1,625 research outputs found

    Promiscuous and lineage-specific roles of cell cycle regulators in haematopoiesis

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    Haematopoietic cell number is maintained by a delicate balance between cell proliferation, differentiation and death. Gene knockout studies in mice have revealed the complex roles of cyclins, CDKs, and CDK inhibitors in regulating cell proliferation and differentiation in the haematopoietic system. These studies point to families of cell cycle regulators which display both redundant and unique roles within a lineage and developmental-stage specific manner. Moreover, the promiscuity of these cell cycle regulators is critical for haematopoietic cell proliferation and differentiation. In this review, we discuss the current evidence from mouse models that the complexity and multifarious nature of the haematopoietic system is critical for its form and function

    Fabrication and material characterization of silver cantilevers via direct surface micromachining

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    Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2008.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.Includes bibliographical references.Microelectromechanical Systems (MEMS) rely heavily on the semiconductor industry's manufacturing paradigm. While the standardized process model allows semiconductor chips to benefit from economy of scale and be sold at low prices, MEMS devices use specialized processes and subsequently have to be sold at higher prices. This severely hinders MEMS development because it is not economically feasible to research and develop specialized devices where only small volumes are needed. As such, tools and processes which divorce MEMS fabrication from this paradigm are needed. Using Hewlett-Packard thermal inkjet technology mounted to an X-Y microcontroller stage, we present a mask-less, or direct, surface micromachining process flow with a 250°C thermal budget. The process uses Cabot Corp.'s silver-based conductive ink for the structural layer and PMMA for the sacrificial layer. Several other materials were tested for use as sacrificial inks in addition to PMMA. Silver cantilevers with dimensions of 200x50[mu]m and 200x100[mu]m were fabricated as a demonstration of the process. The silver cantilevers were mechanically characterized by using force-deflection measurements made by a P-10 contact profilometer or a Hysitron nanoindentor. We present findings of 21.9±1.50GPa or 22±1.5GPa for the silver ink's Young's modulus of elasticity, depending on the characterization method. These measurements were consistent with results measured by nanoindentating Cabot silver films. We hypothesize that the film's porosity is the cause of the silver's reduced material properties. Some preliminary data supporting this hypothesis is provided, and potential methods of improving the material properties and the surface micromachining process are discussed.by Eric W. Lam.S.M

    Rac1 signalling modulates a STAT5/BCL-6 transcriptional switch on cell cycle-associated target gene promoters

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    Gene expression depends on binding of transcriptional regulators to gene promoters, a process controlled by signalling pathways. The transcriptional repressor B-cell lymphoma (BCL)-6 downregulates genes involved in cell-cycle progression and becomes inactivated following phosphorylation by the Rac1 GTPase-activated protein kinase PAK1. Interestingly, the DNA motifs recognized by BCL-6 and signal transducers and activators of transcription 5 (STAT5) are similar. Because STAT5 stimulation in epithelial cells can also be triggered by Rac1 signalling, we asked whether both factors have opposing roles in transcriptional regulation and whether Rac1 signalling may coordinate a transcription factor switch. We used chromatin immunoprecipitation to show that active Rac1 promotes release of the repressor BCL-6 while increasing binding of STAT5A to a BCL-6-regulated reporter gene. We further show in colorectal cell lines that the endogenous activation status of the Rac1/PAK1 pathway correlated with the phosphorylation status of BCL-6 and STAT5A. Three cellular genes (cyclin D2, p15INK4B, small ubiquitin-like modifier 1) were identified to be inversely regulated by BCL-6 and STAT5A and responded to Rac1 signalling with increased expression and corresponding changes in promoter occupancy. Together, our data show that Rac1 signalling controls a group of target genes that are repressed by BCL-6 and activated by STAT5A, providing novel insights into the modulation of gene transcription by GTPase signalling.Fundação para a Ciência e Tecnologia, Portugal [PPCDT/SAU-OBS/57660/2004] (to P.J.), [PTDC/SAU-GMG/119586/2010] (to P.M.), [PEst-OE/BIA/UI4046/2011] (to the BioFig research unit

    Dynamic Microclimate Boundaries across a Sharp Tropical Rainforest–Clearing Edge

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    As landscapes become increasingly fragmented, research into impacts from disturbance and how edges affect vegetation and community structure has become more important. Descriptive studies on how microclimate changes across sharp transition zones have long existed in the literature and recently more attention has been focused on understanding the dynamic patterns of microclimate associated with forest edges. Increasing concern about forest fragmentation has led to new technologies for modeling forest microclimates. However, forest boundaries pose important challenges to not only microclimate modeling but also sampling regimes in order to capture the diurnal and seasonal dynamic aspects of microclimate along forest edges. We measured microclimatic variables across a sharp boundary from a clearing into primary lowland tropical rainforest at La Selva Biological Station in Costa Rica. Dynamic changes in diurnal microclimate were measured along three replicated transects, approximately 30 m in length with data collected every 1 m continuously at 30 min intervals for 24 h with a mobile sensor platform supported by a cable infrastructure. We found that a first-order polynomial fit using piece-wise regression provided the most consistent estimation of the forest edge, relative to the visual edge, although we found no best sensing parameter as all measurements varied. Edge location estimates based on daytime net shortwave radiation had less difference from the visual edge than other shortwave measurements, but estimates made throughout the day with downward-facing or net infrared radiation sensors were more consistent and closer to the visual edge than any other measurement. This research contributes to the relatively small number of studies that have directly measured diurnal temporal and spatial patterns of microclimate variation across forest edges and demonstrates the use of a flexible mobile platform that enables repeated, high-resolution measurements of gradients of microclimate

    Sustained Spindle-Assembly Checkpoint Response Requires De Novo Transcription and Translation of Cyclin B1

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    Background Microtubule-targeting drugs induce mitotic delay at pro-metaphase by preventing the spindle assembly checkpoint to be satisfied. However, especially after prolonged treatments, cells can escape this arrest in a process called mitotic slippage. The mechanisms underlying the spindle assembly checkpoint and slippage are not fully understood. It has been generally accepted that during mitosis there is a temporary shutdown of high-energy-consuming processes, such as transcription and translation. However, the synthesis of specific proteins is maintained or up-regulated since protein synthesis is necessary for entry into and progression through mitosis. Methodology/Principal Findings In this work we investigated whether the mitotic arrest caused by the mitotic checkpoint is independent of transcription and translation. By using immunofluorescent microscopy and western blotting, we demonstrate that inhibition of either of these processes induces a shortening of the mitotic arrest caused by the nocodazole treatment, and ultimately leads to mitotic slippage. Our western blotting and RTQ-PCR results show that inhibition of transcription during mitotic arrest does not affect the expression of the spindle checkpoint proteins, whereas it induces a significant decrease in the mRNA and protein levels of Cyclin B1. The exogenous expression of Cyclin B1 substantially rescued the mitotic phenotype in nocodazole cells treated with the inhibitors of transcription and translation. Conclusions/Significance This work emphasizes the importance of transcription and translation for the maintenance of the spindle assembly checkpoint, suggesting the existence of a mechanism dependent on cyclin B1 gene regulation during mitosis. We propose that continuous transcription of mitotic regulators is required to sustain the activation of the spindle assembly checkpoint

    FOXM1 is overexpressed in B-acute lymphoblastic leukemia (B-ALL) and its inhibition sensitizes B-ALL cells to chemotherapeutic drugs

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    The Forkhead box protein M1 (FOXM1) is a transcription factor that plays a central role in the regulation of cell cycle, proliferation, DNA repair, and apoptosis. FOXM1 is overexpressed in many human tumors and its upregulation has been linked to high proliferation rates and poor prognosis. We therefore studied the role of FOXM1 in B-lymphoblastic leukemia (B-ALL) in order to understand whether FOXM1 could be a key target for leukemia therapy. RT-PCR and western blot analysis were carried out in a small cohort of pediatric B-ALL patients to evaluate FOXM1 levels. To assess its biological relevance, its expression was down-modulated by transient RNA interference in B-ALL cell lines (REH and NALM-6). Our results show that FOXM1 expression is higher in both B-ALL patients and cell lines when compared to PBMC or normal B-cells (CD19+) from healthy donors. Furthermore, blocking FOXM1 activity in two B-ALL cell lines, by either knockdown or treatment with the FOXM1 inhibitor thiostrepton, causes significant decrease in their cell proliferation. This decrease in cell proliferation was coupled with both an induction of the G2/M cell cycle arrest and with a reduction in the S phase population. Finally, we noted how thiostrepton synergises with chemotherapeutic agents commonly used in B-ALL therapy, thus increasing their efficiency. Therefore our results suggest that FOXM1 is highly expressed in both patients and B-ALL cell lines, and that targeting FOXM1 could be an attractive strategy for leukemia therapy and for overcoming drug resistance

    The human papillomavirus type 16 E5 oncoprotein synergizes with EGF-receptor signaling to enhance cell cycle progression and the down-regulation of p27Kip1

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    AbstractE5 oncoprotein activity from high risk human papillomaviruses (HPVs) is associated with growth factor receptor signaling, but the function of this protein is not well understood. In this study, we investigated the role of HPV-16 E5 on the cell cycle progression during EGF-stimulation. Wild-type and NIH 3T3 cells over-expressing human EGF-receptor were transfected with HPV-16 E5 gene and the cell cycle progression was characterized. This analysis showed that the E5-expressing cells increased DNA synthesis (S-phase) by around 40%. Cell cycle protein analysis of E5-expressing cells showed a reduction in the half-life of p27Kip1 protein as compared to control cells (18.4 vs. 12.7 h), an effect that was enhanced in EGF-stimulated cells (12.8 vs. 3.6 h). Blockage of EGF-receptor activity abrogated E5 signals as well as p27Kip1 down-regulation. These results suggest that E5 and the EGF-receptor cooperate to enhance cell cycle entry and progression through regulating p27Kip1 expression at protein level

    SUMOylation inhibits FOXM1 activity and delays mitotic transition

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    The forkhead box transcription factor FOXM1 is an essential effector of G2/M-phase transition, mitosis and the DNA damage response. As such, it is frequently deregulated during tumorigenesis. Here we report that FOXM1 is dynamically modified by SUMO1 but not by SUMO2/3 at multiple sites. We show that FOXM1 SUMOylation is enhanced in MCF-7 breast cancer cells in response to treatment with epirubicin and mitotic inhibitors. Mutation of five consensus conjugation motifs yielded a SUMOylation-deficient mutant FOXM1. Conversely, fusion of the E2 ligase Ubc9 to FOXM1 generated an auto-SUMOylating mutant (FOXM1-Ubc9). Analysis of wild-type FOXM1 and mutants revealed that SUMOylation inhibits FOXM1 activity, promotes translocation to the cytoplasm and enhances APC/Cdh1-mediated ubiquitination and degradation. Further, expression of the SUMOylation-deficient mutant enhanced cell proliferation compared with wild-type FOXM1, whereas the FOXM1-Ubc9 fusion protein resulted in persistent cyclin B1 expression and slowed the time from mitotic entry to exit. In summary, our findings suggest that SUMOylation attenuates FOXM1 activity and causes mitotic delay in cytotoxic drug response
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