RESUMEN
Fear memories are acquired through neuronal plasticity, an orchestrated sequence of events regulated at circuit and cellular levels. The conventional model of fear acquisition assumes unimodal (for example, excitatory or inhibitory) roles of modulatory receptors in controlling neuronal activity and learning. Contrary to this view, we show that protease-activated receptor-1 (PAR1) promotes contrasting neuronal responses depending on the emotional status of an animal by a dynamic shift between distinct G protein-coupling partners. In the basolateral amygdala of fear-naive mice PAR1 couples to Gαq/11 and Gαo proteins, while after fear conditioning coupling to Gαo increases. Concurrently, stimulation of PAR1 before conditioning enhanced, but afterwards it inhibited firing of basal amygdala neurons. An initial impairment of the long-term potentiation (LTP) in PAR1-deficient mice was transformed into an increase in LTP and enhancement of fear after conditioning. These effects correlated with more frequent 2-amino-3-(5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid (AMPA) receptor-mediated miniature post synaptic events and increased neuronal excitability. Our findings point to experience-specific shifts in PAR1-G protein coupling in the amygdala as a novel mechanism regulating neuronal excitability and fear.
Asunto(s)
Amígdala del Cerebelo/fisiología , Miedo/fisiología , Potenciación a Largo Plazo/fisiología , Receptor PAR-1/fisiología , Amígdala del Cerebelo/química , Animales , Condicionamiento Clásico/efectos de los fármacos , Condicionamiento Clásico/fisiología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Conducta Exploratoria/efectos de los fármacos , Conducta Exploratoria/fisiología , Miedo/efectos de los fármacos , Reacción Cataléptica de Congelación/efectos de los fármacos , Reacción Cataléptica de Congelación/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/fisiología , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Potenciación a Largo Plazo/efectos de los fármacos , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Potenciales Postsinápticos Miniatura/efectos de los fármacos , Potenciales Postsinápticos Miniatura/fisiología , Red Nerviosa/efectos de los fármacos , Red Nerviosa/fisiología , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/fisiología , Umbral del Dolor , Técnicas de Placa-Clamp , Pirroles/farmacología , Quinazolinas/farmacología , Receptor PAR-1/antagonistas & inhibidores , Receptor PAR-1/deficiencia , Receptor PAR-1/genética , Reconocimiento en Psicología/efectos de los fármacos , Reconocimiento en Psicología/fisiologíaRESUMEN
In this study the effect of soil type, level of pre-treatment, ponding depth, temperature and sunlight on clogging of soil aquifer treatment (SAT) systems was evaluated over an eight week duration in constant temperature and glasshouse environments. Of the two soil types tested, the more permeable sand media clogged more than the loam, but still retained an order of magnitude higher absolute permeability. A 6- to 8-fold difference in hydraulic loading rates was observed between the four source water types tested (one potable water and three recycled waters), with improved water quality resulting in significantly higher infiltration. Infiltration rates for ponding depths of 30 cm and 50 cm were higher than 10 cm, although for 50 cm clogging rates were higher due to greater compaction of the clogging layer. Overall, physical clogging was more significant than other forms of clogging. Microbial clogging becomes increasingly important when the particulate concentrations in the source waters are reduced through pre-treatment and for finer textured soils due to the higher specific surface area of the media. Clogging by gas binding took place in the glasshouse but not in the lab, and mechanical clogging associated with particle rearrangement was evident in the sand media but not in the loam. These results offer insight into the soil, water quality and operating conditions needed to achieve viable SAT systems.
Asunto(s)
Laboratorios , Suelo/química , Purificación del Agua/métodos , Abastecimiento de Agua/análisis , Bacterias/crecimiento & desarrollo , Biomasa , Fenómenos Químicos , Nefelometría y Turbidimetría , Material Particulado/química , Polisacáridos Bacterianos/análisis , Microbiología del Suelo , Temperatura , Agua/normasRESUMEN
The hotspots of meiotic recombination in the human genome can be localized by genetic techniques. The resolution of these techniques is in the range of kilobases and depends on the density of the physical markers identifying allelic variants of the chromosomal loci. We thought it would be interesting to localize these sites with higher resolution. Assuming that some human chromosomal sites conserve their propensity for recombination when cloned in yeast, we localized the hotspots of recombination in several yeast artificial chromosomes (YACs) carrying human DNA. A number of potential recombination hotspots could be identified in the clones studied. Among them there are two classes of sites that are particularly recombination prone also in human meiotic cells: sites associated with CpG islands and sites located in the vicinity of long minisatellite sequences.
Asunto(s)
ADN Recombinante/genética , Meiosis/genética , Recombinación Genética , Saccharomyces cerevisiae/genética , Secuencia de Bases , Rotura Cromosómica , Mapeo Cromosómico , Cromosomas Artificiales de Levadura/genética , Clonación Molecular , Islas de CpG , Desoxirribonucleasa I , Diploidia , Genoma Humano , Humanos , Plásmidos/genética , Saccharomyces cerevisiae/citologíaRESUMEN
CpG islands are mostly unmethylated GC-, and CpG-rich chromosomal segments overlapping promoter sequences in all housekeeping and many tissue-specific genes in vertebrates. Typically, these islands show an open chromatin structure, low in histone H1 and rich in acetylated histones. We have previously found that the island-like CGCG-rich sites in human DNA are hypersensitive to DNase I upon cloning in Saccharomyces cerevisiae. Here we studied, with a higher resolution, the chromatin formed in yeast by one such site, the CpG island accompanying the human glucose-6-phosphate dehydrogenase gene. We have found two strong hypersensitive sites and several positioned nucleosomes flanking the island despite the absence in yeast of such chromatin fiber-shaping factors as histone H1, methyltransferase, and the tissue-specific transcription factors. This finding, together with similar observations from our laboratories and others supports the idea that variations in GC and/or CpG content substantially contribute to the DNA sequence features modulating the structure of the chromatin. The composition-dependent fluctuations in the accessibility of DNA in the chromatin may constitute an evolutionary advantage and may explain the surprising compositional selection that acts in both the coding and non-coding segments of some genes during mammalian evolution.
Asunto(s)
Cromatina/genética , ADN/genética , Fosfatos de Dinucleósidos , Glucosafosfato Deshidrogenasa/genética , Cromatina/ultraestructura , Cromosomas Artificiales de Levadura , Clonación Molecular , ADN/química , Desoxirribonucleasa I , Histonas/metabolismo , Humanos , Nucleosomas/genética , Nucleosomas/fisiología , Saccharomyces cerevisiae/genética , Factores de Transcripción/metabolismoRESUMEN
In the present study we have investigated the use of recombinant ovine IL-1beta and TNF-alpha both alone and in combination, as natural adjuvants in vaccination trials in sheep. Initial experiments were conducted to investigate the physiological effects of the cytokines in vivo and determine what dose could be administered without adverse pyrogenic effects. Even at the maximum dose tested (100 microg) the only significant physiological effect was a transient increase in body temperature of approximately 2 degrees C in sheep injected with TNF-alpha. Administration of either cytokine had profound effects on the levels of circulating leucocytes for up to 5 days postinjection. The incorporation of either IL-1beta or TNF-alpha in aqueous or Al(OH)3 vaccine formulations enhanced antibody responses to a recombinant antigen from the cestode parasite Taenia ovis. The addition of IL-1beta to aqueous vaccine formulations increased antibody responses 15-20-fold and in Al(OH)3 formulations by three to six fold. TNF-alpha stimulated 1.5 to six-fold and 2.5 to seven-fold increases in antibody levels in aqueous and Al(OH)3-based formulations, respectively, in a dose-dependent manner. The addition of either cytokine to Quil A or IFA vaccines did not enhance the antibody levels elicited. When 10 microg of both IL-1beta and TNF-alpha were incorporated in the aqueous or Al(OH)3 vaccine formulations, increases of 21-fold and 25-fold, respectively, were observed in antibody levels. The adjuvant activity of IL-1beta and TNF-alpha in combination in the Al(OH)3-based vaccine resulted in antibody levels commensurate with those obtained using Quil A or IFA.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Interleucina-1/farmacología , Factor de Necrosis Tumoral alfa/farmacología , Adyuvantes Inmunológicos/administración & dosificación , Factores de Edad , Hidróxido de Aluminio/inmunología , Animales , Ensayo de Inmunoadsorción Enzimática , Interleucina-1/administración & dosificación , Masculino , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Ovinos , Enfermedades de las Ovejas/inmunología , Teniasis/inmunología , Teniasis/veterinaria , Factores de Tiempo , Factor de Necrosis Tumoral alfa/administración & dosificación , Vacunas Sintéticas/inmunologíaRESUMEN
The expression plasmids pGEX-2T and pT7-7 were used to express ovine (Ov) IL-2 cDNA in Escherichia coli. The pGEX-2T vector contained glutathione-S-transferase (GST) as the affinity handle and resulted in high level expression of the GST-IL-2 fusion protein. However, only a small proportion of this fusion protein was present in the soluble fraction. The insoluble fraction was extracted with a detergent, sarkosyl, and even though a large amount of fusion product was obtained, it would not bind to glutathione beads efficiently. Thus, only low yields of biologically active rOvIL-2 were obtained. The yields were not significantly improved when other detergents were used for extraction except for a non-ionic detergent, Zwittergent 3-14, where there was a two- to three-fold increase compared with extraction with sarkosyl. An alternative vector, pT7-7 was used with a 6 x histidine tag followed by a thrombin cleavage site at the amino terminus of the mature ovine IL-2 protein to allow affinity purification by Ni-NTA resin. A large proportion of the rOvIL-2 was partitioned to the insoluble fraction. This expression system was more useful than the pGEX-2T as large quantities of biologically active rOvIL-2 of at least 10 mg l-1 were obtained. The presence of the six histidine residues at the amino end of rOvIL-2 did not reduce its biological activity. Both systems yielded rOvIL-2 with a high specific activity of about 1 x 10(7) U mg-1 as measured by the ability to maintain proliferation of ovine ConA lymphoblasts. Recombinant OvIL-2 was active on bovine but not porcine ConA lymphoblasts.
Asunto(s)
ADN Complementario/biosíntesis , Escherichia coli/genética , Vectores Genéticos/metabolismo , Interleucina-2/genética , Animales , Bovinos , ADN Complementario/aislamiento & purificación , Vectores Genéticos/genética , Interleucina-2/biosíntesis , Interleucina-2/farmacología , Activación de Linfocitos/efectos de los fármacos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/farmacología , Ovinos , Especificidad de la Especie , PorcinosAsunto(s)
Medios de Contraste , Embolización Terapéutica , Arteria Renal , Zeína , Animales , Perros , Radiografía , Arteria Renal/diagnóstico por imagenRESUMEN
The case of a 16 year old boy with Marfan syndrome, recurrent nephrolithiasis, and bilateral Medullary Sponge Kidney (MSKD) is described. This association was not previously described.
Asunto(s)
Síndrome de Marfan/complicaciones , Riñón Esponjoso Medular/complicaciones , Adolescente , Femenino , Humanos , Riñón Esponjoso Medular/etiologíaRESUMEN
Using a TUR-D1001 X-ray unit and electronic image intensifier ZOX 190T-Chirana with an Admira 16 film camera the authors recorded the contractile function of the oesophageal groove in lambs. After an adaptation of the animals to the experimental conditions the lambs were given a contrast medium mixed with milk from a bottle with rubber nipple. The flow of the contrast through the terminal-part of the oesophagus, oesophageal groove and the canal of the omasum was recorded on a film, the rate of film taking was 16 frames per second. The analysis of the film showed that in lambs aged 2 weeks the passage of the contrast medium through oesophageal groove and omasal canal was slower than in adult animals. The film visualizing the function of the groove is of considerable instructive value.
Asunto(s)
Cinerradiografía/métodos , Esófago/diagnóstico por imagen , Animales , Animales Recién Nacidos , Unión Esofagogástrica/diagnóstico por imagen , Peristaltismo , OvinosAsunto(s)
Arterias/anatomía & histología , Bovinos/anatomía & histología , Útero/irrigación sanguínea , Angiografía , Animales , Arterias/patología , Arteriosclerosis/diagnóstico por imagen , Arteriosclerosis/patología , Arteriosclerosis/veterinaria , Enfermedades de los Bovinos/diagnóstico por imagen , Enfermedades de los Bovinos/patología , FemeninoRESUMEN
Studies on the occurrence of spontaneous arteriosclerosis in the arteries of the uterus were conducted on Lowland Black-and-White (LBW) and Simentaler (S) cows. The material studied had been collected from the genital organs of heifers and cows slaughtered in the abattoirs of Gdansk (LBW) and Rzeszów (S). The animals had been found free from disease, both clinically and on post-mortem examination. Pathological changes in the arteries of the uterus were diagnosed by post-mortem angiography, and identified histologically and histochemically. In both breeds studied, high intensity of the occurrence of arteriosclerotic changes was found, both in heifers and in multiparae cows (LBW 81.9%, S 33.3%). Histochemical examination of the walls of pathologically changed vessels failed to reveal any accumulation of fatty bodies.
Asunto(s)
Arteriosclerosis/veterinaria , Enfermedades de los Bovinos/diagnóstico , Angiografía , Animales , Arterias/patología , Arteriosclerosis/diagnóstico , Bovinos , Femenino , Útero/irrigación sanguíneaRESUMEN
The syndrome of hypomagnesemia in patients receiving total parenteral nutrition (TPN) is well known. To determine particular high-risk groups for the development of this syndrome, 26 consecutive patients on TPN were initially evaluated for serum magnesium (Mg) and followed at regular intervals. Seventeen had a diagnosis of solid tumor or hematologic malignancy (CA); nine had inflammatory bowel disease and/or small bowel fistulae (ID). All met the standard criteria for being malnourished--anergy, low serum albumin, and recent weight loss. During TPN, all patients received an average of 24 mEq of magnesium sulfate per day, and all had satisfactory anabolic response in terms of weight gain and increase in serum albumin. Ten patients had at least one magnesium determination below the lower limits of normal, and four of these developed symptoms of tremor which responded to increased amounts of magnesium in their TPN. Eight of these ten (80%) had a diagnosis of CA, and four of four (100%) of those requiring additional magnesium to alleviate symptoms had CA. None of the patients with ID developed symptomatic hypomagnesemia. We conclude that patients with solid tumor malignancy are more likely to develop hypomagnesemia, possibly because of the increased requirements for magnesium in lymphocytolysis of tumor cells., and they must be carefully monitored to prevent this complication.
Asunto(s)
Magnesio/sangre , Neoplasias/terapia , Nutrición Parenteral Total/efectos adversos , Nutrición Parenteral/efectos adversos , Adolescente , Adulto , Anciano , Niño , Citotoxicidad Inmunológica , Femenino , Humanos , Inflamación/terapia , Enfermedades Intestinales/terapia , Linfocitos/fisiología , Masculino , Persona de Mediana Edad , Neoplasias/sangre , Neoplasias/inmunología , Riesgo , SíndromeAsunto(s)
Epífisis/lesiones , Fracturas del Húmero/terapia , Niño , Preescolar , Humanos , Fracturas del Húmero/cirugía , MétodosRESUMEN
Radiological method for investigation of the motor activity of the reticulum in sheep. Acta Physiol. Pol., 1977, 28 (1): 85-91. Using a TUR-D 1001 X-ray apparatus, Chirana electronic image intensifier equipped with an Admira 16 film camera the authors studied the motor activity of the reticulum in sheep. The investigations were carried out on 5 young sheep. Barium suspension was used as contrast medium. The successive phases of reticulum contractions were visualized on X-ray films obtained with a seriograph. The authors determined the time of complete reticulum contraction in films obtained at a rate of 16 frames per second. It was found that the mean time of reticulum contractions in sheep is 6 seconds. The high accuracy and usefulness of radiocinematography for studying reticulum contractions in sheep is stressed.