RESUMEN

The obligate biotrophic pathogen Puccinia horiana is the causal agent of chrysanthemum white rust. Although P. horiana is a quarantine organism, it has been able to spread to most chrysanthemum-producing regions in the world since the 1960s; however, the transfer routes are largely obscure. An extremely low level of allelic diversity was observed in a geographically diverse set of eight isolates using complexity reduction of polymorphic sequences (CRoPS) technology. Only 184 of the 16,196 contigs (1.1%) showed one or more single-nucleotide polymorphisms (SNPs). Thirty-two SNPs and one simple-sequence repeat were translated into molecular markers and used to genotype 45 isolates originating from North and South America, Asia, and Europe. In most cases, phylogenetic clustering was related to geographic origin, indicating local establishment. The European isolates mostly grouped in two major populations that may relate to the two historic introductions previously reported. However, evidence of recent geographic transfer was also observed, including transfer events between Europe and South America and between Southeast Asia and Europe. In contrast with the presumed clonal propagation of this microcyclic rust, strong indications of marker recombination were observed, presumably as a result of anastomosis, karyogamy, and somatic meiosis. Recombination and transfer also explain the geographic dispersal of specific markers. A near-to-significant correlation between the genotypic data and previously obtained pathotype data was observed and one marker was associated with the most virulent pathotype group. In combination with a fast SNP detection method, the markers presented here will be helpful tools to further elucidate the transfer pathways and local survival of this pathogen.

Asunto(s)

Basidiomycota/genética , Chrysanthemum/microbiología , Variación Genética , Enfermedades de las Plantas/microbiología , Recombinación Genética , Análisis del Polimorfismo de Longitud de Fragmentos Amplificados , Asia , Secuencia de Bases , Basidiomycota/clasificación , Basidiomycota/aislamiento & purificación , ADN de Hongos/química , ADN de Hongos/genética , Europa (Continente) , Marcadores Genéticos/genética , Genotipo , Datos de Secuencia Molecular , América del Norte , Filogenia , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN , América del Sur

RESUMEN

Nematodes which have adapted to an anaerobic lifestyle in their adult stages oxidise phosphoenolpyruvate (PEP) to oxaloacetate rather than pyruvate as the final product of glycolysis. This adaptation involves selective expression of the enzyme phosphoenolpyruvate carboxykinase (PEPCK), instead of pyruvate kinase (PK). However, such adaptation is not absolute in aerobic nematode species. We have examined the activity and kinetics of PEPCK and PK in larvae (L(3)) and adults of Teladorsagia circumcincta, a parasite known to exhibit oxygen uptake. Results revealed that PK and PEPCK activity existed in both L(3)s and adults. The enzymes had differing affinity for nucleotide diphosphates: while both can utilise GDP, only PK utilised ADP and only PEPCK utilised IDP. In both life cycle stages, enzymes showed similar affinity for PEP. PK activity was predominant in both stages, although activity of this enzyme was lower in adults. When combined, both the activity levels and the enzyme kinetics showed that pyruvate production is probably favoured in both L(3) and adult stages of T. circumcincta and suggest that metabolism of PEP to oxaloacetate is a minor metabolic pathway in this species.

Asunto(s)

Consumo de Oxígeno/fisiología , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Fosfoenolpiruvato/metabolismo , Piruvato Quinasa/metabolismo , Trichostrongyloidea/metabolismo , Abomaso/parasitología , Adenosina Difosfato/metabolismo , Aerobiosis , Anaerobiosis , Animales , Femenino , Guanosina Difosfato/metabolismo , Inosina Difosfato/metabolismo , Cinética , Larva/enzimología , Larva/crecimiento & desarrollo , Larva/metabolismo , Malato Deshidrogenasa/metabolismo , Ácido Oxaloacético/metabolismo , Ácido Pirúvico/metabolismo , Ovinos , Enfermedades de las Ovejas/parasitología , Trichostrongyloidea/enzimología , Trichostrongyloidea/crecimiento & desarrollo , Tricostrongiloidiasis/parasitología , Tricostrongiloidiasis/veterinaria

RESUMEN

Phosphofructokinase (PFK-1) activity was examined in L(3) and adult Teladorsagia circumcincta, both of which exhibit oxygen consumption. Although activities were higher in the adult stage, the kinetic properties of the enzyme were similar in both life cycle stages. T. circumcincta PFK-1 was subject to allosteric inhibition by high ATP concentration, which increased both the Hill coefficient (from 1.4±0.2 to 1.7±0.2 in L(3)s and 2.0±0.3 to 2.4±0.4 in adults) and the K(½) for fructose 6 phosphate (from 0.35±0.02 to 0.75±0.05mM in L(3)s and 0.40±0.03 to 0.65±0.05mM in adults). The inhibitory effects of high ATP concentration could be reversed by fructose 2,6 bisphosphate and AMP, but glucose 1,6 bisphosphate had no effect on activity. Similarly, phosphoenolpyruvate had no effect on activity, while citrate, isocitrate and malate exerted mild inhibitory effects, but only at concentrations exceeding 2mM. The observed kinetic properties for T. circumcincta PFK-1 were very similar to those reported for purified Ascaris suum PFK-1, though slight differences in sensitivity to ATP concentration suggests there may be subtle variations at the active site. These results are consistent with the conservation of properties of PFK-1 amongst nematode species, despite between species variation in the ability to utilise oxygen.

Asunto(s)

Fosfofructoquinasa-1/metabolismo , Trichostrongyloidea/enzimología , Adenosina Monofosfato/farmacología , Adenosina Trifosfato/farmacología , Animales , Ácido Cítrico/farmacología , Activadores de Enzimas/farmacología , Inhibidores Enzimáticos/farmacología , Fructosadifosfatos/farmacología , Fructosafosfatos/metabolismo , Isocitratos/farmacología , Cinética , Larva/enzimología , Malatos/farmacología , Fosfofructoquinasa-1/antagonistas & inhibidores , Fosforilación , Ovinos

RESUMEN

Nematodes, like other species, derive much of the energy for cellular processes from mitochondrial pathways including the TCA cycle. Previously, we have shown L3 Teladorsagia circumcincta consume oxygen and so may utilise a full TCA cycle for aerobic energy metabolism. We have assessed the relative activity levels and substrate affinities of citrate synthase, aconitase, isocitrate dehydrogenase (both NAD+ and NADP+ specific) and α-ketoglutarate dehydrogenase in homogenates of L3 T. circumcincta. All of these enzymes were present in homogenates. Compared with citrate synthase, low levels of enzyme activity and low catalytic efficiency was observed for NAD+ isocitrate dehydrogenase and especially α-ketoglutarate dehydrogenase. Therefore, it is likely that the activity of these to enzymes regulate overall metabolite flow through the TCA cycle, especially when [NAD+] limits enzyme activity. Of the enzymes tested, only citrate synthase had substrate affinities which were markedly different from values obtained from mammalian species. Overall, the results are consistent with the suggestion that a full TCA cycle exists withinL3 T. circumcincta. While there may subtle variations in enzyme properties, particularly for citrate synthase, the control points for the TCA cycle inL3 T. circumcincta are probably similar to those in the tissues of their host species.

Asunto(s)

Acetilcoenzima A/metabolismo , Ciclo del Ácido Cítrico/fisiología , Enfermedades de las Ovejas/parasitología , Trichostrongyloidea/metabolismo , Tricostrongiloidiasis/veterinaria , Aconitato Hidratasa/metabolismo , Animales , Citrato (si)-Sintasa/metabolismo , Heces/parasitología , Concentración de Iones de Hidrógeno , Isocitrato Deshidrogenasa/metabolismo , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Cinética , Larva/enzimología , Larva/metabolismo , Masculino , Ovinos , Succinato Deshidrogenasa/metabolismo , Trichostrongyloidea/enzimología , Tricostrongiloidiasis/parasitología

RESUMEN

Adult Teladorsagia circumcincta survival and motility in vitro was examined in a range of different cell culture media, supplements and gas mixes. Under optimum conditions, worms survived for 14 days, exhibiting high motility for 9 days and egg production for 72 h. Optimum conditions involved co-culture of worms with a HeLa cell line in a supplemented cell medium (CEM) and an atmosphere containing 10% CO(2), 5% O(2) 85% N(2), 65% humidity at 37 degrees C. The incubation medium consisted of Minimum Essential Medium with 10% fetal calf serum, 1% non-essential amino acids, 1% glutamax and 1% penicillin-neomycin-streptomycin cocktail mix. Compared with optimum conditions, incubation in CEM alone, cell conditioned CEM, RPMI alone, Medium 199 alone, reduced CO(2) or O(2), or when cells were replaced with Escherichia coli, both survival and motility were reduced. Optimum conditions for adult T. circumcincta maintenance for culture, anthelmintic testing or generation of excretory/secretory products are described.

Asunto(s)

Trichostrongyloidea/crecimiento & desarrollo , Animales , Dióxido de Carbono , Línea Celular , Técnicas de Cocultivo , Medios de Cultivo , Medios de Cultivo Condicionados , Femenino , Células HeLa , Humanos , Masculino , Movimiento , Nitrógeno , Oviposición , Oxígeno , Ovinos , Factores de Tiempo , Trichostrongyloidea/fisiología

RESUMEN

Excretion of nitrogenous substances by Teladorsagia circumcincta was investigated during incubation of L3 in phosphate buffer for up to 30h and adult worms for 4-6h. Ammonia was the main excretory product, with about 20% urea. For the first 4-6h, ammonia excretion by L3 was temperature dependent, directly proportional to the number of larvae, but independent of the pH or strength of the phosphate buffer. Later, ammonia excretion slowed markedly in L3 and adults and reversed to net uptake in L3 by 30h. An initial external ammonia concentration of 600 microM did not alter the pattern or magnitude of excretion. Re-uptake of ammonia did not occur at extremes of pH or low buffer strength and was slightly reduced at the highest external concentrations. Ammonium transporters and enzymes of glutamate metabolism, including glutamate dehydrogenase, glutamine synthetase and possibly glutamate synthase, are worthy of further investigation as anthelmintic targets.

Asunto(s)

Compuestos de Nitrógeno/metabolismo , Trichostrongyloidea/metabolismo , Aminoácidos/metabolismo , Amoníaco/metabolismo , Animales , Proteínas del Helminto/metabolismo , Concentración de Iones de Hidrógeno , Larva/metabolismo , Ovinos , Temperatura , Urea/metabolismo , Ácido Úrico/metabolismo

RESUMEN

Nigeria is the only country in West Africa where soybean rust, caused by Phakopsora pachyrhizi, has been officially reported (1). During a disease survey in Ghana during October 2006, soybean (Glycine max) leaves with rust symptoms (tan, angular lesions with erumpent sori exuding urediniospores) were observed in 11 fields in the following districts: Kassena Nankana in the Upper East Region; East Gonja, Central Gonja, and Tolon-Kumbungu in the Northern Region; and Ejisu-Juabeng in the Ashanti Region. Disease incidence in these fields ranged from 50 to 100% and disease severity ranged between 3 and 40% of the leaf area on infected plants. Urediniospores were hyaline, minutely echinulate, and 23 to 31 × 14 to 18 µm. Within a week of collection, leaf samples were sent to the USDA-ARS Foreign Disease-Weed Science Research Unit for verification of pathogen identity. DNA was extracted from leaf pieces containing sori with the Qiagen DNeasy Plant Mini kit (Valencia, CA), and all 11 field samples amplified in a real-time fluorescent PCR with the P. pachyrhizi-specific primers Ppm1 and Ppa2 (2). Sequence alignment of the internal transcribed spacer (ITS) region 2 further confirmed the identification as P. pachyrhizi (2). Infected leaves from three fields were separately washed in sterile water to collect urediniospores that were used to separately inoculate three detached leaves (for each isolate) of susceptible cultivar TGx 1485-1D (3). The abaxial surface of detached leaves was sprayed with 400 µl of spore suspension (1 × 106 spores per ml). A single leaf piece was placed in a 9-cm-diameter petri dish with adaxial side appressed on 1% technical agar amended with 10 µg/ml of kinetin. Lactic acid (1.5 ml/liter) and benomyl (12.5 mg/liter) were added to the agar medium to inhibit growth of saprophytic fungi and bacteria. Petri dishes were incubated at 20°C with a 12-h light/12-h dark cycle. Lesions on inoculated leaves developed 5 to 6 days after inoculation (DAI), and pustules (105 to 120 µm) formed 7 to 8 DAI and erupted 3 days later exuding columns of urediniospores similar in size to the initially collected isolates. Inoculating another set of detached leaves with a spore suspension (1 × 106 spores per ml) from the first set of detached leaves resulted in typical rust symptoms. The PCR assay, alignment of ITS region 2, morphological characters of the isolates, and pathogenicity tests demonstrate that P. pachyrhizi occurs in Ghana. To our knowledge, this is the first report of P. pachyrhizi in Ghana. References: (1) O. A. Akinsanmi et al. Plant Dis. 85:97, 2001. (2) R. D. Frederick et al. Phytopathology 92:217, 2002. (3) M. Twizeyimana et al. Online publication. http://www.plantmanagementnetwork.org/ infocenter/topic/soybeanrust/2006/posters/41.asp. Plant Management Network, 2006.

RESUMEN

Nigeria (1) and Uganda (3) are the closest countries to the Democratic Republic of Congo (DRC) where soybean rust caused by Phakopsora pachyrhizi has been reported. In February 2007, during a disease survey in DRC, soybean (Glycine max) leaves with rust symptoms (tan, angular lesions with erumpent sori exuding urediniospores) were observed in 10 fields in the following areas in Bas Congo Province: Bangu, Kimpese, Kolo-Fuma, Lukala, Mbanza-Ngungu, Mpalukide, Mvuazi, and Ntemo. Rust incidence in these fields ranged from 85 to 100%, while severity ranged between 3 and 35% of the leaf area on infected plants. Urediniospores were hyaline, minutely echinulate, and 23 to 31 × 16 to 20 µm. Within a week of collection, infected leaf samples were sent to the USDA-ARS Foreign Disease-Weed Science Research Unit (FDWSRU) for pathogen identification. DNA was extracted from sections of leaves containing sori with the Qiagen DNeasy Plant Mini kit (Valencia, CA), and all 10 field samples amplified in a real-time fluorescent PCR with the P. pachyrhizi-specific primers Ppm1 and Ppa2 (2). Infected leaves of cultivar Vuangi collected from one field each in the INERA Research Station, Kimpese-Crawford, and Kimpese-Ceco were separately washed in sterile water to collect urediniospores that were used to separately inoculate three detached leaves of susceptible cultivar TGx 1485-1D (4). Lesions on inoculated leaves developed 5 days after inoculation (DAI), and pustules (110 to 130 µm) formed 7 DAI and erupted 2 days later exuding columns of urediniospores similar in size to the initially collected isolates. Inoculation of another set of detached leaves with a spore suspension (1 × 106 spores per ml) from the first set of detached leaves resulted in typical rust symptoms. Seedlings of cultivar Williams also showed typical rust symptoms when inoculated separately with urediniospores collected from nine fields (i.e., all except Kimpese-Ceco, which was infective in the detached leaf assay). Inoculation and incubation were carried out at the FDWSRU Plant Pathogen Containment Facility at Fort Detrick as described earlier (2). The PCR assay, morphological characters of the isolates, and pathogenicity tests demonstrate that P. pachyrhizi occurs in DRC. To our knowledge, this is the first report of P. pachyrhizi infecting soybean in DRC. References: (1) O. A. Akinsanmi et al. Plant Dis. 85:97, 2001. (2) R. D. Frederick et al. Phytopathology 92:217, 2002. (3) E. Kawuki et al. J. Phytopathol. 151:7, 2003. (4) M. Twizeyimana et al. Online publication. http://www.plantmanagementnetwork.org/ infocenter/topic/soybeanrust/2006/posters/41.asp. Plant Management Network, 2006.

RESUMEN

The purpose of this study was to test the hypothesis that the steroid environment affects fluid absorption by the uterine glands. Laser scanning confocal microscopy of the distribution of an extracellular marker (fluorescein isothiocyanate-labelled dextran) within rat uterine glands showed that the endometrial glands change their fluid handling characteristics under different hormonal conditions. Under progesterone dominance, the glands showed an amiloride-sensitive dextran accumulation indicating sodium-dependent fluid absorption; however, this was absent in the oestrogen-dominated state. The rate of fluid uptake in the progesterone-stimulated gland opening was estimated to be approximately 1 x 10(-4) cm s(-1), requiring a suction pressure of between 10 and 20 mm Hg at the mucosal surface. This study provides the first direct evidence of fluid absorption by the uterine glands. Such absorption may provide the mechanism for closure of the uterine lumen and immobilization of the blastocyst necessary for implantation.

Asunto(s)

Líquidos Corporales/fisiología , Implantación del Embrión/fisiología , Glándulas Endocrinas/fisiología , Endometrio/fisiología , Progesterona/fisiología , Absorción , Animales , Dextranos , Femenino , Fluoresceína-5-Isotiocianato/análogos & derivados , Colorantes Fluorescentes , Microscopía Confocal , Embarazo , Ratas , Ratas Wistar

RESUMEN

Strains of the filamentous fungus Cochliobolus carbonum that produce the host-selective compound HC-toxin, a cyclic tetrapeptide, are highly virulent on certain genotypes of maize (Zea mays L.). Production of HC-toxin is under the control of a complex locus, TOX2, which is composed of at least seven linked and duplicated genes that are present only in toxin-producing strains of C. carbonum. One of these genes, TOXE, was earlier shown to be required for the expression of the other TOX2 genes. TOXE has four ankyrin repeats and a basic region similar to those found in basic leucine zipper (bZIP) proteins, but lacks any apparent leucine zipper. Here we show that TOXE is a DNA-binding protein that recognizes a ten-base motif (the "tox-box") without dyad symmetry that is present in the promoters of all of the known TOX2 genes. Both the basic region and the ankyrin repeats are involved in DNA binding. A region of TOXE that includes the first ankyrin repeat is necessary and sufficient for transcriptional activation in yeast. The data indicate that TOXE is the prototype of a new family of transcription factor, so far found only in plant-pathogenic fungi. TOXE plays a specific regulatory role in HC-toxin production and, therefore, pathogenicity by C. carbonum.

Asunto(s)

Ascomicetos/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Péptidos Cíclicos/biosíntesis , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Ascomicetos/genética , Sitios de Unión , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Proteínas Fúngicas/genética , Proteínas Fúngicas/fisiología , Genes Fúngicos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Activación Transcripcional

RESUMEN

1. Fluorescence recovery after photobleaching (FRAP) of fluorescein isothiocyanate (FITC)-labelled 10 and 250 kDa dextran (FITC dextran) in isolated rat descending colonic crypts was measured at 35 degrees C using laser scanning confocal microscopy. 2. FRAP of either 10 or 250 kDa FITC dextran in crypt lumens was almost complete within 2-3 min. 3. In the presence of amiloride (0.1 mM), or in the absence of Na+, the rate of FITC dextran uptake into the crypt lumens was reduced by 70-80 %. 4. The rate of fluid uptake into the crypt lumen, as estimated from the rate of total FITC dextran uptake into the crypt lumen and its adjacent pericryptal region after FRAP, was between 1.3 x 10(-3) and 1.7 x 10(-3) cm x s(-1). 5. Convective flow during FRAP was also determined from the initial rate of FITC dextran advance along the crypt lumen. This effect was almost completely blocked by amiloride (0.1 mM). 6. The permeability of 10 kDa FITC dextran across the descending colonic crypt wall was found to be higher than that of 250 kDa FITC dextran (3.7 (+/- 0.6) x 10(-5) and 1.8 (+/- 0.3) x 10(-6) cm x s(-1), respectively; n = 3 for both, P < 0.01). The permeability of the caecal crypt wall to 10 kDa dextran was higher than that of the descending crypt wall (2.03 (+/- 0.21) x 10(-5) cm x s(-1); n = 3, P < 0.025). 7. Simulation of the flow of Na+, water and FITC dextran into the crypt lumen and across the crypt wall and pericryptal sheath corroborates the observed parameters of water and Na+ flows.

Asunto(s)

Amilorida/farmacología , Colon/metabolismo , Dextranos/farmacocinética , Diuréticos/farmacología , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/farmacocinética , Microscopía Confocal/métodos , Animales , Absorción Intestinal/efectos de los fármacos , Absorción Intestinal/fisiología , Ratas , Ratas Wistar , Sodio/metabolismo , Agua/metabolismo

RESUMEN

How the brain meets its continuous high metabolic demand in light of varying plasma glucose levels and a functional blood-brain barrier (BBB) is poorly understood. GLUT-1, found in high density at the BBB appears to maintain the continuous shuttling of glucose across the blood-brain barrier irrespective of the plasma concentration. We examined the process of glucose transport across a quasi-physiological in vitro blood-brain barrier model. Radiolabeled tracer permeability studies revealed a concentration ratio of abluminal to luminal glucose in this blood-brain barrier model of approximately 0.85. Under conditions where [glucose](lumen) was higher than [glucose](ablumen), influx of radiolabeled 2-deoxyglucose from lumen to the abluminal compartment was approximately 35% higher than efflux from the abluminal side to the lumen. However, when compartmental [glucose] were maintained equal, a reversal of this trend was seen (approximately 19% higher efflux towards the lumen), favoring establishment of a luminal to abluminal concentration gradient. Immunocytochemical experiments revealed that in addition to segregation of GLUT-1 (luminal>abluminal), the intracellular enzyme hexokinase was also asymmetrically distributed (abluminal>luminal). We conclude that glucose transport at the CNS/blood interface appears to be dependent on and regulated by a serial chain of membrane-bound and intracellular transporters and enzymes.

Asunto(s)

Barrera Hematoencefálica/fisiología , Glucosa/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Radioisótopos de Carbono/farmacocinética , Bovinos , Compartimento Celular/efectos de los fármacos , Compartimento Celular/fisiología , Diferenciación Celular/fisiología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/fisiología , Células Cultivadas , Técnicas de Cocultivo , Desoxiglucosa/farmacocinética , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Feto , Transportador de Glucosa de Tipo 1 , Hexoquinasa/metabolismo , Inmunohistoquímica , Membranas Artificiales , Proteínas de Transporte de Monosacáridos/efectos de los fármacos , Fenotipo , Ratas

RESUMEN

Intact G0 nuclei from quiescent mammalian cells initiate DNA synthesis asynchronously in Xenopus egg extracts, despite exposure to the same concentration of replication factors. This indicates that individual nuclei differ in their ability to respond to the inducers of DNA replication. Since the induction of DNA synthesis requires the accumulation of replication factors by active nuclear transport, any variation in the rate of transport among nuclei could contribute to the variability of DNA replication. Using the naturally fluorescent protein allophycocyanin (APC) coupled with the nuclear localization sequence (NLS) of SV40 T antigen, as a marker of nuclear uptake, we show here that individual G0 nuclei differ in their rate of transport over a range of more than 20-fold. Surprisingly, this variation has no direct influence on the timing or extent of DNA synthesis. Similar results were obtained by monitoring the uptake of nucleoplasmin, a nuclear protein present at high levels in egg extracts. These experiments show that the initiation of DNA synthesis is not driven merely by the accumulation of replication factors to some threshold concentration. Instead, some other explanation is needed to account for the timing of initiation.

Asunto(s)

Núcleo Celular/metabolismo , Replicación del ADN , Óvulo/ultraestructura , Secuencia de Aminoácidos , Animales , Microscopía Fluorescente , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Nucleoplasminas , Fosfoproteínas/metabolismo , Xenopus

RESUMEN

BACKGROUND: Therapeutic or accidental exposure to radiation commonly causes gastrointestinal disturbances, including diarrhoea. Rats subjected to whole body ionising radiation at a dose of 8 Gy lose their capacity to absorb fluid via the descending colon after four days. After seven days, fluid absorption recovers to control levels. AIMS: To investigate the effect of ionising radiation on colonic permeability together with its effect on mitochondria dependent apoptotic signals and intercellular adhesion molecules. METHODS: Rats were irradiated with doses of 0-12 Gy. Colonic permeability was measured by accumulation of fluorescein isothiocyanate (FITC) dextran in crypt lumens. Changes in levels of cytochrome c, caspase 3, E and OB cadherin, beta-catenin smooth muscle actin, and collagen IV were assessed using immunocytochemistry with confocal microscopy. RESULTS: Cytosolic cytochrome c increased after 8 Gy (t(1/2) 1.4 (0.6) hours) and peaked at approximately six hours. Caspase 3 increased more slowly, particularly in crypt epithelial cells (t(1/2) 57 (14.5) hours). Pericryptal myofibroblasts disintegrated within 24 hours as was evident from loss of OB cadherin and smooth muscle actin. This coincided with increased crypt permeability to dextran. Intercellular adhesion between crypt luminal cells was not lost until day 4 when both beta-catenin and E-cadherin were minimal. The half maximal dose-response for these effects was in the range 2-4 Gy. Recovery of colonic transport was concurrent with recovery of pericryptal smooth muscle actin and OB cadherin. The pan caspase inhibitor Z-Val-Ala-Asp.fluoromethylketone (1 mg/kg per day) had a small effect in conserving the pericryptal sheath myofibroblasts and sheath permeability but had no systemic therapeutic effects. CONCLUSIONS: These data suggest that radiation damage to the colon may be initiated by mitochondrial events. Loss of crypt fluid absorption and increased permeability coincided with decreased intercellular adhesion between crypt epithelial cells and loss of pericryptal sheath barrier function.

Asunto(s)

Colon/efectos de la radiación , Grupo Citocromo c/efectos de la radiación , Fibroblastos/efectos de la radiación , Absorción Intestinal/efectos de la radiación , Animales , Cadherinas/metabolismo , Inhibidores de Caspasas , Caspasas/metabolismo , Caspasas/fisiología , Adhesión Celular/efectos de la radiación , Colágeno/metabolismo , Colon/fisiología , Grupo Citocromo c/metabolismo , Dextranos/farmacocinética , Relación Dosis-Respuesta en la Radiación , Fibroblastos/metabolismo , Fluoresceína-5-Isotiocianato/farmacocinética , Absorción Intestinal/fisiología , Masculino , Microscopía Confocal , Ratas , Ratas Wistar

RESUMEN

In eukaryotes, the initiation of DNA synthesis requires the assembly of a pre-replicative complex (pre-RC) at origins of replication. This involves the sequential binding of ORC (origin-recognition-complex), Cdc6 and MCM proteins, a process referred to as licensing. After origin firing, the Cdc6 and MCM proteins dissociate from the chromatin, and do not rebind until after the completion of mitosis, thereby restricting replication to a single round in each cell cycle. Although nuclei normally become licensed for replication as they enter G(1), the extent to which the license is retained when cells enter the quiescent state (G(0)) is controversial. Here we show that the replication capacity of nuclei from Swiss 3T3 cells, in Xenopus egg extracts, is not lost abruptly with the onset of quiescence, but instead declines gradually. The decline in replication capacity, which affects both the number of nuclei induced to replicate and their subsequent rate of DNA synthesis, is accompanied by a fall in the level of chromatin-bound MCM2. When quiescent nuclei are incubated in egg extracts, they do not bind further MCMs unless the nuclei are first permeabilized. The residual replication capacity of intact nuclei must therefore be dependent on the remaining endogenous MCMs. Although high levels of Cdk activity are known to block MCM binding, we show that the failure of intact nuclei in egg extracts to increase their bound MCMs is not due to their uptake and accumulation of Cdk complexes. Instead, the failure of binding must be due to exclusion of some other binding factor from the nucleus, or to the presence within nuclei of an inhibitor of binding other than Cdk activity. In contrast to the situation in Xenopus egg extracts, following serum stimulation of intact quiescent cells, the level of bound MCMs does increase before the cells reach S phase, without any disruption of the nuclear envelope.

Asunto(s)

Núcleo Celular/metabolismo , Replicación del ADN/fisiología , Proteínas de Unión al ADN/genética , Factores de Transcripción/genética , Células 3T3 , Adenina/análogos & derivados , Adenina/farmacología , Animales , Proteínas Sanguíneas/farmacología , Núcleo Celular/química , Cromatina/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Mamíferos , Ratones , Proteína 1 de Mantenimiento de Minicromosoma , Componente 2 del Complejo de Mantenimiento de Minicromosoma , Membrana Nuclear/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oocitos/fisiología , Factores de Transcripción/metabolismo , Xenopus

RESUMEN

Vascular cell death is a key feature of atherosclerotic lesions and may contribute to the plaque "necrotic" core, cap rupture, and thrombosis. Oxidatively modified low-density lipoproteins (LDLs) are implicated in the pathogenesis of atherosclerosis, and dietary antioxidants are thought to protect the vasculature against LDL-induced cytotoxicity. Because LDL oxidative modification may vary within atherosclerotic lesions, we examined the effects of defined, oxidatively modified LDL species on human arterial smooth muscle cell apoptosis and the cytoprotective effects of vitamin C. Moderately oxidized LDL (0 to 300 microg protein/mL), which has the highest content of lipid hydroperoxides, induced smooth muscle cell apoptosis within 6 hours, whereas native LDL and mildly and highly oxidized LDL had no effect. Moderately oxidized LDL increased cellular DNA fragmentation, release of fragmented DNA into the culture medium, and annexin V binding and decreased mitochondrial dehydrogenase activity and expression of the antiapoptotic mediator Bcl-x(L). Treatment of cells with native LDL together with the lipid hydroperoxide 13(S)-hydroperoxyoctadeca-9Z,11E-dienoic acid (HPODE, 200 micromol/L, 6 to 24 hours) also induced apoptotic cell death. Pretreatment of smooth muscle cells with vitamin C (0 to 100 micromol/L, 24 hours) attenuated the cytotoxicity and apoptosis induced by both moderately oxidized LDL and HPODE. Our findings suggest that moderately oxidized LDL, with its high lipid hydroperoxide content, rather than mildly or highly oxidized LDL, causes apoptosis of human smooth muscle cells and that vitamin C supplementation may provide protection against plaque instability in advanced atherosclerosis.

Asunto(s)

Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Ácido Ascórbico/farmacología , Ácidos Linoleicos/metabolismo , Peróxidos Lipídicos/metabolismo , Lipoproteínas LDL/metabolismo , Músculo Liso Vascular/citología , Anexina A5/metabolismo , Células Cultivadas , Colorantes , Reacciones Cruzadas , Citotoxinas/metabolismo , Fragmentación del ADN , Humanos , Músculo Liso Vascular/metabolismo , Propidio , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/inmunología

RESUMEN

1. Rat descending colon absorbed fluid against a large hydraulic resistance, imposed by 10 % agarose (w/v) gel plugs inserted in the lumen, by raising the tonicity of the absorbate from the gel to 880 +/- 54 mosmol kg-1; the tonicity of the absorbate from 2.5 % gels was 352 +/- 38 mosmol kg-1. The hypertonic absorbate generated an osmotic pressure which created a fluid tension in the crypt lumen. This was monitored as a suction tension in colonic luminal gels of 45.3 +/- 3 cmH2O with 2.5 % gels and 725 +/- 145 cmH2O with 10 % gels. The caecum was unable to absorb fluid against a significant hydraulic resistance. 2. Fluorescein isothiocyanate-labelled dextran (FITC dextran; molecular mass 10000 Da) accumulated within descending colonic crypt lumens by concentration polarization. Maximal accumulation at a depth of 20-40 micrometer below the mucosal surface was 5.68 +/- 0.2-fold above control levels. Caecal crypts accumulated dextran to a maximum of 1.8 +/- 0.17-fold above control levels. 3. The relationship between crypt luminal tension and suction tension of the distal colon was also demonstrated using paraffin, which occluded the crypt lumens with microscopic droplets and completely inhibited fluid absorption from high resistance luminal gels. 4. Reduction in dietary Na+ intake raised plasma aldosterone and the capacity of the distal colon to dehydrate against a high luminal hydraulic resistance. The caecum did not respond in this way to varied Na+ intake.

Asunto(s)

Colon/metabolismo , Deshidratación/metabolismo , Equilibrio Hidroelectrolítico/fisiología , Animales , Ciego/metabolismo , Dextranos/farmacocinética , Dieta Hiposódica , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/farmacocinética , Geles , Presión Hidrostática , Absorción Intestinal/efectos de los fármacos , Absorción Intestinal/fisiología , Microscopía Confocal , Parafina/farmacología , Excipientes Farmacéuticos/farmacología , Ratas , Ratas Wistar , Sefarosa , Sodio/farmacología , Equilibrio Hidroelectrolítico/efectos de los fármacos

RESUMEN

1. Confocal microscopic studies of rat colonic mucosa showed that the pericryptal sheath surrounding distal colonic crypts is an effective barrier both to dextran and NaCl movement, whereas no such structure surrounds the caecal crypts. 2. The distal colonic pericryptal barrier was functionally demonstrated by accumulation of Sodium Green within the pericryptal space. After exposure to benzamil, Sodium Green accumulation was decreased. Fluorescein isocyanate-labelled dextran (FITC dextran; molecular mass 10000 Da) was accumulated in the crypt lumens and pericryptal spaces. Both dextran and Sodium Green accumulation were absent from the pericryptal zone surrounding caecal crypts. 3. Low dietary Na+ intake raised rat plasma aldosterone and stimulated distal pericryptal sheath growth and adhesiveness as shown by increased amounts of F-actin, smooth muscle actin, beta-catenin and E-cadherins in the pericryptal zone. It also raised the capacity of the distal colon to dehydrate against a high luminal hydraulic resistance. This linkage indicates that trophic effects on the colon resulting from a low Na+ diet are not confined solely to effects on transepithelial Na+ transport, but are observed in the pericryptal sheath. 4. A computer model of crypt function confirms that a pericryptal sheath with low permeability to NaCl is an essential component of the crypt dehydrating mechanism.

Asunto(s)

Absorción Intestinal/fisiología , Intestino Grueso/química , Intestino Grueso/metabolismo , Transactivadores , Actinas/análisis , Aldosterona/sangre , Amilorida/análogos & derivados , Amilorida/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Compuestos de Boro , Cadherinas/análisis , Ciego/química , Ciego/citología , Ciego/metabolismo , Proteínas del Citoesqueleto/análisis , Dextranos/farmacocinética , Dieta Hiposódica , Colorantes Fluorescentes , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/química , Mucosa Intestinal/metabolismo , Intestino Grueso/citología , Músculo Liso/química , Compuestos Orgánicos , Faloidina , Ratas , Ratas Wistar , Cloruro de Sodio/farmacocinética , beta Catenina

RESUMEN

Fusion of human red cells through the action of polyethylene glycol gives rise to pairs or higher clusters with a common membrane envelope, in which a barrier at the position of the original interface can be seen in phase contrast. At early times this septum contains lipids, as judged by labelling with a fluorescent lipophile, and transmembrane protein; this was shown by the presence of the preponderant component, band 3, detected by a fluorescent label, covalently attached before fusion at an extracellular site, or by immunofluorescence with anti-band 3 antibody. Ankyrin, which is bound to band 3, is also observed in the septum. The lipid thereafter disappears from the interface, carrying most of the band 3 with it. A continuous membrane skeletal network, defined by the presence of spectrin (detected by immunofluorescent staining in epifluorescence and confocal microscopy) appears to persist for long periods, but in many cells interruptions develop in the septum. In other fused pairs, particularly at longer times, the interface is seen to have vanished completely. Protease inhibitors have no discernible effect on any of these observations. The results suggest a model for the events that follow fusion. Covalent cross-linking of membrane proteins beyond a critical level causes inhibition of fusion, suggesting that proteins, probably the membrane skeletal network, regulate the fusion process. The efficiency of fusion is strikingly dependent on the composition of the isotonic medium, being relatively high at an orthophosphate concentration of 5 mM and minimal at 20 mM.

Asunto(s)

Fusión Celular/efectos de los fármacos , Membrana Eritrocítica/efectos de los fármacos , Proteínas de la Membrana/efectos de los fármacos , Polietilenglicoles/farmacología , Proteína 1 de Intercambio de Anión de Eritrocito/efectos de los fármacos , Ancirinas/efectos de los fármacos , Membrana Eritrocítica/química , Membrana Eritrocítica/ultraestructura , Humanos , Lípidos de la Membrana/sangre , Microscopía Confocal , Microscopía Fluorescente , Modelos Químicos , Espectrina/efectos de los fármacos

RESUMEN

Fluorescence microscopy has become a powerful tool for both the localization of cellular components in fixed cells, using target-specific fluorescent probes and labeled antibodies, and the fluorescence imaging of ions in single living cells. Despite its markedly lower spatial resolution when compared with electron microscopy, the essentially non-invasive nature of light microscopy provides a unique tool for examining cell behaviour at the level of the single cell bringing new insight into both cellular heterogeneities and cell-cell interactions. Further developments in both fluorescent probes and instrumentation should provide even more powerful tools to probe the mechanisms of cell function, both in vitro and in vivo.

Asunto(s)

Islotes Pancreáticos/citología , Microscopía Confocal , Microscopía Fluorescente , Animales , Colorantes Fluorescentes , Humanos , Factores de Tiempo