Abnormal regulation of Na,K-ATPase in Glucose Intolerant Rats.

Introduction: Glucose is the most important physiological insulin secretagogue. However, the mechanisms underlying glucose-induced insulin release are not fully understood. The role of electrogenic systems such as ionic pumps, to these events remains essentially uninvestigated. Na,K-ATPase, responsible for maintaining Na+ and K+ gradients across the plasma membrane and generates a net outward current, thus changes in its activity may contribute to the early ionic events regulating insulin secretion (Therien and Blostein, 2000). Objective: The aim of this work was to evaluate the regulation of Na,K-ATPase activity by glucose in intact -cells of normal and glucose intolerant (GI) rats and its putative contribution to the regulation of insulin secretion. Material and Methods: Pancreatic -cells, from normal or control or GI rats, were isolated and cultured (48h). Cell batches were pre-incubated (30min) with 2mM glucose to reach basal. Afterwards cells were challenged with glucose in the interval 0-11mM for 60min, for dose-dependence evaluation, or with 8mM glucose for 5-120min, for time-dependence evaluation. ATPase activity was assessed in intact cells by colorimetric quantification of Pi formed in 30min. Na,K-ATPase activity was calculated by the difference between the activities obtained in the absence and in presence the of 1mM ouabain (Costa et al., 2009). Results: In β-cells from normal rats, glucose induced a bimodal regulation of Na,K-ATPase. In the absence of glucose, Na,K-ATPase activity was 0.056±0.015 U/mg. Stimulation with 2mM glucose induced an increase of Na,K-ATPase activity of ~4 fold whereas for [glucose] above 2mM it was observed a significant inhibition of Na,K-ATPase activity (0.061±0.013, 0.080±0.009 and 0.064±0.005 U/mg for 5.6, 8.4 and 11mM glucose, respectively, compared to 0.188±0.035 U/mg observed in 2mM G; n=3-8). β-cells from GI rats does not present this profile; in the absence of glucose, Na,K-ATPase activity was 0.202±0.036 U/mg and no significant differences from this value were observed with the other glucose concentration tested. Addicionally, in β-cells from normal rats, glucose (8mM) induced a time-dependent inhibition, with a biphasic profile, of Na,K-ATPase - it was observed a decrease in the pump activity between 0 and 20min stimulation where it reached a minimum value (77%). For incubation periods over 20min, the pump activity slowly and partially recovered (54%, 55% and 52%, for 30, 60 and 120min, respectively; n=7). In β-cells from GI animals, an less accentuated decrease of Na,K-ATPase activity between 0 ans 20min was also observed (34%), and is not observed further recover in activity. Conclusions: This work demonstrates there Na,K-ATPase is strictly regulated by glucose in pancreatic β-cell. This regulation is unpaired in GI animals. Na,K-ATPase contribution to glucose-induced ionic events and insulin secretion might be relevant and must be explored as a possible therapeutic target in TD2 . 1. Therien AG, Blostein R (2000) Mechanisms of sodium pump regulation. Am J Physiol Cell Physiol 279:C541-C566 2. Costa AR, Real J, Antunes CM, Cruz-Morais J (2009) A new approach for determination of Na,K-ATPase activity: application to intact pancreatic beta-cells. In Vitro Cell Dev Biol Ani

Implication of AMPK in glucose-evoked modulation of Na,K-ATPase

Background and aims: Na,K-ATPase is an integral membrane protein that maintains the gradients of Na+ and K+, using the energy of ATP hydrolysis, maintaining the ionic gradients that allow electrical activity to occur. It has been demonstrated that, in pancreatic β-cells, Na,K-ATPase is regulated by glucose and that this phenomenon is impaired in glucose intolerant subjects. However, the mechanism underlying glucose-induced modulation of Na,K-ATPase is still unclear. The AMP-activated protein kinase (AMPK) is a molecular key player in energy homeostasis, providing exquisite sensitivity to small changes in intracellular AMP levels and thus to intracellular [ATP]/[ADP] ratio, that is known to activate protein regulatory pathways. Since in pancreatic β-cell, glucose has marked effects on oxidative metabolism and total intracellular ATP and AMP levels, the involvement of AMPK in the cascade of events regulating Na,K-ATPase regulation in pancreatic β-cells was postulated. The aim of this work was to evaluate the putative role of AMPK in the glucose-evoked regulation of Na,K-ATPase activity in the pancreatic β-cell. Materials and methods: Pancreatic -cells from normal (control) or glucose-intolerant Wistar rats (GIR) were isolated and cultured (48h). Cell batches were pre-incubated (30min) with 2.1mM glucose to reach basal activity. Afterwards cells were challenged to 8.4mM glucose for 20min, in the presence or absence of AMPK agonists (AICAR) and antagonists (compound C; CC). ATPase activity was assessed in intact cells by colorimetric quantification of Pi formed in 30min. Na,K-ATPase activity was calculated by the difference between the activities obtained in the absence and in presence the of 1mM ouabain. Results: In basal conditions the activity of Na,K-ATPase from normal and GIR pancreatic β-cell was similar (0.184±0.030 and 0.186±0.020 molPi/min/mgProt, respectively). Challenging the control β-cells with glucose 8.4mM evoked a 62% reduction of Na,K-ATPase activity whereas in GIR β-cells a significantly lower inhibition (40%) was observed. The addition of AICAR 1mM abolished glucose-induced Na,K-ATPase inhibition (0,166±0.011 molPi/min/mg). In control β-cell, the addition of CC 10 μM had no effect on glucose-induced inhibition of Na,K-ATPase. In the contrary, in GIR β-cells it significantly potentiated glucose-evoked inhibition of Na,K-ATPase reaching values similar to that observed in the controls (66%). Conclusions: The AMPK agonist AICAR counteracts the inhibitory action of glucose on Na,K-ATPase of control β-cells whereas CC amplified the glucose-induced inhibition of Na,K-ATPase in GIR β-cells. These results suggest that AMPK plays a central role in the cascade of events underlying glucose-induced modulation of Na,K-ATPase and that the defect must be upstream of AMPK. Finally, abnormal glucose-induced regulation of Na,K-ATPase occurs prior to overt type 2 diabetes and might be a feature in the disease development

The Effect of Clothing Upon Perceptions of Source Credibility

This study investigated the effect of clothing upon perceptions of credibility. A male an female model were dressed either formally (in a suit) or informally (in casual slacks). Subjects were 399 undergraduatee students from the University of Nebraska at Omaha. Each suject viewed one of four photographs (male dressed formally, male dressed informally, female dressed formally, female dressed informally) and read a narrative accompanying each photograph. The narrative described the situational context, which included high-reputed characteristics (education, managerial occupation, and expertise in the topic of communication) for each model. Subjects completed McCroskey and Jenson\u27s 25 bipolar adjectives to measure five dimensions of credibility (competency, character, sociability, composure and extroversion)

Chemical composition and anti-diabetic properties of Cytisus multiflorus

Bakground and aims: The interest on plants with potential medicinal properties has been increasing worldwide. In the Iberian Peninsula there are some endemic species known by the population for their pharmacologic activity with valorization potential that have not been yet characterized. The white Spanish broom (Cytisus multiflorus) is described as having anti-diabetic effect [1] and in a preliminary the hypoglycemic and hyper-insulinemic effect of an aqueous extract has been shown [2]. The aim of this work was to fractionate and analyse the composition of the aqueous extract of C. multiflorus flowering parts and evaluate its potential as an anti-diabetic agent. Materials and methods: The aqueous extract was primarily fractionated by SPE using water:methanol (W:Me) eluent (a 10% step-wise gradient W:Me from 100:0 to 0:100) followed by high performance liquid chromatography with diode array detector (HPLC-DAD). The most relevant fraction were analysed by LC-MS to determine the chemical composition. Total fenol content was determined by a modified Folin-Ciocalteau method and the anti-oxidant activity was evaluated by the DPPH mehod. Finally, the hipoglicemic potential was evaluated in vivo using glucose intolerant rats (GIR). Results: Eleven fractions of the bulk extract were obtained. Seven of these fractions (10, 30, 40, 50, 60, 70 e 80% Me) were found to have a relevant compounds, mostly flavonoid compounds, namely, rutin (50, 60 and 70% Me fractions), ferrulic acid (30% Me), referred as having hypoglicemic effect. The fractions obtained with 50 and 70% Me showed the highest content in phenol equivalents and the highest anti-oxidant effect were found in the 50 and 60% Me fractions. The 30 and 60% Me fraction had no effect on the post-prandial glicemia. Conclusions: The 30, 50, 60 and 70% Me fractions, due to their chemical composition and anti-oxidant effects were the most promising to have anti-diabetic effect. However, the 30 and 60% Me were found to be ineffective. The 50% Me fraction showed both a high content of flavonoid compounds and the highest anti-oxidant power which suggest that it may constitute the most promising one. The anti-diabetic properties of this fraction should be investigated. [1] Camejo-Rodrigues J. et al. (2003). J. Ethnopharmacol, 89, 199-209 [2] Célia M. Antunes, Laurinda R. Areias, Inês P. Vieira, Ana C. Costa, M. Teresa Tinoco, & Júlio Cruz-Morais (2009). Rev. Fitoterapia 9 (Supl.1): 91

Packing of R3 by crosses

The existence of tilings of R^n by crosses, a cluster of unit cubes comprising a central one and 2n arms, has been studied by several authors. We have completely solved the problem for R^2, characterizing the crosses which lattice tile R^2, as well as determining the maximum packing density for the crosses which do not lattice tile the plane. In this paper we motivate a similar approach to study lattice packings of R^3 by crosses.The existence of tilings of Rn by crosses, a cluster of unit cubes comprising a central one and 2n arms, has been studied by several authors. We have completely solved the problem for R2 characterizing the crosses which lattice tile R2 as well as determining the maximum packing density for the crosses which do not lattice tile the plane. In this paper we motivate a similar approach to study lattice packings of R3 by crosses.publishe

Effects of environmental contaminants in inflammatory bowel disease: a systematic review

Introduction: The Inflammatory Bowel Disease (IBD) is characterized by a chronic inflammation of the gastrointestinal tract and it has two important forms: Crohn’s Disease and Ulcerative Colitis. Although the pathophysiology of the disease is mostly unknown, an interaction between the immune system, genetics, microbiome and environmental factors seems to be responsible for the disease onset. The role of environmental factors in the pathophysiology of this disease is reinforced if we consider the rapid growth in incidence and prevalence rates of IBD in areas where the incidence was previously low as, for example, Asia. However, the literature on the role of environmental factors in IBD is reduced and is not systematized. Hence, in this work a Systematic Review of the literature was performed. Materials and methods: For this systematic review the methodology “Navigation Guide Method for Grading Human Evidence” was used [1]. The research was carried out in three databases (Scopus, Web of Science, PubMed / MEDLINE). The inclusion criteria used were: research articles with original human data, clinically diagnosed inflammatory bowel disease, quantification of environmental contaminants and study of the association between environmental contaminants and inflammatory bowel disease. Only articles written in Portuguese, English, French or Spanish were included. Results: Sixteen studies were included in this systematic review. Of these, 13 studied Inflammatory Bowel Disease in general, 2 studies included only patients with Crohn’s Disease and 1 study included only patients with Ulcerative Colitis. The most studied environmental contaminants were sulfur dioxide, nitrogen dioxide, carbon monoxide, particulate matter (PM), perfluorooctanoic acid, aluminum, iron, zinc, copper, titanium dioxide, silicates and endotoxins. We also identified different categories of studies, 4 studies on the effects of air pollution, 3 on the intake of contaminated water and 3 on the effects of diet. Additionally, 2 studies evaluated the serum concentrations of contaminants, 1 studied the impact of exposure to house dust, 2 attempted a cell characterization of IBD patients and in another colitis was induced. Overall, the quality of evidence was rated “moderate to low” and the strength of evidence as “Inadequate evidence on toxicity”. Discussion and conclusions: In this systematic review, we have identified different classes of environmental contaminants associated with IBD, but studies reporting their concentration in biological matrices are rare. Based on a moderate to low level of quality, the available evidence gathered in this Systematic Review is insufficient to access the role of environmental contaminants in inflammatory bowel disease. New epidemiological studies are necessary

Alterações na modulação da Na,K-ATPase pela glicose em célula β-pancreática de animais intolerantes à glicose.

Alterações na modulação da Na,K-ATPase pela glicose em célula β-pancreática de animais intolerantes à glicose AR Costa1,2, CM Antunes1,3, J Cruz-Morais1,2 1Departamento de Química, 2Instituto de Ciências Agrárias e Ambientais Mediterrânicas (ICAAM), Universidade de Évora, Largo dos Colegiais 2, 7000 Évora; 3Centro de Neurociências e Biologia Celular (CNC), Universidade de Coimbra, 3004-517 Coimbra;[email protected] A diabetes tipo 2 é uma síndrome metabólica, que se caracteriza por alterações da secreção ou acção da insulina, cujas bases bioquímicas não estão completamente esclarecidas. Na célula-, a resposta fisiológica à glicose envolve a despolarização, resultante principalmente do fecho de canais de K+ sensíveis ao ATP, seguido do influxo de Ca2+ por canais sensíveis à voltagem tipo-L e consequente aumento da concentração intracelular de Ca2+, necessário à exocitose da insulina, hormona responsável pela homeostasia da glicose. A Na,K-ATPase (ou bomba de sódio), é o sistema de transporte activo responsável por manter o gradiente transmembranar de Na+ e K+. Estudos recentes têm sugerindo que a inibição da Na,K-ATPase possa contribuir para a despolarização induzida por glicose. Como o acoplamento estímulo-secreção se encontra alterado em célula- de animais diabéticos, proposemo-nos estudar a participação da Na,K-ATPase neste mecanismo utilizando um modelo de pré-diabetes que se caracteriza essencialmente por apresentar alterações na curva de tolerância à glicose. Para isso, desenvolveu-se um método que permite avaliar a actividade da bomba em células β-pancreáticas primárias, mantidas em culturas aderentes, que preservam os processos de transdução de sinal. Com este método observámos que a glicose inibe a Na,K-ATPase em animais normais, estando este processo significativamente atenuado em animais pré-diabéticos. Observámos ainda que a secreção de insulina estimulada por glicose, nas células de pré-diabéticos, é cerca de 4 vezes menor relativamente aos controlos. Estes dados permitem-nos sugerir que a regulação da bomba por glicose se encontra modificada em animais pré-diabéticos, podendo contribuir para as alterações dos mecanismos iónicos responsáveis pelo acoplamento estímulo-secreção, nomeadamente na cinética da despolarização, comprometendo a secreção de insulina. O trabalho prossegue no sentido de esclarecer os mecanismos através dos quais a glicose induz inibição na Na,K-ATPase, bem como no levantamento dos factores que podem contribuir para as diferenças observadas entre os animais normais e pré-diabéticos, nomeadamente ao nível da expressão isoenzimática

Automatic neural generalized font identification

The final publication is available at Springer via http://dx.doi.org/10.1007/978-1-4471-1599-1_116Proceedings of the 8th International Conference on Artificial Neural Networks, Skövde, Sweden, 2–4 September 1998Neural methods are gaining a steady acceptance as powerful tools in a variety of pattern detection problems, OCR certainly being one of them. The concrete implementation of these neural OCR systems is of course a well guarded corporate secret, but in broad terms it can be said that in most of the cases, multilayer perceptrons (MLPs) are used. There are several reasons for the MLPs’ success. To begin with, they are based in well understood mathematical and statistical principles and there are efficient tools and methodologies for their training and evaluation. Furthermore they have good generalization properties.With partial support of grant TIC 95-965 of Spain's CICy

Achados metálicos de cobre no Baixo Vouga (Centro-Norte de Portugal)

O objetivo deste trabalho é o de dar a conhecer dois objetos metálicos, à base de cobre, encontrados em contextos arqueológicos distintos do curso inferior do rio Vouga e desta forma contribuir para o estudo das primeiras produções metalúrgicas na fachada ocidental do Centro-Norte português. O primeiro artefacto, um punhal com chanfraduras na zona de encaixe, foi detetado no sítio da Tapada do Espinheiro, freguesia de Sepins, concelho de Cantanhede, num terraço localizado a NW da plataforma de Murtede-Cordinhã, sobranceira à Vala Real/rio da Tapada (rio da Ponte), tributária do rio Cértima, afluente da margem sul do Vouga. O achado, detetado em trabalhos de prospeção, inseria-se numa área com cerca de 1.000 m2 onde ocorriam manchas de terra escura, concentração de cerâmica, de artefactos em pedra polida e talhada e alguns calhaus e blocos calcários resultantes da destruição de estruturas pétreas. A segunda peça, um machado plano, foi encontrado no topo da referida plataforma, a norte da freguesia de Sepins, concelho de Cantanhede, numa estação arqueológica de grandes dimensões sobranceira ao rio da Ponte, afluente do Cértima. Em ambos os contextos há fragmentos cerâmicos profusamente decorados com decorações incisas metopadas de tipo Penha, típicos do Calcolítico do Noroeste português, assim como decorações penteadas e espinhadas, comuns no Calcolítico do Nordeste e no Calcolítico e Inícios da Idade do Bronze do Alto Douro e da Beira Alta, balizas cronológicas onde estas peças se poderão inserir. Ao contrário do acervo cerâmico que indicia contactos com o Noroeste Peninsular e áreas mais interiores da bacia do Mondego, o punhal de chanfradura, de tradição meridional calcolítica, permite admitir que o curso inferior da bacia do Vouga foi uma zona charneira no encontro de diferentes tradições culturais, durante o III milénio a.C. A composição química dos objectos metálicos, determinada por espectrometria de fluorescência de raios X, contribui para a caracterização das primeiras produções metalúrgicas na fachada ocidental Centro – Norte do país, bem como a sua contextualização na arqueometalurgia peninsular.Fundação para a Ciência e a Tecnologia (FCT

Bozepinib: A Promising Selective Derivative Targeting Breast Cancer Stem Cells

Bozepinib is a potent antitumour compound that shows an IC50 of 0.166 μM against MDA-MB-231 human breast cancer cell line. It is also a very selective drug that presents a therapeutic index (TI) of 11.0 against MDA-MB-231 in relation to the normal MCF-10A. It is important to identify new cancer stem-like cells (CSCs) anticancer drugs to struggle against the resistance and the high risk of relapse in patients. In the present chapter, we show how bozepinib demonstrated selectivity on cancer cells and showed an inhibitory effect over kinases involved in carcinogenesis, proliferation and angiogenesis. Bozepinib inhibits HER-2 signaling pathway and JNK and ERK kinases. In addition, it has an inhibitory effect on AKT and VEGF together with anti-angiogenic and anti-migratory activities. Interestingly, bozepinib suppresses the formation of both mammo- and colonospheres and eliminated ALDH+ CSC subpopulations at a low micromolar range similar to salinomycin. It also induces the downregulation of SOX2, c-MYC and β-CATENIN and upregulation of the GLI-3 Hedgehog signaling repressor. Finally, bozepinib shows in vivo antitumor and anti-metastatic efficacy in xenotransplanted nude mice without presenting subacute toxicity. However, further studies in cancer patients are needed to confirm the therapeutic potential of bozepinib