Disturbed Clockwork Resetting in Sharp-1 and Sharp-2 Single and Double Mutant Mice

BACKGROUND: The circadian system provides the basis to anticipate and cope with daily recurrent challenges to maintain the organisms' homeostasis. De-synchronization of circadian feedback oscillators in humans causes 'jet lag', likely contributes to sleep-, psychiatric-, metabolic disorders and even cancer. However, the molecular mechanisms leading to the disintegration of tissue-specific clocks are complex and not well understood. METHODOLOGY/PRINCIPAL FINDINGS: Based on their circadian expression and cell culture experiments, the basic Helix-Loop-Helix (bHLH) transcription factors SHARP-1(Dec2) and SHARP-2(Stra13/Dec1) were proposed as novel negative regulators of the molecular clock. To address their function in vivo, we generated Sharp-1 and Sharp-2 single and double mutant mice. Our experiments reveal critical roles for both factors in regulating period length, tissue-specific control of clock gene expression and entrainment to external cues. Light-pulse experiments and rapid delays of the light-dark cycle (experimental jet lag) unravel complementary functions for SHARP-1 and SHARP-2 in controlling activity phase resetting kinetics. Moreover, we show that SHARP-1 and 2 can serve dual functions as repressors and co-activators of mammalian clock gene expression in a context-specific manner. This correlates with increased amplitudes of Per2 expression in the cortex and liver and a decrease in the suprachiasmatic nucleus (SCN) of double mutant mice. CONCLUSIONS/SIGNIFICANCE: The existence of separate mechanisms regulating phase of entrainment, rhythm amplitude and period length has been postulated before. The differential effects of Sharp-deficiency on rhythmicity and behavioral re-entrainment, coupled to tissue-dependent regulatory functions, provide a new mechanistic basis to further understand the complex process of clock synchronizations

Aurora-A overexpression enhances cell-aggregation of Ha-ras transformants through the MEK/ERK signaling pathway

<p>Abstract</p> <p>Background</p> <p>Overexpression of Aurora-A and mutant Ras (Ras^V12) together has been detected in human bladder cancer tissue. However, it is not clear whether this phenomenon is a general event or not. Although crosstalk between Aurora-A and Ras signaling pathways has been reported, the role of these two genes acting together in tumorigenesis remains unclear.</p> <p>Methods</p> <p>Real-time PCR and sequence analysis were utilized to identify Ha- and Ki-<it>ras </it>mutation (Gly -> Val). Immunohistochemistry staining was used to measure the level of Aurora-A expression in bladder and colon cancer specimens. To reveal the effect of overexpression of the above two genes on cellular responses, mouse NIH3T3 fibroblast derived cell lines over-expressing either Ras^V12and wild-type Aurora-A (designated WT) or Ras^V12and kinase-inactivated Aurora-A (KD) were established. MTT and focus formation assays were conducted to measure proliferation rate and focus formation capability of the cells. Small interfering RNA, pharmacological inhibitors and dominant negative genes were used to dissect the signaling pathways involved.</p> <p>Results</p> <p>Overexpression of wild-type Aurora-A and mutation of Ras^V12were detected in human bladder and colon cancer tissues. Wild-type Aurora-A induces focus formation and aggregation of the Ras^V12transformants. Aurora-A activates Ral A and the phosphorylation of AKT as well as enhances the phosphorylation of MEK, ERK of WT cells. Finally, the Ras/MEK/ERK signaling pathway is responsible for Aurora-A induced aggregation of the Ras^V12transformants.</p> <p>Conclusion</p> <p>Wild-type-Aurora-A enhances focus formation and aggregation of the Ras^V12transformants and the latter occurs through modulating the Ras/MEK/ERK signaling pathway.</p

Aurora kinases are expressed in medullary thyroid carcinoma (MTC) and their inhibition suppresses in vitro growth and tumorigenicity of the MTC derived cell line TT

International audienceBACKGROUND: The Aurora kinase family members, Aurora-A, -B and -C, are involved in the regulation of mitosis, and alterations in their expression are associated with cell malignant transformation. To date no information on the expression of these proteins in medullary thyroid carcinoma (MTC) are available. We here investigated the expression of the Aurora kinases in human MTC tissues and their potential use as therapeutic targets. METHODS: The expression of the Aurora kinases in 26 MTC tissues at different TNM stages was analyzed at the mRNA level by quantitative RT-PCR. We then evaluated the effects of the Aurora kinase inhibitor MK-0457 on the MTC derived TT cell line proliferation, apoptosis, soft agar colony formation, cell cycle and ploidy. RESULTS: The results showed the absence of correlation between tumor tissue levels of any Aurora kinase and tumor stage indicating the lack of prognostic value for these proteins. Treatment with MK-0457 inhibited TT cell proliferation in a time- and dose-dependent manner with IC50 = 49.8 ± 6.6 nM, as well as Aurora kinases phosphorylation of substrates relevant to the mitotic progression. Time-lapse experiments demonstrated that MK-0457-treated cells entered mitosis but were unable to complete it. Cytofluorimetric analysis confirmed that MK-0457 induced accumulation of cells with ≥ 4N DNA content without inducing apoptosis. Finally, MK-0457 prevented the capability of the TT cells to form colonies in soft agar. CONCLUSIONS: We demonstrate that Aurora kinases inhibition hampered growth and tumorigenicity of TT cells, suggesting its potential therapeutic value for MTC treatment

Extracellular signal-regulated kinase 1/2 activity is not required in mammalian cells during late G2 for timely entry into or exit from mitosis

Author Posting. © American Society for Cell Biology, 2006. This article is posted here by permission of American Society for Cell Biology for personal use, not for redistribution. The definitive version was published in Molecular Biology of the Cell 17 (2006): 5227-5240, doi:10.1091/mbc.E06-04-0284.Extracellular signal-regulated kinase (ERK)1/2 activity is reported to be required in mammalian cells for timely entry into and exit from mitosis (i.e., the G2-mitosis [G2/M] and metaphase-anaphase [M/A] transitions). However, it is unclear whether this involvement reflects a direct requirement for ERK1/2 activity during these transitions or for activating gene transcription programs at earlier stages of the cell cycle. To examine these possibilities, we followed live cells in which ERK1/2 activity was inhibited through late G2 and mitosis. We find that acute inhibition of ERK1/2 during late G2 and through mitosis does not affect the timing of the G2/M or M/A transitions in normal or transformed human cells, nor does it impede spindle assembly, inactivate the p38 stress-activated checkpoint during late G2 or the spindle assembly checkpoint during mitosis. Using CENP-F as a marker for progress through G2, we also show that sustained inhibition of ERK1/2 transiently delays the cell cycle in early/mid-G2 via a p53-dependent mechanism. Together, our data reveal that ERK1/2 activity is required in early G2 for a timely entry into mitosis but that it does not directly regulate cell cycle progression from late G2 through mitosis in normal or transformed mammalian cells.This research was supported by National Institutes of Health Grant GMS-40198 to C.L.R., by National Institutes of Health/National Cancer Institute Grant CA109182, and Samuel Waxman Cancer Research Foundation grants to J.A.A.-G

Rasd1 Modulates the Coactivator Function of NonO in the Cyclic AMP Pathway

All living organisms exhibit autonomous daily physiological and behavioural rhythms to help them synchronize with the environment. Entrainment of circadian rhythm is achieved via activation of cyclic AMP (cAMP) and mitogen-activated protein kinase signaling pathways. NonO (p54nrb) is a multifunctional protein involved in transcriptional activation of the cAMP pathway and is involved in circadian rhythm control. Rasd1 is a monomeric G protein implicated to play a pivotal role in potentiating both photic and nonphotic responses of the circadian rhythm. In this study, we have identified and validated NonO as an interacting partner of Rasd1 via affinity pulldown, co-immunoprecipitation and indirect immunofluorescence studies. The GTP-hydrolysis activity of Rasd1 is required for the functional interaction. Functional interaction of Rasd1-NonO in the cAMP pathway was investigated via reporter gene assays, chromatin immunoprecipitation and gene knockdown. We showed that Rasd1 and NonO interact at the CRE-site of specific target genes. These findings reveal a novel mechanism by which the coregulator activity of NonO can be modulated

CELLA DI CARICO CON SENSORI IN FIBRA OTTICA A RETICOLO DI BRAGG

Dispositivo per misurare un carico. Il dispositivo comprende un primo elemento ed un secondo elemento per ricevere un carico. Un elemento elastico è interposto tra il primo elemento e il secondo elemento, e vincolato a questi mediante due estremità tra loro opposte, in modo tale da permettere la deformazione a flessione di detto elemento elastico quando il carico è applicato al primo elemento o al secondo elemento. Almeno un sensore in fibra ottica è fissato ad una faccia dell’elemento elastico rivolta verso uno tra il primo ed il secondo elemento, per misurare una deformazione dell’elemento elastico. L’elemento elastico è sagomato in modo tale che, applicando il carico al primo elemento o al secondo elemento, la faccia che porta il sensore subisce una deformazione sostanzialmente costante lungo tutta la lunghezza del sensore. Viene anche descritto un veicolo ferroviario che fa uso di un tale dispositivo per misurare la forza scambiata tra il pantografo del treno ed una catenaria

Direct Measurement of Power on a Gravity Independent Flywheel-based ErgometerSpecial Topics in Structural Dynamics, Volume 6

YoYo Technology™ commercializes a training instrument called YoYo Squat™, a gravity independent flywheel-based ergometer, that allows to train quadriceps and adductors as well as supporting trunk and core muscle groups unloading the shoulder and spine. This ergometer, however, is equipped only with an optical sensor (encoder) to measure the rotation of the flywheel thus making it almost impossible to determine the instantaneous power generated by the athlete as well as the force as a function of the number of repetitions. It was therefore decided to equip the YoYo Squat™ with miniaturized single axis load cells, a linear position transducer parallel to the strap and strain gauge force plates for both feet. This has allowed to assess not only the above mentioned quantities but also the point of application of the normal load on the two feet thus further improving the training output and the feedback to both the athlete and the trainee for improved performance and/or more focused training