The antilipolytic effect of insulin and epidermal growth factor in rat adipocytes are mediated by different mechanisms

Epidermal growth factor (EGF) and insulin induced similar effects in isolated rat adipocytes. To determine whether EGF and insulin produced similar effects through the same mechanisms, we focused on lipolysis. Insulin inhibited the lipolysis stimulated by isoproterenol, glucagon (either alone or in combination with adenosine deaminase), adenosine deaminase itself, or forskolin. In contrast, EGF did not inhibit the lipolysis stimulated by forskolin or by hormones when the cells were also incubated with adenosine deaminase. The effect of insulin, but not that of EGF, on isoproterenol-stimulated lipolysis disappeared when adipocytes were incubated with 1 microM wortmannin. These results indicate that EGF and insulin affected lipolysis through different mechanisms. We observed that EGF, but not insulin, increased cytosolic Ca2+. The effect of EGF, but not that of insulin, disappeared when the cells were incubated in a Ca2+-free medium. We suggest that EGF, but not insulin, mediate its antilipolytic effect through a Ca2+-dependent mechanism which, however, do not involve Ca2+-activated protein kinase C isoforms. This is based on the following: 1) phorbol 12-myristate 13-acetate affected lipolysis in an opposite way to that of EGF; and 2) the protein kinase C inhibitor bisindolylmaleimide GF 109203X did not affect the antilipolytic action of EGF. Our results indicate that the antilipolytic effect of EGF resembles more that of vasopressin than that of insulin.

Registro da ocorrência de Meloidogyne incognita no porta-enxerto 'IAC 766-Campinas' no estado de Pernambuco e reação de porta-enxertos e de cultivares copa de videira a Meloidogyne spp.

Relata-se a ocorrência de Meloidogyne incognita (Est. I2) em pomar de videira na cidade de Petrolina, Pernambuco. Plantas da cultivar Festival enxertadas sobre 'IAC 766-Campinas', atacadas por Meloidogyne incognita, apresentavam sintomas de menor vigor, folhas amarelecidas e folhagem mais esparsa, e raízes com numerosas galhas em meio ao cultivo de feijão-de-porco (Canavalia ensiformis), também atacado pelo nematoide. Com o objetivo de avaliar a resistência de quatro porta-enxertos tropicais, oito de clima temperado, e as cultivares Niágara Rosada e Chardonnay quanto à reação a M. incognita e M. arenaria, em casa de vegetação, realizou-se este trabalho. Mudas das videiras, mantidas em vaso com solo esterilizado, foram inoculadas com 10.000 ovos + J2 de M. incognita ou M. arenaria por planta. Decorridos oito meses da inoculação, cada planta foi avaliada quanto ao número de galhas e número de ovos por planta, determinando-se, a seguir, o fator de reprodução de cada espécie do nematoide (FR=população inicial/população final) nos diferentes materiais testados. Entre os porta-enxertos avaliados, 'Harmony', 'Salt Creek', '1103 Paulsen', 'IAC 572-Jales', 'IAC 313-Tropical', 'K5BB Kober' e 'SO4', foram resistentes (FR<1,00) e 'Solferino', imune (FR=0,00) a M. incognita, sendo os demais suscetíveis ao nematoide. Quando se avaliou a reação da videira a M. arenaria, exceto '106-Traviú', '420 A', 'Rupestris du Lot' e '1103 Paulsen', os demais porta-enxertos foram resistentes (FR>1,00) ao nematoide. No entanto, ambas as cultivares copa foram suscetíveis às duas espécies de Meloidogyne testadas.

Extra Forces induced by wide-pulse, high-frequency electrical stimulation: Occurrence, magnitude, variability and underlying mechanisms.

OBJECTIVE: In contrast to conventional (CONV) neuromuscular electrical stimulation (NMES), the use of "wide-pulse, high-frequencies" (WPHF) can generate higher forces than expected by the direct activation of motor axons alone. We aimed at investigating the occurrence, magnitude, variability and underlying neuromuscular mechanisms of these "Extra Forces" (EF). METHODS: Electrically-evoked isometric plantar flexion force was recorded in 42 healthy subjects. Additionally, twitch potentiation, H-reflex and M-wave responses were assessed in 13 participants. CONV (25Hz, 0.05ms) and WPHF (100Hz, 1ms) NMES consisted of five stimulation trains (20s on-90s off). RESULTS: K-means clustering analysis disclosed a responder rate of almost 60%. Within this group of responders, force significantly increased from 4% to 16% of the maximal voluntary contraction force and H-reflexes were depressed after WPHF NMES. In contrast, non-responders showed neither EF nor H-reflex depression. Twitch potentiation and resting EMG data were similar between groups. Interestingly, a large inter- and intrasubject variability of EF was observed. CONCLUSION: The responder percentage was overestimated in previous studies. SIGNIFICANCE: This study proposes a novel methodological framework for unraveling the neurophysiological mechanisms involved in EF and provides further evidence for a central contribution to EF in responders.

New genes for focal epilepsies with speech and language disorders.

The last 2 years have seen exciting advances in the genetics of Landau-Kleffner syndrome and related disorders, encompassed within the epilepsy-aphasia spectrum (EAS). The striking finding of mutations in the N-methyl-D-aspartate (NMDA) receptor subunit gene GRIN2A as the first monogenic cause in up to 20 % of patients with EAS suggests that excitatory glutamate receptors play a key role in these disorders. Patients with GRIN2A mutations have a recognizable speech and language phenotype that may assist with diagnosis. Other molecules involved in RNA binding and cell adhesion have been implicated in EAS; copy number variations are also found. The emerging picture highlights the overlap between the genetic determinants of EAS with speech and language disorders, intellectual disability, autism spectrum disorders and more complex developmental phenotypes.

Mechanisms underlying cytotoxicity induced by engineered nanomaterials: a review of in vitro studies

Engineered nanomaterials are emerging functional materials with technologically interesting properties and a wide range of promising applications, such as drug delivery devices, medical imaging and diagnostics, and various other industrial products. However, concerns have been expressed about the risks of such materials and whether they can cause adverse effects. Studies of the potential hazards of nanomaterials have been widely performed using cell models and a range of in vitro approaches. In the present review, we provide a comprehensive and critical literature overview on current in vitro toxicity test methods that have been applied to determine the mechanisms underlying the cytotoxic effects induced by the nanostructures. The small size, surface charge, hydrophobicity and high adsorption capacity of nanomaterial allow for specific interactions within cell membrane and subcellular organelles, which in turn could lead to cytotoxicity through a range of different mechanisms. Finally, aggregating the given information on the relationships of nanomaterial cytotoxic responses with an understanding of its structure and physicochemical properties may promote the design of biologically safe nanostructures.

Ewing sarcoma cancer stem cells : from mechanisms to therapeutic targeting

Le sarcome d'Ewing (SE) est la 2ème tumeur des os la plus fréquente chez les enfants, et le pronostic est sombre au stade métastatique. La pathogenèse du SE repose sur une translocation, provocant la fusion du domaine activateur du facteur de transcription EWS, avec la partie liant l'ADN de la protéine FLI-1. Les cellules souches cancéreuses (CSC) sont supposées être les moteurs de la croissance tumorale, et représente de ce fait des cibles thérapeutiques préférentielles. Dans ce travail nous nous sommes efforcés de comprendre, ainsi que de cibler les mécanismes liés à l'émergence des CSC dans le sarcome d'Ewing. La formation des CSC du ES est liée à un défaut de maturation des miRNAs provoqué par une sous-expression d'un gène, TARBP2, dans les CSC. Ce défaut de maturation peut être corrigé par un traitement des cellules avec de l'enoxacine, une fluoroquinolone utilisée pour traiter les infections urinaires. L'enoxacine seule n'étant pas suffisante pour éradiquer les tumeurs in vivo, nous avons testé la combinaison d'une thérapie ciblée sur les CSC avec une chimiothérapie classique, la doxorubicine, ciblant les cellules différentiées. In vitro l'enoxacine induit l'apoptose dans les CCS sans affecter les cellules différentiées, alors que à l'inverse, la doxorubicine n'affecte que les cellules de la « masse » tumorale. In vivo la combinaison de ces deux drogues inhibe la croissance de tumeurs provenant de cellules primaires xenotranplantées et éradique les CSCs. Nos résultats mettent en lumière une nouvelle approche thérapeutique directement applicable pour le sarcome d'Ewing, et pourraient ainsi rapidement déboucher sur des essais cliniques. Dans la deuxième partie de ce travail nous avons essayé de comprendre comment EWS-FLI1, la protéine de fusion issue de la translocation chromosomique du sarcome d'Ewing conduit à la génération des CSC. Pour cela nous avons effectué des ChIPseq (immunoprecipitation de la chromatine suivi de séquençage) pour EWS-FLI1 ainsi que pour certaines modifications histoniques. -- Ewing sarcoma family tumors (ESFT) are the second most frequent bone tumors in children and have a high rate of recurrence when metastatic at presentation. The pathogenesis of Ewing sarcoma is underlayed by a translocation, leading to the fusion of the trans-activating domain of EWS with the FLU DNA binding domain. Cancer stem cells (CSCs) are thought to be the driving force of tumor growth. In this work we focused on understanding the mechanisms underlying ESFT CSC emergence as well as defining targeted therapeutic strategies. Emergence of CSCs in ESFT has been shown to arise from a defect in TARBP2-dependent microRNA maturation, which can be corrected by exposure to the fluoroquinolone enoxacin. As enoxacin alone is not sufficient to reverse tumor growth in vivo, we assessed the effect of combining a drug that abrogates CSC properties with doxorubicin, a standard-of-care therapy in ESFT. Primary ESFT CSCs and bulk tumor cells were treated with different concentration of drugs and displayed divergent responses to doxorubicin and enoxacin. Doxorubicin, which targets the tumor bulk, displayed toxicity toward primary adherent ESFT cells in culture but not to CSC-enriched ESFT spheres. Conversely, enoxacin induced apoptosis but only in ESFT spheres and specifically on the CD133+ population. In combination, the two drugs markedly depleted CSC and strongly reduced primary growth in xenograft assays of two primary ESFT. Our results identify a potentially attractive therapeutic strategy for ESFT that combines mechanism-based targeting of CSC using a low toxicity antibiotic with a standard-of-care cytotoxic drug, offering immediate applications for clinical evaluation. In the second part of this work we performed chromatin immunopercipitation on CSCs and bulk cells for EWS-FLI1 binding as well as some chromatin modifications, and concluded that EWS-FLI1 shows cell context dependent binding.

Metabolic gene expression changes in astrocytes in Multiple Sclerosis cerebral cortex are indicative of immune-mediated signaling.

Emerging as an important correlate of neurological dysfunction in Multiple Sclerosis (MS), extended focal and diffuse gray matter abnormalities have been found and linked to clinical manifestations such as seizures, fatigue and cognitive dysfunction. To investigate possible underlying mechanisms we analyzed the molecular alterations in histopathological normal appearing cortical gray matter (NAGM) in MS. By performing a differential gene expression analysis of NAGM of control and MS cases we identified reduced transcription of astrocyte specific genes involved in the astrocyte-neuron lactate shuttle (ANLS) and the glutamate-glutamine cycle (GGC). Additional quantitative immunohistochemical analysis demonstrating a CX43 loss in MS NAGM confirmed a crucial involvement of astrocytes and emphasizes their importance in MS pathogenesis. Concurrently, a Toll-like/IL-1β signaling expression signature was detected in MS NAGM, indicating that immune-related signaling might be responsible for the downregulation of ANLS and GGC gene expression in MS NAGM. Indeed, challenging astrocytes with immune stimuli such as IL-1β and LPS reduced their ANLS and GGC gene expression in vitro. The detected upregulation of IL1B in MS NAGM suggests inflammasome priming. For this reason, astrocyte cultures were treated with ATP and ATP/LPS as for inflammasome activation. This treatment led to a reduction of ANLS and GGC gene expression in a comparable manner. To investigate potential sources for ANLS and GGC downregulation in MS NAGM, we first performed an adjuvant-driven stimulation of the peripheral immune system in C57Bl/6 mice in vivo. This led to similar gene expression changes in spinal cord demonstrating that peripheral immune signals might be one source for astrocytic gene expression changes in the brain. IL1B upregulation in MS NAGM itself points to a possible endogenous signaling process leading to ANLS and GGC downregulation. This is supported by our findings that, among others, MS NAGM astrocytes express inflammasome components and that astrocytes are capable to release Il-1β in-vitro. Altogether, our data suggests that immune signaling of immune- and/or central nervous system origin drives alterations in astrocytic ANLS and GGC gene regulation in the MS NAGM. Such a mechanism might underlie cortical brain dysfunctions frequently encountered in MS patients.

Compostos antioxidantes em frutos de acessos de caramboleira em diferentes ambientes de Pernambuco

A polpa da carambola contém compostos químicos com propriedades antioxidantes importantes à saúde humana, contra o envelhecimento celular e a prevenção de doenças. Este trabalho objetivou avaliar o fruto de acessos de caramboleira quanto às concentrações de compostos antioxidantes. Foram analisadas as concentrações de ácido ascórbico, carotenoides totais, flavonoides, fenólicos totais e taninos em cinco acessos selecionados de carambola, cultivada em três distintas regiões edafoclimáticas de Pernambuco. Os acessos de carambola diferiram significativamente entre si, quanto às concentrações de ácido ascórbico, fenólicos totais e taninos. A significância para os efeitos de ambiente confirmou que os locais de cultivo são heterogêneos, em relação ao potencial produtivo de frutos com diferentes concentrações médias de ácido ascórbico, flavonóis, fenólicos totais e taninos. A interação significativa entre genótipos e ambientes indica que os acessos de carambola apresentam respostas diferenciadas às concentrações de ácido ascórbico, flavonóis, fenólicos totais e taninos, quando cultivadas em diferentes locais. As condições de cultivo da Estação Experimental do IPA Itambé e os acessos IPA-7.2, IPA-22.3 proporcionaram as maiores concentrações de ácido ascórbico, fenólicos totais e taninos.

Dynamic single cell measurements of kinase activity by synthetic kinase activity relocation sensors.

BACKGROUND: Mitogen activated protein kinases (MAPK) play an essential role in integrating extra-cellular signals and intra-cellular cues to allow cells to grow, adapt to stresses, or undergo apoptosis. Budding yeast serves as a powerful system to understand the fundamental regulatory mechanisms that allow these pathways to combine multiple signals and deliver an appropriate response. To fully comprehend the variability and dynamics of these signaling cascades, dynamic and quantitative single cell measurements are required. Microscopy is an ideal technique to obtain these data; however, novel assays have to be developed to measure the activity of these cascades. RESULTS: We have generated fluorescent biosensors that allow the real-time measurement of kinase activity at the single cell level. Here, synthetic MAPK substrates were engineered to undergo nuclear-to-cytoplasmic relocation upon phosphorylation of a nuclear localization sequence. Combination of fluorescence microscopy and automated image analysis allows the quantification of the dynamics of kinase activity in hundreds of single cells. A large heterogeneity in the dynamics of MAPK activity between individual cells was measured. The variability in the mating pathway can be accounted for by differences in cell cycle stage, while, in the cell wall integrity pathway, the response to cell wall stress is independent of cell cycle stage. CONCLUSIONS: These synthetic kinase activity relocation sensors allow the quantification of kinase activity in live single cells. The modularity of the architecture of these reporters will allow their application in many other signaling cascades. These measurements will allow to uncover new dynamic behaviour that previously could not be observed in population level measurements.

DIVERSIDADE GENOTÍPICA E PATOGÊNICA DE Colletotrichum musae NO ESTADO DE PERNAMBUCO

Sessenta isolados de oriundos de Colletotrichumcampos de produção de banana dos Municípios de Vicência, São VicenteFérrer e Machados, no Estado de Pernambuco, foram avaliados quanto a características morfológicas,moleculares, culturais, de virulência e de diversidade genética. Os isolados foram identificados como C. musae, tendo a maioria conídios retos, oblongos, com ápices arredondados. A taxa de crescimento micelial variou de1,36 a 1,91 cm/dia. Foram encontrados três grupos de coloração para as colônias: branca, creme e salmão,enquanto a presença de setores variou de 0 a 8 por isolado e, na maioria dos isolados (73,3%), houve a presença de microescleródios. A diferença em virulência foi significativa para a área abaixo da curva de progresso da doença, indicando variabilidade entre os isolados. O dendrograma gerado pela análise UPGMA dos marcadores ISSR–PCR revelou a formação de três grupos pelo coeficiente de similaridade de Dice, os quais correspondem, na sua maioria, às três áreas amostradas.

Manipulation of arthropod sex determination by endosymbionts: diversity and molecular mechanisms.

Arthropods exhibit a large variety of sex determination systems both at the chromosomal and molecular level. Male heterogamety, female heterogamety, and haplodiploidy occur frequently, but partially different genes are involved. Endosymbionts, such as Wolbachia, Cardinium,Rickettsia, and Spiroplasma, can manipulate host reproduction and sex determination. Four major reproductive manipulation types are distinguished: cytoplasmic incompatibility, thelytokous parthenogenesis, male killing, and feminization. In this review, the effects of these manipulation types and how they interfere with arthropod sex determination in terms of host developmental timing, alteration of sex determination, and modification of sexual differentiation pathways are summarized. Transitions between different manipulation types occur frequently which suggests that they are based on similar molecular processes. It is also discussed how mechanisms of reproductive manipulation and host sex determination can be informative on each other, with a special focus on haplodiploidy. Future directions on how the study of endosymbiotic manipulation of host reproduction can be key to further studies of arthropod sex determination are shown.

The alpha1-adrenergic receptors in cardiac hypertrophy: Signaling mechanisms and functional implications.

Cardiac hypertrophy is a complex remodeling process of the heart induced by physiological or pathological stimuli resulting in increased cardiomyocyte size and myocardial mass. Whereas cardiac hypertrophy can be an adaptive mechanism to stressful conditions of the heart, prolonged hypertrophy can lead to heart failure which represents the primary cause of human morbidity and mortality. Among G protein-coupled receptors, the α1-adrenergic receptors (α1-ARs) play an important role in the development of cardiac hypertrophy as demonstrated by numerous studies in the past decades, both in primary cardiomyocyte cultures and genetically modified mice. The results of these studies have provided evidence of a large variety of α1-AR-induced signaling events contributing to the defining molecular and cellular features of cardiac hypertrophy. Recently, novel signaling mechanisms have been identified and new hypotheses have emerged concerning the functional role of the α1-adrenergic receptors in the heart. This review will summarize the main signaling pathways activated by the α1-AR in the heart and their functional implications in cardiac hypertrophy.

Sisäinen muutosviestintä fuusiotilanteessa - Kansallis-Osake-Pankki Oy:n ja Suomen Yhdyspankki Oy:n fuusion käsittely KOP:n sisäisessä henkilöstöviestinnässä

Tutkielma pyrkii selvittämään millaista viestinnällistä järjestelmää käytettiin kerrottaessa Kansallis-Osake-Pankin henkilöstölle Kansallis-Osake-Pankin ja Suomen Yhdyspankin fuusiosta. Näkökulmana on henkilöstön asema viestin vastaanottajana. Tutkielma hahmottaa johdon taholta tulleita sisäisen viestinnässä käytettyjä äänenpainoja ja sävyjä. Lisäksi tutkielma etsii kahden pankin pääjohtajien muutospuheista sisällöllisiä eroja ja kunkin organisaation totuttuja viestintäkäytänteitä. Tutkimusmenetelmänä käytetään kvalitatiivista tutkimusta ja tutkimusinstrumenttina tässä tutkielmassa toimii diskurssianalyysi. Diskurssianalyysi on teoreettinen viitekehys, jonka tarkoituksena on etsiä mitä merkityksiä teksteissä tuotetaan ja nähdä kieli todellisuuden rakentajana. Tutkimusaineistona on Kansallis-Osake-Pankin henkilöstölehdet, Kansallis-Osake-Pankin ja Suomen Yhdyspankin vuosikertomukset, fuusioesite ja valikoidut aiheesta kirjoitetut lehtiartikkelit. Kansallis-Osake-Pankin sisäistä viestintää leimasi fuusiouutisen julkitultua kriisiviestinnän piirteet: huhujen karkoittaminen ja virallisen tiedon voimakas levittäminen. Fuusiouutisen alkushokin jälkeen KOP:n viestinnästä nousi esiin viisi teemaa: viestien henkilöityminen pääjohtajaan, empatia henkilöstöä kohtaan vaikeissa tilanteissa, yhteisöllisyys, henkilön huomioonottaminen ja läheisyys.

Multicontrast MRI Quantification of Focal Inflammation and Degeneration in Multiple Sclerosis.

INTRODUCTION: Local microstructural pathology in multiple sclerosis patients might influence their clinical performance. This study applied multicontrast MRI to quantify inflammation and neurodegeneration in MS lesions. We explored the impact of MRI-based lesion pathology in cognition and disability. METHODS: 36 relapsing-remitting MS subjects and 18 healthy controls underwent neurological, cognitive, behavioural examinations and 3 T MRI including (i) fluid attenuated inversion recovery, double inversion recovery, and magnetization-prepared gradient echo for lesion count; (ii) T1, T2, and T2(*) relaxometry and magnetisation transfer imaging for lesion tissue characterization. Lesions were classified according to the extent of inflammation/neurodegeneration. A generalized linear model assessed the contribution of lesion groups to clinical performances. RESULTS: Four lesion groups were identified and characterized by (1) absence of significant alterations, (2) prevalent inflammation, (3) concomitant inflammation and microdegeneration, and (4) prevalent tissue loss. Groups 1, 3, 4 correlated with general disability (Adj-R (2) = 0.6; P = 0.0005), executive function (Adj-R (2) = 0.5; P = 0.004), verbal memory (Adj-R (2) = 0.4; P = 0.02), and attention (Adj-R (2) = 0.5; P = 0.002). CONCLUSION: Multicontrast MRI provides a new approach to infer in vivo histopathology of plaques. Our results support evidence that neurodegeneration is the major determinant of patients' disability and cognitive dysfunction.