Resistance to EGF receptor inhibitors in glioblastoma mediated by phosphorylation of the PTEN tumor suppressor at tyrosine 240

Glioblastoma multiforme (GBM) is the most aggressive of the astrocytic malignancies and the most common intracranial tumor in adults. Although the epidermal growth factor receptor (EGFR) is overexpressed and/or mutated in at least 50% of GBM cases and is required for tumor maintenance in animal models, EGFR inhibitors have thus far failed to deliver significant responses in GBM patients. One inherent resistance mechanism in GBM is the coactivation of multiple receptor tyrosine kinases, which generates redundancy in activation of phosphoinositide-3'-kinase (PI3K) signaling. Here we demonstrate that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is frequently phosphorylated at a conserved tyrosine residue, Y240, in GBM clinical samples. Phosphorylation of Y240 is associated with shortened overall survival and resistance to EGFR inhibitor therapy in GBM patients and plays an active role in mediating resistance to EGFR inhibition in vitro. Y240 phosphorylation can be mediated by both fibroblast growth factor receptors and SRC family kinases (SFKs) but does not affect the ability of PTEN to antagonize PI3K signaling. These findings show that, in addition to genetic loss and mutation of PTEN, its modulation by tyrosine phosphorylation has important implications for the development and treatment of GBM.

Eye enucleation activates the transcription nuclear factor kappa-B in the rat superior colliculus

Ocular enucleation produces significant morphological and physiological changes in central visual areas. However, our knowledge of the molecular events resulting from eye enucleation in visual brain areas remains elusive. We characterized here the transcription nuclear factor kappa-B (NF-kappa B) activation induced by ocular enucleation in the rat superior colliculus (SC). We also tested the effectiveness of the synthetic glucocorticoid dexamethasone in inhibiting its activation. Electrophoretic mobility shift assays to detect NF-kappa B indicated that this transcription factor is activated in the SC from 1 h to day 15 postlesion. The expression of p65 and p50 proteins in the nuclear extracts was also increased. Dexamethasone treatment was able to significantly inhibit NF-kappa B activation. These findings suggest that this transcriptional factor is importantly involved in the visual system short-term processes that ensue after retinal lesions in the adult brain. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Both adiponectin and interleukin-10 inhibit LPS-induced activation of the NF-kappa B pathway in 3T3-L1 adipocytes

Adiponectin and interleukin 10 (IL-10) are adipokines that are predominantly secreted by differentiated adipocytes and are involved in energy homeostasis, insulin sensitivity, and the anti-inflammatory response. These two adipokines are reduced in obese subjects, which favors increased activation of nuclear factor kappa B (NF-kappa B) and leads to elevation of pro-inflammatory adipokines. However, the effects of adiponectin and IL-10 on NF-kappa B DNA binding activity (NF-kappa Bp50 and NF-kappa Bp65) and proteins involved with the toll-like receptor (TLR-2 and TLR-4) pathway, such as MYD88 and TRAF6 expression, in lipopolysaccharide-treated 3T3-L1 adipocytes are unknown. Stimulation of lipopolysaccharide-treated 3T3-L1 adipocytes for 24 h elevated IL-6 levels; activated the NF-kappa B pathway cascade; increased protein expression of IL-6R, TLR-4, MYD88, and TRAF6; and increased the nuclear activity of NF-kappa B (p50 and p65) DNA binding. Adiponectin and IL-10 inhibited the elevation of IL-6 levels and activated NF-kappa B (p50 and p65) DNA binding. Taken together, the present results provide evidence that adiponectin and IL-10 have an important role in the anti-inflammatory response in adipocytes. In addition, inhibition of NF-kappa B signaling pathways may be an excellent strategy for the treatment of inflammation in obese individuals. (C) 2011 Elsevier Ltd. All rights reserved.

Mutation Spectrum in the Large GTPase Dynamin 2, and Genotype-Phenotype Correlation in Autosomal Dominant Centronuclear Myopathy

Centronuclear myopathy (CNM) is a genetically heterogeneous disorder associated with general skeletal muscle weakness, type I fiber predominance and atrophy, and abnormally centralized nuclei. Autosomal dominant CNM is due to mutations in the large GTPase dynamin 2 (DNM2), a mechanochemical enzyme regulating cytoskeleton and membrane trafficking in cells. To date, 40 families with CNM-related DNM2 mutations have been described, and here we report 60 additional families encompassing a broad genotypic and phenotypic spectrum. In total, 18 different mutations are reported in 100 families and our cohort harbors nine known and four new mutations, including the first splice-site mutation. Genotype-phenotype correlation hypotheses are drawn from the published and new data, and allow an efficient screening strategy for molecular diagnosis. In addition to CNM, dissimilar DNM2 mutations are associated with Charcot-Marie-Tooth (CMT) peripheral neuropathy (CMTD1B and CMT2M), suggesting a tissue-specific impact of the mutations. In this study, we discuss the possible clinical overlap of CNM and CMT, and the biological significance of the respective mutations based on the known functions of dynamin 2 and its protein structure. Defects in membrane trafficking due to DNM2 mutations potentially represent a common pathological mechanism in CNM and CMT. Hum Mutat 33: 949-959, 2012. (C) 2012 Wiley Periodicals, Inc.

Simcluster: clustering enumeration gene expression data on the simplex space

Abstract Background Transcript enumeration methods such as SAGE, MPSS, and sequencing-by-synthesis EST "digital northern", are important high-throughput techniques for digital gene expression measurement. As other counting or voting processes, these measurements constitute compositional data exhibiting properties particular to the simplex space where the summation of the components is constrained. These properties are not present on regular Euclidean spaces, on which hybridization-based microarray data is often modeled. Therefore, pattern recognition methods commonly used for microarray data analysis may be non-informative for the data generated by transcript enumeration techniques since they ignore certain fundamental properties of this space. Results Here we present a software tool, Simcluster, designed to perform clustering analysis for data on the simplex space. We present Simcluster as a stand-alone command-line C package and as a user-friendly on-line tool. Both versions are available at: http://xerad.systemsbiology.net/simcluster. Conclusion Simcluster is designed in accordance with a well-established mathematical framework for compositional data analysis, which provides principled procedures for dealing with the simplex space, and is thus applicable in a number of contexts, including enumeration-based gene expression data.

Cocaine induces cell death and activates the transcription nuclear factor kappa-b in pc12 cells

Cocaine is a worldwide used drug and its abuse is associated with physical, psychiatric and social problems. The mechanism by which cocaine causes neurological damage is very complex and involves several neurotransmitter systems. For example, cocaine increases extracellular levels of dopamine and free radicals, and modulates several transcription factors. NF-κB is a transcription factor that regulates gene expression involved in cellular death. Our aim was to investigate the toxicity and modulation of NF-κB activity by cocaine in PC 12 cells. Treatment with cocaine (1 mM) for 24 hours induced DNA fragmentation, cellular membrane rupture and reduction of mitochondrial activity. A decrease in Bcl-2 protein and mRNA levels, and an increase in caspase 3 activity and cleavage were also observed. In addition, cocaine (after 6 hours treatment) activated the p50/p65 subunit of NF-κB complex and the pretreatment of the cells with SCH 23390, a D1 receptor antagonist, attenuated the NF-κB activation. Inhibition of NF-κB activity by using PDTC and Sodium Salicilate increased cell death caused by cocaine. These results suggest that cocaine induces cell death (apoptosis and necrosis) and activates NF-κB in PC12 cells. This activation occurs, at least partially, due to activation of D1 receptors and seems to have an anti-apoptotic effect on these cells.

Valor energético total e contribuição percentual de calorias por macronutrientes da alimentação de idosos domiciliados em Fortaleza - CE

OBJETIVO: Descrever o valor energético total e contribuição percentual de calorias por macronutrientes da alimentação de idosos domiciliados em Fortaleza/CE. MÉTODOS: Este estudo é populacional, transversal e domiciliar. Participaram deste estudo 458 idosos (66,6% mulheres). As variáveis avaliadas foram: valor energético total (VET) da alimentação e contribuição percentual de calorias por proteínas, carboidratos e lipídios. Os resultados são apresentados sob a forma de médias, desvios-padrão e distribuição percentilar (P5, P10, P25, P50, P75, P90, P95). Os testes t-Student e análise de variância (ANOVA de uma via) com o teste Post Hoc LSD foram utilizados para verificar a diferença estatística das médias entre dois grupos e entre três grupos ou mais, respectivamente. RESULTADOS: Ao comparar os valores médios do VET entre as categorias das variáveis socioeconômico-demográficas foi encontrada diferença estatisticamente significativa entre as mulheres para cor, anos de estudo e nível socioeconômico. Entre os homens foram encontradas diferenças para anos de estudo e nível socioeconômico. O valor energético médio da alimentação dos homens foi significativamente superior ao das mulheres. Entre as mulheres o valor médio foi 1.236,4 kcal e entre os homens foi 1.475,8 kcal. Os valores médios da contribuição percentual de calorias por proteínas, carboidratos e lipídios foram semelhantes entre homens e mulheres. CONCLUSÃO: Os idosos deste estudo apresentam diferenças significativas nas médias do valor energético da alimentação entre os sexos e entre as categorias de anos de estudo e nível socioeconómico. As mulheres apresentaram, ainda, diferenças significativas entre as categorias de cor. A contribuição relativa média dos macronutrientes no valor energético da alimentação foi semelhante entre os sexos e grupos etários.

Elaboração de tabelas de percentis através de parâmetros antropométricos, de desempenho, bioquímicos, hematológicos, hormonais e psicológicos em futebolistas profissionais

INTRODUÇÃO: A carência de valores de referência de parâmetros antropométricos, de desempenho, bioquímicos, hematológicos, hormonais e psicológicos é uma limitação importante nas investigações envolvendo futebolistas profissionais. OBJETIVO: Elaborar tabelas de percentis para servirem como referencial de comparação para estudos posteriores. MÉTODOS: Foram utilizados 82 jogadores profissionais de futebol que foram avaliados aproximadamente 30 dias após o início da principal competição disputada pelas equipes. No primeiro dia de avaliação foram coletadas amostras de sangue (25mL) em jejum para determinação dos parâmetros hematológicos (eritrócitos, hemoglobina, hematócrito, volume corpuscular médio - VCM, hemoglobina corpuscular média - HCM, concentração de hemoglobina corpuscular média - CHCM, leucócitos, neutrófilos, eosinófilos, linfócitos, monócitos e plaquetas) e das concentrações de adrenalina, cortisol, creatina quinase, creatinina, noradrenalina, testosterona e ureia. Posteriormente, os atletas foram submetidos à avaliação antropométrica e psicológica. Em seguida, a avaliação da eficiência do sistema anaeróbio lático foi realizada em pista oficial de atletismo. No segundo dia foram realizadas as avaliações para determinação da eficiência do sistema anaeróbio alático e aeróbio. RESULTADOS: A distribuição de percentis (P0, P15, P30, P50, P70, P85 e P100) foi utilizada para apresentação dos resultados. CONCLUSÃO: A elaboração de tabelas de percentis pode ser utilizada como referencial de comparação para investigações posteriores.

Norepinephrine activates NF-κB transcription factor in cultured rat pineal gland

AIMS: The circadian rhythm in mammalian pineal melatonin secretion is modulated by norepinephrine (NE) released at night. NE interaction with β1-adrenoceptors activates PKA that phosphorylates the transcription factor CREB, leading to the transcription and translation of the arylalkylamine-N-acetyltransferase (AANAT) enzyme. Several studies have reported the interplay between CREB and the nuclear factor-κB (NF-κB) and a circadian rhythm for this transcription factor was recently described in the rat pineal gland. In this work we studied a direct effect of NE on NF-κB activation and the role played by this factor on melatonin synthesis and Aanat transcription and activity. MAIN METHODS: Cultured rat pineal glands were incubated in the presence of two different NF-κB inhibitors, pyrrolidine-dithiocarbamate or sodium salicylate, and stimulated with NE. Melatonin content was quantified by HPLC with electrochemical detection. AANAT activity was measured by a radiometric assay and the expression of Aanat mRNA was analyzed by real-time PCR. Gel shift assay was performed to study the NF-κB activation in cultured rat pineal glands stimulated by NE. KEY FINDINGS: Our results showed that the p50/p50 homodimer of NF-κB is activated by NE and that it has a role in melatonin synthesis, acting on Aanat transcription and activity. SIGNIFICANCE: Here we present evidence that NF-κB is an important transcription factor that acts, directly or indirectly, on Aanat transcription and activity leading to a modulation of melatonin synthesis. NE plays a role in the translocation of NF-κB p50/p50 homodimer to the nucleus of pinealocytes, thus probably influencing the nocturnal pineal melatonin synthesis

Why do arms extract less oxygen than legs during exercise?

[EN] To determine whether conditions for O2 utilization and O2 off-loading from the hemoglobin are different in exercising arms and legs, six cross-country skiers participated in this study. Femoral and subclavian vein blood flow and gases were determined during skiing on a treadmill at approximately 76% maximal O2 uptake (V(O2)max) and at V(O2)max with different techniques: diagonal stride (combined arm and leg exercise), double poling (predominantly arm exercise), and leg skiing (predominantly leg exercise). The percentage of O2 extraction was always higher for the legs than for the arms. At maximal exercise (diagonal stride), the corresponding mean values were 93 and 85% (n = 3; P < 0.05). During exercise, mean arm O2 extraction correlated with the P(O2) value that causes hemoglobin to be 50% saturated (P50: r = 0.93, P < 0.05), but for a given value of P50, O2 extraction was always higher in the legs than in the arms. Mean capillary muscle O2 conductance of the arm during double poling was 14.5 (SD 2.6) ml.min(-1).mmHg(-1), and mean capillary P(O2) was 47.7 (SD 2.6) mmHg. Corresponding values for the legs during maximal exercise were 48.3 (SD 13.0) ml.min(-1).mmHg(-1) and 33.8 (SD 2.6) mmHg, respectively. Because conditions for O2 off-loading from the hemoglobin are similar in leg and arm muscles, the observed differences in maximal arm and leg O2 extraction should be attributed to other factors, such as a higher heterogeneity in blood flow distribution, shorter mean transit time, smaller diffusing area, and larger diffusing distance, in arms than in legs.

Strahlungsinduzierte Mukositis als Risiko der Raumfahrt : Modelluntersuchungen an Röntgen- und Schwerionen-bestrahlten organotypischen Mundschleimhaut-Modellen

Während ausgedehnter Weltraumflüge zum Mond und Mars wäre die Besatzung eines Raumschiffs dicht ionisierender Schwerionenstrahlung, der sogenannten kosmischen Strahlung, ausgesetzt. Da bei vielen Krebspatienten die orale Mukositis als eine gravierende Nebenwirkung bei der herkömmlichen Strahlentherapie auftritt, könnte diese Erkrankung ein medizinisches Problem während ausgedehnter Weltraumflüge darstellen. Allerdings liegen bislang keine Untersuchungen über eine mögliche Mukositis-induzierte Wirkung von Schwerionenstrahlung vor. Um das Risiko strahlungsinduzierter oraler Mukositis durch kosmische Strahlung abzuschätzen, wurden dreidimensionale organotypische Mundschleimhaut-Modelle 12C Schwerionen- bzw. Röntgenstrahlung ausgesetzt und nach definierten Inkubationszeiten kryokonserviert. Aus dem Material wurden Kryoschnitte gefertigt, mit denen anschließend histologische und immunhistologische Fluoreszenzfärbungen zum Nachweis von zum Beispiel Kompaktheitsverlust, Doppelstrangbrüchen der DNA und NF-κB-Aktivierung durchgeführt wurden. Die Ausschüttung von Tumornekrosefaktor α, Interleukin 1β, Interleukin 6 und Interleukin 8 wurde in Probenüberständen mittels ELISA analysiert. Das Hauptaugenmerk dieser Studie lag dabei auf den frühen durch Strahlung verursachten Effekten. rnIm Rahmen dieser Dissertation wurde ein dreidimensionales Mundschleimhaut-Modell etabliert, das verglichen mit humaner Mundschleimhaut viele organotypische Differenzierungsmarker und in vivo-ähnliche Reaktionen auf bestimmte Reize zeigte. Nach einer Bestrahlung mit schweren Kohlenstoffionen bzw. mit Röntgenstrahlung wurden strahlungsinduzierte Effekte auf mehreren Ebenen detektiert. Bereits 1 h nach Bestrahlung konnten dosisabhängig vermehrte Doppelstrangbrüche in der DNA detektiert werden, wobei bei gleicher Dosis Schwerionenstrahlung im Durchschnitt doppelt so viele DSB verursachte wie Röntgenstrahlung. Das Mundschleimhaut-Modell reagierte auf beide Strahlungsarten mit einer Aktivierung des Transkriptionsfaktors NF-κB p50, wobei Röntgenstrahlung diese Aktivierung früher induzierte als Schwerionen. Eine strahlungsinduzierte Aktivierung von NF-κB p65 wurde in den organotypischen Kulturen zu den untersuchten Zeitpunkten nicht beobachtet. Im Vergleich zu nicht bestrahlten Kulturen waren die Konzentrationen von Interleukin 6 und Interleukin 8 unabhängig von der Strahlungsqualität nach Bestrahlung signifikant erhöht. Interleukin-1β dagegen wurde nur nach Röntgenbestrahlung signifikant vermehrt ausgeschüttet. In Schwerionen-bestrahlten Proben wurden zwar tendenziell höhere Konzentrationen beobachtet, statistische Signifikanzen ergaben sich aber nicht. Die Analysen zur TNF-α- und IFN-γ-Ausschüttung in bestrahlten organotypischen Kulturen ergaben innerhalb des gewählten Beobachtungszeitraums von 48 h keine strahlungsinduzierten Effekte. Bei den Untersuchungen der Proliferation in den Zellen der bestrahlten organotypischen Kulturen wurde bereits 24 h nach Röntgenstrahlung und 3 Tage nach Schwerionenstrahlung eine deutliche verminderte Proliferation beobachtet. Des Weiteren zeigte das Mundschleimhaut-Modell unabhängig von Strahlungsqualitäten einen eindeutigen Verlust in der Zellintegrität und daraus resultierenden Kompaktheitsverlust des Gewebes, was in der in vivo-Situation wahrscheinlich einer Gewebedegeneration entspricht. rnNach Bestrahlung mit sowohl Röntgenstrahlung als auch schweren Ionen wurden im gewählten Mukosa-Modell innerhalb von 48 h nach Behandlung strahlungsinduzierte proinflammatorische Marker eindeutig und reproduzierbar detektiert. Dies deutet darauf hin, dass während langer extraterrestrischer Expeditionen das Risiko der oralen Mukositis einkalkuliert und mit den daraus folgenden Konsequenzen gerechnet werden muss.rn

Reduced nuclear translocation of nuclear factor (NF)-kappaB p65 in the footpad epidermis of dogs infected with distemper virus

Infection of canine footpads with the canine distemper virus (CDV) can cause massive epidermal thickening (hard pad disease), as a consequence of increased proliferation of keratinocytes and hyperkeratosis. Keratinocytes of canine footpad epidermis containing detectable CDV nucleoprotein antigen and CDV mRNA were shown previously to have increased proliferation indices. Because various proteins that play a role in the proliferation of epidermal cells are viral targets, the potential participation of such proteins in CDV-associated keratinocyte proliferation was investigated. Transforming growth factor-alpha (TGF-alpha), cell cycle regulatory proteins p21, p27 and p53, and nuclear factor (NF)-kappaB transcription factor components p50 and p65 were studied in the footpad epidermis from the following groups of dogs inoculated with CDV: group 1, consisting of seven dogs with clinical distemper and CDV in the footpad epidermis; group 2, consisting of four dogs with clinical distemper but no CDV in the footpad epidermis; group 3, consisting of eight dogs with neither clinical distemper nor CDV in the footpad epithelium. Group 4 consisted of two uninoculated control dogs. The expression of TGF-alpha, p21, p27 and p53, and p50 in the basal layer, lower and upper spinous layers, and in the granular layer did not differ statistically between CDV-positive (group 1) and CDV-negative (groups 2-4) footpad epidermis. However, there were differences in the levels of nuclear and cytoplasmic p65 expression between group 1 dogs and the other three groups. Thus, footpads from group 1 dogs had more keratinocytes containing p65 in the cytoplasm and, conversely, fewer nuclei that were positive for p65. These findings indicate that p65 translocation into the nucleus is reduced in CDV-infected footpad epidermis. Such decreased translocation of p65 may help to explain increased keratinocyte proliferation in hard pad disease and suggests interference of CDV with the NF-kappaB pathway.

Association of alcohol craving and proximal outcomes of a residential treatment program for patients with alcohol use disorders

Background: Alcohol craving is an essential construct in research and treatment of alcohol use disorders (AUD). Craving is mostly investigated in association with concurrent variables or distal treatment outcomes at follow-up. Objectives: The aim of this study is to examine craving at admission and its relevance for essential proximal outcomes at discharge from AUD treatment such as positive alcohol expectancy, abstinent-related self-efficacy, and substance-related coping, as well as patients’ demographic and AUD characteristics. Methods: In total, 36 patients were recruited within an inpatient treatment AUD program. Results: An association between craving and positive alcohol expectancies at discharge was found in the regression model even when the respective expectancies, age, gender, and severity of alcohol dependence at admission were controlled for (F(2,29)1⁄432.71, p50.001). Craving explained 2.3% of the variance of change in positive alcohol expectancy. Conclusion: The results suggest a low predictive value of craving for positive alcohol expectancy. In addition, we found significant associations between the craving and the severity of AUD and alcohol consumption before admission. Future studies should include proximal outcomes related to treatment efficacy as well as distal outcomes.

Investigation of the deregulation of interleukin-6 in two non-Hodgkin's lymphoma cell lines

Increased serum interleukin-6 (IL-6) is a poor prognostic factor for patients with lymphoma. This may be related to the fact that IL-6 has been shown to be an autocrine and paracrine growth factor for lymphoma cells. We have investigated the regulation of IL-6 in two lymphoma cell lines which produce IL-6 as an autocrine growth factor. The cell lines, LY3 and LY12, were established from two patients with non-Hodgkin's lymphoma. One patient had diffuse large cell lymphoma (LY3), whereas the other had small noncleaved cell lymphoma (LY12). There was no rearrangement or amplification of the IL-6 gene, but we detected IL-1 alpha and TNF production in addition to IL-6. We investigated the effect of inhibitors of IL-1 and TNF on IL-6 production in LY3 and LY12. Our results show that IL-6 production is mainly secondary to endogenous IL-1 production in LY3 cells, however LY12 cells produce IL-6 via a different mechanism since neither anti-IL-1 nor anti-TNF significantly inhibited IL-6 production.^ Transfection of LY12 cells with wildtype and mutant IL-6 promoter-chloramphenicol acetyl transferase constructs, showed increased activity of a trans-acting factor that binds to the NF-kB motif. Therefore, we determined whether there were abnormalities in members of the NF-kB family of transcription factors, such as p65, p50, p52/lyt-10 or rel, which bind to kB motifs. We found increased expression of the p52/lyt-10 transcription factor and activation of the NF-kB pathway in LY12. However, expression of p50, p65 and rel was not increased in LY12 cells. Future investigations could be aimed at determining the effect of inhibitors of NF-kB on IL-6 production. ^