion or insertion mutants. Here we use bacterial artificial chromosome (BAC)-mediated gene-dosage analysis in transgenic mice to reveal novel genetic functions that are not evident from conventional loss-of-function mutations. We demonstrate a role for the zinc-finger transcription factor Zipro1 (formerly Ru49 and Zfp38) in the proliferation of granule cell precursors in the developing cerebellum, and document the contribution of this process to the final stages of cerebellar morphogenesis. We also show that Zipro1 is expressed in skin, and increased Zipro1 dosage results in a hair-loss phenotype associated with increased epithelial cell proliferation and abnormal hair follicle development.
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Cerebelo/citología , Cromosomas Bacterianos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Dosificación de Gen , Piel/citología , Células Madre/metabolismo , Transactivadores/genética , Transactivadores/fisiología , Animales , Recuento de Células , Muerte Celular , División Celular , Cerebelo/anatomía & histología , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/metabolismo , Técnicas Genéticas , Cabello/metabolismo , Ratones , Ratones Transgénicos , Modelos Genéticos , Fenotipo , Transactivadores/análisis , Transactivadores/metabolismoRESUMEN
Low-grade serous ovarian cancer (LGSOC) poses a specific clinical challenge due to advanced presentation at diagnosis and the lack of effective systemic treatments. The aim of this study was to use a precision medicine approach to identify clinically actionable mutations in a patient with recurrent LGSOC. Primary, metastatic and recurrence tissue, and blood samples were collected from a stage IV LGSOC patient. Single-gene testing for clinically actionable mutations (BRAF V600, KRAS and NRAS) and subsequent whole-exome sequencing (WES) were performed. Droplet digital PCR was used to evaluate the presence of an identified BRAF D594G mutation in the matched plasma cell-free DNA (cfDNA). No clinically actionable mutations were identified using single-gene testing. WES identified a BRAF D594G mutation in six of seven tumor samples. The patient was commenced on a MEK inhibitor, trametinib, but with minimal clinical response. A newly designed ddPCR assay detected the BRAF alteration in the matched tissues and liquid biopsy cfDNA. The identification and sensitive plasma detection of a common "druggable" target emphasises the impact of precision medicine on the management of rare tumors and its potential contribution to novel monitoring regimens in this field.
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BACKGROUND: The purpose of this review was to evaluate the consistency of central corneal thickness (CCT) values reported with use of Topcon SP-2000 P and SP-3000 P non-contact specular microscopes since their introduction in 1999 with the two microscopes having been commonly used in a wide range of studies. METHODS: As a primary resource, PubMed was used to search for peer-reviewed articles in any language that included CCT values obtained with non-contact specular microscopy reported for humans with nominally healthy corneas. Relevant articles were obtained and any cited publications also checked. RESULTS: A total of 76 articles were identified which reported CCT on different small-to-moderate sized groups of individuals, published between 1999 and 2019. From these, an overall group mean CCT value of 0.525 ± 0.013 mm (median 0.525 mm) can be calculated. An estimated 95 % confidence interval (CI, based on 1.96 SD) would be between 0.500 and 0.550 mm. For the two Topcon models, the group mean ± SD values were 0.529 ± 0.013 mm and 0.517 ± 010 mm respectively. An assessment of the CCT data sets in relation to the reported average age indicated no statistically significant effect (p = 0.289, r = -0.129). Very similar average CCT values were also encountered in 4 other reports where these microscopes were used in large-scale population studies as well as in 2 other reports using the newer Topcon SP-1 P model. CONCLUSIONS: The Topcon stand-alone non-contact specular microscopes have yielded consistent and predictable corneal thickness measures over many years.
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Córnea , Microscopía , Córnea/diagnóstico por imagen , Humanos , Reproducibilidad de los ResultadosRESUMEN
OBJECTIVE: To assess the level of agreement for assignment of a normal grade for human ocular surface bulbar conjunctival cells as collected by conjunctival impression cytology. METHODS: Suitable images from published articles that included a scale marker were subjected to the same planimetry method to assess cell area, nucleus area, long (L) and short (S) dimensions of the cells and the nucleus. These measures, along with the nucleus-to-cytoplasmic (N/C) ratio, were compared. RESULTS: Area measures on normal grade images indicated a broad unimodal distribution (250-275 µm(2)), and distribution of nucleus area values was heterogeneous, with neither being normally distributed. The inter-sample variance for any particular area value ranged from 11 to 90% (group averages of 49%). The L:S dimension ratio averaged 1.288, consistently showing these cells are not round. N/C values showed a wide range (from 0.151 to 0.655), as did nucleus-to-cytoplasm dimensions (from 0.332 to 0.603). CONCLUSIONS: Current criteria for subjective assignment of a normal grade to bulbar conjunctival cells do not appear to be robust enough to allow for inter-sample comparison. Quantitative measures need to be developed further.
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Conjuntiva/citología , Técnicas Citológicas/métodos , Células Epiteliales/citología , Núcleo Celular , Citodiagnóstico/métodos , Citoplasma , Femenino , Humanos , Valores de Referencia , Adulto JovenRESUMEN
The basic helix-loop-helix (bHLH) transcription factors Ptf1a and Math1 are necessary for the specification of gamma-aminobutyric acid-ergic and glutamatergic cell lineages in the cerebellum, respectively. Recent evidence suggests cascades of bHLH factor activities drive cell type specificity in Ptf1a(+ve) and Math1(+ve) lineages. In this manuscript, we reveal cell lineages in the cerebellar cortex but not deep cerebellar nuclei express the pro-neural bHLH factor Neurogenin1 (Ngn1). Ngn1 is expressed in ventricular zone progenitors and in newly generated neurons in the caudal cerebellar primordium. In later embryonic and postnatal developmental stages, Ngn1 is expressed in progenitors and in migrating interneurons in the prospective white matter. Transgenic fate-mapping reveals Ngn1 reporter-gene expression in Purkinje cells, multiple inhibitory interneuron cell types, and in unipolar brush cells of the cortex. The data suggest Ngn1 is a component of the bHLH factor code regulating cell type specification in the cerebellar cortex.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Linaje de la Célula/genética , Corteza Cerebelosa/embriología , Corteza Cerebelosa/crecimiento & desarrollo , Corteza Cerebelosa/metabolismo , Proteínas del Tejido Nervioso/genética , Animales , Animales Recién Nacidos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular/genética , Movimiento Celular/genética , Embrión de Mamíferos , Femenino , Regulación del Desarrollo de la Expresión Génica , Edad Gestacional , Masculino , Ratones , Ratones Transgénicos , Mitosis/genética , Modelos Biológicos , Proteínas del Tejido Nervioso/metabolismo , Células Madre/metabolismo , Células Madre/fisiología , Distribución TisularRESUMEN
A fibre optic spectrophotometer front-end system for measuring corneas to overcome shortcomings associated with existing instruments was tested. The system allowed prompt measurement postmortem, minimizing beam pathlength to reduce the effects of scatter and unwanted refraction and eliminated optical interfaces and cuvette media. Rabbit corneas were excised immediately postmortem and placed on a detecting fibre optic coupled to an Ocean Optics spectrophotometer and illuminated by a deuterium-halogen source. The compact instrument with its small beam size allowed tissue profiling at test points across the corneal surface and efficient interchange for comparison of different tissues. This simplified system operation allowed rapid tissue altering to study induced changes on transmittance. The corneal transmittance data showed a consistent sharp cut-off at 320 nm in the ultraviolet radiation (UVR) spectrum, which decayed rapidly from postmortem swelling. Inter- and intra-corneal consistency was demonstrated by comparing data from different regions of the same cornea and those from opposite eyes. Changes to the spectra, particularly in the UVB below 300 nm, were evident when the corneal epithelium was removed, indicating that this layer is not the only corneal UVR filter. The new system reduced much of the variability associated with previous methods, as it rapidly measured corneal transmittance postmortem. Data are in broad agreement with published transmittance curves. The removal of the corneal epithelium revealed a substantial stromal contribution to the overall corneal UVR absorption, suggesting that corneas with pathologically or iatrogenically thinned stromas are less effective UVR blockers.
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Córnea/fisiología , Animales , Calibración , Epitelio Corneal/fisiología , Tecnología de Fibra Óptica , Luz , Conejos , Espectrofotometría Infrarroja , Espectrofotometría Ultravioleta , Rayos UltravioletaRESUMEN
Goblet cells were visualized in impression cytology specimens from bulbar conjunctiva of the rabbit eye using Giemsa staining. Highly magnified images were used to generate outlines of the goblet cells and their characteristic eccentric nuclei. Using sets of 10 cells from 15 cytology specimens, I found that the longest dimension of the goblet cells averaged 16.7 ± 2.3 µm, the shortest dimension averaged 14.4 ± 1.8 µm and the nucleus averaged 6.3 ± 0.8 µm. The goblet cells were ellipsoid in shape and the longest:shortest cell dimension ratio averaged 1.169 ± 0.091. The goblet cell areas ranged from 108 to 338 µm2 (average 193 ± 50 µm2). The area could be predicted reliably from the longest and shortest dimensions (r2 = 0.903). The areas of goblet cell nuclei were 15-58 µm2 (average 33 ± µm2) and the nucleus:cytoplasm area fraction was predictably greater in smaller goblet cells and less in the larger goblet cells (Spearman correlation = 0.817). The nuclei were estimated to occupy an average of 9.5% of the cell volume. The differences in size, shape and nucleus:cytoplasm ratio may reflect differences in goblet cell maturation.
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Núcleo Celular/metabolismo , Tamaño de la Célula , Conjuntiva/metabolismo , Citoplasma/metabolismo , Células Epiteliales/citología , Células Caliciformes/metabolismo , Animales , ConejosRESUMEN
Lurcher (Lc) is a gain-of-function mutation in the delta2 glutamate receptor (GRID2) that results in the cell-autonomous death of cerebellar Purkinje cells in heterozygous lurcher (+/Lc) mice. This in turn triggers the massive loss of afferent granule cells during the first few postnatal weeks. Evidence suggests that the death of Purkinje cells as a direct consequence of GRID2(Lc) activation and the secondary death of granule cells because of target deprivation occur by apoptosis. We have used mice carrying null mutations of both the Bax and p53 genes to examine the roles of these genes in cell loss in lurcher animals. The absence of Bax delayed Purkinje cell death in response to the GRID2(Lc) mutation and permanently rescued the secondary death of granule cells. In contrast, the p53 deletion had no effect on either cell death pathway. Our results demonstrate that target deprivation induces a Bax-dependent, p53-independent cell death response in cerebellar granule cells in vivo. In contrast, Bax plays a minor role in GRID2(Lc)-mediated Purkinje cell death.
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Apoptosis/genética , Ratones Mutantes Neurológicos/genética , Degeneración Nerviosa/patología , Proteínas Proto-Oncogénicas c-bcl-2 , Animales , Caspasa 3 , Caspasas/metabolismo , Supervivencia Celular/genética , Eliminación de Gen , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/enzimología , Neuronas/patología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Células de Purkinje/metabolismo , Células de Purkinje/patología , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Proteína X Asociada a bcl-2RESUMEN
The Lurcher (Lc) mutation in the delta2 glutamate receptor gene leads to the presence of a constitutive inward current in the cerebellar Purkinje cells of Lurcher heterozygous mice and to the postnatal degeneration of these neurons. In addition, cerebellar granule cells and olivary neurons of Lc/+ mice die as an indirect effect of the mutation after the loss of their target Purkinje cells. The apoptotic nature of Lc/+ Purkinje cell death remains controversial. To address this question, we studied the involvement of caspase-3, a key effector of apoptosis, in the neurodegenerative processes occurring in Lc/+ cerebellum. Several antibodies recognizing different regions of caspase-3 were used in immunoblotting and immunohistochemical experiments. We demonstrate that pro-caspase-3 is specifically upregulated in the dying Lc/+ Purkinje cells, but not in granule cells and olivary neurons, suggesting that different death-inducing signals trigger variant apoptotic pathways in the CNS. The subcellular localization of pro-caspase-3 was shown to be cytoplasmic and mitochondrial. Active caspase-3 as well as DNA fragmentation was found in numerous granule cells and some Purkinje cells of the Lc/+ cerebellum. Thus, caspase-3 activation is involved in both the direct and indirect neuronal death induced by the Lurcher mutation, strongly supporting the idea that the Lc/+ Purkinje cell dies by apoptosis.
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Caspasas/metabolismo , Ratones Mutantes Neurológicos/fisiología , Neuronas/fisiología , Animales , Caspasa 3 , Muerte Celular/fisiología , Fragmentación del ADN , Activación Enzimática/fisiología , Precursores Enzimáticos/metabolismo , Ratones , Células de Purkinje/enzimología , Células de Purkinje/fisiología , Células de Purkinje/ultraestructura , Valores de Referencia , Distribución TisularRESUMEN
Many solutions have been used to investigate the swelling properties of the mammalian corneal stroma but few of the solutions resemble the expected extracellular matrix fluid of the corneal stroma, and little information is available on whether incubation ex vivo causes significant changes in the gross composition of the stroma. From quality-selected recent post-mortem eyes of adult cattle, stroma preparations were cut from the central part of the cornea. The time-dependent changes in wet mass were assessed over 9 h at 37 degrees C, and the preparations then dried. Various solutions of known pH (6.88-8.32) and osmolality (<50-327 mosmol/kg) were used, and were assayed for protein and proteoglycan after the incubation. The rates and extent of stromal swelling were lowest in a glucose-supplemented mixed salts solution containing 35 mM bicarbonate (0.5% CO2) solution, marginally greater in a mixed salts solution containing 35 mM bicarbonate (5% CO2) or similar non-bicarbonate mixed salts solutions (including BSS), and progressively greater in phosphate-buffered saline (PBS), various phosphate buffers (10-67 mM) and saline solutions (0.025-1%), and greatest in water. The initial rates of swelling ranged from 44 to 451 mg/h and the secondary rates from 9 to 106 mg/h. In all solutions, protein and proteoglycans were detected, but these ranged from around 1 to 10% of the samples with the bicarbonate-buffered solutions, to around 30% with the use of some phosphate buffers or saline.
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Colágeno/química , Córnea/química , Córnea/citología , Proteínas del Ojo/química , Proteoglicanos/química , Animales , Bicarbonatos , Tampones (Química) , Bovinos , Matriz Extracelular/química , Espacio Extracelular/química , Técnicas In Vitro , Soluciones Isotónicas , Sales (Química) , Soluciones , Agua/análisisRESUMEN
The effects of cationic surfactants on the time-dependent increases in hydration of the corneal stroma were investigated to assess if the contribution of the proteoglycans could be titrated and how it might relate to the maximum and minimum swelling properties of the corneal stroma. From recent post-mortem eyes from adult sheep, square (8 x 8 mm) samples of corneal stroma were prepared and incubated in isotonic neutral pH mixed salts solution with added glucose, or pure water, at 37 degrees C. The time-dependent changes in wet mass were assessed over 24 h in the absence or presence of 0. 001-2% w/v cetylpyridium chloride (CPC) or benzalkonium chloride (BAC). The rate and magnitude of stromal swelling was reduced in a concentration-dependent fashion by the surfactants. In mixed salts solution, 100% inhibition of swelling could be achieved at 2% CPC and BAC. In pure water, the relative swelling was much more substantial and could only be attenuated by CPC.
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Cetilpiridinio/química , Colágeno/análisis , Sustancia Propia/química , Detergentes/química , Matriz Extracelular/química , Agua/análisis , Animales , Soluciones Isotónicas , Ovinos , Factores de TiempoRESUMEN
The pH sensitivity of the swelling of the mammalian corneal stroma was reinvestigated to assess whether or not there were detectable differences in the hydration properties of this collagen-keratocyte matrix within a physiologically relevant range (as opposed to extremes of acid or alkaline pH) and at a physiologically relevant temperature. From recent post-mortem eyes of adult cows, square (8 x 8 mm) samples of corneal stroma were prepared and incubated in an isotonic, buffered (HEPES etc.), mixed salts solution with added glucose at 37 degrees C. The time-dependent changes in wet mass were assessed over 24 h. The rate and magnitude of stromal swelling were different within the range of pH 6.5-8.5. The wet mass of stromal samples increased almost 2-fold within 1 h, and then at lesser rates to realise 3.25-3.75-fold and 4-5-fold increases in wet mass by 9 h and 24 h respectively. The maximum increases were observed at pH 7.25-7.5, with most of the effect being the result of differences in the initial rate of swelling. The discontinuous swelling and the pH effect on the rates of swelling were also evident when the data were fitted to a previous kinetic model (Elliott et al., J. Physiol. (Lond.) 298 (1980) 453-470). It is concluded that pH changes in the physiological range can have a small but reproducible impact on the swelling kinetics of the isolated mammalian corneal stroma ex vivo.
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Córnea/metabolismo , Concentración de Iones de Hidrógeno , Células del Estroma/metabolismo , Agua/metabolismo , Animales , Tampones (Química) , Bovinos , Córnea/citología , Glucosa , Técnicas In Vitro , MasculinoRESUMEN
(1) Evidence is presented which indicates that the carbocyanine dye (3,3'-dipropyl thiadicarbocyanine) can be used as a spectroscopic probe for monitoring the resting potential across tha plasma membrane of the ciliated protozoan Paramecium. (2) The dye at low concentrations (less than or equal to 1 muM) does not affect either the viability or the motility of the cells, nor does it induce a chemotactic response. (3) The fluorescence of the dye bound to the cells alters as the potential across the membrane is changed by increasing the external cation concentration. (4) The absorbance of the bound dye also changes in response to an alteration of the membrane potential. (5) The membrane potential changes as measured by the fluorescence method have been correlated with the measurements of the potential estimated by microelectrode methods. (6) Both cations which induce a negative chemotactic response in Paramecium (K+, Na+, Ba2+) and several non-toxic cations bring about a rapid depolarization of the plasma membrane. The significance of these rapid changes in relation to the swimming behaviour of the ciliate is discussed.
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Quimiotaxis , Paramecium/fisiología , Animales , Bario/farmacología , Sitios de Unión , Carbocianinas , Membrana Celular/fisiología , Colina/farmacología , Cinética , Potenciales de la Membrana/efectos de los fármacos , Membranas Artificiales , Paramecium/efectos de los fármacos , Fosfolípidos , Potasio/farmacología , Compuestos de Amonio Cuaternario/farmacología , Sodio/farmacología , Especificidad de la Especie , Espectrometría de Fluorescencia , Valinomicina/farmacologíaRESUMEN
1. The flagellated alga, Euglena gracilis, swims forward essentially in a straight path under constant light intensity. Strong motility of the cells can be supported by Mg2+ alone but optimum motility is found in the presence of Mg2+, Ca2+ and K+. 2. Ca2+, Co2+, Mn2+ and Ba2+ induce a concentration-dependent increase in the rate at which the cells change the direction of their swimming path (a klinokinesis). Ni2+ immobilizes the flagellum. 3. On perception of a reduction ('step-down stimulus') in blue light intensity in their environment, Euglena rotate in place (tumble) for a finite period (the step-down photophobic response). 4. The duration of the tumbling is enhanced in the presence of divalent cations following the series Ca2+ greater than Ba2+ greater than Mn2+ greater than Co2+ greater than Mg2+ = Ni2+ = 0. 5. Neither the tumbling response in the presence of low concentrations of Ca2+ or the Ca2+-stimulated response is altered by verapamil (a Ca2+ conductance antagonist). The Ca2+ conductance/active transport antagonist, ruthenium red, is also inactive. 6. The Ca2+ ionophore, A23187, has little effect on flagellar activity in the absence of extracellular Ca2+. However, in the presence of A23187, Ca2+ induces a specific light-independent, concentration-dependent discontinuous tumbling response of the cells. 7. The data support a role for Ca2+ and Mg2+ in control of flagellar activity. However, blue light-induced tumbling behavior would not appear to be the direct result of a light-mediated alteration in the Ca2+ conductance of the flagellar membrane to affect flagellar reorientation. The results are discussed in connection with previous theories on control of flagella activity in green alga.
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Antibacterianos/farmacología , Calcimicina/farmacología , Calcio/metabolismo , Cationes Bivalentes/farmacología , Euglena gracilis/metabolismo , Luz , Euglena gracilis/efectos de los fármacos , Euglena gracilis/efectos de la radiación , Flagelos/fisiología , Movimiento , Rojo de Rutenio/farmacología , Verapamilo/farmacologíaRESUMEN
1. The blue light-induced cell tumbling behavior (the step-down photophobic response) and the accumulation of cells into a blue light trap (photoaccumulation) were investigated in Euglena. Dose response plots for these phenomena which we collectively term 'photobehavior' show both threshold and saturation characteristics. 2. NaCl effects apparent elevation in the photosensitivity of the cell as evidenced by alteration of the dose response plot character and lowering of the light intensity saturation level. 3. NaCl and ouabain enhance the duration of the photophobic responses and the rate of photoaccumulation. KCl and NH4Cl have lesser or inhibitory effects. 4. Choline chloride reduces the duration of the photophobic responses and the rate of photoaccumulation. 5. KCl reduces the enhancement of photobehavior induced by NaCl and at constant chloride concentration, photobehavior is unaffected by the relative KCl and NaCl concentrations. 6. Antagonists of voltage-dependent, monovalent cation fluxes in membranes (tetrodotoxin, procaine, tetraethylammonium, 4-aminopyridine) do not alter photobehavior. 7. The results suggest a role for a photoreceptor membrane-located transport system for Na+/K+ as a key step in control of the intraflagellar free Ca/+ levels that determine the photobehavior mediated by flagellar reorientation.
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Euglena gracilis/efectos de la radiación , Canales Iónicos/metabolismo , Potasio/metabolismo , Sodio/metabolismo , Movimiento Celular/efectos de los fármacos , Movimiento Celular/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Euglena gracilis/fisiología , Luz , Potenciales de la Membrana/efectos de los fármacos , Ouabaína/farmacología , Cloruro de Potasio/farmacología , Cloruro de Sodio/farmacologíaRESUMEN
Lurcher is an autosomal semidominant murine mutation. Lurcher heterozygotes (+/Lc) lose all their cerebellar Purkinje cells by adulthood. Explants from 2 days postnatal (P2) wild-type (+/+) and +/Lc cerebellar cortex were grown in vitro to investigate the role of local neuronal environment and afferent input on the degenerating +/Lc Purkinje cell. In Lurcher explants, Purkinje cells were maintained for up to 25 days in vitro. No significant difference was observed between +/+ and +/Lc Purkinje cell numbers from 10 to 20 days in vitro, as revealed by calbindin-D immunoreactivity. Growing +/Lc explants in association with +/+ explants resulted in no significant difference in Purkinje cell survival (10-20 days in vitro). Image analysis of the gross morphology of calbindin-D-immunostained Purkinje cells from +/+ and +/Lc explants grown in vitro revealed a significant decrease in the total area and dendritic lengths of +/Lc Purkinje cells (15 and 20 days in vitro). The fine structure of +/Lc and +/+ Purkinje cells was examined under the electron microscope (10-25 days in vitro). No difference in ultrastructure was observed between +/Lc and +/+ Purkinje cells grown in vitro, and many features similar to normal Purkinje cell development in vivo were present. These included monosynaptic parallel fibre synapses with Purkinje cell dendritic spines, other interneuron synapses with Purkinje cell dendrites and soma, astroglial investment, and minimal extracellular space in the neuropil. Unusual features observed included a persistence of the perisomatic spines in some Purkinje cells, an absence of Nissl bodies in the Purkinje cell perikaryon, naked Purkinje cell dendritic spines, and occasional heterologous synapses. The results are discussed in the light of previous chimeric analysis of the Lurcher mutation, and a hypothesis is put forward to explain the survival of +/Lc Purkinje cells in vitro.
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Cerebelo/crecimiento & desarrollo , Células de Purkinje/ultraestructura , Animales , Células Cultivadas , Cerebelo/citología , Femenino , Ratones , Ratones Endogámicos C3H , Ratones Mutantes Neurológicos , Microscopía , Microscopía Electrónica , Degeneración Nerviosa/fisiología , Valores de ReferenciaRESUMEN
Cerebellar pattern formation was investigated in rats treated with DNA modifying agents. Animals were subjected to combinations of daily injections of methylazoxymethanol acetate (MAM) for the last 6 days gestation and/or localised X-irradiation of the hindbrain on postnatal days 1 and 5 (P1 and P5). Animals were analysed on embryonic day 18 (E18), P0, P3, P7, and P14. Five parameters of the cerebellum were recorded from midsagittal sections: the number of primary lobules; the thickness of the external germinal layer (EGL); the density of cells in the internal granule cell layer (IGL) region; and the midsagittal area and perimeter. In addition, the laterolateral cerebellar distance was calculated. The data demonstrate that pre- and postnatal reduction of the EGL results in reduced cerebellar growth and folding. Cessation of the treatment at birth results in a recovery and eventual overproduction of EGL, but cerebellar growth and the development of fissures lags behind that of normal rats. Pre- and postnatal destruction of the EGL severely limited cerebellar growth and fissuration, and the cerebella contained only five primary lobules at P14. Rats subjected to postnatal X-irradiation alone had a similar low density of granule cells relative to those treated with a combination of prenatal MAM injections and postnatal X-irradiation, and yet the cerebella contained deeper fissures and more lobules (nine at P14). The data indicate that there are two phases of cerebellar folding: the establishment of five lobules that arise independent of granule cell production, and the granule cell-dependent expansion and partitioning of these five principal lobules during postnatal development. We propose that the lack of correlation between the severity of the granule cell loss and degree of lobulation in agranular rats indicates that granule cells exert an inductive influence over lobulation that is in part independent of the forces generated by their production and differentiation.
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Anomalías Inducidas por Medicamentos/patología , Enfermedades Cerebelosas/patología , Cerebelo/embriología , Cerebelo/patología , Ratas Wistar/embriología , Alquilantes , Animales , Enfermedades Cerebelosas/inducido químicamente , Cerebelo/efectos de la radiación , Femenino , Masculino , Acetato de Metilazoximetanol/análogos & derivados , Embarazo , Efectos Tardíos de la Exposición Prenatal , Traumatismos Experimentales por Radiación/patología , RatasRESUMEN
Analogs of N,N'-bis[6-[(2-methoxybenzyl)amino]hex-1-yl]cystamine (benextramine, BXT, 2) were synthesized using solution-phase peptide synthesis methodology and analyzed for activity in displacing specifically bound 1 nM N-[propionyl-3H]neuropeptide Y([3H]NPY) from benextramine-sensitive neuropeptide Y (NPY) binding sites in rat brain. Our new synthetic approach to these analogs began with the acylation of cystamine with the N-hydroxysuccinimide ester of tert-butyloxycarbonyl (t-Boc) protected 6-aminohexanoic acid, followed by deprotection of the t-Boc groups with 4 N HCl in dioxane. Acylation of this symmetric diamine with N-hydroxysuccinimide esters of appropriately substituted benzoic acids, followed by reduction of the resultant tetraamides with diborane in refluxing THF, afforded the target compounds. The BXT analog lacking the benzylic group (i.e., compound 11) had no [3H]NPY displacement activity at concentrations up to 1.4 x 10(-3) M. The 9-fold range in activities observed for the ortho, meta, and para regioisomers of the methoxy, chloro, and hydroxy benextramine analogs at benextramine-sensitive NPY rat brain binding sites does not differ from the range of potencies observed at alpha-adrenoceptors. However, the order of potencies at [3H]NPY sites differs from the order of potencies at alpha-adrenoceptors, with the m-methoxyphenyl (9a), m-hydroxyphenyl (10b), and 2-naphthyl (9f) analogs being the most active at [3H]NPY binding sites. The present results demonstrate the importance of the benzylic moiety for BXT's NPY antagonist activity, and suggest that the BXT binding site on the NPY receptor is significantly distinct from that on the alpha-adrenoceptor.
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Antagonistas Adrenérgicos alfa/síntesis química , Cistamina/análogos & derivados , Neuropéptido Y/farmacología , Receptores de Neuropéptido Y/efectos de los fármacos , Antagonistas Adrenérgicos alfa/metabolismo , Antagonistas Adrenérgicos alfa/farmacología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Cistamina/metabolismo , Cistamina/farmacología , Interacciones Farmacológicas , Masculino , Datos de Secuencia Molecular , Neuropéptido Y/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores de Neuropéptido Y/metabolismo , Relación Estructura-ActividadRESUMEN
N alpha-Acetyl (Ac), N-terminal deletion fragments of porcine neuropeptide Y (NPY) have been synthesized and characterized for solution conformation properties by circular dichroism and for receptor binding activity at benextramine-sensitive Y1 binding sites in rat brain cortex. Sequential deletion of Tyr1, Pro2, and Ser3 had no effect on the structural (alpha-helical content of 32.5, 30.6, and 30.7%, respectively, at 1 x 10(-5) M) or aggregation (monomer to dimer transition for N alpha-Ac-NPY3-36 and N alpha-Ac-NPY4-36) properties of NPY. In contrast, deletion of Tyr1 decreased receptor binding activity in rat brain cortex by 4-fold (IC50 = 13.0 nM versus 3.75 nM for NPY), but further deletion of Pro2-Ser3 had no additional detrimental effect on receptor binding activity relative to the desTyr1 analog. Thus, Pro2 and Ser3 do not contribute either to the stability of the NPY tertiary structure nor directly to the receptor-ligand interactions. Additional removal of N-terminal amino acids Lys4-Pro5 decreased the helical content and abolished aggregation to a dimeric form of the resultant analog, results suggesting that the residues around Pro5 are important for formation of NPY's compact, pancreatic polypeptide (PP)-fold structure. This loss in structure also correlated with a further 2-3 fold drop in receptor binding activity. These structure-activity correlations provide evidence for the importance of the PP-fold structure in the activity of NPY at Y1 receptors in rat brain cortex.