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1.
Tijdschr Psychiatr ; 65(7): 411-417, 2023.
Artículo en Holandés | MEDLINE | ID: mdl-37756025

RESUMEN

BACKGROUND: Sleep gets little attention in mental health care treatments. Epidemiological research with regards to the association between sleep problems and anxiety and mood disorders can contribute to good clinical decision making. AIM: Based on data from the Netherlands Mental Health Survey and Incidence Study-2 (NEMESIS-2), we examined the relation between sleep problems and first onset, recurrence and persistence of anxiety and mood disorders within a 3 year period. METHOD: Different groups of respondents were selected to examine the relation between sleep problems and different stages of anxiety and mood disorders within three years. DSM-IV diagnoses were determined using the Composite International Diagnostic Interview (CIDI 3.0) and sleep problems with the Women’s Health Initiative Insomnia Rating Scale (IRS; ≥ 9). Logistic regression was performed. Multivariable analysis took into account a large number of potentially confounding variables. RESULTS: Almost a quarter of the respondents without an anxiety or mood disorder and almost half of the respondents with an anxiety or mood disorder experience sleep problems. In the multivariable analysis, sleep problems were associated with recurrence of an anxiety disorder (OR 2.10; 95% CI 1.31-3.38), but not with the first onset and persistence of an anxiety disorder. Furthermore, sleep problems appear to be associated with the first onset of a mood disorder (OR 2.18; 95% CI 1.27-3.74) and with the persistence of a mood disorder (OR 2.51; 95% CI 1.17-5.37), but not with recurrence of this disorder. CONCLUSION: The results underline the importance of identifying sleep problems of people with (an increased risk of) anxiety and mood disorders. The treatment of sleep problems may contribute to a reduced incidence of these mental disorders and a better and sustainable recovery.


Asunto(s)
Trastornos del Humor , Trastornos del Sueño-Vigilia , Femenino , Humanos , Trastornos del Humor/epidemiología , Ansiedad , Trastornos de Ansiedad/epidemiología , Manual Diagnóstico y Estadístico de los Trastornos Mentales , Trastornos del Sueño-Vigilia/epidemiología
2.
Genet Mol Res ; 16(3)2017 Sep 21.
Artículo en Inglés | MEDLINE | ID: mdl-28973773

RESUMEN

Although PCR-based techniques have become an essential tool in the field of molecular and genetic research, the amplification of repetitive DNA sequences is limited. This is due to the truncated nature of the amplified sequences, which are also prone to errors during DNA polymerase-based amplification. The complex structure of repetitive DNA can form hairpin loops, which promote dissociation of the polymerase from the template, impairing complete amplification, and leading to the formation of incomplete fragments that serve as megaprimers. These megaprimers anneal with other sequences, generating unexpected fragments in each PCR cycle. Our gene model, MaSp1, is 1037-bp long, with 68% GC content, and its amino acid sequence is characterized by poly-alanine-glycine motifs, which represent the repetitive codon consensus. We describe the amplification of the MaSp1 gene through minor changes in the PCR program. The results show that a denaturation temperature of 98°C is the key determinant in the amplification of the MaSp1 partial gene sequence.


Asunto(s)
ADN/química , Reacción en Cadena de la Polimerasa/métodos , Secuencias Repetitivas de Aminoácido , Composición de Base , Fibroínas/genética , Secuencias Invertidas Repetidas , Desnaturalización de Ácido Nucleico , Reacción en Cadena de la Polimerasa/normas
3.
Appetite ; 107: 144-151, 2016 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-27470098

RESUMEN

Even when individuals are aware of long-term health effects of their diet, and form healthy intentions, they often engage in relatively unhealthy snacking habits. Some individuals fall back on unhealthy habits more easily than others. We aim to explore whether time perspective can explain why some individuals are more prone to rely on habits and others on intentions. Study 1 (N = 1503) provides a first exploration of the role of time perspective by exploring individual differences in perception of long-term and short-term consequences. In accordance with our hypotheses, Study 1 shows that habits are associated with short-term consequences and intentions with long-term consequences. Study 2 (N = 1497) shows that the effects of habits on snacking behaviour are strengthened by a present time perspective, whereas the effects of intentions on snacking behaviour are strengthened by a future time perspective. These findings imply that there is a fundamental difference in the guiding function of intentions and habits which might explain individual differences in following intentions versus habits. Individuals with a long-term perspective are more inclined to follow intentions and individuals with a short-term perspective are more inclined to follow habits.


Asunto(s)
Conducta Alimentaria/psicología , Hábitos , Individualidad , Intención , Bocadillos , Adulto , Estudios Transversales , Dieta , Femenino , Conductas Relacionadas con la Salud , Humanos , Masculino , Persona de Mediana Edad , Motivación
4.
Nanotechnology ; 24(48): 484014, 2013 Dec 06.
Artículo en Inglés | MEDLINE | ID: mdl-24196842

RESUMEN

Highly conductive screen printed metallic (silver) structures (current collecting grids) combined with poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) are a viable replacement for indium tin oxide (ITO) and inkjet printed silver as transparent electrode materials. To provide successful integration into organic photovoltaic (OPV) devices, screen printed silver current collecting grids should be embedded into a substrate to avoid topology issues. In this study micron-thick conductive structures are embedded and integrated into OPV devices. The embedded structures are produced roll-to-roll with optimized process settings and materials. Topology measurements show that the embedded grids are well suited for integration into OPV devices since the surface is almost without spikes and has low surface roughness. JV measurements of OPV devices with embedded structures on a polyethylene terephthalate/silicon nitride (PET/SiN) substrate show an efficiency of 2.15%, which is significantly higher than identical flexible devices with ITO (1.02%) and inkjet printed silver (1.48%). The use of embedded screen printed silver instead of ITO and inkjet printed silver in OPV devices will allow for higher efficiency devices which can be produced with larger design and process freedom.

5.
Acta Biomater ; 9(5): 6653-62, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23415750

RESUMEN

Introducing nanoroughness on various biomaterials has been shown to profoundly effect cell-material interactions. Similarly, physical forces act on a diverse array of cells and tissues. Particularly in bone, the tissue experiences compressive or tensile forces resulting in fluid shear stress. The current study aimed to develop an experimental setup for bone cell behavior, combining a nanometrically grooved substrate (200 nm wide, 50 nm deep) mimicking the collagen fibrils of the extracellular matrix, with mechanical stimulation by pulsatile fluid flow (PFF). MC3T3-E1 osteoblast-like cells were assessed for morphology, expression of genes involved in cell attachment and osteoblastogenesis and nitric oxide (NO) release. The results showed that both nanotexture and PFF did affect cellular morphology. Cells aligned on nanotexture substrate in a direction parallel to the groove orientation. PFF at a magnitude of 0.7 Pa was sufficient to induce alignment of cells on a smooth surface in a direction perpendicular to the applied flow. When environmental cues texture and flow were interacting, PFF of 1.4 Pa applied parallel to the nanogrooves initiated significant cellular realignment. PFF increased NO synthesis 15-fold in cells attached to both smooth and nanotextured substrates. Increased collagen and alkaline phosphatase mRNA expression was observed on the nanotextured substrate, but not on the smooth substrate. Furthermore, vinculin and bone sialoprotein were up-regulated after 1 h of PFF stimulation. In conclusion, the data show that interstitial fluid forces and structural cues mimicking extracellular matrix contribute to the final bone cell morphology and behavior, which might have potential application in tissue engineering.


Asunto(s)
Modelos Biológicos , Nanoestructuras , Osteoblastos/citología , Células 3T3 , Animales , Secuencia de Bases , Cartilla de ADN , Ratones , Microscopía de Fuerza Atómica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
6.
Eur Cell Mater ; 23: 182-93; discussion 193-4, 2012 Mar 13.
Artículo en Inglés | MEDLINE | ID: mdl-22415804

RESUMEN

Organised nanotopography mimicking the natural extracellular matrix can be used to control morphology, cell motility, and differentiation. However, it is still unknown how specific cell types react with specific patterns. Both initial adhesion and preferential cell migration may be important to initiate and increase cell locomotion and coverage with cells, and thus achieve an enhanced wound healing response around an implantable material. Therefore, the aim of this study was to evaluate how MC3T3-E1 osteoblast initial adhesion and directional migration are influenced by nanogrooves with pitches ranging from 150 nm up to 1000 nm. In this study, we used a multi-patterned substrate with five different groove patterns and a smooth area with either a concentric or radial orientation. Initial cell adhesion measurements after 10 s were performed using atomic force spectroscopy-assisted single-cell force spectroscopy, and demonstrated that nascent cell adhesion was highly induced by a 600 nm pitch and reduced by a 150 nm pitch. Addition of RGD peptide significantly reduced adhesion, indicating that integrins and cell adhesive proteins (e.g. fibronectin or vitronectin) are key factors in specific cell adhesion on nanogrooved substrates. Also, cell migration was highly dependent on the groove pitch; the highest directional migration parallel to the grooves was observed on a 600 nm pitch, whereas a 150 nm pitch restrained directional cell migration. From this study, we conclude that grooves with a pitch of 600 nm may be favourable to enhance fast wound closure, thereby promoting tissue regeneration.


Asunto(s)
Materiales Biocompatibles/química , Moléculas de Adhesión Celular/metabolismo , Adhesión Celular , Movimiento Celular , Integrinas/metabolismo , Nanoestructuras/química , Osteoblastos/citología , Animales , Células Cultivadas , Ratones , Microscopía de Fuerza Atómica/métodos , Oligopéptidos , Osteoblastos/metabolismo , Silicio/química , Propiedades de Superficie , Ingeniería de Tejidos/métodos , Cicatrización de Heridas/fisiología
7.
Eur Cell Mater ; 20: 329-43, 2010 Nov 09.
Artículo en Inglés | MEDLINE | ID: mdl-21061239

RESUMEN

The natural environment of a living cell is not only organized on a micrometer, but also on a nanometer scale. Mimicking such a nanoscale topography in implantable biomaterials is critical to guide cellular behavior. Also, a correct positioning of cells on biomaterials is supposed to be very important for promoting wound healing and tissue regeneration. The exact mechanism by which nanotextures can control cellular behavior are thus far not well understood and it is thus far unknown how cells recognize and respond to certain surface patterns, whereas a directed response appears to be absent on other pattern types. Focal adhesions (FAs) are known to be involved in the process of specific pattern recognition and subsequent response by cells. In this study, we used a high throughput screening "Biochip" containing 40 different nanopatterns to evaluate the influence of several nanotopographical cues like depth, width, (an)isotropy and spacing (ridge-groove ratio) on osteoblast behavior. Microscopical analysis and time lapse imaging revealed that an isotropic topography did not alter cell morphology, but it highly induced cell motility. Cells cultured on anisotropic topographies on the other hand, were highly elongated and aligned. Time-lapse imaging revealed that cell motility is highly dependent on the ridge-groove ratio of anisotropic patterns. The highest motility was observed on grooves with a ratio of 1:3, whereas the lowest motility was observed on ratios of 1:1 and 3:1. FA measurements demonstrated that FA-length decreased with increasing motility. From the study it can be concluded that osteoblast behavior is tightly controlled by nanometer surface features.


Asunto(s)
Movimiento Celular , Nanoestructuras/química , Osteoblastos/citología , Animales , Anisotropía , Células Cultivadas , Adhesiones Focales/metabolismo , Osteoblastos/fisiología , Osteoblastos/ultraestructura , Ratas , Propiedades de Superficie , Imagen de Lapso de Tiempo , Ingeniería de Tejidos/métodos
8.
Artículo en Inglés | MEDLINE | ID: mdl-19680869

RESUMEN

An ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed for the determination of fluoroquinolones-ciprofloxacin (CIPRO), danofloxacin (DANO), enrofloxacin (ENRO) and sarafloxacin (SARA)-in aquaculture products, specifically salmon, shrimp and tilapia. After initial sample extraction with an acidic acetonitrile solution, the extract was diluted with dichloromethane and centrifuged, then an aliquot was concentrated and applied to a C18 solid-phase extraction cartridge and concentrated for a second time. The resultant residue was dissolved in acetonitrile, diluted with water, and then further defatted with hexane. The fluoroquinolone residues were determined by UPLC with an HSS T3 C18 reverse-phase column using an ammonium hydroxide-formic acid buffer in an acetonitrile gradient with MS/MS detection using multiple reaction monitoring. Average recoveries for salmon tissue ranged from 73% for DANO to 95% for SARA, for shrimp from 71% for DANO to 109% for SARA, and from 62% for DANO to 111% for SARA in tilapia, fortified at the 1.0 ng g(-1) level. Standard curves were linear between 0.002 and 0.5 ng injected for all compounds. Detection limits of 0.2 ng g(-1) for CIPRO, DANO, ENRO, and SARA were easily obtainable. The operational errors, interferences, and recoveries for fortified samples demonstrate that this described method is suitable for routine use in a regulatory programme. The recommended method is simple, rapid, specific and reliable for the routine monitoring of fluoroquinolone residues in aquatic species such as salmon, tilapia and shrimp.


Asunto(s)
Antibacterianos/análisis , Acuicultura , Residuos de Medicamentos/análisis , Fluoroquinolonas/análisis , Alimentos Marinos/análisis , Animales , Cromatografía Liquida/métodos , Análisis de los Alimentos/métodos , Contaminación de Alimentos/análisis , Penaeidae/química , Salmón/metabolismo , Espectrometría de Masas en Tándem , Tilapia/metabolismo
9.
Biomaterials ; 28(27): 3944-51, 2007 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-17576010

RESUMEN

The differences in morphological behaviour between fibroblasts cultured on smooth and nanogrooved substrata (groove depth: 5-350 nm, width: 20-1000 nm) have been evaluated in vitro. The aim of the study was to clarify to what extent cell guidance occurs on increasingly smaller topographies. Pattern templates were made using electron beam lithography, and were subsequently replicated in polystyrene cell culture material using solvent casting. The replicates were investigated with atomic force microscopy (AFM). After seeding with fibroblasts, morphological characteristics were investigated using scanning electron microscopy (SEM) and light microscopy, in order to obtain qualitative and quantitative information on cell alignment. AFM revealed that the nanogroove/ridge widths were replicated perfectly, although at deeper levels the grooves became more concave. The smooth substrata had no distinguishable pattern other than a roughness amplitude of 1 nm. Interestingly, microscopy and image analysis showed that fibroblast after 4 h had adjusted their shape according to nanotopographical features down to cut-off values of 100 nm width and 75 nm depth. After 24 h culturing time, fibroblasts would even align themselves on groove depths as shallow as 35 nm. It appears depth is the most essential parameter in cellular alignment on groove patterns with a pitch ratio of 1:1. On the smooth substrata, cells always spread out in a random fashion. Analysis of variance (ANOVA) demonstrated that both main parameters, topography and culturing time, were significant. We conclude that fibroblast cells cultured on nanotopography experience a threshold feature size of 35 nm, below this value contact guidance does no longer exist.


Asunto(s)
Materiales Biocompatibles/química , Técnicas de Cultivo de Célula/métodos , Fibroblastos/citología , Fibroblastos/fisiología , Nanoestructuras/química , Nanoestructuras/ultraestructura , Ingeniería de Tejidos/métodos , Animales , Células Cultivadas , Masculino , Tamaño de la Partícula , Ratas , Ratas Wistar , Propiedades de Superficie
11.
Food Addit Contam ; 24(1): 14-20, 2007 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-17164212

RESUMEN

Thirty shrimp, marine fish, freshwater fish, and canned fish composite samples collected and prepared as part of the Canadian Total Diet Study were analysed for 39 different veterinary drug residues. The analyses were undertaken to obtain baseline data that could be used to estimate the dietary exposure of Canadians to these residues. The most frequently observed residue was AOZ (four out of 30 samples), the metabolite of furazolidone, at a range of 0.50 to 2.0 ng g(-1) wet weight. Other residues detected included enrofloxacin (three samples; 0.3-0.73 ng g(-1)), leucomalachite green (three samples; 0.73-1.2 ng g(-1)), oxolinic acid (two samples; 0.3-4.3 ng g(-1)), AMOZ (the metabolite of furaltadone; one sample; 0.40 ng g(-1)), chloramphenicol (one sample; 0.40 ng g(-1)), and SEM (the metabolite of nitrofurazone; one sample; 0.8 ng g(-1)). The results of this survey indicate that Canadians are exposed to low ng g-1 concentrations of some banned and unapproved veterinary drug residues via the consumption of certain fish and shrimp.


Asunto(s)
Acuicultura , Residuos de Medicamentos/análisis , Contaminación de Alimentos/análisis , Alimentos Marinos/análisis , Drogas Veterinarias/análisis , Animales , Canadá , Peces/metabolismo , Análisis de los Alimentos/métodos , Penaeidae/metabolismo
12.
Ned Tijdschr Geneeskd ; 150(33): 1839-43, 2006 Aug 19.
Artículo en Holandés | MEDLINE | ID: mdl-16967596

RESUMEN

A 35-year-old man and his partner were referred for intracytoplasmic sperm injection treatment (ICSI) because of secondary infertility due to severe oligoasthenoteratospermia. Three years earlier he had presented elsewhere with left unilateral gynaecomastia. A hypertrophic mammary gland had been excised one year later. Histopathological investigation showed benign hypertrophy. One year later he developed gynaecomastia on the other side. Physical examination and incomplete hormonal screening showed no abnormalities. The couple were referred to our tertiary clinic for ICSI treatment. The patient still had unilateral gynaecomastia. Hormonal screening showed not only severe oligoasthenoteratospermia, but also an elevated serum oestrogen level. Scrotal ultrasound revealed a 17 mm mass in his right testicle. Subsequently unilateral orchidectomy was performed. Histology showed a benign Leydig cell tumour for which no further therapy was required. Four months after surgery the gynaecomastia diminished, oestrogen levels became normal and improvement in semen parameters followed. Patients with severe male infertility or gynaecomastia are at a higher risk of developing a testicular neoplasm. Besides history taking, physical examination of breasts and testicles, hormonal screening and scrotal sonography should be performed as some testicular neoplasms are not apparent on palpation.


Asunto(s)
Estrógenos/sangre , Ginecomastia/diagnóstico , Tumor de Células de Leydig/diagnóstico , Neoplasias Testiculares/diagnóstico , Adulto , Diagnóstico Diferencial , Ginecomastia/complicaciones , Ginecomastia/cirugía , Humanos , Infertilidad Masculina/diagnóstico , Infertilidad Masculina/etiología , Tumor de Células de Leydig/complicaciones , Tumor de Células de Leydig/cirugía , Masculino , Orquiectomía , Neoplasias Testiculares/complicaciones , Neoplasias Testiculares/cirugía , Testículo/diagnóstico por imagen , Ultrasonografía
13.
J AOAC Int ; 88(3): 761-72, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-16001850

RESUMEN

A method that uses liquid chromatography with tandem mass spectrometry (LC/MS/MS) has been developed for the highly sensitive and specific determination of amnesic shellfish poisoning toxins, diarrhetic shellfish poisoning toxins, and other lipophilic algal toxins and metabolites in shellfish. The method was subjected to a full single-laboratory validation and a limited interlaboratory study. Tissue homogenates are blended with methanol-water (9 + 1), and the centrifuged extract is cleaned up with a hexane wash. LC/MS/MS (triple quadrupole) is used for quantitative analysis with reversed-phase gradient elution (acidic buffer), electrospray ionization (positive and negative ion switching), and multiple-reaction monitoring. Ester forms of dinophysis toxins are detected as the parent toxins after hydrolysis of the methanolic extract. The method is quantitative for 6 key toxins when reference standards are available: azaspiracid-1 (AZA1), domoic acid (DA), gymnodimine (GYM), okadaic acid (OA), pectenotoxin-2 (PTX2), and yessotoxin (YTX). Relative response factors are used to estimate the concentrations of other toxins: azaspiracid-2 and -3 (AZA2 and AZA3), dinophysis toxin-1 and -2 (DTX1 and DTX2), other pectenotoxins (PTX1, PTX6, and PTX11), pectenotoxin secoacid metabolites (PTX2-SA and PTX11-SA) and their 7-epimers, spirolides, and homoYTX and YTX metabolites (45-OHYTX and carboxyYTX). Validation data have been gathered for Greenshell mussel, Pacific oyster, cockle, and scallop roe via fortification and natural contamination. For the 6 key toxins at fortification levels of 0.05-0.20 mg/kg, recoveries were 71-99% and single laboratory reproducibilities, relative standard deviations (RSDs), were 10-24%. Limits of detection were <0.02 mg/kg. Extractability data were also obtained for several toxins by using successive extractions of naturally contaminated mussel samples. A preliminary interlaboratory study was conducted with a set of toxin standards and 4 mussel extracts. The data sets from 8 laboratories for the 6 key toxins plus DTX1 and DTX2 gave within-laboratories repeatability (RSD(R)) of 8-12%, except for PTX-2. Between-laboratories reproducibility (RSDR) values were compared with the Horwitz criterion and ranged from good to adequate for 7 key toxins (HorRat values of 0.8-2.0).


Asunto(s)
Cromatografía Liquida/métodos , Análisis de los Alimentos/métodos , Espectrometría de Masas/métodos , Toxinas Biológicas/análisis , Animales , Bioensayo , Éteres Cíclicos/análisis , Furanos/análisis , Furanos/metabolismo , Compuestos Heterocíclicos con 3 Anillos/análisis , Hidrocarburos Cíclicos/análisis , Hidrólisis , Iminas/análisis , Ácido Kaínico/análogos & derivados , Ácido Kaínico/análisis , Macrólidos , Toxinas Marinas/análisis , Metanol/química , Ratones , Moluscos , Venenos de Moluscos , Ácido Ocadaico/análisis , Oxocinas/análisis , Piranos/análisis , Piranos/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Mariscos , Compuestos de Espiro/análisis , Factores de Tiempo
15.
J AOAC Int ; 84(5): 1358-62, 2001.
Artículo en Inglés | MEDLINE | ID: mdl-11601454

RESUMEN

A liquid chromatographic (LC) method for determining residues of the antiparasitic drugs emamectin (EMA) and ivermectin (IVR) in fish tissues has been developed. EMA and IVR residues are extracted with acetonitrile and cleaned up on a C18 solid-phase extraction column. Extracts are derivatized with 1-methylimidazole and trifluoroacetic anhydride and the components are determined by LC on a C18 reversed-phase column with fluorescence detection (excitation: 365 nm, emission: 470 nm). The mobile phase is 94% acetonitrile-water run isocratically. Calibration curves were linear between 1 and 32 ng injected for both EMA and IVR. The limit of detection for both analytes was 0.5 ng/g, with a limit of quantitation of 1.5 ng/g. Recoveries of EMA and IVR added to salmon muscle averaged 96 +/- 9% and 86 +/- 6%, respectively, at levels between 5 and 80 ng/g. The percent relative standard deviation for the described method was less than 7% over the range of concentrations studied. The operational errors, interferences, and recoveries for fortified samples compare favorably with an established IVR method. The recommended method is simple, rapid, and specific for monitoring residues of EMA and IVR in Atlantic salmon muscle.


Asunto(s)
Antihelmínticos/análisis , Disacáridos/análisis , Residuos de Medicamentos/análisis , Ivermectina/análisis , Músculos/química , Salmo salar/metabolismo , Animales , Cromatografía Liquida , Indicadores y Reactivos , Ivermectina/análogos & derivados , Estándares de Referencia , Reproducibilidad de los Resultados , Espectrometría de Fluorescencia
16.
RNA ; 7(6): 896-903, 2001 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-11421364

RESUMEN

In yeast, the 3' end of mature 18S rRNA is generated by endonucleolytic cleavage of the 20S precursor at site D. Available data indicate that the major cis-acting elements required for this processing step are located in relatively close proximity to the cleavage site. To identify these elements, we have studied the effect of mutations in the mature 18S and ITS1 sequences neighboring site D on pre-rRNA processing in vivo. Using clustered point mutations, we found that alterations in the sequence spanning site D from position -5 in 18S rRNA to +6 in ITS1 reduced the efficiency of processing at this site to different extents as demonstrated by the lower level of the mature 18S rRNA and the increase in 20S pre-rRNA in cells expressing only mutant rDNA units. More detailed analysis revealed an important role for the residue located 2 nt upstream from site D (position -2), whereas sequence changes at position -1, +1, and +2 relative to site D had no effect. The data further demonstrate that the proposed base pairing between the 3' end of 18S rRNA and the 5' end of ITS1 is not important for efficient and accurate processing at site D, nor for the formation of functional 40S ribosomal subunits. These results were confirmed by analyzing the accumulation of the D-A2 fragment derived from the mutant 20S pre-rRNA in cells that lack the Xrn1p exonuclease responsible for its degradation. The latter results also showed that the accuracy of cleavage was affected by altering the spacer sequence directly downstream of site D but not by mutations in the 18S rRNA sequence preceding this site.


Asunto(s)
ARN Ribosómico 18S/química , Saccharomyces cerevisiae/genética , Secuencia de Bases , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , ARN Ribosómico 18S/genética
17.
Nucleic Acids Res ; 29(24): 5001-8, 2001 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-11812830

RESUMEN

Mutational analysis has shown that the integrity of the region in domain III of 25S rRNA that is involved in binding of ribosomal protein L25 is essential for the production of mature 25S rRNA in the yeast Saccharomyces cerevisiae. However, even structural alterations that do not noticeably affect recognition by L25, as measured by an in vitro assay, strongly reduced 25S rRNA formation by inhibiting the removal of ITS2 from the 27S(B) precursor. In order to analyze the role of L25 in yeast pre-rRNA processing further we studied the effect of genetic depletion of the protein or mutation of each of its three previously identified functional domains, involved in nuclear import (N-terminal), RNA binding (central) and 60S subunit assembly (C-terminal), respectively. Depletion of L25 or mutating its (pre-)rRNA-binding domain blocked conversion of the 27S(B) precursor to 5.8S/25S rRNA, confirming that assembly of L25 is essential for ITS2 processing. However, mutations in either the N- or the C-terminal domain of L25, which only marginally affect its ability to bind to (pre-)rRNA, also resulted in defective ITS2 processing. Furthermore, in all cases there was a notable reduction in the efficiency of processing at the early cleavage sites A0, A1 and A2. We conclude that the assembly of L25 is necessary but not sufficient for removal of ITS2, as well as for fully efficient cleavage at the early sites. Additional elements located in the N- as well as C-terminal domains of L25 are required for both aspects of pre-rRNA processing.


Asunto(s)
Precursores del ARN/metabolismo , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/metabolismo , Saccharomyces cerevisiae/genética , Sitios de Unión/genética , Mutación , Precursores del ARN/genética , Procesamiento Postranscripcional del ARN , ARN Ribosómico/genética , Proteínas Ribosómicas/genética , Saccharomyces cerevisiae/metabolismo
18.
J Mol Biol ; 296(1): 7-17, 2000 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-10656814

RESUMEN

Domain III of Saccharomyces cerevisiae 25 S rRNA contains the recognition site for the primary rRNA-binding ribosomal protein L25, which belongs to the functionally conserved EL23/L25 family of ribosomal proteins. The EL23/L25 binding region is very complex, consisting of several irregular helices held together by long-distance secondary and tertiary interactions. Moreover, it contains the eukaryote-specific V9 (D7a) expansion segment. Functional characterisation of the structural elements of this site by a detailed in vitro and in vivo mutational analysis indicates the presence of two separate regions that are directly involved in L25 binding. In particular, mutation of either of two conserved nucleotides in the loop of helix 49 significantly reduces in vitro L25 binding, thus strongly supporting their role as attachment sites for the r-protein. Two other helices appear to be primarily required for the correct folding of the binding site. Mutations that abolish in vitro binding of L25 block accumulation of 25 S rRNA in vivo because they stall pre-rRNA processing at the level of its immediate precursor, the 27 S(B) pre-rRNA. Surprisingly, several mutations that do not significantly affect L25 binding in vitro cause the same lethal defect in 27 S(B) pre-rRNA processing. Deletion of the V9 expansion segment also leads to under-accumulation of mature 25 S rRNA and a twofold reduction in growth rate. We conclude that an intact domain III, including the V9 expansion segment, is essential for normal processing and assembly of 25 S rRNA.


Asunto(s)
Conformación de Ácido Nucleico , ARN Ribosómico/química , ARN Ribosómico/metabolismo , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Secuencia de Bases , Sitios de Unión , División Celular , Secuencia Conservada/genética , Genes Letales/genética , Datos de Secuencia Molecular , Mutación/genética , Unión Proteica , Precursores del ARN/química , Precursores del ARN/genética , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN/genética , ARN de Hongos/química , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN Ribosómico/genética , Proteínas de Unión al ARN/metabolismo , Ribosomas/química , Ribosomas/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo
19.
FEBS Lett ; 452(3): 335-40, 1999 Jun 11.
Artículo en Inglés | MEDLINE | ID: mdl-10386617

RESUMEN

Nuclear import usually relies on the presence of nuclear localization sequences (NLSs). NLSs are recognized by NLS receptors (importins), which target their substrates to the nuclear pore. We identified the NLSs of the yeast ribosomal proteins S22 and S25 and studied the former by mutational analysis. Furthermore, in S25 the nucleolar targeting information was found to overlap with its NLS. Comparison with previously published data on yeast ribosomal protein NLSs and computer analysis indicates the existence of a novel type of ribosomal protein-specific NLS that differs from the classical Chelsky and bipartite NLSs. The existence of such a ribosomal protein-specific NLS is in accordance with the recent identification of ribosomal protein-specific importins.


Asunto(s)
Nucléolo Celular/ultraestructura , Núcleo Celular/ultraestructura , Proteínas Ribosómicas/análisis , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citología , Secuencia de Aminoácidos , Inmunohistoquímica , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/química , Proteínas Ribosómicas/química , Proteínas Ribosómicas/genética , Saccharomyces cerevisiae/ultraestructura , beta-Galactosidasa/análisis , beta-Galactosidasa/química
20.
Am J Obstet Gynecol ; 180(4): 1024-9, 1999 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-10203673

RESUMEN

The medical literature was searched for publications between 1966 and September 1997 for data on the association of Apgar score, umbilical blood pH, or Sarnat grading of encephalopathy with long-term adverse outcome. Odds ratios for these associations were combined to calculate common odds ratios with 95% confidence intervals. Our search identified abstracts of 1312 studies and 81 articles with sufficient numeric data to formulate contingency tables. Forty-two of these qualified for inclusion in our meta-analysis. The strongest associations in the prediction of neonatal death were found by comparing umbilical artery pH <7 with pH >/=7 (common odds ratio 43; 95% confidence interval 15-124) and by comparing Sarnat grade III with grade II (common odds ratio 24; 95% confidence interval 13-45). In the prediction of cerebral palsy, the strongest associations were found for Sarnat grade III versus grade II (common odds ratio 20; 95% confidence interval 6-70) and for 20-minute Apgar score 0 to 3 versus 4 to 6 (common odds ratio 15; 95% confidence interval 5-50).


Asunto(s)
Mortalidad Infantil , Examen Físico , Puntaje de Apgar , Parálisis Cerebral/epidemiología , Sangre Fetal/química , Estudios de Seguimiento , Humanos , Concentración de Iones de Hidrógeno , Recién Nacido , Oportunidad Relativa , Factores de Riesgo
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