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https://www.readbyqxmd.com/read/28049774/synchronization-and-arrest-of-the-budding-yeast-cell-cycle-using-chemical-and-genetic-methods
#1
Adam P Rosebrock
The cell cycle of budding yeast can be arrested at specific positions by different genetic and chemical methods. These arrests enable study of cell cycle phase-specific phenotypes that would be missed during examination of asynchronous cultures. Some methods for arrest are reversible, with kinetics that enable release of cells back into a synchronous cycling state. Benefits of chemical and genetic methods include scalability across a large range of culture sizes from a few milliliters to many liters, ease of execution, the absence of specific equipment requirements, and synchronization and release of the entire culture...
January 3, 2017: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/28017899/nocodazole-treatment-interrupted-brucella-abortus-invasion-in-raw-264-7-cells-and-successfully-attenuated-splenic-proliferation-with-enhanced-inflammatory-response-in-mice
#2
Alisha Wehdnesday Bernardo Reyes, Huynh Tan Hop, Lauren Togonon Arayan, Tran Xuan Ngoc Huy, Wongi Min, Hu Jang Lee, Hong Hee Chang, Suk Kim
Brucellosis is one of the most important and widespread zoonosis worldwide responsible for serious economic losses and considerable public health burden. In this study, we investigated the modulatory effect of a microtubule-inhibitor, nocodazole, on B. abortus infection in murine macrophages and in a mouse model. Nocodazole activated macrophages and directly inhibited the growth of Brucella in a dose-dependent manner. Nocodazole increased adhesion but reduced invasion and intracellular growth of Brucella in macrophages although it did not affect co-localization of Brucella with LAMP-1...
December 23, 2016: Microbial Pathogenesis
https://www.readbyqxmd.com/read/28011649/newcastle-disease-virus-induces-stable-formation-of-bona-fide-stress-granules-to-facilitate-viral-replication-through-manipulating-host-protein-translation
#3
Yingjie Sun, Luna Dong, Shengqing Yu, Xiaoxu Wang, Hang Zheng, Pin Zhang, Chunchun Meng, Yuan Zhan, Lei Tan, Cuiping Song, Xusheng Qiu, Guijun Wang, Ying Liao, Chan Ding
Mammalian cells respond to various environmental stressors to form stress granules (SGs) by arresting cytoplasmic mRNA, protein translation element, and RNA binding proteins. Virus-induced SGs function in different ways, depending on the species of virus; however, the mechanism of SG regulation of virus replication is not well understood. In this study, Newcastle disease virus (NDV) triggered stable formation of bona fide SGs on HeLa cells through activating the protein kinase R (PKR)/eIF2α pathway. NDV-induced SGs contained classic SG markers T-cell internal antigen (TIA)-1, Ras GTPase-activating protein-binding protein (G3BP)-1, eukaryotic initiation factors, and small ribosomal subunit, which could be disassembled in the presence of cycloheximide...
December 23, 2016: FASEB Journal: Official Publication of the Federation of American Societies for Experimental Biology
https://www.readbyqxmd.com/read/28004475/structure-based-approaches-for-the-design-of-benzimidazole-2-carbamate-derivatives-as-tubulin-polymerization-inhibitors
#4
Rodrigo Aguayo-Ortiz, Lucia Cano-González, Rafael Castillo, Alicia Hernández-Campos, Laura Domínguez
Microtubules are highly dynamic assemblies of α/β-tubulin heterodimers whose polymerization inhibition is among one of the most successful approaches for anticancer drug development. Overexpression of the class I (βI) and class III (βIII) β-tubulin isotypes in breast and lung cancers and the highly expressed class VI (βVI) β-tubulin isotype in normal blood cells have increased the interest for designing specific tubulin-binding anticancer therapies. To this end, we employed our previously proposed model of the β-tubulin-nocodazole complex, supported by the recently determined X-ray structure, to identify the fundamental structural differences between β-tubulin isotypes...
December 22, 2016: Chemical Biology & Drug Design
https://www.readbyqxmd.com/read/28004015/influenza-infection-modulates-vesicular-trafficking-and-induces-golgi-complex-disruption
#5
Vibha Yadav, Antonito T Panganiban, Kerstin Honer Zu Bentrup, Thomas G Voss
Influenza A virus (IFV) replicates its genome in the nucleus of infected cells and uses the cellular protein transport system for genome trafficking from the nucleus to the plasma membrane. However, many details of the mechanism of this process, and its relationship to subsequent cytoplasmic virus trafficking, have not been elucidated. We examined the effect of nuclear transport inhibitors Leptomycin B (LB), 5,6 dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB), the vesicular transport inhibitor Brefeldin A (BFA), the caspase inhibitor ZWEHD, and microtubule inhibitor Nocodazole (NOC) on virus replication and intracellular trafficking of viral nucleoprotein (NP) from the nucleus to the ER and Golgi...
December 2016: Virusdisease
https://www.readbyqxmd.com/read/27997747/dynamics-of-the-sealing-zone-in-cultured-osteoclasts
#6
Sarit Batsir, Benjamin Geiger, Zvi Kam
Bone resorption by osteoclasts depends on the formation and stability of the sealing zone (SZ), a peripheral belt of actin and integrin-based podosomes. Recent studies demonstrated that the SZ is a highly dynamic structure, undergoing cycles of assembly and disassembly. In this study, we explored the mechanisms underlying the regulation of SZ stability and reorganization in osteoclasts cultured on glass slides, and forming an SZ-like podosome belt (SZL). By monitoring this belt in cultured RAW264.7 cells expressing GFP-tagged actin, we show here that SZL stability is usually locally regulated, and its dissociation, occurring mostly in concave segments, is manifested in the loss of both podosome coherence, and actin belt continuity...
December 20, 2016: Cytoskeleton
https://www.readbyqxmd.com/read/27994056/inducible-inhibition-of-g%C3%AE-%C3%AE-reveals-localization-dependent-functions-at-the-plasma-membrane-and-golgi
#7
Lauren M Klayman, Philip B Wedegaertner
Heterotrimeric G proteins signal at a variety of endomembrane locations, in addition to their canonical function at the cytoplasmic surface of the plasma membrane (PM) where they are activated by cell-surface G protein-coupled receptors (GPCRs). Here we focus on βγ signaling at the Golgi, where βγ activates a signaling cascade ultimately resulting in vesicle fission from the trans Golgi network (TGN). To develop a novel molecular tool for inhibiting endogenous βγ in a spatial-temporal manner, we take advantage of a lipid-association mutant of the widely-used βγ inhibitor GRK2ct (GRK2ct-KERE) and the FRB/FKBP heterodimerization system...
December 19, 2016: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/27991556/kif2a-regulates-the-spindle-assembly-and-the-metaphase-i-anaphase-i-transition-in-mouse-oocyte
#8
Ming-Huang Chen, Yu Liu, Ya-Long Wang, Rui Liu, Bai-Hui Xu, Fei Zhang, Fei-Ping Li, Lin Xu, Yan-Hong Lin, Shu-Wen He, Bao-Qiong Liao, Xian-Pei Fu, Xiao-Xue Wang, Xiang-Jun Yang, Hai-Long Wang
KIF2A, a member of the kinesin-13 family, has been reported to play a role in spindle assembly in mitosis. However, its function in mammalian meiosis remains unknown. In this research, we examined the expression, localization and function of KIF2A during mouse oocyte meiosis. KIF2A was expressed in some key stages in mouse oocyte meiosis. Immunofluorescent staining showed that KIF2A distributed in the germinal vesicle at the germinal vesicle stage and as the spindle assembling after meiosis resumption, KIF2A gradually accumulated to the entire spindle...
December 19, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27942359/cytolethal-distending-toxin-from-campylobacter-jejuni-requires-the-cytoskeleton-for-toxic-activity
#9
Estela T Méndez-Olvera, Jaime A Bustos-Martínez, Yolanda López-Vidal, Antonio Verdugo-Rodríguez, Daniel Martínez-Gómez
BACKGROUND: Campylobacter jejuni is one of the major causes of infectious diarrhea worldwide. The distending cytolethal toxin (CDT) of Campylobacter spp. interferes with normal cell cycle progression. This toxic effect is considered a result of DNase activity that produces chromosomal DNA damage. To perform this event, the toxin must be endocytosed and translocated to the nucleus. OBJECTIVES: The aim of this study was to evaluate the role of the cytoskeleton in the translocation of CDT to the nucleus...
October 2016: Jundishapur Journal of Microbiology
https://www.readbyqxmd.com/read/27941677/the-enrichment-of-survivin-in-exosomes-from-breast-cancer-cells-treated-with-paclitaxel-promotes-cell-survival-and-chemoresistance
#10
Bridget T Kreger, Eric R Johansen, Richard A Cerione, Marc A Antonyak
The generation and release of membrane-enclosed packets from cancer cells, called extracellular vesicles (EVs), play important roles in propagating transformed phenotypes, including promoting cell survival. EVs mediate their effects by transferring their contents, which include specific proteins and nucleic acids, to target cells. However, how the cargo and function of EVs change in response to different stimuli remains unclear. Here, we discovered that treating highly aggressive MDAMB231 breast cancer cells with paclitaxel (PTX), a chemotherapy that stabilizes microtubules, causes them to generate a specific class of EV, namely exosomes, that are highly enriched with the cell survival protein and cancer marker, Survivin...
December 9, 2016: Cancers
https://www.readbyqxmd.com/read/27885657/cdc25b-is-involved-in-the-centrosomal-microtubule-nucleation-in-two-cell-stage-mouse-embryos
#11
Cheng Cui, Tianxia Zang, Yu Cao, Xin Qin, Xuewei Zhang
CDC25B has been demonstrated to activate the complex of CDK1/Cyclin B and trigger mitosis. We have recently demonstrated that p-CDC25B-Ser351 is located at the centrosomes of mouse oocytes and contributes to the release of mouse oocytes from prophase I arrest. But much less is known about CDC25B function at the centrosome in two-cell stage mouse embryos. Here we investigate the effect of CDC25B regulating the microtubules nucleation. Microinjection of anti-CDC25B antibody caused aberrant microtubule nucleation...
December 2016: Development, Growth & Differentiation
https://www.readbyqxmd.com/read/27875556/mxa-is-a-novel-regulator-of-endosome-associated-transcriptional-signaling-by-bone-morphogenetic-proteins-4-and-9-bmp4-and-bmp9
#12
Huijuan Yuan, Pravin B Sehgal
There is confusion about the role that IFN-α plays in the pathogenesis of pulmonary arterial hypertension (PAH) with different investigators reporting a causative or a protective role. There is now clear evidence in PAH pathogenesis for the involvement of BMP4 and BMP9 signaling, and its disruption by mutations in BMPR2. In the present study, we investigated MxA, an IFN-α-inducible cytoplasmic dynamin-family GTPase for effects on BMP4/9 signaling, including in the presence of PAH-disease-associated mutants of BMPR2...
2016: PloS One
https://www.readbyqxmd.com/read/27833610/tcr-triggering-induces-the-formation-of-lck-rack1-actinin-1-multiprotein-network-affecting-lck-redistribution
#13
Ondřej Ballek, Jan Valečka, Martina Dobešová, Adéla Broučková, Jasper Manning, Pavel Řehulka, Jiří Stulík, Dominik Filipp
The initiation of T-cell signaling is critically dependent on the function of the member of Src family tyrosine kinases, Lck. Upon T-cell antigen receptor (TCR) triggering, Lck kinase activity induces the nucleation of signal-transducing hubs that regulate the formation of complex signaling network and cytoskeletal rearrangement. In addition, the delivery of Lck function requires rapid and targeted membrane redistribution, but the mechanism underpinning this process is largely unknown. To gain insight into this process, we considered previously described proteins that could assist in this process via their capacity to interact with kinases and regulate their intracellular translocations...
2016: Frontiers in Immunology
https://www.readbyqxmd.com/read/27815904/synchronization-of-hela-cells
#14
Hoi Tang Ma, Randy Y C Poon
HeLa is one of the oldest and most commonly used cell lines in biomedical research. Owing to the ease of which they can be effectively synchronized by various methods, HeLa cells have been used extensively for studying the cell cycle. Here, we describe several protocols for synchronizing HeLa cells from different phases of the cell cycle, including G1 phase using the HMG-CoA reductase inhibitor lovastatin, S phase with a double thymidine block procedure, and G2 phase with the CDK1 inhibitor RO-3306. Cells can also be enriched in mitosis using nocodazole and mechanical shake-off...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27815897/synchronization-and-desynchronization-of-cells-by-interventions-on-the-spindle-assembly-checkpoint
#15
Mohamed Jemaà, Gwenola Manic, Ilio Vitale
Cell cycle checkpoints are surveillance mechanisms that sequentially and continuously monitor cell cycle progression thereby contributing to the preservation of genetic stability. Among them, the spindle assembly checkpoint (SAC) prevents the occurrence of abnormal divisions by halting the metaphase to anaphase transition following the detection of erroneous microtubules-kinetochore attachment(s). Most synchronization strategies are based on the activation of cell cycle checkpoints to enrich the population of cells in a specific phase of the cell cycle...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27798705/defective-autophagy-mitochondrial-clearance-and-lipophagy-in-niemann-pick-type-b-lymphocytes
#16
Barbara Canonico, Erica Cesarini, Sara Salucci, Francesca Luchetti, Elisabetta Falcieri, Gianna Di Sario, Fulvio Palma, Stefano Papa
Niemann-Pick disease type A (NP-A) and type B (NP-B) are lysosomal storage diseases (LSDs) caused by sphingomyelin accumulation in lysosomes relying on reduced or absent acid sphingomyelinase. A considerable body of evidence suggests that lysosomal storage in many LSD impairs autophagy, resulting in the accumulation of poly-ubiquitinated proteins and dysfunctional mitochondria, ultimately leading to cell death. Here we test this hypothesis in a cellular model of Niemann-Pick disease type B, in which autophagy has never been studied...
2016: PloS One
https://www.readbyqxmd.com/read/27791030/mitotic-golgi-disassembly-is-required-for-bipolar-spindle-formation-and-mitotic-progression
#17
Gianni Guizzunti, Joachim Seemann
During mitosis, the mammalian Golgi vesiculates and, upon partitioning, reassembles in each daughter cell; however, it is not clear whether the disassembly process per se is important for partitioning or is merely an outcome of mitotic entry. Here, we show that Golgi vesiculation is required for progression to metaphase. To prevent Golgi disassembly, we expressed HRP linked to a Golgi-resident protein and acutely triggered the polymerization of 3,3'-diaminobenzidine (DAB) in the Golgi lumen. The DAB polymer does not affect interphase cell viability, but inhibits Golgi fragmentation by nocodazole and brefeldin A and also halts cells in early mitosis...
October 25, 2016: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/27783035/the-microtubule-inhibitor-podofilox-inhibits-an-early-entry-step-of-human-cytomegalovirus
#18
Tobias Cohen, Toni M Schwarz, Frederic Vigant, Thomas J Gardner, Rosmel E Hernandez, Benhur Lee, Domenico Tortorella
Human cytomegalovirus is a ubiquitous β-herpesvirus that infects many different cell types through an initial binding to cell surface receptors followed by a fusion event at the cell membrane or endocytic vesicle. A recent high-throughput screen to identify compounds that block a step prior to viral gene expression identified podofilox as a potent and nontoxic inhibitor. Time-of-addition studies in combination with quantitative-PCR analysis demonstrated that podofilox limits an early step of virus entry at the cell surface...
October 24, 2016: Viruses
https://www.readbyqxmd.com/read/27752143/%C3%AE-tat1-controls-longitudinal-spreading-of-acetylation-marks-from-open-microtubules-extremities
#19
Nathalie Ly, Nadia Elkhatib, Enzo Bresteau, Olivier Piétrement, Mehdi Khaled, Maria M Magiera, Carsten Janke, Eric Le Cam, Andrew D Rutenberg, Guillaume Montagnac
Acetylation of the lysine 40 of α-tubulin (K40) is a post-translational modification occurring in the lumen of microtubules (MTs) and is controlled by the α-tubulin acetyl-transferase αTAT1. How αTAT1 accesses the lumen and acetylates α-tubulin there has been an open question. Here, we report that acetylation starts at open ends of MTs and progressively spreads longitudinally from there. We observed acetylation marks at the open ends of in vivo MTs re-growing after a Nocodazole block, and acetylated segments growing in length with time...
October 18, 2016: Scientific Reports
https://www.readbyqxmd.com/read/27715397/depletion-of-jmjd5-sensitizes-tumor-cells-to-microtubule-destabilizing-agents-by-altering-microtubule-stability
#20
Junyu Wu, Zhimin He, Da-Liang Wang, Fang-Lin Sun
Microtubules play essential roles in mitosis, cell migration, and intracellular trafficking. Drugs that target microtubules have demonstrated great clinical success in cancer treatment due to their capacity to impair microtubule dynamics in both mitotic and interphase stages. In a previous report, we demonstrated that JMJD5 associated with mitotic spindle and was required for proper mitosis. However, it remains elusive whether JMJD5 could regulate the stability of cytoskeletal microtubules and whether it affects the efficacy of microtubule-targeting agents...
November 2016: Cell Cycle
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