RESUMEN
We studied simultaneously the (4)He(e,e'p), (4)He(e,e'pp), and (4)He(e,e'pn) reactions at Q(2)=2(GeV/c)(2) and x(B)>1, for an (e,e'p) missing-momentum range of 400 to 830 MeV/c. The knocked-out proton was detected in coincidence with a proton or neutron recoiling almost back to back to the missing momentum, leaving the residual A=2 system at low excitation energy. These data were used to identify two-nucleon short-range correlated pairs and to deduce their isospin structure as a function of missing momentum, in a region where the nucleon-nucleon (NN) force is expected to change from predominantly tensor to repulsive. The abundance of neutron-proton pairs is reduced as the nucleon momentum increases beyond â¼500 MeV/c. The extracted fraction of proton-proton pairs is small and almost independent of the missing momentum. Our data are compared with calculations of two-nucleon momentum distributions in (4)He and discussed in the context of probing the elusive repulsive component of the NN force.
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Survival of juvenile freshwater mussels (Echyridella menziesii (Gray, 1843) formerly known as Hyridella menziesi) and crayfish (Paranephrops planifrons, White, 1842) decreased after four days exposure to microcystin-containing cell-free extracts (MCFE) of Microcystis sp. at concentrations typical of severe cyanobacterial blooms. Crayfish survival was 100, 80, and 50% in microcystin concentrations of 1339, 2426, and 11146 µg L(-1) respectively, and shade- and shelter-seeking behavior was negatively affected when concentrations were ≥2426 µg L(-1) . Mussel survival decreased to 92% and reburial rates decreased to 16% after exposure for 96 h to MCFE containing microcystins at concentrations of 5300 µg L(-1) . Crayfish survival was 100% when fed freeze-dried Microcystis sp. incorporated into an artificial diet (6-100 µg microcystin kg(-1) ww) at dietary doses from 0.03 to 0.55 µg g(-1) body weight d(-1) for 27 days. Specific growth rate was significantly lower in crayfish fed ≥0.15 µg g(-1) body weight day(-1) compared with controls, but not compared with a diet incorporating nontoxic cyanobacteria. Microcystins accumulated preferentially in crayfish hepatopancreas and mussel digesta as MCFE or dietary concentrations increased. These laboratory data indicate that, assuming dissolved oxygen concentrations remain adequate, and no simultaneous exposure to live Microcystis sp. cells, cell-free microcystins will only be a significant stressor to juvenile crayfish and mussels in severe Microcystis sp. blooms. In contrast, crayfish were negatively affected by relatively low concentrations of microcystins in artificial diets compared with those measured locally in benthic cyanobacterial mats.
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Astacoidea/fisiología , Bivalvos/fisiología , Floraciones de Algas Nocivas , Microcistinas/toxicidad , Animales , Conducta Animal , Cadena Alimentaria , Agua Dulce , MicrocystisRESUMEN
The cyanobacterial toxins, nodularin and microcystin, are highly efficient inhibitors of cellular protein phosphatases. Toxicity primarily evolves following ingestion of cyanobacterial material or toxins and results in liver and renal pathology. Ingestion is the main route of exposure in the World Health Organizations current risk assessment of nodularin and microcystins. Nasally applied microcystin appears to have a 10-fold higher availability and toxicity than orally ingested toxins, suggesting that aerosolized toxins could represent a major risk for human populations close to lakes with cyanobacterial blooms. In this study, nodularin and microcystin levels in aerosols were assessed using high and low volume air samplers for 4, 12 and 24 h periods at lakes Forsyth and Rotorua (South Island, New Zealand). These lakes were experiencing blooms of Nodularia spumigena and Microcystis sp., respectively. Using the high volume samplers up to 16.2 pg m(-3) of nodularin and 1.8 pg m(-3) of microcystins were detected in the air. Aerosolized nodularin and microcystins do not appear to represent an acute or chronic hazard to humans. The latter was concluded based on calculations using average human air intakes, the highest nodularin or microcystin concentrations measured in the air in this study, and assuming inhalatory toxicities comparable to toxicological data obtained following intraperitoneal applications in mice. However, as the toxin concentrations in the air were calculated over extended sampling periods, peak values may be underestimated. Aerosolized toxins should be considered when developing risk assessments particularly for lakeside populations and recreational users where inhalation of cyanotoxins may be a secondary exposure source to a primary oral exposure.
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Aerosoles/análisis , Contaminantes Atmosféricos/análisis , Agua Dulce/química , Microcistinas/análisis , Péptidos Cíclicos/análisis , Microbiología del Aire , Atmósfera/química , Cianobacterias/crecimiento & desarrollo , Monitoreo del Ambiente , Agua Dulce/microbiología , Exposición por Inhalación/análisis , Exposición por Inhalación/estadística & datos numéricos , Nueva ZelandaRESUMEN
Flow cytometry has potential as a rapid assessment technique to evaluate phytoplankton biomass and species composition. It facilitates for multi-parameter analysis of individual cells on the basis of light scattering effects induced from cellular constituents, as well as auto-fluorescence. Fluorescence emission characteristics may be especially useful in classifying cyanobacteria as they contain phycoerythrin which emits light predominantly in the 550-600 nm waveband, chlorophyll-a (650-700 nm emission) and allophycocyanin (660 nm emission). The objective of our study was to assess the utility of flow cytometry for the rapid identification and sorting of freshwater algae and cyanobacteria species. Using a selection of laboratory-cultured freshwater algae and cyanobacteria species, this study demonstrated unique light scatter and fluorescent characteristics for each species examined, allowing for rapid species identification and sorting of mixed populations of laboratory cultures and samples from two lakes in the Rotorua region (New Zealand). Analysis of lake water samples collected over seven months demonstrated changes in abundance and community composition of phytoplankton in the two lakes and demonstrates that flow cytometry may be a useful technique for examining seasonal changes in phytoplankton composition.
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Eutrofización , Lagos , Fitoplancton/aislamiento & purificación , Separación Celular , Citometría de FlujoRESUMEN
Research on harmful algal and cyanobacterial blooms (HABs and CHABs) has risen dramatically due to their increasing global distribution, frequency, and intensity. These blooms jeopardize public health, ecosystem function, sustainability and can have negative economic impacts. Numerous monitoring programs have been established using light microscopy, liquid chromatography coupled to mass spectrometry (LC-MS), ELISA, and spectrophotometry to monitor HABs/CHABs outbreaks. Recently, DNA/RNA-based molecular methods have been integrated into these programs to replace or complement traditional methods through analyzing environmental DNA and RNA (eDNA/eRNA) with techniques such as quantitative polymerase chain reaction (qPCR), fluorescent in situ hybridization (FISH), sandwich hybridization assay (SHA), isothermal amplification methods, and microarrays. These have enabled the detection of rare or cryptic species, enhanced sample throughput, and reduced costs and the need for visual taxonomic expertise. However, these methods have limitations, such as the need for high capital investment in equipment or detection uncertainties, including determining whether organisms are viable. In this review, we discuss the potential of newly developed molecular diagnosis technology based on Clustered Regularly Interspaced Short Palindromic Repeats/Cas proteins (CRISPR/Cas), which utilizes the prokaryotic adaptative immune systems of bacteria and archaea. Cas12 and Cas13-based platforms can detect both DNA and RNA with attomolar sensitivity within an hour. CRISPR/Cas diagnostic is a rapid, inexpensive, specific, and ultrasensitive technology that, with some further development, will provide many new platforms that can be used for HABs/CHABs biomonitoring and research.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Floraciones de Algas Nocivas , Monitoreo Biológico , Ecosistema , Hibridación Fluorescente in SituRESUMEN
AIMS: The purpose of this study was to determine the variability in anatoxin-a (ATX) and homoanatoxin-a (HTX) concentrations in benthic cyanobacterial mats within sampling sites and to assess the applicability of using a PCR-based approach to determine ATX- and HTX-production potential. METHODS AND RESULTS: ATX and HTX variability was investigated by collecting 15 samples from 10 × 10 m grids in seven rivers. ATX and HTX concentrations were determined using liquid chromatography-mass spectrometry (LC-MS). Samples from two sites contained no ATX or HTX and at one site ATX and HTX were detected in all samples. At four sites, both toxic and nontoxic samples co-occurred and these samples were sometimes spaced less than 1 m apart. PCR amplification of a region of a polyketide synthase (ks2, putatively involved in the biosynthetic pathway of ATX and HTX) successfully distinguished ATX-and-HTX- and non-ATX-and-HTX-producing cultured Phormidium strains. Results from environmental samples were more variable, and the results were in congruence with the LC-MS data in only 58% of samples. CONCLUSIONS: Fine-scale spatial variability in ATX and HTX concentrations occurs among benthic cyanobacterial mats. SIGNIFICANCE AND IMPACT OF THE STUDY: Multiple benthic cyanobacterial mat samples must be collected at a sampling site to provide an accurate assessment of ATX and HTX concentrations at that location. The PCR-based technique offers the potential to be a useful early warning technique.
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Toxinas Bacterianas/análisis , Compuestos Bicíclicos Heterocíclicos con Puentes/análisis , Cianobacterias/química , Monitoreo del Ambiente/métodos , Tropanos/análisis , Microbiología del Agua , Cromatografía Liquida , Cianobacterias/genética , Toxinas de Cianobacterias , Espectrometría de Masas , Nueva Zelanda , Sintasas Poliquetidas/genética , Ríos/microbiologíaRESUMEN
Harmful cyanobacterial blooms (=cyanoHABs) are an increasing feature of many waterbodies throughout the world. Many bloom-forming species produce toxins, making them of particular concern for drinking water supplies, recreation and fisheries in waterbodies along the freshwater to marine continuum. Global changes resulting from human impacts, such as climate change, over-enrichment and hydrological alterations of waterways, are major drivers of cyanoHAB proliferation and persistence. This review advocates that to better predict and manage cyanoHABs in a changing world, researchers need to leverage studies undertaken to date, but adopt a more complex and definitive suite of experiments, observations, and models which can effectively capture the temporal scales of processes driven by eutrophication and a changing climate. Better integration of laboratory culture and field experiments, as well as whole system and multiple-system studies are needed to improve confidence in models predicting impacts of climate change and anthropogenic over-enrichment and hydrological modifications. Recent studies examining adaptation of species and strains to long-term perturbations, e.g. temperature and carbon dioxide (CO2) levels, as well as incorporating multi-species and multi-stressor approaches emphasize the limitations of approaches focused on single stressors and individual species. There are also emerging species of concern, such as toxic benthic cyanobacteria, for which the effects of global change are less well understood, and require more detailed study. This review provides approaches and examples of studies tackling the challenging issue of understanding how global changes will affect cyanoHABs, and identifies critical information needs for effective prediction and management.
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Cianobacterias , Cambio Climático , Eutrofización , Explotaciones Pesqueras , Agua Dulce , HumanosRESUMEN
Brefeldin A (BFA) induces the formation of an extensively fused network of membranes derived from the trans-Golgi network (TGN) and early endosomes (EE). We describe in detail here the unaffected passage of endocytosed material through the fused TGN/EE compartments to lysosomes in BFA-treated cells. We also confirmed that BFA caused the formation of tubular lysosomes, although the kinetics and extent of tubulation varied greatly between different cell types. The BFA-induced tubular lysosomes were often seen to form simple networks. Formation of tubular lysosomes was microtubule-mediated and energy-dependent; interestingly, however, maintenance of the tubulated lysosomes only required microtubules and was insensitive to energy poisons. Upon removal of BFA, the tubular lysosomes rapidly recovered in an energy-dependent process. In most cell types examined, the extensive TGN/EE network is ephemeral, eventually collapsing into a compact cluster of tubulo-vesicular membranes in a process that precedes the formation of tubular lysosomes. However, in primary bovine testicular cells, the BFA-induced TGN/EE network was remarkably stable (for > 12 h). During this time, the TGN/EE network coexisted with tubular lysosomes, however, the two compartments remained completely separate. These results show that BFA has multiple, profound effects on the morphology of various compartments of the endosome-lysosome system. In spite of these changes, endocytic traffic can continue through the altered compartments suggesting that transport occurs through noncoated vesicles or through vesicles that are insensitive to BFA.
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Antibacterianos/farmacología , Ciclopentanos/farmacología , Orgánulos/fisiología , Orgánulos/ultraestructura , Adenosina Trifosfato/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Biomarcadores , Brefeldino A , Catepsina D/aislamiento & purificación , Compartimento Celular , Células Cultivadas , Endocitosis/efectos de los fármacos , Endocitosis/fisiología , Metabolismo Energético , Humanos , Inmunohistoquímica , Cinética , Lisosomas/efectos de los fármacos , Lisosomas/fisiología , Lisosomas/ultraestructura , Microtúbulos/fisiología , Orgánulos/efectos de los fármacos , Fracciones SubcelularesRESUMEN
Recent in vivo studies with the fungal metabolite, brefeldin A (BFA), have shown that in the absence of vesicle formation, membranes of the Golgi complex and the trans-Golgi network (TGN) are nevertheless able to extend long tubules which fuse with selected target organelles. We report here that the ability to form tubules (> 7 microns long) could be reproduced in vitro by treatment of isolated, intact Golgi membranes with BFA under certain conditions. Surprisingly, an even more impressive degree of tubulation could be achieved by incubating Golgi stacks with an ATP-reduced cytosolic fraction, without any BFA at all. Similarly, tubulation of Golgi membranes in vivo occurred after treatment of cells with intermediate levels of NaN3 and 2-deoxyglucose. The formation of tubules in vitro, either by BFA treatment or low-ATP cytosol, correlated precisely with a loss of the vesicle-associated coat protein beta-COP from Golgi membranes. After removal of BFA or addition of ATP, membrane tubules served as substrates for the rebinding of beta-COP and for the formation of vesicles in vitro. These results provide support for the idea that a reciprocal relationship exists between tubulation and vesiculation (Klausner, R. D., J. G. Donaldson, and J. Lippincott-Schwartz. 1992. J. Cell Biol. 116:1071-1080). Moreover, they show that tubulation is an inherent property of Golgi membranes, since it occurs without the aid of microtubules or BFA treatment. Finally the results indicate the presence of cytosolic factors, independent of vesicle-associated coat proteins, that mediate the budding/tubulation of Golgi membranes.
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Ciclopentanos/farmacología , Aparato de Golgi/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Animales , Transporte Biológico , Brefeldino A , Proteína Coatómero , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Membranas Intracelulares/ultraestructura , Hígado/ultraestructura , Masculino , Microscopía Electrónica , Proteínas Asociadas a Microtúbulos/metabolismo , RatasRESUMEN
The Ras target AF-6 has been shown to serve as one of the peripheral components of cell-cell adhesions, and is thought to participate in cell-cell adhesion regulation downstream of Ras. We here purified an AF-6-interacting protein with a molecular mass of approximately 220 kD (p220) to investigate the function of AF-6 at cell-cell adhesions. The peptide sequences of p220 were identical to the amino acid sequences of mouse Fam. Fam is homologous to a deubiquitinating enzyme in Drosophila, the product of the fat facets gene. Recent genetic analyses indicate that the deubiquitinating activity of the fat facets product plays a critical role in controlling the cell fate. We found that Fam accumulated at the cell-cell contact sites of MDCKII cells, but not at free ends of plasma membranes. Fam was partially colocalized with AF-6 and interacted with AF-6 in vivo and in vitro. We also showed that AF-6 was ubiquitinated in intact cells, and that Fam prevented the ubiquitination of AF-6.
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Adhesión Celular/fisiología , Proteínas de Drosophila , Endopeptidasas/fisiología , Genes ras/genética , Cinesinas/fisiología , Miosinas/fisiología , Animales , Encéfalo/metabolismo , Bovinos , Línea Celular , Cisteína Endopeptidasas/fisiología , Técnica del Anticuerpo Fluorescente , Inmunohistoquímica , Leupeptinas/farmacología , Complejos Multienzimáticos/fisiología , Complejo de la Endopetidasa Proteasomal , Proteínas Recombinantes de Fusión/metabolismo , Transfección/genética , Ubiquitina Tiolesterasa , Ubiquitinas/metabolismoRESUMEN
An association between serum concentrations of persistent organic pollutants (POPs), such as 2,2',4,4',5,5'-hexachlorobiphenyl (PCB-153), and risk of type 2 diabetes mellitus (T2DM) has been reported. Conditional on body mass index (BMI) and waist circumference (WC), a higher serum PCB-153 concentration may be a marker of T2DM risk because it reflects other aspects of obesity that are related to T2DM risk and to PCB-153 clearance. To estimate the amount of residual confounding by other aspects of obesity, we performed a quantitative bias analysis on the results of a specific study. A physiologically-based pharmacokinetic (PBPK) model was developed to predict serum levels of PCB-153 for a simulated population. T2DM status was assigned to simulated subjects based on age, sex, BMI, WC, and visceral adipose tissue mass. The distributions of age, BMI, WC, and T2DM prevalence of the simulated population were tailored to closely match the target population. Analysis of the simulated data showed that a small part of the observed association appeared to be due to residual confounding. For example, the predicted odds ratio of T2DM that would have been obtained had the results been adjusted for visceral adipose tissue mass, for the ≥90th percentile of PCB-153 serum concentration, was 6.60 (95% CI 2.46-17.74), compared with an observed odds ratio of 7.13 (95% CI 2.65-19.13). Our results predict that the association between PCB-153 and risk of type 2 diabetes mellitus would not be substantially changed by additional adjustment for visceral adipose tissue mass in epidemiologic analyses. Confirmation of these predictions with longitudinal data would be reassuring.
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Diabetes Mellitus Tipo 2/inducido químicamente , Contaminantes Ambientales/toxicidad , Bifenilos Policlorados/toxicidad , Adulto , Anciano , Sesgo , Índice de Masa Corporal , Simulación por Computador , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/epidemiología , Contaminantes Ambientales/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Modelos Biológicos , Obesidad/sangre , Obesidad/complicaciones , Bifenilos Policlorados/sangre , Prevalencia , Circunferencia de la Cintura , Adulto JovenRESUMEN
Genetic background affects animal phenotype and therefore is of particular relevance to studies using genetically manipulated mice. Strain differences in hypothalamic-pituitary-adrenocortical (HPA) axis activity may contribute to background-specificity of some mutations. Here, we analysed components of the HPA axis in mice lacking a functional neurokinin-1 receptor (NK1-/-) on two backgrounds: backcrossed C57BL/6 (B6) and mixed C57BL/6 x 129/sv (129B6). We hypothesized that HPA axis activity would vary between these strains, leading to differences in the NK1-/- phenotype. We compared levels of plasma corticosterone between the groups, and found 129B6 mice exhibited elevated levels of stress-induced corticosterone compared with B6 mice, regardless of genotype. Although the level of basal corticotrophin-releasing factor and stress-induced c-fos mRNAs did not differ between the genotypes of either strain, examination of glucocorticoid receptor immunoreactivity within the hippocampus revealed that NK1-/- mice on the 129B6 background had elevated expression compared with wild-type, whilst there was no difference between genotypes in the B6 strain. Similarly, hippocampal neurogenesis in NK1-/- mice was greater than in wild-type on the 129B6 strain, and did not differ between genotypes on the B6 background. Finally, novelty- and morphine-induced locomotion were assessed. NK1-/- mice on the 129B6 background exhibited hyperlocomotion in response to novelty and greater sensitivity to the locomotor-stimulating properties of morphine than wild-type. In contrast, in B6 mice, no differences were observed between genotypes for either locomotor behaviour. In summary, we find that HPA axis activity differs between the strains and that there are profoundly background-specific effects of the NK1 receptor mutation.
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Encéfalo/metabolismo , Sistema Hipotálamo-Hipofisario/metabolismo , Receptores de Neuroquinina-1/genética , Estrés Fisiológico/metabolismo , Animales , Encéfalo/citología , Proliferación Celular , Corticosterona/sangre , Corticosterona/metabolismo , Resistencia a Medicamentos/genética , Conducta Exploratoria/fisiología , Predisposición Genética a la Enfermedad/genética , Genotipo , Hipocampo/citología , Hipocampo/metabolismo , Sistema Hipotálamo-Hipofisario/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Morfina/farmacología , Actividad Motora/efectos de los fármacos , Actividad Motora/genética , Mutación/genética , Fenotipo , Receptores de Glucocorticoides/metabolismo , Especificidad de la Especie , Estrés Fisiológico/genética , Estrés Fisiológico/fisiopatología , Taquicininas/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
The activity of the hypothalamic-pituitary-adrenal (HPA) axis is characterised both by an ultradian pulsatile pattern of glucocorticoid secretion and an endogenous diurnal rhythm. Glucocorticoid feedback plays a major role in regulating HPA axis activity and this mechanism occurs via two different receptors: mineralocorticoid (MR) and glucocorticoid receptors (GR). In the present study, the effects of both acute and subchronic treatment with the GR antagonist Org 34850 on basal and stress-induced HPA axis activity in male rats were evaluated. To investigate the effect of Org 34850 on basal diurnal corticosterone rhythm over the 24-h cycle, an automated blood sampling system collected samples every 10 min. Acute injection of Org 34850 (10 mg/kg, s.c.) did not affect basal or stress-induced corticosterone secretion, but was able to antagonise the inhibitory effect of the glucocorticoid agonist methylprednisolone on stress-induced corticosterone secretion. However, 5 days of treatment with Org 34850 (10 mg/kg, s.c., two times a day), compared to rats treated with vehicle (5% mulgofen in 0.9% saline, 1 ml/kg, s.c.), increased corticosterone secretion over the 24-h cycle and resulted in changes in the pulsatile pattern of hormone release, but had no significant effect on adrenocorticotrophic hormone secretion or on stress-induced corticosterone secretion. Subchronic treatment with Org 34850 did not alter GR mRNA expression in the hippocampus, paraventricular nucleus of the hypothalamus or anterior-pituitary, or MR mRNA expression in the hippocampus. Our data suggest that a prolonged blockade of GRs is required to increase basal HPA axis activity. The changes observed here with ORG 34850 are consistent with inhibition of GR-mediated negative feedback of the HPA axis. In light of the evidence showing an involvement of dysfunctional HPA axis in the pathophysiology of depression, Org 34850 could be a potential treatment for mood disorders.
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Corticosterona/metabolismo , Receptores de Glucocorticoides/antagonistas & inhibidores , Esteroides/metabolismo , Estrés Psicológico , Sulfonas/metabolismo , Hormona Adrenocorticotrópica/metabolismo , Animales , Ritmo Circadiano/efectos de los fármacos , Ritmo Circadiano/fisiología , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Sistema Hipotálamo-Hipofisario/fisiología , Hibridación in Situ , Masculino , Sistema Hipófiso-Suprarrenal/efectos de los fármacos , Sistema Hipófiso-Suprarrenal/fisiología , Ratas , Ratas Sprague-Dawley , Esteroides/farmacología , Sulfonas/farmacologíaRESUMEN
Basal activity of the rat hypothalamic-pituitary-adrenal (HPA) axis is highly dynamic and displays both circadian and ultradian rhythmicity in corticosterone secretion. This study investigated the relationship between basal corticosterone pulsatility and the corticosterone response to noise during the early light phase when there are no endogenous corticosterone pulses and during the early dark phase when there are hourly pulses of corticosterone. An automated blood sampling system was used to collect blood in conscious male rats at 5-min intervals before, during and after exposure to 10-min periods of white noise (104 dB). Behavioural responses to noise were also monitored during these periods. During the early light phase (morning), there was a consistent corticosteroid response to noise with corticosterone concentrations rising rapidly and reaching peak values 10-15 min after the noise had ceased, following which circulating concentrations declined at a rate comparable to the hormones half-life. A second noise stress, 80 min later, resulted in adaptation of the corticosterone response. During the early dark phase (evening), the corticosterone response to the noise was similar to that seen in the morning, although there was no adaptation to a second stimulus. During the evening, the inhibition of endogenous HPA activity after the sound was limited to 40 min following stress.
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Ritmo Circadiano/fisiología , Corticosterona/sangre , Sistema Hipotálamo-Hipofisario/metabolismo , Ruido/efectos adversos , Sistema Hipófiso-Suprarrenal/metabolismo , Estrés Psicológico/metabolismo , Estimulación Acústica , Adaptación Fisiológica , Animales , Corticosterona/metabolismo , Masculino , Periodicidad , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVES: Truncating mutations in USP9X have been identified in oral squamous cell carcinoma patients. The aim of this study was to determine USP9X's functional role, if any, in head and neck cancer cells. MATERIALS AND METHODS: USP9X was depleted/overexpressed in head and neck cancer cell line: SCC15 (tongue), CAL27 (tongue), FaDu (pharynx) and Detroit 562 (pharynx). Cell proliferation was monitored using the CyQUANT assay, and cell cycle distribution was determined by flow cytometry. Immunoblot assays were conducted to assess protein levels. RT-qPCR was performed to determine Notch and Wnt pathway target gene expression. RESULTS: Our data showed a direct correlation between USP9X protein levels and proliferation, as well as Notch pathway activity in head and neck cancer cells. However, at least in FaDu, USP9X did not appear to regulate proliferation through the Notch pathway. Immunoblotting revealed a dramatic reduction in downstream targets of mTOR complex 1, namely total ribosomal protein (S6) and its phosphorylated form (pS6), when USP9X was depleted in FaDu cells. In contrast, in immortalized but non-tumorigenic HaCaT keratinocytes, USP9X depletion led to increase in cell proliferation, maintaining direct regulation of Notch activity. CONCLUSIONS: The functional role of USP9X was found to be context dependent. USP9X possibly promotes head and neck cancer cell proliferation through the mTOR pathway.
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Proliferación Celular , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Ubiquitina Tiolesterasa/metabolismo , Línea Celular , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/genética , Humanos , Receptores Notch/metabolismo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Ubiquitina Tiolesterasa/genéticaRESUMEN
Parkinson's disease is a complex age-related neurodegenerative disorder. Approximately 90% of Parkinson's disease cases are idiopathic, of unknown origin. The aetiology of Parkinson's disease is not fully understood but increasing evidence implies a failure in fundamental cellular processes including mitochondrial dysfunction and increased oxidative stress. To dissect the cellular events underlying idiopathic Parkinson's disease, we use primary cell lines established from the olfactory mucosa of Parkinson's disease patients. Previous metabolic and transcriptomic analyses identified deficiencies in stress response pathways in patient-derived cell lines. The aim of this study was to investigate whether these deficiencies manifested as increased susceptibility, as measured by cell viability, to a range of extrinsic stressors. We identified that patient-derived cells are more sensitive to mitochondrial complex I inhibition and hydrogen peroxide induced oxidative stress, than controls. Exposure to low levels (50 nM) of rotenone led to increased apoptosis in patient-derived cells. We identified an endogenous deficit in mitochondrial complex I in patient-derived cells, but this did not directly correlate with rotenone-sensitivity. We further characterized the sensitivity to rotenone and identified that it was partly associated with heat shock protein 27 levels. Finally, transcriptomic analysis following rotenone exposure revealed that patient-derived cells express a diminished response to rotenone-induced stress compared with cells from healthy controls. Our cellular model of idiopathic Parkinson's disease displays a clear susceptibility phenotype to mitochondrial stress. The determination of molecular mechanisms underpinning this susceptibility may lead to the identification of biomarkers for either disease onset or progression.
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Apoptosis/efectos de los fármacos , Complejo I de Transporte de Electrón/antagonistas & inhibidores , Proteínas de Choque Térmico HSP27/metabolismo , Mitocondrias/metabolismo , Mucosa Olfatoria/citología , Enfermedad de Parkinson/patología , Rotenona/farmacología , Supervivencia Celular , Células Cultivadas , Humanos , Peróxido de Hidrógeno/toxicidad , Mucosa Olfatoria/metabolismo , Estrés Oxidativo/efectos de los fármacos , Enfermedad de Parkinson/etiologíaRESUMEN
Organizational effects of testosterone during a critical period of neonatal life have major irreversible effects on adult sexual behavior. We have investigated whether perinatal androgen changes also affect another major sexually differentiated system, the hypothalamo-pituitary-adrenal axis. This was assessed in male rats who had been exposed to perinatal flutamide or 1,4,6-androstatriene-3,17-dione (ATD). Once the animals reached adulthood, an automated sampling system was used to collect blood from freely moving animals at 10-min intervals over 24 h, followed by a noise stress and then the administration of lipopolysaccharide (LPS). Perinatal flutamide- and ATD-treated rats not only had higher mean corticosterone levels and increased frequency and amplitude of corticosterone pulses over the 24 h compared with vehicle-injected controls, but they also showed markedly increased corticosterone responses to both noise and LPS. All parameters of increased hypothalamo-pituitary-adrenal activity resembled the normal physiological state of the intact adult female rather than that of the intact adult male rat. Furthermore, 3 h after LPS administration, both flutamide- and ATD-treated animals had markedly higher levels of corticotropin-releasing factor mRNA in the parvocellular paraventricular nucleus (PVN) and proopiomelanocortin mRNA in the adenohypophysis. Flutamide-treated rats also had a greater level of PVN arginine vasopressin mRNA. PVN glucocorticoid receptor mRNA levels were significantly lower in both the flutamide- and the ATD-treated male rats. These data highlight the importance of perinatal exposure to both testosterone and estrogen(s) on the development of a masculinized circadian corticosterone profile and stress-induced hypothalamo-pituitary-adrenal axis activity in the adult male rat.
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Estrógenos/fisiología , Sistema Hipotálamo-Hipofisario/fisiología , Sistema Hipófiso-Suprarrenal/fisiología , Testosterona/fisiología , Hormona Adrenocorticotrópica/sangre , Androstatrienos/farmacología , Animales , Arginina Vasopresina/genética , Corticosterona/sangre , Hormona Liberadora de Corticotropina/genética , Femenino , Flutamida/farmacología , Lipopolisacáridos/farmacología , Masculino , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Transcortina/análisisRESUMEN
The Drosophila fat facets and canoe genes regulate non-neural cell fate decisions during ommatidium formation. We have shown previously that the FAM (fat facets in mouse) de-ubiquitinating enzyme regulates the function of AF-6, (mammalian canoe homologue), in the MDCK epithelial cell line (Taya et al., 1998. The Ras target AF-6 is a substrate of the fam de-ubiquitinating enzyme. J. Cell Biol. 142, 1053-1062). We report here that the expression of the FAM and AF-6 proteins overlaps extensively in the mouse eye from embryogenesis to maturity, especially in the non-neural epithelia including the retinal pigment epithelium, subcapsular epithelium of the lens and corneal epithelium. Expression is not limited to the epithelia however, as FAM and AF-6 also co-localize during lens fibre development as well as in sub-populations of the neural retina.
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Proteínas de Drosophila , Endopeptidasas/análisis , Ojo/embriología , Cinesinas/análisis , Miosinas/análisis , Secuencia de Aminoácidos , Animales , Drosophila , Endopeptidasas/inmunología , Proteínas de Insectos , Cinesinas/inmunología , Ratones , Datos de Secuencia Molecular , Miosinas/inmunología , Ubiquitina TiolesterasaRESUMEN
FAM is a developmentally regulated substrate-specific deubiquitylating enzyme. It binds the cell adhesion and signalling molecules beta-catenin and AF-6 in vitro, and stabilises both in mammalian cell culture. To determine if FAM is required at the earliest stages of mouse development we examined its expression and function in preimplantation mouse embryos. FAM is expressed at all stages of preimplantation development from ovulation to implantation. Exposure of two-cell embryos to FAM-specific antisense, but not sense, oligodeoxynucleotides resulted in depletion of the FAM protein and failure of the embryos to develop to blastocysts. Loss of FAM had two physiological effects, namely, a decrease in cleavage rate and an inhibition of cell adhesive events. Depletion of FAM protein was mirrored by a loss of beta-catenin such that very little of either protein remained following 72h culture. The residual beta-catenin was localised to sites of cell-cell contact suggesting that the cytoplasmic pool of beta-catenin is stabilised by FAM. Although AF-6 levels initially decreased they returned to normal. However, the nascent protein was mislocalised at the apical surface of blastomeres. Therefore FAM is required for preimplantation mouse embryo development and regulates beta-catenin and AF-6 in vivo.
Asunto(s)
Embrión de Mamíferos/fisiología , Endopeptidasas/metabolismo , Endopeptidasas/fisiología , Transactivadores , Animales , Blastocisto/metabolismo , Western Blotting , Adhesión Celular , Comunicación Celular , División Celular , Células Cultivadas , Citoplasma/metabolismo , Proteínas del Citoesqueleto/metabolismo , Regulación del Desarrollo de la Expresión Génica , Cinesinas/metabolismo , Ratones , Microscopía Fluorescente , Miosinas/metabolismo , Oligonucleótidos/farmacología , Oligonucleótidos Antisentido/farmacología , Unión Proteica , Factores de Tiempo , Ubiquitina Tiolesterasa , beta CateninaRESUMEN
The Drosophila fat facets (faf) gene is a ubiquitin-specific protease necessary for the normal development of the eye and of the syncytial stage embryo in the fly. Using a gene trap approach in embryonic stem cells we have isolated a murine gene with extensive sequence similarity to the Drosophila faf gene and called it Fam (fat facets in mouse). The putative mouse protein shows colinearity and a high degree of sequence identity to the Drosophila protein over almost its entire length of 2554 amino acids. The two enzymatic sites characteristic of ubiquitin-specific proteases are very highly conserved between mice and Drosophila and this conservation extends to yeast. Fam is expressed in a complex pattern during postimplantation development. In situ hybridisation detected Fam transcripts in the rapidly expanding cell populations of gastrulating and neurulating embryos, in post-mitotic cells of the CNS as well as in the apoptotic regions between the digits, indicating that it is not associated with a single developmental or cellular event. The strong sequence similarity to faf and the developmentally regulated expression pattern suggest that Fam and the ubiquitin pathway may play a role in determining cell fate in mammals, as has been established for Drosophila.