Multiple interactions between RNA polymerase I, TIF-IA and TAF(I) subunits regulate preinitiation complex assembly at the ribosomal gene promoter.

PubMed

Yuan, Xuejun; Zhao, Jian; Zentgraf, Hanswalter; Hoffmann-Rohrer, Urs; Grummt, Ingrid

2002-11-01

In mammals, growth-dependent regulation of rRNA synthesis is brought about by the transcription initiation factor TIF-IA. TIF-IA is associated with a fraction of the TBP-containing factor TIF-IB/SL1 and the initiation-competent form of RNA polymerase I (Pol I). We investigated the mechanisms that down-regulate cellular pre-rRNA synthesis and demonstrate that nutrient starvation, density arrest and protein synthesis inhibitors inactivate TIF-IA and impair the association of TIF-IA with Pol I. Moreover, we used a panel of TIF-IA deletion mutants to map the domains that mediate the interaction of TIF-IA with Pol I and TIF-IB/SL1. We found that amino acids 512-609 interact with two subunits of Pol I, RPA43 and PAF67, whereas a short, conserved motif (LARAK, amino acids 411-415) is required for the association of TIF-IA with TAF(I)95 and TAF(I)68. The results uncover an interphase for essential protein-protein interactions that facilitate Pol I preinitiation complex formation.

The MAP kinase pathway coordinates crossover designation with disassembly of synaptonemal complex proteins during meiosis

PubMed Central

Nadarajan, Saravanapriah; Mohideen, Firaz; Tzur, Yonatan B; Ferrandiz, Nuria; Crawley, Oliver; Montoya, Alex; Faull, Peter; Snijders, Ambrosius P; Cutillas, Pedro R; Jambhekar, Ashwini; Blower, Michael D; Martinez-Perez, Enrique; Harper, J Wade; Colaiacovo, Monica P

2016-01-01

Asymmetric disassembly of the synaptonemal complex (SC) is crucial for proper meiotic chromosome segregation. However, the signaling mechanisms that directly regulate this process are poorly understood. Here we show that the mammalian Rho GEF homolog, ECT-2, functions through the conserved RAS/ERK MAP kinase signaling pathway in the C. elegans germline to regulate the disassembly of SC proteins. We find that SYP-2, a SC central region component, is a potential target for MPK-1-mediated phosphorylation and that constitutively phosphorylated SYP-2 impairs the disassembly of SC proteins from chromosomal domains referred to as the long arms of the bivalents. Inactivation of MAP kinase at late pachytene is critical for timely disassembly of the SC proteins from the long arms, and is dependent on the crossover (CO) promoting factors ZHP-3/RNF212/Zip3 and COSA-1/CNTD1. We propose that the conserved MAP kinase pathway coordinates CO designation with the disassembly of SC proteins to ensure accurate chromosome segregation. DOI: http://dx.doi.org/10.7554/eLife.12039.001 PMID:26920220

Mediator independently orchestrates multiple steps of preinitiation complex assembly in vivo

PubMed Central

Eyboulet, Fanny; Wydau-Dematteis, Sandra; Eychenne, Thomas; Alibert, Olivier; Neil, Helen; Boschiero, Claire; Nevers, Marie-Claire; Volland, Hervé; Cornu, David; Redeker, Virginie; Werner, Michel; Soutourina, Julie

2015-01-01

Mediator is a large multiprotein complex conserved in all eukaryotes, which has a crucial coregulator function in transcription by RNA polymerase II (Pol II). However, the molecular mechanisms of its action in vivo remain to be understood. Med17 is an essential and central component of the Mediator head module. In this work, we utilised our large collection of conditional temperature-sensitive med17 mutants to investigate Mediator's role in coordinating preinitiation complex (PIC) formation in vivo at the genome level after a transfer to a non-permissive temperature for 45 minutes. The effect of a yeast mutation proposed to be equivalent to the human Med17-L371P responsible for infantile cerebral atrophy was also analyzed. The ChIP-seq results demonstrate that med17 mutations differentially affected the global presence of several PIC components including Mediator, TBP, TFIIH modules and Pol II. Our data show that Mediator stabilizes TFIIK kinase and TFIIH core modules independently, suggesting that the recruitment or the stability of TFIIH modules is regulated independently on yeast genome. We demonstrate that Mediator selectively contributes to TBP recruitment or stabilization to chromatin. This study provides an extensive genome-wide view of Mediator's role in PIC formation, suggesting that Mediator coordinates multiple steps of a PIC assembly pathway. PMID:26240385

Genome-wide uniformity of human ‘open’ pre-initiation complexes

PubMed Central

Lai, William K.M.; Pugh, B. Franklin

2017-01-01

Transcription of protein-coding and noncoding DNA occurs pervasively throughout the mammalian genome. Their sites of initiation are generally inferred from transcript 5′ ends and are thought to be either locally dispersed or focused. How these two modes of initiation relate is unclear. Here, we apply permanganate treatment and chromatin immunoprecipitation (PIP-seq) of initiation factors to identify the precise location of melted DNA separately associated with the preinitiation complex (PIC) and the adjacent paused complex (PC). This approach revealed the two known modes of transcription initiation. However, in contrast to prevailing views, they co-occurred within the same promoter region: initiation originating from a focused PIC, and broad nucleosome-linked initiation. PIP-seq allowed transcriptional orientation of Pol II to be determined, which may be useful near promoters where sufficient sense/anti-sense transcript mapping information is lacking. PIP-seq detected divergently oriented Pol II at both coding and noncoding promoters, as well as at enhancers. Their occupancy levels were not necessarily coupled in the two orientations. DNA sequence and shape analysis of initiation complex sites suggest that both sequence and shape contribute to specificity, but in a context-restricted manner. That is, initiation sites have the locally “best” initiator (INR) sequence and/or shape. These findings reveal a common core to pervasive Pol II initiation throughout the human genome. PMID:27927716

Modeling operational behavior of a disassembly line

NASA Astrophysics Data System (ADS)

Kizilkaya, Elif A.; Gupta, Surendra M.

2004-12-01

In this paper we present a dynamic kanban (pull) system specifically developed for disassembly lines. This type of kanban system is much more complex than the traditional kanban system used in assembly lines. For instance, unlike the assembly line where the external demand occurs only at the last station, the demands in the disassembly case also occur at any of the intermittent stations. The reason is that as a product moves on the disassembly line, various parts are disassembled at every station and accumulated at that station. Therefore, there are as many demand sources as there are number of parts. We consider a case example involving the end-of-life products. Based on the precedence relationships and other criteria such as hazardous properties of the parts, we balance the disassembly line. The results of the disassembly line-balancing problem (DLBP) are used as input to the proposed dynamic kanban system for disassembly line (DKSDL). We compare the performance of the DKSDL to the modified kanban system for disassembly line (MKSDL), which was previously introduced by the authors. We show, via simulation, that the DKSDL is far superior to MKSDL considered.

RNA polymerase II components and Rrn7 form a preinitiation complex on the HomolD box to promote ribosomal protein gene expression in Schizosaccharomyces pombe.

PubMed

Montes, Matías; Moreira-Ramos, Sandra; Rojas, Diego A; Urbina, Fabiola; Käufer, Norbert F; Maldonado, Edio

2017-02-01

In Schizosaccharomyces pombe, ribosomal protein gene (RPG) promoters contain a TATA box analog, the HomolD box, which is bound by the Rrn7 protein. Despite the importance of ribosome biogenesis for cell survival, the mechanisms underlying RPG transcription remain unknown. In this study, we found that components of the RNA polymerase II (RNAPII) system, consisting of the initiation or general transcription factors (GTFs) TFIIA, IIB, IIE, TATA-binding protein (TBP) and the RNAPII holoenzyme, interacted directly with Rrn7 in vitro, and were able to form a preinitiation complex (PIC) on the HomolD box. PIC complex formation follows an ordered pathway on these promoters. The GTFs and RNAPII can also be cross-linked to HomolD-containing promoters in vivo. In an in vitro reconstituted transcription system, RNAPII components and Rrn7 were necessary for HomolD-directed transcription. The Mediator complex was required for basal transcription from those promoters in whole cell extract (WCE). The Med17 subunit of Mediator also can be cross-linked to the promoter region of HomolD-containing promoters in vivo, suggesting the presence of the Mediator complex on HomolD box-containing promoters. Together, these data show that components of the RNAPII machinery and Rrn7 participate in the PIC assembly on the HomolD box, thereby directing RPG transcription. © 2017 Federation of European Biochemical Societies.

Promoter Melting Plays Critical Role in Lymphocyte Activation | Center for Cancer Research

Cancer.gov

Transcription in eukaryotic cells is a precisely timed ballet that consists of RNA polymerase II (pol II) recruitment to gene promoters, assembly of the multiprotein preinitiation complex, opening of the DNA, escape of pol II from the promoter, pol II pausing downstream, mRNA elongation, and, eventually, termination. The two main points of regulation are thought to be

In vitro fluorescence studies of transcription factor IIB-DNA interaction.

PubMed

Górecki, Andrzej; Figiel, Małgorzata; Dziedzicka-Wasylewska, Marta

2015-01-01

General transcription factor TFIIB is one of the basal constituents of the preinitiation complex of eukaryotic RNA polymerase II, acting as a bridge between the preinitiation complex and the polymerase, and binding promoter DNA in an asymmetric manner, thereby defining the direction of the transcription. Methods of fluorescence spectroscopy together with circular dichroism spectroscopy were used to observe conformational changes in the structure of recombinant human TFIIB after binding to specific DNA sequence. To facilitate the exploration of the structural changes, several site-directed mutations have been introduced altering the fluorescence properties of the protein. Our observations showed that binding of specific DNA sequences changed the protein structure and dynamics, and TFIIB may exist in two conformational states, which can be described by a different microenvironment of W52. Fluorescence studies using both intrinsic and exogenous fluorophores showed that these changes significantly depended on the recognition sequence and concerned various regions of the protein, including those interacting with other transcription factors and RNA polymerase II. DNA binding can cause rearrangements in regions of proteins interacting with the polymerase in a manner dependent on the recognized sequences, and therefore, influence the gene expression.

Quantifying cadherin mechanotransduction machinery assembly/disassembly dynamics using fluorescence covariance analysis.

PubMed

Vedula, Pavan; Cruz, Lissette A; Gutierrez, Natasha; Davis, Justin; Ayee, Brian; Abramczyk, Rachel; Rodriguez, Alexis J

2016-06-30

Quantifying multi-molecular complex assembly in specific cytoplasmic compartments is crucial to understand how cells use assembly/disassembly of these complexes to control function. Currently, biophysical methods like Fluorescence Resonance Energy Transfer and Fluorescence Correlation Spectroscopy provide quantitative measurements of direct protein-protein interactions, while traditional biochemical approaches such as sub-cellular fractionation and immunoprecipitation remain the main approaches used to study multi-protein complex assembly/disassembly dynamics. In this article, we validate and quantify multi-protein adherens junction complex assembly in situ using light microscopy and Fluorescence Covariance Analysis. Utilizing specific fluorescently-labeled protein pairs, we quantified various stages of adherens junction complex assembly, the multiprotein complex regulating epithelial tissue structure and function following de novo cell-cell contact. We demonstrate: minimal cadherin-catenin complex assembly in the perinuclear cytoplasm and subsequent localization to the cell-cell contact zone, assembly of adherens junction complexes, acto-myosin tension-mediated anchoring, and adherens junction maturation following de novo cell-cell contact. Finally applying Fluorescence Covariance Analysis in live cells expressing fluorescently tagged adherens junction complex proteins, we also quantified adherens junction complex assembly dynamics during epithelial monolayer formation.

Interactome disassembly during apoptosis occurs independent of caspase cleavage.

PubMed

Scott, Nichollas E; Rogers, Lindsay D; Prudova, Anna; Brown, Nat F; Fortelny, Nikolaus; Overall, Christopher M; Foster, Leonard J

2017-01-12

Protein-protein interaction networks (interactomes) define the functionality of all biological systems. In apoptosis, proteolysis by caspases is thought to initiate disassembly of protein complexes and cell death. Here we used a quantitative proteomics approach, protein correlation profiling (PCP), to explore changes in cytoplasmic and mitochondrial interactomes in response to apoptosis initiation as a function of caspase activity. We measured the response to initiation of Fas-mediated apoptosis in 17,991 interactions among 2,779 proteins, comprising the largest dynamic interactome to date. The majority of interactions were unaffected early in apoptosis, but multiple complexes containing known caspase targets were disassembled. Nonetheless, proteome-wide analysis of proteolytic processing by terminal amine isotopic labeling of substrates (TAILS) revealed little correlation between proteolytic and interactome changes. Our findings show that, in apoptosis, significant interactome alterations occur before and independently of caspase activity. Thus, apoptosis initiation includes a tight program of interactome rearrangement, leading to disassembly of relatively few, select complexes. These early interactome alterations occur independently of cleavage of these protein by caspases. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Designing and verifying a disassembly line approach to cope with the upsurge of end-of-life vehicles in China.

PubMed

Zhang, Chunliang; Chen, Ming

2018-06-01

An upsurge of end-of-life vehicles (ELVs) is emerging in China, which means a potential monumental environmental crisis. The approach of disassembly line is expected to be an effective solution to such increasing volumes. Due to the complexity of vehicle product and uncertainties of disassembly processes, a complete set of disassembly line system should be taken into detailed consideration. We have designed and constructed a novel disassembly line using a flexible transition technique with the objective of complete disassembly. Prior to productivity testing, comparative Arena-based simulations on four scenarios have been performed and finally a best scenario is selected. The results show that the guarantee of cycle time is the key to meet the productivity target of 30,000 vehicles for one year. To achieve it, some constructive measures such as forcible entry tools are given. Copyright © 2018 Elsevier Ltd. All rights reserved.

Facilitated recycling protects human RNA polymerase III from repression by Maf1 in vitro.

PubMed

Cabart, Pavel; Lee, JaeHoon; Willis, Ian M

2008-12-26

Yeast cells synthesize approximately 3-6 million molecules of tRNA every cell cycle at a rate of approximately 2-4 transcripts/gene/s. This high rate of transcription is achieved through many rounds of reinitiation by RNA polymerase (pol) III on stable DNA-bound complexes of the initiation factor TFIIIB. Studies in yeast have shown that the rate of reinitiation is increased by facilitated recycling, a process that involves the repeated reloading of the polymerase on the same transcription unit. However, when nutrients become limiting or stress conditions are encountered, RNA pol III transcription is rapidly repressed through the action of the conserved Maf1 protein. Here we examine the relationship between Maf1-mediated repression and facilitated recycling in a human RNA pol III in vitro system. Using an immobilized template transcription assay, we demonstrate that facilitated recycling is conserved from yeast to humans. We assessed the ability of recombinant human Maf1 to inhibit different steps in transcription before and after preinitiation complex assembly. We show that recombinant Maf1 can inhibit the recruitment of TFIIIB and RNA pol III to immobilized templates. However, RNA pol III bound to preinitiation complexes or in elongation complexes is protected from repression by Maf1 and can undergo several rounds of initiation. This indicates that recombinant Maf1 is unable to inhibit facilitated recycling. The data suggest that additional biochemical steps may be necessary for rapid Maf1-dependent repression of RNA pol III transcription.

Conformational changes in the P site and mRNA entry channel evoked by AUG recognition in yeast translation preinitiation complexes

PubMed Central

Zhang, Fan; Saini, Adesh K.; Shin, Byung-Sik; Nanda, Jagpreet; Hinnebusch, Alan G.

2015-01-01

The translation preinitiation complex (PIC) is thought to assume an open conformation when scanning the mRNA leader, with AUG recognition evoking a closed conformation and more stable P site interaction of Met-tRNAi; however, physical evidence is lacking that AUG recognition constrains interaction of mRNA with the 40S binding cleft. We compared patterns of hydroxyl radical cleavage of rRNA by Fe(II)-BABE tethered to unique sites in eIF1A in yeast PICs reconstituted with mRNA harboring an AUG or near-cognate (AUC) start codon. rRNA residues in the P site display reduced cleavage in AUG versus AUC PICs; and enhanced cleavage in the AUC complexes was diminished by mutations of scanning enhancer elements of eIF1A that increase near-cognate recognition in vivo. This suggests that accessibility of these rRNA residues is reduced by accommodation of Met-tRNAi in the P site (PIN state) and by their interactions with the anticodon stem of Met-tRNAi. Our cleavage data also provide evidence that AUG recognition evokes dissociation of eIF1 from its 40S binding site, ejection of the eIF1A-CTT from the P-site and rearrangement to a closed conformation of the entry channel with reduced mobility of mRNA. PMID:25670678

Mediator independently orchestrates multiple steps of preinitiation complex assembly in vivo.

PubMed

Eyboulet, Fanny; Wydau-Dematteis, Sandra; Eychenne, Thomas; Alibert, Olivier; Neil, Helen; Boschiero, Claire; Nevers, Marie-Claire; Volland, Hervé; Cornu, David; Redeker, Virginie; Werner, Michel; Soutourina, Julie

2015-10-30

Mediator is a large multiprotein complex conserved in all eukaryotes, which has a crucial coregulator function in transcription by RNA polymerase II (Pol II). However, the molecular mechanisms of its action in vivo remain to be understood. Med17 is an essential and central component of the Mediator head module. In this work, we utilised our large collection of conditional temperature-sensitive med17 mutants to investigate Mediator's role in coordinating preinitiation complex (PIC) formation in vivo at the genome level after a transfer to a non-permissive temperature for 45 minutes. The effect of a yeast mutation proposed to be equivalent to the human Med17-L371P responsible for infantile cerebral atrophy was also analyzed. The ChIP-seq results demonstrate that med17 mutations differentially affected the global presence of several PIC components including Mediator, TBP, TFIIH modules and Pol II. Our data show that Mediator stabilizes TFIIK kinase and TFIIH core modules independently, suggesting that the recruitment or the stability of TFIIH modules is regulated independently on yeast genome. We demonstrate that Mediator selectively contributes to TBP recruitment or stabilization to chromatin. This study provides an extensive genome-wide view of Mediator's role in PIC formation, suggesting that Mediator coordinates multiple steps of a PIC assembly pathway. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

TP53INP2/DOR, a mediator of cell autophagy, promotes rDNA transcription via facilitating the assembly of the POLR1/RNA polymerase I preinitiation complex at rDNA promoters.

PubMed

Xu, Yinfeng; Wan, Wei; Shou, Xin; Huang, Rui; You, Zhiyuan; Shou, Yanhong; Wang, Lingling; Zhou, Tianhua; Liu, Wei

2016-07-02

Cells control their metabolism through modulating the anabolic and catabolic pathways. TP53INP2/DOR (tumor protein p53 inducible nuclear protein 2), participates in cell catabolism by serving as a promoter of autophagy. Here we uncover a novel function of TP53INP2 in protein synthesis, a major biosynthetic and energy-consuming anabolic process. TP53INP2 localizes to the nucleolus through its nucleolar localization signal (NoLS) located at the C-terminal domain. Chromatin immunoprecipitation (ChIP) assays detected an association of TP53INP2 with the ribosomal DNA (rDNA), when exclusion of TP53INP2 from the nucleolus repressed rDNA promoter activity and the production of ribosomal RNA (rRNA) and proteins. The removal of TP53INP2 also impaired the association of the POLR1/RNA polymerase I preinitiation complex (PIC) with rDNA. Further, TP53INP2 interacts directly with POLR1 PIC, and is required for the assembly of the complex. These data indicate that TP53INP2 promotes ribosome biogenesis through facilitating rRNA synthesis at the nucleolus, suggesting a dual role of TP53INP2 in cell metabolism, assisting anabolism on the nucleolus, and stimulating catabolism off the nucleolus.

Risk of Contamination in Assembled vs Disassembled Instruments in Hip Arthroplasty Surgery.

PubMed

Mayer, Ryan R; Bederman, S Samuel; Colin, Vincent M; Berger, Martina M; Cesario, Thomas C; Schwarzkopf, Ran

2016-08-01

Periprosthetic joint infection (PJI) is one of the most common causes of revision total hip arthroplasty (THA) and associated with higher costs, prolonged pain, and worse clinical outcomes. Many factors have been linked to increased infection rates, one being the operative equipment and instrumentation used during the surgical procedure. With few arthroplasty instruments designed for complete disassembly and increasingly complex instrument designs, this study seeks to understand the effect that instrument disassembly plays on infection using disassembled and assembled standard femoral broach handles (BHs). Two BHs, not designed for disassembly, were modified and then contaminated in the disassembled state with Geobacillus stearothermophilus vegetative-form bacteria and spores. Using both flash and standard sterilization cycles, the BHs were steam sterilized in the disassembled or assembled state and then analyzed for remaining bacteria and spores. At all target locations after either a flash sterilization cycle or a standard sterilization cycle, complete eradication of both the vegetative-form and spore-form of G stearothermophilus was achieved. This study demonstrates that adequate decontamination of the tested BHs can be achieved after steam sterilization in either the disassembled or assembled state, without an increased risk of infection transmission. Copyright © 2016 Elsevier Inc. All rights reserved.

Risk of Contamination in Assembled vs Disassembled Instruments in Hip Arthroplasty Surgery

PubMed Central

Mayer, Ryan R.; Bederman, S. Samuel; Colin, Vincent M.; Berger, Martina M.; Cesario, Thomas C.; Schwarzkopf, Ran

2018-01-01

Background Periprosthetic joint infection (PJI) is one of the most common causes of revision total hip arthroplasty (THA) and associated with higher costs, prolonged pain, and worse clinical outcomes. Many factors have been linked to increased infection rates, one being the operative equipment and instrumentation used during the surgical procedure. With few arthroplasty instruments designed for complete disassembly and increasingly complex instrument designs, this study seeks to understand the effect that instrument disassembly plays on infection using disassembled and assembled standard femoral broach handles (BHs). Methods Two BHs, not designed for disassembly, were modified and then contaminated in the disassembled state with Geobacillus stearothermophilus vegetative-form bacteria and spores. Using both flash and standard sterilization cycles, the BHs were steam sterilized in the disassembled or assembled state and then analyzed for remaining bacteria and spores. Results At all target locations after either a flash sterilization cycle or a standard sterilization cycle, complete eradication of both the vegetative-form and spore-form of G stearothermophilus was achieved. Conclusion This study demonstrates that adequate decontamination of the tested BHs can be achieved after steam sterilization in either the disassembled or assembled state, without an increased risk of infection transmission. PMID:26948131

Regulated assembly and disassembly of the yeast telomerase quaternary complex

PubMed Central

Tucey, Timothy M.

2014-01-01

The enzyme telomerase, which elongates chromosome termini, is a critical factor in determining long-term cellular proliferation and tissue renewal. Hence, even small differences in telomerase levels can have substantial consequences for human health. In budding yeast, telomerase consists of the catalytic Est2 protein and two regulatory subunits (Est1 and Est3) in association with the TLC1 RNA, with each of the four subunits essential for in vivo telomerase function. We show here that a hierarchy of assembly and disassembly results in limiting amounts of the quaternary complex late in the cell cycle, following completion of DNA replication. The assembly pathway, which is driven by interaction of the Est3 telomerase subunit with a previously formed Est1–TLC1–Est2 preassembly complex, is highly regulated, involving Est3-binding sites on both Est2 and Est1 as well as an interface on Est3 itself that functions as a toggle switch. Telomerase subsequently disassembles by a mechanistically distinct pathway due to dissociation of the catalytic subunit from the complex in every cell cycle. The balance between the assembly and disassembly pathways, which dictate the levels of the active holoenzyme in the cell, reveals a novel mechanism by which telomerase (and hence telomere homeostasis) is regulated. PMID:25240060

The effect of sudden server breakdown on the performance of a disassembly line

NASA Astrophysics Data System (ADS)

Udomsawat, Gun; Gupta, Surendra M.

2005-11-01

Product and material recovery relies on the disassembly process to separate target components or materials from the end-of-life (EOL) products. Disassembly line is especially effective when products in large quantity are disassembled. Unlike an assembly line, a disassembly line is more complex and is subjected to numerous uncertainties including stochastic and multi-level arrivals of component demands, stochastic arrival times for EOL products, and process interruption due to equipment failure. These factors seriously impair the control mechanism in the disassembly line. A common production control mechanism is the traditional push system (TPS). TPS responds to the aforementioned complications by carrying substantial amounts of inventories. An alternative control mechanism is a newly developed multi-kanban pull system (MKS) that relies on dynamic routing of kanbans, which tends to minimize the system's inventories while maintaining demand serviceability. In this paper we explore the impact of sudden breakdown of server on the performance of a disassembly line. We compare the overall performances of the TPS and MKS by considering two scenarios. We present the solution procedure and results for these cases.

Eukaryotic translation initiation factor 3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex

PubMed Central

Aitken, Colin Echeverría; Beznosková, Petra; Vlčkova, Vladislava; Chiu, Wen-Ling; Zhou, Fujun; Valášek, Leoš Shivaya; Hinnebusch, Alan G; Lorsch, Jon R

2016-01-01

Eukaryotic translation initiation factor 3 (eIF3) is a central player in recruitment of the pre-initiation complex (PIC) to mRNA. We probed the effects on mRNA recruitment of a library of S. cerevisiae eIF3 functional variants spanning its 5 essential subunits using an in vitro-reconstituted system. Mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment to a native yeast mRNA. Alterations to the eIF3a CTD and eIF3b/i/g significantly slow mRNA recruitment, and mutations within eIF3b/i/g destabilize eIF2•GTP•Met-tRNAi binding to the PIC. Using model mRNAs lacking contacts with the 40S entry or exit channels, we uncovered a critical role for eIF3 requiring the eIF3a NTD, in stabilizing mRNA interactions at the exit channel, and an ancillary role at the entry channel requiring residues of the eIF3a CTD. These functions are redundant: defects at each channel can be rescued by filling the other channel with mRNA. DOI: http://dx.doi.org/10.7554/eLife.20934.001 PMID:27782884

Hmo1 directs pre-initiation complex assembly to an appropriate site on its target gene promoters by masking a nucleosome-free region

PubMed Central

Kasahara, Koji; Ohyama, Yoshifumi; Kokubo, Tetsuro

2011-01-01

Saccharomyces cerevisiae Hmo1 binds to the promoters of ∼70% of ribosomal protein genes (RPGs) at high occupancy, but is observed at lower occupancy on the remaining RPG promoters. In Δhmo1 cells, the transcription start site (TSS) of the Hmo1-enriched RPS5 promoter shifted upstream, while the TSS of the Hmo1-limited RPL10 promoter did not shift. Analyses of chimeric RPS5/RPL10 promoters revealed a region between the RPS5 upstream activating sequence (UAS) and core promoter, termed the intervening region (IVR), responsible for strong Hmo1 binding and an upstream TSS shift in Δhmo1 cells. Chromatin immunoprecipitation analyses showed that the RPS5-IVR resides within a nucleosome-free region and that pre-initiation complex (PIC) assembly occurs at a site between the IVR and a nucleosome overlapping the TSS (+1 nucleosome). The PIC assembly site was shifted upstream in Δhmo1 cells on this promoter, indicating that Hmo1 normally masks the RPS5-IVR to prevent PIC assembly at inappropriate site(s). This novel mechanism ensures accurate transcriptional initiation by delineating the 5′- and 3′-boundaries of the PIC assembly zone. PMID:21288884

C. elegans Nuclear Envelope Proteins Emerin, MAN1, Lamin, and Nucleoporins Reveal Unique Timing of Nuclear Envelope Breakdown during Mitosis

PubMed Central

Lee, Kenneth K.; Gruenbaum, Yosef; Spann, Perah; Liu, Jun; Wilson, Katherine L.

2000-01-01

Emerin, MAN1, and LAP2 are integral membrane proteins of the vertebrate nuclear envelope. They share a 43-residue N-terminal motif termed the LEM domain. We found three putative LEM domain genes in Caenorhabditis elegans, designated emr-1, lem-2, and lem-3. We analyzed emr-l, which encodes Ce-emerin, and lem-2, which encodes Ce-MAN1. Ce-emerin and Ce-MAN1 migrate on SDS-PAGE as 17- and 52-kDa proteins, respectively. Based on their biochemical extraction properties and immunolocalization, both Ce-emerin and Ce-MAN1 are integral membrane proteins localized at the nuclear envelope. We used antibodies against Ce-MAN1, Ce-emerin, nucleoporins, and Ce-lamin to determine the timing of nuclear envelope breakdown during mitosis in C. elegans. The C. elegans nuclear envelope disassembles very late compared with vertebrates and Drosophila. The nuclear membranes remained intact everywhere except near spindle poles during metaphase and early anaphase, fully disassembling only during mid-late anaphase. Disassembly of pore complexes, and to a lesser extent the lamina, depended on embryo age: pore complexes were absent during metaphase in >30-cell embryos but existed until anaphase in 2- to 24-cell embryos. Intranuclear mRNA splicing factors disassembled after prophase. The timing of nuclear disassembly in C. elegans is novel and may reflect its evolutionary position between unicellular and more complex eukaryotes. PMID:10982402

Orai1 as New Therapeutic Target for Inhibiting Breast Tumor Metastasis

DTIC Science & Technology

2009-09-01

includes focal adhesion assembly (formation of focal complex) and focal adhesion disassembly, we used live - cell imaging to quantify the rates of assembly...A and B) Live cell imaging of paxillin-GFP transfected MEF cells in the absence (A) or presence (B) of SKF96365. Scale bar: 10 µm. (C and D...includes focal adhesion assembly (formation of focal complexes) and focal adhesion disassembly, we used live - cell imaging to quantify the rates of focal

The Mediator complex: a central integrator of transcription

PubMed Central

Allen, Benjamin L.; Taatjes, Dylan J.

2016-01-01

The RNA polymerase II (pol II) enzyme transcribes all protein-coding and most non-coding RNA genes and is globally regulated by Mediator, a large, conformationally flexible protein complex with variable subunit composition (for example, a four-subunit CDK8 module can reversibly associate). These biochemical characteristics are fundamentally important for Mediator's ability to control various processes important for transcription, including organization of chromatin architecture and regulation of pol II pre-initiation, initiation, re-initiation, pausing, and elongation. Although Mediator exists in all eukaryotes, a variety of Mediator functions appear to be specific to metazoans, indicative of more diverse regulatory requirements. PMID:25693131

The CD63-Syntenin-1 Complex Controls Post-Endocytic Trafficking of Oncogenic Human Papillomaviruses.

PubMed

Gräßel, Linda; Fast, Laura Aline; Scheffer, Konstanze D; Boukhallouk, Fatima; Spoden, Gilles A; Tenzer, Stefan; Boller, Klaus; Bago, Ruzica; Rajesh, Sundaresan; Overduin, Michael; Berditchevski, Fedor; Florin, Luise

2016-08-31

Human papillomaviruses enter host cells via a clathrin-independent endocytic pathway involving tetraspanin proteins. However, post-endocytic trafficking required for virus capsid disassembly remains unclear. Here we demonstrate that the early trafficking pathway of internalised HPV particles involves tetraspanin CD63, syntenin-1 and ESCRT-associated adaptor protein ALIX. Following internalisation, viral particles are found in CD63-positive endosomes recruiting syntenin-1, a CD63-interacting adaptor protein. Electron microscopy and immunofluorescence experiments indicate that the CD63-syntenin-1 complex controls delivery of internalised viral particles to multivesicular endosomes. Accordingly, infectivity of high-risk HPV types 16, 18 and 31 as well as disassembly and post-uncoating processing of viral particles was markedly suppressed in CD63 or syntenin-1 depleted cells. Our analyses also present the syntenin-1 interacting protein ALIX as critical for HPV infection and CD63-syntenin-1-ALIX complex formation as a prerequisite for intracellular transport enabling viral capsid disassembly. Thus, our results identify the CD63-syntenin-1-ALIX complex as a key regulatory component in post-endocytic HPV trafficking.

Promoter Melting Plays Critical Role in Lymphocyte Activation | Center for Cancer Research

Cancer.gov

Transcription in eukaryotic cells is a precisely timed ballet that consists of RNA polymerase II (pol II) recruitment to gene promoters, assembly of the multiprotein preinitiation complex, opening of the DNA, escape of pol II from the promoter, pol II pausing downstream, mRNA elongation, and, eventually, termination. The two main points of regulation are thought to be polymerase recruitment and pause release, but most studies investigating these regulatory processes involved actively cycling cells.

Stress Granule-Inducing Eukaryotic Translation Initiation Factor 4A Inhibitors Block Influenza A Virus Replication

PubMed Central

Slaine, Patrick D.; Kleer, Mariel; Smith, Nathan K.; Khaperskyy, Denys A.

2017-01-01

Eukaryotic translation initiation factor 4A (eIF4A) is a helicase that facilitates assembly of the translation preinitiation complex by unwinding structured mRNA 5′ untranslated regions. Pateamine A (PatA) and silvestrol are natural products that disrupt eIF4A function and arrest translation, thereby triggering the formation of cytoplasmic aggregates of stalled preinitiation complexes known as stress granules (SGs). Here we examined the effects of eIF4A inhibition by PatA and silvestrol on influenza A virus (IAV) protein synthesis and replication in cell culture. Treatment of infected cells with either PatA or silvestrol at early times post-infection resulted in SG formation, arrest of viral protein synthesis and failure to replicate the viral genome. PatA, which irreversibly binds to eIF4A, sustained long-term blockade of IAV replication following drug withdrawal, and inhibited IAV replication at concentrations that had minimal cytotoxicity. By contrast, the antiviral effects of silvestrol were fully reversible; drug withdrawal caused rapid SG dissolution and resumption of viral protein synthesis. IAV inhibition by silvestrol was invariably associated with cytotoxicity. PatA blocked replication of genetically divergent IAV strains, suggesting common dependence on host eIF4A activity. This study demonstrates that the core host protein synthesis machinery can be targeted to block viral replication. PMID:29258238

Heat-shock inactivation of the TFIIH-associated kinase and change in the phosphorylation sites on the C-terminal domain of RNA polymerase II.

PubMed

Dubois, M F; Vincent, M; Vigneron, M; Adamczewski, J; Egly, J M; Bensaude, O

1997-02-15

The C-terminal domain (CTD) of the RNA polymerase II largest subunit (RPB1) plays a central role in transcription. The CTD is unphosphorylated when the polymerase assembles into a preinitiation complex of transcription and becomes heavily phosphorylated during promoter clearance and entry into elongation of transcription. A kinase associated to the general transcription factor TFIIH, in the preinitiation complex, phosphorylates the CTD. The TFIIH-associated CTD kinase activity was found to decrease in extracts from heat-shocked HeLa cells compared to unstressed cells. This loss of activity correlated with a decreased solubility of the TFIIH factor. The TFIIH-kinase impairment during heat-shock was accompanied by the disappearance of a particular phosphoepitope (CC-3) on the RPB1 subunit. The CC-3 epitope was localized on the C-terminal end of the CTD and generated in vitro when the RPB1 subunit was phosphorylated by the TFIIH-associated kinase but not by another CTD kinase such as MAP kinase. In apparent discrepancy, the overall RPB1 subunit phosphorylation increased during heat-shock. The decreased activity in vivo of the TFIIH kinase might be compensated by a stress-activated CTD kinase such as MAP kinase. These results also suggest that heat-shock gene transcription may have a weak requirement for TFIIH kinase activity.

Heat-shock inactivation of the TFIIH-associated kinase and change in the phosphorylation sites on the C-terminal domain of RNA polymerase II.

PubMed Central

Dubois, M F; Vincent, M; Vigneron, M; Adamczewski, J; Egly, J M; Bensaude, O

1997-01-01

The C-terminal domain (CTD) of the RNA polymerase II largest subunit (RPB1) plays a central role in transcription. The CTD is unphosphorylated when the polymerase assembles into a preinitiation complex of transcription and becomes heavily phosphorylated during promoter clearance and entry into elongation of transcription. A kinase associated to the general transcription factor TFIIH, in the preinitiation complex, phosphorylates the CTD. The TFIIH-associated CTD kinase activity was found to decrease in extracts from heat-shocked HeLa cells compared to unstressed cells. This loss of activity correlated with a decreased solubility of the TFIIH factor. The TFIIH-kinase impairment during heat-shock was accompanied by the disappearance of a particular phosphoepitope (CC-3) on the RPB1 subunit. The CC-3 epitope was localized on the C-terminal end of the CTD and generated in vitro when the RPB1 subunit was phosphorylated by the TFIIH-associated kinase but not by another CTD kinase such as MAP kinase. In apparent discrepancy, the overall RPB1 subunit phosphorylation increased during heat-shock. The decreased activity in vivo of the TFIIH kinase might be compensated by a stress-activated CTD kinase such as MAP kinase. These results also suggest that heat-shock gene transcription may have a weak requirement for TFIIH kinase activity. PMID:9016617

Redox-regulated Export of the Major Histocompatibility Complex Class I-Peptide Complexes from the Endoplasmic Reticulum

PubMed Central

Lee, Sungwook; Park, Boyoun; Kang, Kwonyoon

2009-01-01

In contrast to the fairly well-characterized mechanism of assembly of MHC class I-peptide complexes, the disassembly mechanism by which peptide-loaded MHC class I molecules are released from the peptide-loading complex and exit the endoplasmic reticulum (ER) is poorly understood. Optimal peptide binding by MHC class I molecules is assumed to be sufficient for triggering exit of peptide-filled MHC class I molecules from the ER. We now show that protein disulfide isomerase (PDI) controls MHC class I disassembly by regulating dissociation of the tapasin-ERp57 disulfide conjugate. PDI acts as a peptide-dependent molecular switch; in the peptide-bound state, it binds to tapasin and ERp57 and induces dissociation of the tapasin-ERp57 conjugate. In the peptide-free state, PDI is incompetent to bind to tapasin or ERp57 and fails to dissociate the tapasin-ERp57 conjugates, resulting in ER retention of MHC class I molecules. Thus, our results indicate that even after optimal peptide loading, MHC class I disassembly does not occur by default but, rather, is a regulated process involving PDI-mediated interactions within the peptide-loading complex. PMID:19477919

Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating

PubMed Central

Böcking, Till; Aguet, François; Harrison, Stephen C.; Kirchhausen, Tomas

2010-01-01

Heat shock cognate protein 70 (Hsc70) supports remodeling of protein complexes -- for example, disassembly of clathrin coats on endocytic coated vesicles. To understand how a simple ATP driven molecular clamp catalyzes a large-scale disassembly reaction, we have used single-particle fluorescence imaging to track the dynamics of Hsc70 and its clathrin substrate in real time. Hsc70 accumulates to a critical level, determined by kinetic modeling to be one Hsc70 for every two functional attachment sites; rapid, all-or-none uncoating then ensues. We propose that Hsc70 traps conformational distortions, seen previously by electron cryomicroscopy, in the vicinity of each occupied site and that accumulation of local strains destabilises the clathrin lattice. Capture of conformational fluctuations may be a general mechanism for chaperone-driven disassembly of protein complexes. PMID:21278753

Mutations in CDC45, Encoding an Essential Component of the Pre-initiation Complex, Cause Meier-Gorlin Syndrome and Craniosynostosis.

PubMed

Fenwick, Aimee L; Kliszczak, Maciej; Cooper, Fay; Murray, Jennie; Sanchez-Pulido, Luis; Twigg, Stephen R F; Goriely, Anne; McGowan, Simon J; Miller, Kerry A; Taylor, Indira B; Logan, Clare; Bozdogan, Sevcan; Danda, Sumita; Dixon, Joanne; Elsayed, Solaf M; Elsobky, Ezzat; Gardham, Alice; Hoffer, Mariette J V; Koopmans, Marije; McDonald-McGinn, Donna M; Santen, Gijs W E; Savarirayan, Ravi; de Silva, Deepthi; Vanakker, Olivier; Wall, Steven A; Wilson, Louise C; Yuregir, Ozge Ozalp; Zackai, Elaine H; Ponting, Chris P; Jackson, Andrew P; Wilkie, Andrew O M; Niedzwiedz, Wojciech; Bicknell, Louise S

2016-07-07

DNA replication precisely duplicates the genome to ensure stable inheritance of genetic information. Impaired licensing of origins of replication during the G1 phase of the cell cycle has been implicated in Meier-Gorlin syndrome (MGS), a disorder defined by the triad of short stature, microtia, and a/hypoplastic patellae. Biallelic partial loss-of-function mutations in multiple components of the pre-replication complex (preRC; ORC1, ORC4, ORC6, CDT1, or CDC6) as well as de novo stabilizing mutations in the licensing inhibitor, GMNN, cause MGS. Here we report the identification of mutations in CDC45 in 15 affected individuals from 12 families with MGS and/or craniosynostosis. CDC45 encodes a component of both the pre-initiation (preIC) and CMG helicase complexes, required for initiation of DNA replication origin firing and ongoing DNA synthesis during S-phase itself, respectively, and hence is functionally distinct from previously identified MGS-associated genes. The phenotypes of affected individuals range from syndromic coronal craniosynostosis to severe growth restriction, fulfilling diagnostic criteria for Meier-Gorlin syndrome. All mutations identified were biallelic and included synonymous mutations altering splicing of physiological CDC45 transcripts, as well as amino acid substitutions expected to result in partial loss of function. Functionally, mutations reduce levels of full-length transcripts and protein in subject cells, consistent with partial loss of CDC45 function and a predicted limited rate of DNA replication and cell proliferation. Our findings therefore implicate the preIC as an additional protein complex involved in the etiology of MGS and connect the core cellular machinery of genome replication with growth, chondrogenesis, and cranial suture homeostasis. Copyright © 2016 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Function of the growth-regulated transcription initiation factor TIF-IA in initiation complex formation at the murine ribosomal gene promoter.

PubMed

Schnapp, A; Schnapp, G; Erny, B; Grummt, I

1993-11-01

Alterations in the rate of cell proliferation are accompanied by changes in the transcription of rRNA genes. In mammals, this growth-dependent regulation of transcription of genes coding for rRNA (rDNA) is due to reduction of the amount or activity of an essential transcription factor, called TIF-IA. Extracts prepared from quiescent cells lack this factor activity and, therefore, are transcriptionally inactive. We have purified TIF-IA from exponentially growing cells and have shown that it is a polypeptide with a molecular mass of 75 kDa which exists as a monomer in solution. Using a reconstituted transcription system consisting of purified transcription factors, we demonstrate that TIF-IA is a bona fide transcription initiation factor which interacts with RNA polymerase I. Preinitiation complexes can be assembled in the absence of TIF-IA, but formation of the first phosphodiester bonds of nascent rRNA is precluded. After initiation, TIF-IA is liberated from the initiation complex and facilitates transcription from templates bearing preinitiation complexes which lack TIF-IA. Despite the pronounced species specificity of class I gene transcription, this growth-dependent factor has been identified not only in mouse but also in human cells. Murine TIF-IA complements extracts from both growth-inhibited mouse and human cells. The analogous human activity appears to be similar or identical to that of TIF-IA. Therefore, despite the fact that the RNA polymerase transcription system has evolved sufficiently rapidly that an rDNA promoter from one species will not function in another species, the basic mechanisms that adapt ribosome synthesis to cell proliferation have been conserved.

Cape Canaveral Air Force Station, Launch Complex 39, Solid Rocket ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

Cape Canaveral Air Force Station, Launch Complex 39, Solid Rocket Booster Disassembly & Refurbishment Complex, Thrust Vector Control Deservicing Facility, Hangar Road, Cape Canaveral, Brevard County, FL

The Prolyl Isomerase Pin1 Promotes the Herpesvirus-Induced Phosphorylation-Dependent Disassembly of the Nuclear Lamina Required for Nucleocytoplasmic Egress.

PubMed

Milbradt, Jens; Hutterer, Corina; Bahsi, Hanife; Wagner, Sabrina; Sonntag, Eric; Horn, Anselm H C; Kaufer, Benedikt B; Mori, Yasuko; Sticht, Heinrich; Fossen, Torgils; Marschall, Manfred

2016-08-01

The nuclear lamina lines the inner nuclear membrane providing a structural framework for the nucleus. Cellular processes, such as nuclear envelope breakdown during mitosis or nuclear export of large ribonucleoprotein complexes, are functionally linked to the disassembly of the nuclear lamina. In general, lamina disassembly is mediated by phosphorylation, but the precise molecular mechanism is still not completely understood. Recently, we suggested a novel mechanism for lamina disassembly during the nuclear egress of herpesviral capsids which involves the cellular isomerase Pin1. In this study, we focused on mechanistic details of herpesviral nuclear replication to demonstrate the general importance of Pin1 for lamina disassembly. In particular, Ser22-specific lamin phosphorylation consistently generates a Pin1-binding motif in cells infected with human and animal alpha-, beta-, and gammaherpesviruses. Using nuclear magnetic resonance spectroscopy, we showed that binding of Pin1 to a synthetic lamin peptide induces its cis/trans isomerization in vitro. A detailed bioinformatic evaluation strongly suggests that this structural conversion induces large-scale secondary structural changes in the lamin N-terminus. Thus, we concluded that a Pin1-induced conformational change of lamins may represent the molecular trigger responsible for lamina disassembly. Consistent with this concept, pharmacological inhibition of Pin1 activity blocked lamina disassembly in herpesvirus-infected fibroblasts and consequently impaired virus replication. In addition, a phospho-mimetic Ser22Glu lamin mutant was still able to form a regular lamina structure and overexpression of a Ser22-phosphorylating kinase did not induce lamina disassembly in Pin1 knockout cells. Intriguingly, this was observed in absence of herpesvirus infection proposing a broader importance of Pin1 for lamina constitution. Thus, our results suggest a functional model of similar events leading to disassembly of the nuclear lamina in response to herpesviral or inherent cellular stimuli. In essence, Pin1 represents a regulatory effector of lamina disassembly that promotes the nuclear pore-independent egress of herpesviral capsids.

The Prolyl Isomerase Pin1 Promotes the Herpesvirus-Induced Phosphorylation-Dependent Disassembly of the Nuclear Lamina Required for Nucleocytoplasmic Egress

PubMed Central

Milbradt, Jens; Hutterer, Corina; Bahsi, Hanife; Wagner, Sabrina; Sonntag, Eric; Kaufer, Benedikt B.; Mori, Yasuko; Sticht, Heinrich; Fossen, Torgils; Marschall, Manfred

2016-01-01

The nuclear lamina lines the inner nuclear membrane providing a structural framework for the nucleus. Cellular processes, such as nuclear envelope breakdown during mitosis or nuclear export of large ribonucleoprotein complexes, are functionally linked to the disassembly of the nuclear lamina. In general, lamina disassembly is mediated by phosphorylation, but the precise molecular mechanism is still not completely understood. Recently, we suggested a novel mechanism for lamina disassembly during the nuclear egress of herpesviral capsids which involves the cellular isomerase Pin1. In this study, we focused on mechanistic details of herpesviral nuclear replication to demonstrate the general importance of Pin1 for lamina disassembly. In particular, Ser22-specific lamin phosphorylation consistently generates a Pin1-binding motif in cells infected with human and animal alpha-, beta-, and gammaherpesviruses. Using nuclear magnetic resonance spectroscopy, we showed that binding of Pin1 to a synthetic lamin peptide induces its cis/trans isomerization in vitro. A detailed bioinformatic evaluation strongly suggests that this structural conversion induces large-scale secondary structural changes in the lamin N-terminus. Thus, we concluded that a Pin1-induced conformational change of lamins may represent the molecular trigger responsible for lamina disassembly. Consistent with this concept, pharmacological inhibition of Pin1 activity blocked lamina disassembly in herpesvirus-infected fibroblasts and consequently impaired virus replication. In addition, a phospho-mimetic Ser22Glu lamin mutant was still able to form a regular lamina structure and overexpression of a Ser22-phosphorylating kinase did not induce lamina disassembly in Pin1 knockout cells. Intriguingly, this was observed in absence of herpesvirus infection proposing a broader importance of Pin1 for lamina constitution. Thus, our results suggest a functional model of similar events leading to disassembly of the nuclear lamina in response to herpesviral or inherent cellular stimuli. In essence, Pin1 represents a regulatory effector of lamina disassembly that promotes the nuclear pore-independent egress of herpesviral capsids. PMID:27556400

Nuclear import of viral DNA genomes.

PubMed

Greber, Urs F; Fassati, Ariberto

2003-03-01

The genomes of many viruses traffic into the nucleus, where they are either integrated into host chromosomes or maintained as episomal DNA and then transcriptionally activated or silenced. Here, we discuss the existing evidence on how the lentiviruses, adenoviruses, herpesviruses, hepadnaviruses and autonomous parvoviruses enter the nucleus. Depending on the size of the capsid enclosing the genome, three principles of viral nucleic acids import are discussed. The first principle is that the capsid disassembles in the cytosol or in a docked state at the nuclear pore complex and a subviral genomic complex is trafficked through the pore. Second, the genome is injected from a capsid that is docked to the pore complex, and third, import factors are recruited to cytosolic capsids to increase capsid affinity to the pore complex, mediate translocation and allow disassembly in the nucleoplasm.

TFIIB Co-Localizes and Interacts with α-Tubulin during Oocyte Meiosis in the Mouse and Depletion of TFIIB Causes Arrest of Subsequent Embryo Development

PubMed Central

Shen, Qi-Yuan; Wei, Zhu-Ying; Li, Xin-Xin; Liang, Hao; Bou, Shorgan; Li, Guang-Peng

2013-01-01

TFIIB (transcription factor IIB) is a transcription factor that provides a bridge between promoter-bound TFIID and RNA polymerase II, and it is a target of various transcriptional activator proteins that stimulate the pre-initiation complex assembly. The localization and/or attachment matrix of TFIIB in the cytoplast is not well understood. This study focuses on the function of TFIIB and its interrelationship with α-tubulins in a mouse model. During oocyte maturation TFIIB distributes throughout the entire nucleus of the germinal vesicle (GV). After progression to GV breakdown (GVBD), TFIIB and α-tubulin co-localize and accumulate in the vicinity of the condensed chromosomes. During the MII stage, the TFIIB signals are more concentrated at the equatorial plate and the kinetochores. Colcemid treatment of oocytes disrupts the microtubule (MT) system, although the TFIIB signals are still present with the altered MT state. Injection of oocytes with TFIIB antibodies and siRNAs causes abnormal spindle formation and irregular chromosome alignment. These findings suggest that TFIIB dissociates from the condensed chromatids and then tightly binds to microtubules from GVBD to the MII phase. The assembly and disassembly of TFIIB may very well be associated with and driven by microtubules. TFIIB maintains its contact with the α-tubulins and its co-localization forms a unique distribution pattern. Depletion of Tf2b in oocytes results in a significant decrease in TFIIB expression, although polar body extrusion does not appear to be affected. Knockdown of Tf2b dramatically affects subsequent embryo development with more than 85% of the embryos arrested at the 2-cell stage. These arrested embryos still maintain apparently normal morphology for at least 96h without any obvious degeneration. Analysis of the effects of TFIIB in somatic cells by co-transfection of BiFC plasmids pHA-Tf2b and pFlag-Tuba1α further confirms a direct interaction between TFIIB and α-tubulins. PMID:24244602

Assembly and disassembly of the nucleolus during the cell cycle.

PubMed

Hernandez-Verdun, Danièle

2011-01-01

The nucleolus is a large nuclear domain in which transcription, maturation and assembly of ribosomes take place. In higher eukaryotes, nucleolar organization in three sub-domains reflects the compartmentation of the machineries related to active or inactive transcription of the ribosomal DNA, ribosomal RNA processing and assembly with ribosomal proteins of the two (40S and 60S) ribosomal subunits. The assembly of the nucleoli during telophase/early G(1) depends on pre-existing machineries inactivated during prophase (the transcription machinery and RNP processing complexes) and on partially processed 45S rRNAs inherited throughout mitosis. In telophase, the 45S rRNAs nucleate the prenucleolar bodies and order the dynamics of nucleolar assembly. The assembly/disassembly processes of the nucleolus depend on the equilibrium between phosphorylation/dephosphorylation of the transcription machinery and on the RNP processing complexes under the control of the CDK1-cyclin B kinase and PP1 phosphatases. The dynamics of assembly/disassembly of the nucleolus is time and space regulated.

Tethering of SCFDia2 to the Replisome Promotes Efficient Ubiquitylation and Disassembly of the CMG Helicase

PubMed Central

Maculins, Timurs; Nkosi, Pedro Junior; Nishikawa, Hiroko; Labib, Karim

2015-01-01

Summary Disassembly of the Cdc45-MCM-GINS (CMG) DNA helicase, which unwinds the parental DNA duplex at eukaryotic replication forks, is the key regulated step during replication termination but is poorly understood [1, 2]. In budding yeast, the F-box protein Dia2 drives ubiquitylation of the CMG helicase at the end of replication, leading to a disassembly pathway that requires the Cdc48 segregase [3]. The substrate-binding domain of Dia2 comprises leucine-rich repeats, but Dia2 also has a TPR domain at its amino terminus that interacts with the Ctf4 and Mrc1 subunits of the replisome progression complex [4, 5], which assembles around the CMG helicase at replication forks [6]. Previous studies suggested two disparate roles for the TPR domain of Dia2, either mediating replisome-specific degradation of Mrc1 and Ctf4 [4] or else tethering SCFDia2 (SCF [Skp1/cullin/F-box protein]) to the replisome to increase its local concentration at replication forks [5]. Here, we show that SCFDia2 does not mediate replisome-specific degradation of Mrc1 and Ctf4, either during normal S phase or in response to replication stress. Instead, the tethering of SCFDia2 to the replisome progression complex increases the efficiency of ubiquitylation of the Mcm7 subunit of CMG, both in vitro and in vivo. Correspondingly, loss of tethering reduces the efficiency of CMG disassembly in vivo and is synthetic lethal in combination with a disassembly-defective allele of CDC48. Residual ubiquitylation of Mcm7 in dia2-ΔTPR cells is still CMG specific, highlighting the complex regulation of the final stages of chromosome replication, about which much still remains to be learned. PMID:26255844

Ran-dependent nuclear export mediators: a structural perspective

PubMed Central

Güttler, Thomas; Görlich, Dirk

2011-01-01

Nuclear export is an essential eukaryotic activity. It proceeds through nuclear pore complexes (NPCs) and is mediated by soluble receptors that shuttle between nucleus and cytoplasm. RanGTPase-dependent export mediators (exportins) constitute the largest class of these carriers and are functionally highly versatile. All of these exportins load their substrates in response to RanGTP binding in the nucleus and traverse NPCs as ternary RanGTP–exportin–cargo complexes to the cytoplasm, where GTP hydrolysis leads to export complex disassembly. The different exportins vary greatly in their substrate range. Recent structural studies of both protein- and RNA-specific exporters have illuminated how exportins bind their cargoes, how Ran triggers cargo loading and how export complexes are disassembled in the cytoplasm. Here, we review the current state of knowledge and highlight emerging principles as well as prevailing questions. PMID:21878989

eIF1 Loop 2 interactions with Met-tRNAi control the accuracy of start codon selection by the scanning preinitiation complex.

PubMed

Thakur, Anil; Hinnebusch, Alan G

2018-05-01

The eukaryotic 43S preinitiation complex (PIC), bearing initiator methionyl transfer RNA (Met-tRNA i ) in a ternary complex (TC) with eukaryotic initiation factor 2 (eIF2)-GTP, scans the mRNA leader for an AUG codon in favorable context. AUG recognition evokes rearrangement from an open PIC conformation with TC in a "P OUT " state to a closed conformation with TC more tightly bound in a "P IN " state. eIF1 binds to the 40S subunit and exerts a dual role of enhancing TC binding to the open PIC conformation while antagonizing the P IN state, necessitating eIF1 dissociation for start codon selection. Structures of reconstituted PICs reveal juxtaposition of eIF1 Loop 2 with the Met-tRNA i D loop in the P IN state and predict a distortion of Loop 2 from its conformation in the open complex to avoid a clash with Met-tRNA i We show that Ala substitutions in Loop 2 increase initiation at both near-cognate UUG codons and AUG codons in poor context. Consistently, the D71A-M74A double substitution stabilizes TC binding to 48S PICs reconstituted with mRNA harboring a UUG start codon, without affecting eIF1 affinity for 40S subunits. Relatively stronger effects were conferred by arginine substitutions; and no Loop 2 substitutions perturbed the rate of TC loading on scanning 40S subunits in vivo. Thus, Loop 2-D loop interactions specifically impede Met-tRNA i accommodation in the P IN state without influencing the P OUT mode of TC binding; and Arg substitutions convert the Loop 2-tRNA i clash to an electrostatic attraction that stabilizes P IN and enhances selection of poor start codons in vivo.

Actin-interacting Protein 1 Promotes Disassembly of Actin-depolymerizing Factor/Cofilin-bound Actin Filaments in a pH-dependent Manner.

PubMed

Nomura, Kazumi; Hayakawa, Kimihide; Tatsumi, Hitoshi; Ono, Shoichiro

2016-03-04

Actin-interacting protein 1 (AIP1) is a conserved WD repeat protein that promotes disassembly of actin filaments when actin-depolymerizing factor (ADF)/cofilin is present. Although AIP1 is known to be essential for a number of cellular events involving dynamic rearrangement of the actin cytoskeleton, the regulatory mechanism of the function of AIP1 is unknown. In this study, we report that two AIP1 isoforms from the nematode Caenorhabditis elegans, known as UNC-78 and AIPL-1, are pH-sensitive in enhancement of actin filament disassembly. Both AIP1 isoforms only weakly enhance disassembly of ADF/cofilin-bound actin filaments at an acidic pH but show stronger disassembly activity at neutral and basic pH values. However, a severing-defective mutant of UNC-78 shows pH-insensitive binding to ADF/cofilin-decorated actin filaments, suggesting that the process of filament severing or disassembly, but not filament binding, is pH-dependent. His-60 of AIP1 is located near the predicted binding surface for the ADF/cofilin-actin complex, and an H60K mutation of AIP1 partially impairs its pH sensitivity, suggesting that His-60 is involved in the pH sensor for AIP1. These biochemical results suggest that pH-dependent changes in AIP1 activity might be a novel regulatory mechanism of actin filament dynamics. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Dual-responsive polyplexes with enhanced disassembly and endosomal escape for efficient delivery of siRNA.

PubMed

Zhu, Jia; Qiao, Mingxi; Wang, Qi; Ye, Yuqing; Ba, Shuang; Ma, Jingjing; Hu, Haiyang; Zhao, Xiuli; Chen, Dawei

2018-04-01

Despite the extracellular barriers for siRNA delivery have been overcome by utilizing advanced nanoparticle delivery systems, the key intracellular barriers after internalization including efficient disassembly of siRNA and endosomal escape still remains challenging. To address the issues, we developed a unique pH- and redox potential-responsive polyplex delivery system based on the copolymer of mPEG-b-PLA-PHis-ssPEI1.8 k, which is composed of a pH-responsive copolymer of PEG-b-PLA-PHis (Mw 5 k) and a branched PEI (Mw1.8 k) linked with redox cleavable disulfide bond. The copolymer showed excellent siRNA complexation and protection abilities against endogenous substances at the relatively low N/P ratio of 6. The siRNA release from the polyplexes (N/P 6) was markedly increased from 13.62% to 58.67% under conditions simulating the endosomal microenvironment. Fluorescence resonance energy transfer (FRET) test also indicated a higher disassembly extent of siRNA from the copolymer. The accelerated siRNA release from the polyplexes was markedly restrained when the N/P ratio was raised above 10 due to the increasing of electrostatic interactions. The efficient endosomal escape of siRNA after internalization was confirmed by confocal microscopy, which was attributed to the cleavaged PEI chains inducing membrane destabilization, the "proton sponge effect" of PHis and PEI as well as the relative small size of after disassembly. The enhanced disassembly and endosomal escape were elucidated as the leading cause for polyplexes (N/P 6) showed more efficient Bcl-2 silencing (85.45%) than those polyplexes with higher N/P ratios (N/P 10 and 15). In vivo results further demonstrated that polyplexes (N/P 6) delivery of siBcl-2 significantly inhibited the MCF-7 breast tumor growth as compared to its counterparts. The incorporation of convertible non-electrical interactions at a balance with electrostatic interactions in complexation siRNA has been demonstrated as an effective strategy to achieve efficient disassembly from stable polyplexes. Moreover, polyplexes equipped with the enhanced disassembly and endosomal escape provides a new potential way to tackle the intracellular delivery bottleneck for siRNA delivery. Copyright © 2018 Elsevier Ltd. All rights reserved.

Retrospective Analysis of Dose Titration and Serum Testosterone Level Assessments in Patients Treated With Topical Testosterone.

PubMed

Muram, David; Kaltenboeck, Anna; Boytsov, Natalie; Hayes-Larson, Eleanor; Ivanova, Jasmina; Birnbaum, Howard G; Swindle, Ralph

2015-11-01

Patterns of care following topical testosterone agent (TTA) initiation are poorly understood. This study aimed to characterize care following TTA initiation and compare results between patients with and without a serum testosterone (T) assay within 30 days before and including TTA initiation. Adult men (N=4,146) initiating TTAs from January 1, 2011, to March 31, 2012, were identified from a commercially insured database. Patients were included if they initiated at recommended starting dose (RSD) and had ≥12 and ≥6 months of continuous eligibility preinitiation (baseline) and postinitiation (study period), respectively. Patients were stratified by preinitiation T assay. Maintenance dose attainment month was determined using unadjusted generalized estimating equations regression to compare dose relative to RSD month by month. Outcomes included maintenance dose attainment month, time to stopping of index TTA refills or a claim for nonindex testosterone replacement therapy (TRT), and proportion of patients with study period T assay or diagnosis of hypogonadism (HG) or another low testosterone condition, and were compared using chi-square and Wilcoxon rank-sum tests for categorical and continuous variables, respectively. Maintenance dose was attained in Month 4 postinitiation, at 115.2% of RSD. Approximately 46% of patients had a preinitiation T assay; these men were more likely to receive a diagnosis of HG or another low testosterone condition, to have a follow-up T assay, to continue treatment by filling a nonindex TRT, and less likely to stop refilling treatment with their index TTA. Differences in care following TTA initiation suggest that preinitiation T assays (i.e., guideline-based care) may be helpful in ensuring treatment benefits. © The Author(s) 2014.

Disassemblability modeling technology of configurable product based on disassembly constraint relation weighted design structure matrix(DSM)

NASA Astrophysics Data System (ADS)

Qiu, Lemiao; Liu, Xiaojian; Zhang, Shuyou; Sun, Liangfeng

2014-05-01

The current research of configurable product disassemblability focuses on disassemblability evaluation and disassembly sequence planning. Little work has been done on quantitative analysis of configurable product disassemblability. The disassemblability modeling technology for configurable product based on disassembly constraint relation weighted design structure matrix (DSM) is proposed. Major factors affecting the disassemblability of configurable product are analyzed, and the disassembling degrees between components in configurable product are obtained by calculating disassembly entropies such as joint type, joint quantity, disassembly path, disassembly accessibility and material compatibility. The disassembly constraint relation weighted DSM of configurable product is constructed and configuration modules are formed by matrix decomposition and tearing operations. The disassembly constraint relation in configuration modules is strong coupling, and the disassembly constraint relation between modules is weak coupling, and the disassemblability configuration model is constructed based on configuration module. Finally, taking a hydraulic forging press as an example, the decomposed weak coupling components are used as configuration modules alone, components with a strong coupling are aggregated into configuration modules, and the disassembly sequence of components inside configuration modules is optimized by tearing operation. A disassemblability configuration model of the hydraulic forging press is constructed. By researching the disassemblability modeling technology of product configuration design based on disassembly constraint relation weighted DSM, the disassembly property in maintenance, recycling and reuse of configurable product are optimized.

Emerging players in the initiation of eukaryotic DNA replication

PubMed Central

2012-01-01

Faithful duplication of the genome in eukaryotes requires ordered assembly of a multi-protein complex called the pre-replicative complex (pre-RC) prior to S phase; transition to the pre-initiation complex (pre-IC) at the beginning of DNA replication; coordinated progression of the replisome during S phase; and well-controlled regulation of replication licensing to prevent re-replication. These events are achieved by the formation of distinct protein complexes that form in a cell cycle-dependent manner. Several components of the pre-RC and pre-IC are highly conserved across all examined eukaryotic species. Many of these proteins, in addition to their bona fide roles in DNA replication are also required for other cell cycle events including heterochromatin organization, chromosome segregation and centrosome biology. As the complexity of the genome increases dramatically from yeast to human, additional proteins have been identified in higher eukaryotes that dictate replication initiation, progression and licensing. In this review, we discuss the newly discovered components and their roles in cell cycle progression. PMID:23075259

Real-time observation of the initiation of RNA polymerase II transcription.

PubMed

Fazal, Furqan M; Meng, Cong A; Murakami, Kenji; Kornberg, Roger D; Block, Steven M

2015-09-10

Biochemical and structural studies have shown that the initiation of RNA polymerase II transcription proceeds in the following stages: assembly of the polymerase with general transcription factors and promoter DNA in a 'closed' preinitiation complex (PIC); unwinding of about 15 base pairs of the promoter DNA to form an 'open' complex; scanning downstream to a transcription start site; synthesis of a short transcript, thought to be about 10 nucleotides long; and promoter escape. Here we have assembled a 32-protein, 1.5-megadalton PIC derived from Saccharomyces cerevisiae, and observe subsequent initiation processes in real time with optical tweezers. Contrary to expectation, scanning driven by the transcription factor IIH involved the rapid opening of an extended transcription bubble, averaging 85 base pairs, accompanied by the synthesis of a transcript up to the entire length of the extended bubble, followed by promoter escape. PICs that failed to achieve promoter escape nevertheless formed open complexes and extended bubbles, which collapsed back to closed or open complexes, resulting in repeated futile scanning.

LRRK2 phosphorylates pre-synaptic N-ethylmaleimide sensitive fusion (NSF) protein enhancing its ATPase activity and SNARE complex disassembling rate.

PubMed

Belluzzi, Elisa; Gonnelli, Adriano; Cirnaru, Maria-Daniela; Marte, Antonella; Plotegher, Nicoletta; Russo, Isabella; Civiero, Laura; Cogo, Susanna; Carrion, Maria Perèz; Franchin, Cinzia; Arrigoni, Giorgio; Beltramini, Mariano; Bubacco, Luigi; Onofri, Franco; Piccoli, Giovanni; Greggio, Elisa

2016-01-13

Lrrk2, a gene linked to Parkinson's disease, encodes a large scaffolding protein with kinase and GTPase activities implicated in vesicle and cytoskeletal-related processes. At the presynaptic site, LRRK2 associates with synaptic vesicles through interaction with a panel of presynaptic proteins. Here, we show that LRRK2 kinase activity influences the dynamics of synaptic vesicle fusion. We therefore investigated whether LRRK2 phosphorylates component(s) of the exo/endocytosis machinery. We have previously observed that LRRK2 interacts with NSF, a hexameric AAA+ ATPase that couples ATP hydrolysis to the disassembling of SNARE proteins allowing them to enter another fusion cycle during synaptic exocytosis. Here, we demonstrate that NSF is a substrate of LRRK2 kinase activity. LRRK2 phosphorylates full-length NSF at threonine 645 in the ATP binding pocket of D2 domain. Functionally, NSF phosphorylated by LRRK2 displays enhanced ATPase activity and increased rate of SNARE complex disassembling. Substitution of threonine 645 with alanine abrogates LRRK2-mediated increased ATPase activity. Given that the most common Parkinson's disease LRRK2 G2019S mutation displays increased kinase activity, our results suggest that mutant LRRK2 may impair synaptic vesicle dynamics via aberrant phosphorylation of NSF.

Active control of complex, multicomponent self-assembly processes

NASA Astrophysics Data System (ADS)

Schulman, Rebecca

The kinetics of many complex biological self-assembly processes such as cytoskeletal assembly are precisely controlled by cells. Spatiotemporal control over rates of filament nucleation, growth and disassembly determine how self-assembly occurs and how the assembled form changes over time. These reaction rates can be manipulated by changing the concentrations of the components needed for assembly by activating or deactivating them. I will describe how we can use these principles to design driven self-assembly processes in which we assemble and disassemble multiple types of components to create micron-scale networks of semiflexible filaments assembled from DNA. The same set of primitive components can be assembled into many different, structures depending on the concentrations of different components and how designed, DNA-based chemical reaction networks manipulate these concentrations over time. These chemical reaction networks can in turn interpret environmental stimuli to direct complex, multistage response. Such a system is a laboratory for understanding complex active material behaviors, such as metamorphosis, self-healing or adaptation to the environment that are ubiquitous in biological systems but difficult to quantitatively characterize or engineer.

Generation of control sequences for a pilot-disassembly system

NASA Astrophysics Data System (ADS)

Seliger, Guenther; Kim, Hyung-Ju; Keil, Thomas

2002-02-01

Closing the product and material cycles has emerged as a paradigm for industry in the 21st century. Disassembly plays a key role in a life cycle economy since it enables the recovery of resources. A partly automated disassembly system should adapt to a large variety of products and different degrees of devaluation. Also the amounts of products to be disassembled can vary strongly. To cope with these demands an approach to generate on-line disassembly control sequences will be presented. In order to react on these demands the technological feasibility is considered within a procedure for the generation of disassembly control sequences. Procedures are designed to find available and technologically feasible disassembly processes. The control system is formed by modularised and parameterised control units in the cell level within the entire control architecture. In the first development stage product and process analyses at the sample product washing machine were executed. Furthermore a generalized disassembly process was defined. Afterwards these processes were structured in primary and secondary functions. In the second stage the disassembly control at the technological level was investigated. Factors were the availability of the disassembly tools and the technological feasibility of the disassembly processes within the disassembly system. Technical alternative disassembly processes are determined as a result of availability of the tools and technological feasibility of processes. The fourth phase was the concept for the generation of the disassembly control sequences. The approach will be proved in a prototypical disassembly system.

Molecular Architecture of the 40S⋅eIF1⋅eIF3 Translation Initiation Complex

PubMed Central

Erzberger, Jan P.; Stengel, Florian; Pellarin, Riccardo; Zhang, Suyang; Schaefer, Tanja; Aylett, Christopher H.S.; Cimermančič, Peter; Boehringer, Daniel; Sali, Andrej; Aebersold, Ruedi; Ban, Nenad

2014-01-01

Summary Eukaryotic translation initiation requires the recruitment of the large, multiprotein eIF3 complex to the 40S ribosomal subunit. We present X-ray structures of all major components of the minimal, six-subunit Saccharomyces cerevisiae eIF3 core. These structures, together with electron microscopy reconstructions, cross-linking coupled to mass spectrometry, and integrative structure modeling, allowed us to position and orient all eIF3 components on the 40S⋅eIF1 complex, revealing an extended, modular arrangement of eIF3 subunits. Yeast eIF3 engages 40S in a clamp-like manner, fully encircling 40S to position key initiation factors on opposite ends of the mRNA channel, providing a platform for the recruitment, assembly, and regulation of the translation initiation machinery. The structures of eIF3 components reported here also have implications for understanding the architecture of the mammalian 43S preinitiation complex and the complex of eIF3, 40S, and the hepatitis C internal ribosomal entry site RNA. PMID:25171412

Hsc70-induced Changes in Clathrin-Auxilin Cage Structure Suggest a Role for Clathrin Light Chains in Cage Disassembly

PubMed Central

Young, Anna; Stoilova-McPhie, Svetla; Rothnie, Alice; Vallis, Yvonne; Harvey-Smith, Phillip; Ranson, Neil; Kent, Helen; Brodsky, Frances M; Pearse, Barbara M F; Roseman, Alan; Smith, Corinne J

2013-01-01

The molecular chaperone, Hsc70, together with its co-factor, auxilin, facilitates the ATP-dependent removal of clathrin during clathrin-mediated endocytosis in cells. We have used cryo-electron microscopy to determine the 3D structure of a complex of clathrin, auxilin401-910 and Hsc70 at pH 6 in the presence of ATP, frozen within 20 seconds of adding Hsc70 in order to visualize events that follow the binding of Hsc70 to clathrin and auxilin before clathrin disassembly. In this map, we observe density beneath the vertex of the cage that we attribute to bound Hsc70. This density emerges asymmetrically from the clathrin vertex, suggesting preferential binding by Hsc70 for one of the three possible sites at the vertex. Statistical comparison with a map of whole auxilin and clathrin previously published by us reveals the location of statistically significant differences which implicate involvement of clathrin light chains in structural rearrangements which occur after Hsc70 is recruited. Clathrin disassembly assays using light scattering suggest that loss of clathrin light chains reduces the efficiency with which auxilin facilitates this reaction. These data support a regulatory role for clathrin light chains in clathrin disassembly in addition to their established role in regulating clathrin assembly. PMID:23710728

Artificial biofilms establish the role of matrix interactions in staphylococcal biofilm assembly and disassembly.

PubMed

Stewart, Elizabeth J; Ganesan, Mahesh; Younger, John G; Solomon, Michael J

2015-08-14

We demonstrate that the microstructural and mechanical properties of bacterial biofilms can be created through colloidal self-assembly of cells and polymers, and thereby link the complex material properties of biofilms to well understood colloidal and polymeric behaviors. This finding is applied to soften and disassemble staphylococcal biofilms through pH changes. Bacterial biofilms are viscoelastic, structured communities of cells encapsulated in an extracellular polymeric substance (EPS) comprised of polysaccharides, proteins, and DNA. Although the identity and abundance of EPS macromolecules are known, how these matrix materials interact with themselves and bacterial cells to generate biofilm morphology and mechanics is not understood. Here, we find that the colloidal self-assembly of Staphylococcus epidermidis RP62A cells and polysaccharides into viscoelastic biofilms is driven by thermodynamic phase instability of EPS. pH conditions that induce phase instability of chitosan produce artificial S. epidermidis biofilms whose mechanics match natural S. epidermidis biofilms. Furthermore, pH-induced solubilization of the matrix triggers disassembly in both artificial and natural S. epidermidis biofilms. This pH-induced disassembly occurs in biofilms formed by five additional staphylococcal strains, including three clinical isolates. Our findings suggest that colloidal self-assembly of cells and matrix polymers produces biofilm viscoelasticity and that biofilm control strategies can exploit this mechanism.

Artificial biofilms establish the role of matrix interactions in staphylococcal biofilm assembly and disassembly

PubMed Central

Stewart, Elizabeth J.; Ganesan, Mahesh; Younger, John G.; Solomon, Michael J.

2015-01-01

We demonstrate that the microstructural and mechanical properties of bacterial biofilms can be created through colloidal self-assembly of cells and polymers, and thereby link the complex material properties of biofilms to well understood colloidal and polymeric behaviors. This finding is applied to soften and disassemble staphylococcal biofilms through pH changes. Bacterial biofilms are viscoelastic, structured communities of cells encapsulated in an extracellular polymeric substance (EPS) comprised of polysaccharides, proteins, and DNA. Although the identity and abundance of EPS macromolecules are known, how these matrix materials interact with themselves and bacterial cells to generate biofilm morphology and mechanics is not understood. Here, we find that the colloidal self-assembly of Staphylococcus epidermidis RP62A cells and polysaccharides into viscoelastic biofilms is driven by thermodynamic phase instability of EPS. pH conditions that induce phase instability of chitosan produce artificial S. epidermidis biofilms whose mechanics match natural S. epidermidis biofilms. Furthermore, pH-induced solubilization of the matrix triggers disassembly in both artificial and natural S. epidermidis biofilms. This pH-induced disassembly occurs in biofilms formed by five additional staphylococcal strains, including three clinical isolates. Our findings suggest that colloidal self-assembly of cells and matrix polymers produces biofilm viscoelasticity and that biofilm control strategies can exploit this mechanism. PMID:26272750

Intracellular accumulation and oligosaccharide processing of alkaline phosphatase under disassembly of the Golgi complex caused by brefeldin A.

PubMed

Takami, N; Oda, K; Fujiwara, T; Ikehara, Y

1990-12-27

Electron microscopic observations showed that the fungal metabolite brefeldin A caused disassembly of the Golgi complex in human choriocarcinoma cells and accumulation of alkaline phosphatase (ALP) in the endoplasmic reticulum (ER) and nuclear envelope, where ALP was not apparently detectable in control cells. Pulse/chase experiments with [35S]methionine demonstrated that in the control cells, ALP synthesized as a 63-kDa precursor form was rapidly converted to a 66-kDa form, by processing of its N-linked oligosaccharides from the high-mannose type to the complex type, which was expressed on the cell surface after 30 min of chase. In contrast, in the brefeldin-A-treated cells the precursor was gradually converted to a 65-kDa form, slightly smaller than the control mature form, which was not expressed on the cell surface even after a prolonged time of chase. Kinetics of the ALP processing in the brefeldin-A-treated cells demonstrated that the precursor was initially converted to an intermediate form, partially sensitive to endo-beta-N-acetylglucosaminidase H (endo H), then to an endo-H-resistant 65-kDa form. In addition, this form was found to be sensitive to neuraminidase digestion, though its sialylation was not so complete as that of the control mature form. Taken together, these results suggest that under disassembly of the Golgi complex caused by brefeldin A, oligosaccharide-processing enzymes including sialyltransferase, an enzyme in the trans Golgi cisterna(e) and/or the trans Golgi network, might be redistributed into the ER and involved in processing of the oligosaccharides of ALP accumulating there.

The assembly and disassembly of ecological networks.

PubMed

Bascompte, Jordi; Stouffer, Daniel B

2009-06-27

Global change has created a severe biodiversity crisis. Species are driven extinct at an increasing rate, and this has the potential to cause further coextinction cascades. The rate and shape of these coextinction cascades depend very much on the structure of the networks of interactions across species. Understanding network structure and how it relates to network disassembly, therefore, is a priority for system-level conservation biology. This process of network collapse may indeed be related to the process of network build-up, although very little is known about both processes and even less about their relationship. Here we review recent work that provides some preliminary answers to these questions. First, we focus on network assembly by emphasizing temporal processes at the species level, as well as the structural building blocks of complex ecological networks. Second, we focus on network disassembly as a consequence of species extinctions or habitat loss. We conclude by emphasizing some general rules of thumb that can help in building a comprehensive framework to understand the responses of ecological networks to global change.

Asymmetric ring structure of Vps4 required for ESCRT-III disassembly

NASA Astrophysics Data System (ADS)

Caillat, Christophe; Macheboeuf, Pauline; Wu, Yuanfei; McCarthy, Andrew A.; Boeri-Erba, Elisabetta; Effantin, Gregory; Göttlinger, Heinrich G.; Weissenhorn, Winfried; Renesto, Patricia

2015-12-01

The vacuolar protein sorting 4 AAA-ATPase (Vps4) recycles endosomal sorting complexes required for transport (ESCRT-III) polymers from cellular membranes. Here we present a 3.6-Å X-ray structure of ring-shaped Vps4 from Metallosphera sedula (MsVps4), seen as an asymmetric pseudohexamer. Conserved key interface residues are shown to be important for MsVps4 assembly, ATPase activity in vitro, ESCRT-III disassembly in vitro and HIV-1 budding. ADP binding leads to conformational changes within the protomer, which might propagate within the ring structure. All ATP-binding sites are accessible and the pseudohexamer binds six ATP with micromolar affinity in vitro. In contrast, ADP occupies one high-affinity and five low-affinity binding sites in vitro, consistent with conformational asymmetry induced on ATP hydrolysis. The structure represents a snapshot of an assembled Vps4 conformation and provides insight into the molecular motions the ring structure undergoes in a concerted action to couple ATP hydrolysis to ESCRT-III substrate disassembly.

Transcription coactivator SAYP combines chromatin remodeler Brahma and transcription initiation factor TFIID into a single supercomplex

PubMed Central

Vorobyeva, Nadezhda E.; Soshnikova, Nataliya V.; Nikolenko, Julia V.; Kuzmina, Julia L.; Nabirochkina, Elena N.; Georgieva, Sofia G.; Shidlovskii, Yulii V.

2009-01-01

Transcription activation by RNA polymerase II is a complicated process driven by combined, precisely coordinated action of a wide array of coactivator complexes, which carry out chromatin-directed activities and nucleate the assembly of the preinitiation complex on the promoter. Using various techniques, we have shown the existence of a stable coactivator supercomplex consisting of the chromatin-remodeling factor Brahma (SWI/SNF) and the transcription initiation factor TFIID, named BTFly (Brahma and TFIID in one assembly). The coupling of Brahma and TFIID is mediated by the SAYP factor, whose evolutionarily conserved activation domain SAY can directly bind to both BAP170 subunit of Brahma and TAF5 subunit of TFIID. The integrity of BTFly is crucial for its ability to activate transcription. BTFly is distributed genome-wide and appears to be a means of effective transcription activation. PMID:19541607

Mediator-dependent Nuclear Receptor Functions

PubMed Central

Chen, Wei; Roeder, Robert

2011-01-01

As gene-specific transcription factors, nuclear hormone receptors are broadly involved in many important biological processes. Their function on target genes requires the stepwise assembly of different coactivator complexes that facilitate chromatin remodeling and subsequent preinitiation complex (PIC) formation and function. Mediator has proved to be a crucial, and general, nuclear receptor-interacting coactivator, with demonstrated functions in transcription steps ranging from chromatin remodeling to subsequent PIC formation and function. Here we discuss (i) our current understanding of pathways that nuclear receptors and other interacting cofactors employ to recruit Mediator to target gene enhancers and promoters, including conditional requirements for the strong NR-Mediator interactions mediated by the NR AF2 domain and the MED1 LXXLLL motifs and (ii) mechanisms by which Mediator acts to transmit signals from enhancer-bound nuclear receptors to the general transcription machinery at core promoters to effect PIC formation and function. PMID:21854863

Stimulation of ribosomal RNA gene promoter by transcription factor Sp1 involves active DNA demethylation by Gadd45-NER pathway.

PubMed

Rajput, Pallavi; Pandey, Vijaya; Kumar, Vijay

2016-08-01

The well-studied Pol II transcription factor Sp1 has not been investigated for its regulatory role in rDNA transcription. Here, we show that Sp1 bound to specific sites on rDNA and localized into the nucleoli during the G1 phase of cell cycle to activate rDNA transcription. It facilitated the recruitment of Pol I pre-initiation complex and impeded the binding of nucleolar remodeling complex (NoRC) to rDNA resulting in the formation of euchromatin active state. More importantly, Sp1 also orchestrated the site-specific binding of Gadd45a-nucleotide excision repair (NER) complex resulting in active demethylation and transcriptional activation of rDNA. Interestingly, knockdown of Sp1 impaired rDNA transcription due to reduced engagement of the Gadd45a-NER complex and hypermethylation of rDNA. Thus, the present study unveils a novel role of Sp1 in rDNA transcription involving promoter demethylation. Copyright © 2016 Elsevier B.V. All rights reserved.

Conformational Differences between Open and Closed States of the Eukaryotic Translation Initiation Complex

PubMed Central

Llácer, Jose L.; Hussain, Tanweer; Marler, Laura; Aitken, Colin Echeverría; Thakur, Anil; Lorsch, Jon R.; Hinnebusch, Alan G.; Ramakrishnan, V.

2015-01-01

Summary Translation initiation in eukaryotes begins with the formation of a pre-initiation complex (PIC) containing the 40S ribosomal subunit, eIF1, eIF1A, eIF3, ternary complex (eIF2-GTP-Met-tRNAi), and eIF5. The PIC, in an open conformation, attaches to the 5′ end of the mRNA and scans to locate the start codon, whereupon it closes to arrest scanning. We present single particle cryo-electron microscopy (cryo-EM) reconstructions of 48S PICs from yeast in these open and closed states, at 6.0 Å and 4.9 Å, respectively. These reconstructions show eIF2β as well as a configuration of eIF3 that appears to encircle the 40S, occupying part of the subunit interface. Comparison of the complexes reveals a large conformational change in the 40S head from an open mRNA latch conformation to a closed one that constricts the mRNA entry channel and narrows the P site to enclose tRNAi, thus elucidating key events in start codon recognition. PMID:26212456

Downstream DNA Tension Regulates the Stability of the T7 RNA Polymerase Initiation Complex

PubMed Central

Skinner, Gary M.; Kalafut, Bennett S.; Visscher, Koen

2011-01-01

Gene transcription by the enzyme RNA polymerase is tightly regulated. In many cases, such as in the lac operon in Escherichia coli, this regulation is achieved through the action of protein factors on DNA. Because DNA is an elastic polymer, its response to enzymatic processing can lead to mechanical perturbations (e.g., linear stretching and supercoiling) that can affect the operation of other DNA processing complexes acting elsewhere on the same substrate molecule. Using an optical-tweezers assay, we measured the binding kinetics between single molecules of bacteriophage T7 RNA polymerase and DNA, as a function of tension. We found that increasing DNA tension under conditions that favor formation of the open complex results in destabilization of the preinitiation complex. Furthermore, with zero ribonucleotides present, when the closed complex is favored, we find reduced tension sensitivity, implying that it is predominantly the open complex that is sensitive. This result strongly supports the “scrunching” model for T7 transcription initiation, as the applied tension acts against the movement of the DNA into the scrunched state, and introduces linear DNA tension as a potential regulatory quantity for transcription initiation. PMID:21320448

Meiotic Clade AAA ATPases: Protein Polymer Disassembly Machines.

PubMed

Monroe, Nicole; Hill, Christopher P

2016-05-08

Meiotic clade AAA ATPases (ATPases associated with diverse cellular activities), which were initially grouped on the basis of phylogenetic classification of their AAA ATPase cassette, include four relatively well characterized family members, Vps4, spastin, katanin and fidgetin. These enzymes all function to disassemble specific polymeric protein structures, with Vps4 disassembling the ESCRT-III polymers that are central to the many membrane-remodeling activities of the ESCRT (endosomal sorting complexes required for transport) pathway and spastin, katanin p60 and fidgetin affecting multiple aspects of cellular dynamics by severing microtubules. They share a common domain architecture that features an N-terminal MIT (microtubule interacting and trafficking) domain followed by a single AAA ATPase cassette. Meiotic clade AAA ATPases function as hexamers that can cycle between the active assembly and inactive monomers/dimers in a regulated process, and they appear to disassemble their polymeric substrates by translocating subunits through the central pore of their hexameric ring. Recent studies with Vps4 have shown that nucleotide-induced asymmetry is a requirement for substrate binding to the pore loops and that recruitment to the protein lattice via MIT domains also relieves autoinhibition and primes the AAA ATPase cassettes for substrate binding. The most striking, unifying feature of meiotic clade AAA ATPases may be their MIT domain, which is a module that is found in a wide variety of proteins that localize to ESCRT-III polymers. Spastin also displays an adjacent microtubule binding sequence, and the presence of both ESCRT-III and microtubule binding elements may underlie the recent findings that the ESCRT-III disassembly function of Vps4 and the microtubule-severing function of spastin, as well as potentially katanin and fidgetin, are highly coordinated. Copyright © 2015 Elsevier Ltd. All rights reserved.

The histone shuffle: histone chaperones in an energetic dance

PubMed Central

Das, Chandrima; Tyler, Jessica K.; Churchill, Mair E.A.

2014-01-01

Our genetic information is tightly packaged into a rather ingenious nucleoprotein complex called chromatin in a manner that enables it to be rapidly accessed during genomic processes. Formation of the nucleosome, which is the fundamental unit of chromatin, occurs via a stepwise process that is reversed to enable the disassembly of nucleosomes. Histone chaperone proteins have prominent roles in facilitating these processes as well as in replacing old histones with new canonical histones or histone variants during the process of histone exchange. Recent structural, biophysical and biochemical studies have begun to shed light on the molecular mechanisms whereby histone chaperones promote chromatin assembly, disassembly and histone exchange to facilitate DNA replication, repair and transcription. PMID:20444609

Kin28 regulates the transient association of Mediator with core promoters.

PubMed

Jeronimo, Célia; Robert, François

2014-05-01

Mediator is an essential, broadly used eukaryotic transcriptional coactivator. How and what Mediator communicates from activators to RNA polymerase II (RNAPII) remains an open question. Here we performed genome-wide location profiling of Saccharomyces cerevisiae Mediator subunits. Mediator is not found at core promoters but rather occupies the upstream activating sequence, upstream of the pre-initiation complex. In the absence of Kin28 (CDK7) kinase activity or in cells in which the RNAPII C-terminal domain is mutated to replace Ser5 with alanine, however, Mediator accumulates at core promoters together with RNAPII. We propose that Mediator is released quickly from promoters after phosphorylation of Ser5 by Kin28 (CDK7), which also allows for RNAPII to escape from the promoter.

Structural basis for nuclear import complex dissociation by RanGTP.

PubMed

Lee, Soo Jae; Matsuura, Yoshiyuki; Liu, Sai Man; Stewart, Murray

2005-06-02

Nuclear protein import is mediated mainly by the transport factor importin-beta that binds cytoplasmic cargo, most often via the importin-alpha adaptor, and then transports it through nuclear pore complexes. This active transport is driven by disassembly of the import complex by nuclear RanGTP. The switch I and II loops of Ran change conformation with nucleotide state, and regulate its interactions with nuclear trafficking components. Importin-beta consists of 19 HEAT repeats that are based on a pair of antiparallel alpha-helices (referred to as the A- and B-helices). The HEAT repeats stack to yield two C-shaped arches, linked together to form a helicoidal molecule that has considerable conformational flexibility. Here we present the structure of full-length yeast importin-beta (Kap95p or karyopherin-beta) complexed with RanGTP, which provides a basis for understanding the crucial cargo-release step of nuclear import. We identify a key interaction site where the RanGTP switch I loop binds to the carboxy-terminal arch of Kap95p. This interaction produces a change in helicoidal pitch that locks Kap95p in a conformation that cannot bind importin-alpha or cargo. We suggest an allosteric mechanism for nuclear import complex disassembly by RanGTP.

Research on automated disassembly technology for waste LCD

NASA Astrophysics Data System (ADS)

Qin, Qin; Zhu, Dongdong; Wang, Jingwei; Dou, Jianfang; Wang, Sujuan; Tu, Zimei

2017-11-01

In the field of Waste LCD disassembling and recycling, there are existing two major problems: 1) disassembling waste LCD mainly depends on manually mechanical crushing; 2) the resource level is not high. In order to deal with the above problems, in this paper, we develop an efficient, safe and automated waste LCD disassembling assembly line technology. This technology can disassembly and classify mainstream LCD into four components, which are liquid crystal display panels, housings and metal shield, PCB assembly. It can also disassembly many kinds of waste LCD. Compared with the traditional cooperation of manual labor and electric tools method, our proposed technology can significantly improve disassembling efficiency and demonstrate good prospects and promotional value.

29 CFR 1926.1407 - Power line safety (up to 350 kV)-assembly and disassembly.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 29 Labor 8 2011-07-01 2011-07-01 false Power line safety (up to 350 kV)-assembly and disassembly... Cranes and Derricks in Construction § 1926.1407 Power line safety (up to 350 kV)—assembly and disassembly... area of assembly/disassembly, closer than 20 feet to a power line during the assembly/disassembly...

Low cytoplasmic pH reduces ER-Golgi trafficking and induces disassembly of the Golgi apparatus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Soonthornsit, Jeerawat; Yamaguchi, Yoko; Tamura, Daisuke

The Golgi apparatus was dramatically disassembled when cells were incubated in a low pH medium. The cis-Golgi disassembled quickly, extended tubules and spread to the periphery of cells within 30 min. In contrast, medial- and trans-Golgi were fragmented in significantly larger structures of smaller numbers at a slower rate and remained largely in structures distinct from the cis-Golgi. Electron microscopy revealed the complete disassembly of the Golgi stack in low pH treated cells. The effect of low pH was reversible; the Golgi apparatus reassembled to form a normal ribbon-like structure within 1–2 h after the addition of a control medium.more » The anterograde ER to Golgi transport and retrograde Golgi to ER transport were both reduced under low pH. Phospholipase A{sub 2} inhibitors (ONO, BEL) effectively suppressed the Golgi disassembly, suggesting that the phospholipase A{sub 2} was involved in the Golgi disassembly. Over-expression of Rab1, 2, 30, 33 and 41 also suppressed the Golgi disassembly under low pH, suggesting that they have protective role against Golgi disassembly. Low pH treatment reduced cytoplasmic pH, but not the luminal pH of the Golgi apparatus, strongly suggesting that reduction of the cytoplasmic pH triggered the Golgi disassembly. Because a lower cytoplasmic pH is induced in physiological or pathological conditions, disassembly of the Golgi apparatus and reduction of vesicular transport through the Golgi apparatus may play important roles in cell physiology and pathology. Furthermore, our findings indicated that low pH treatment can serve as an important tool to analyze the molecular mechanisms that support the structure and function of the Golgi apparatus. - Highlights: • The Golgi apparatus reversibly disassembles by low pH treatment. • The cis-Golgi disassembles quickly generating tubular structures. • Both anterograde and retrograde transport between the ER and the Golgi apparatus are reduced. • Phospholipase A{sub 2} inhibitors (ONO-RS082, BEL) prevented the low pH induced Golgi disassembly. • Rab1, 2, 30, 33 and 41 suppress low pH induced Golgi disassembly.« less

Decapping activators in Saccharomyces cerevisiae act by multiple mechanisms.

PubMed

Nissan, Tracy; Rajyaguru, Purusharth; She, Meipei; Song, Haiwei; Parker, Roy

2010-09-10

Eukaryotic mRNA degradation often occurs in a process whereby translation initiation is inhibited and the mRNA is targeted for decapping. In yeast cells, Pat1, Scd6, Edc3, and Dhh1 all function to promote decapping by an unknown mechanism(s). We demonstrate that purified Scd6 and a region of Pat1 directly repress translation in vitro by limiting the formation of a stable 48S preinitiation complex. Moreover, while Pat1, Edc3, Dhh1, and Scd6 all bind the decapping enzyme, only Pat1 and Edc3 enhance its activity. We also identify numerous direct interactions between Pat1, Dcp1, Dcp2, Dhh1, Scd6, Edc3, Xrn1, and the Lsm1-7 complex. These observations identify three classes of decapping activators that function to directly repress translation initiation and/or stimulate Dcp1/2. Moreover, Pat1 is identified as critical in mRNA decay by first inhibiting translation initiation, then serving as a scaffold to recruit components of the decapping complex, and finally activating Dcp2. Copyright © 2010 Elsevier Inc. All rights reserved.

Structure of the initiation-competent RNA polymerase I and its implication for transcription

NASA Astrophysics Data System (ADS)

Pilsl, Michael; Crucifix, Corinne; Papai, Gabor; Krupp, Ferdinand; Steinbauer, Robert; Griesenbeck, Joachim; Milkereit, Philipp; Tschochner, Herbert; Schultz, Patrick

2016-07-01

Eukaryotic RNA polymerase I (Pol I) is specialized in rRNA gene transcription synthesizing up to 60% of cellular RNA. High level rRNA production relies on efficient binding of initiation factors to the rRNA gene promoter and recruitment of Pol I complexes containing initiation factor Rrn3. Here, we determine the cryo-EM structure of the Pol I-Rrn3 complex at 7.5 Å resolution, and compare it with Rrn3-free monomeric and dimeric Pol I. We observe that Rrn3 contacts the Pol I A43/A14 stalk and subunits A190 and AC40, that association re-organizes the Rrn3 interaction interface, thereby preventing Pol I dimerization; and Rrn3-bound and monomeric Pol I differ from the dimeric enzyme in cleft opening, and localization of the A12.2 C-terminus in the active centre. Our findings thus support a dual role for Rrn3 in transcription initiation to stabilize a monomeric initiation competent Pol I and to drive pre-initiation complex formation.

A selective decoy-doxorubicin complex for targeted co-delivery, STAT3 probing and synergistic anti-cancer effect.

PubMed

Wang, Shao-Jen; Hou, Yung-Te; Chen, Lin-Chi

2015-09-04

A novel selective decoy oligodeoxynucleotide (dODN)-doxorubicin (DOX) complex is reported for cancer theranostics. It eliminates the use of a ligand or carrier for targeted delivery and disassembles into therapeutic dODN and DOX upon encountering over-activated STAT3 in cancer cells. Hence, in situ STAT3 probing and synergistic anti-cancer effect are attained at the same time.

Inositol hexakisphosphate kinase-1 mediates assembly/disassembly of the CRL4–signalosome complex to regulate DNA repair and cell death

PubMed Central

Rao, Feng; Xu, Jing; Khan, A. Basit; Gadalla, Moataz M.; Cha, Jiyoung Y.; Xu, Risheng; Tyagi, Richa; Dang, Yongjun; Chakraborty, Anutosh; Snyder, Solomon H.

2014-01-01

Inositol polyphosphates containing an energetic pyrophosphate bond are formed primarily by a family of three inositol hexakisphosphate (IP6) kinases (IP6K1–3). The Cullin-RING ubiquitin ligases (CRLs) regulate diverse biological processes through substrate ubiquitylation. CRL4, comprising the scaffold Cullin 4A/B, the E2-interacting Roc1/2, and the adaptor protein damage-specific DNA-binding protein 1, is activated by DNA damage. Basal CRL4 activity is inhibited by binding to the COP9 signalosome (CSN). UV radiation and other stressors dissociate the complex, leading to E3 ligase activation, but signaling events that trigger signalosome dissociation from CRL4 have been unclear. In the present study, we show that, under basal conditions, IP6K1 forms a ternary complex with CSN and CRL4 in which IP6K1 and CRL4 are inactive. UV dissociates IP6K1 to generate IP7, which then dissociates CSN–CRL4 to activate CRL4. Thus, IP6K1 is a novel CRL4 subunit that transduces UV signals to mediate disassembly of the CRL4–CSN complex, thereby regulating nucleotide excision repair and cell death. PMID:25349427

29 CFR 1926.1403 - Assembly/Disassembly-selection of manufacturer or employer procedures.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 29 Labor 8 2013-07-01 2013-07-01 false Assembly/Disassembly-selection of manufacturer or employer... CONSTRUCTION Cranes and Derricks in Construction § 1926.1403 Assembly/Disassembly—selection of manufacturer or... applicable to assembly and disassembly, or (b) Employer procedures for assembly and disassembly. Employer...

29 CFR 1926.1403 - Assembly/Disassembly-selection of manufacturer or employer procedures.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 29 Labor 8 2012-07-01 2012-07-01 false Assembly/Disassembly-selection of manufacturer or employer... CONSTRUCTION Cranes and Derricks in Construction § 1926.1403 Assembly/Disassembly—selection of manufacturer or... applicable to assembly and disassembly, or (b) Employer procedures for assembly and disassembly. Employer...

29 CFR 1926.1403 - Assembly/Disassembly-selection of manufacturer or employer procedures.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 29 Labor 8 2014-07-01 2014-07-01 false Assembly/Disassembly-selection of manufacturer or employer... CONSTRUCTION Cranes and Derricks in Construction § 1926.1403 Assembly/Disassembly—selection of manufacturer or... applicable to assembly and disassembly, or (b) Employer procedures for assembly and disassembly. Employer...

29 CFR 1926.1403 - Assembly/Disassembly-selection of manufacturer or employer procedures.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 29 Labor 8 2011-07-01 2011-07-01 false Assembly/Disassembly-selection of manufacturer or employer... CONSTRUCTION Cranes and Derricks in Construction § 1926.1403 Assembly/Disassembly—selection of manufacturer or... applicable to assembly and disassembly, or (b) Employer procedures for assembly and disassembly. Employer...

Impact of different disassembly line balancing algorithms on the performance of dynamic kanban system for disassembly line

NASA Astrophysics Data System (ADS)

Kizilkaya, Elif A.; Gupta, Surendra M.

2005-11-01

In this paper, we compare the impact of different disassembly line balancing (DLB) algorithms on the performance of our recently introduced Dynamic Kanban System for Disassembly Line (DKSDL) to accommodate the vagaries of uncertainties associated with disassembly and remanufacturing processing. We consider a case study to illustrate the impact of various DLB algorithms on the DKSDL. The approach to the solution, scenario settings, results and the discussions of the results are included.

The AAA+ ATPase TRIP13 remodels HORMA domains through N-terminal engagement and unfolding

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ye, Qiaozhen; Kim, Dong Hyun; Dereli, Ihsan

Proteins of the conserved HORMA domain family, including the spindle assembly checkpoint protein MAD2 and the meiotic HORMADs, assemble into signaling complexes by binding short peptides termed “closure motifs”. The AAA+ ATPase TRIP13 regulates both MAD2 and meiotic HORMADs by disassembling these HORMA domain–closure motif complexes, but its mechanisms of substrate recognition and remodeling are unknown. Here, we combine X-ray crystallography and crosslinking mass spectrometry to outline how TRIP13 recognizes MAD2 with the help of the adapter protein p31comet. We show that p31comet binding to the TRIP13 N-terminal domain positions the disordered MAD2 N-terminus for engagement by the TRIP13 “poremore » loops”, which then unfold MAD2 in the presence of ATP. N-terminal truncation of MAD2 renders it refractory to TRIP13 action in vitro, and in cells causes spindle assembly checkpoint defects consistent with loss of TRIP13 function. Similar truncation of HORMAD1 in mouse spermatocytes compromises its TRIP13-mediated removal from meiotic chromosomes, highlighting a conserved mechanism for recognition and disassembly of HORMA domain–closure motif complexes by TRIP13.« less

Kin28 regulates the transient association of Mediator with core promoters

PubMed Central

Jeronimo, Célia; Robert, François

2014-01-01

Mediator is an essential, broadly utilized eukaryotic transcriptional co-activator. How and what it communicates from activators to RNA polymerase II (RNAPII) remains an open question. Here we performed genome-wide location profiling of Saccharomyces cerevisiae Mediator subunits. Mediator is not found at core promoters but rather occupies the upstream activating sequence (UAS), upstream of the pre-initiation complex. In the absence of Kin28 (CDK7) kinase activity, or in cells where the RNAPII C-terminal domain (CTD) is mutated to replace Ser5 with alanines, however, Mediator accumulates at core promoters together with RNAPII. We propose that Mediator is quickly released from promoters upon Ser5 phosphorylation by Kin28 (CDK7), which also allows for RNAPII to escape from the promoter. PMID:24704787

The eIF4E-binding proteins are modifiers of cytoplasmic eIF4E relocalization during the heat shock response.

PubMed

Sukarieh, R; Sonenberg, N; Pelletier, J

2009-05-01

Stress granules (SGs) arise as a consequence of cellular stress, contain stalled translation preinitiation complexes, and are associated with cell survival during environmental insults. SGs are dynamic entities with proteins relocating into and out of them during stress. Among the repertoire of proteins present in SGs is eukaryotic initiation factor 4E (eIF4E), a translation factor required for cap-dependent translation and that regulates a rate-limiting step for protein synthesis. Herein, we demonstrate that localization of eIF4E to SGs is dependent on the presence of a family of repressor proteins, eIF4E-binding proteins (4E-BPs). Our results demonstrate that 4E-BPs regulate the SG localization of eIF4E.

MCM-BP regulates unloading of the MCM2–7 helicase in late S phase

PubMed Central

Nishiyama, Atsuya; Frappier, Lori; Méchali, Marcel

2011-01-01

Origins of DNA replication are licensed by recruiting MCM2–7 to assemble the prereplicative complex (pre-RC). How MCM2–7 is inactivated or removed from chromatin at the end of S phase is still unclear. Here, we show that MCM-BP can disassemble the MCM2–7 complex and might function as an unloader of MCM2–7 from chromatin. In Xenopus egg extracts, MCM-BP exists in a stable complex with MCM7, but is not associated with the MCM2–7 hexameric complex. MCM-BP accumulates in nuclei in late S phase, well after the loading of MCM2–7 onto chromatin. MCM-BP immunodepletion in Xenopus egg extracts inhibits replication-dependent MCM dissociation without affecting pre-RC formation and DNA replication. When excess MCM-BP is incubated with Xenopus egg extracts or immunopurified MCM2–7, it binds to MCM proteins and promotes disassembly of the MCM2–7 complex. Recombinant MCM-BP also releases MCM2–7 from isolated late-S-phase chromatin, but this activity is abolished when DNA replication is blocked. MCM-BP silencing in human cells also delays MCM dissociation in late S phase. We propose that MCM-BP plays a key role in the mechanism by which pre-RC is cleared from replicated DNA in vertebrate cells. PMID:21196493

Maternal Dead-end 1 promotes translation of nanos1 by binding the eIF3 complex.

PubMed

Aguero, Tristan; Jin, Zhigang; Chorghade, Sandip; Kalsotra, Auinash; King, Mary Lou; Yang, Jing

2017-10-15

In the developing embryo, primordial germ cells (PGCs) represent the exclusive progenitors of the gametes, and their loss results in adult infertility. During early development, PGCs are exposed to numerous signals that specify somatic cell fates. To prevent somatic differentiation, PGCs must transiently silence their genome, an early developmental process that requires Nanos activity. However, it is unclear how Nanos translation is regulated in developing embryos. We report here that translation of nanos1 after fertilization requires Dead-end 1 (Dnd1), a vertebrate-specific germline RNA-binding protein. We provide evidence that Dnd1 protein, expression of which is low in oocytes, but increases dramatically after fertilization, directly interacts with, and relieves the inhibitory function of eukaryotic initiation factor 3f, a repressive component in the 43S preinitiation complex. This work uncovers a novel translational regulatory mechanism that is fundamentally important for germline development. © 2017. Published by The Company of Biologists Ltd.

Near-atomic resolution visualization of human transcription promoter opening

PubMed Central

He, Yuan; Yan, Chunli; Fang, Jie; Inouye, Carla; Tjian, Robert; Ivanov, Ivaylo; Nogales, Eva

2016-01-01

In eukaryotic transcription initiation, a large multi-subunit pre-initiation complex (PIC) that assembles at the core promoter is required for the opening of the duplex DNA and identification of the start site for transcription by RNA polymerase II. Here we use cryo-electron microscropy (cryo-EM) to determine near-atomic resolution structures of the human PIC in a closed state (engaged with duplex DNA), an open state (engaged with a transcription bubble), and an initially transcribing complex (containing six base pairs of DNA–RNA hybrid). Our studies provide structures for previously uncharacterized components of the PIC, such as TFIIE and TFIIH, and segments of TFIIA, TFIIB and TFIIF. Comparison of the different structures reveals the sequential conformational changes that accompany the transition from each state to the next throughout the transcription initiation process. This analysis illustrates the key role of TFIIB in transcription bubble stabilization and provides strong structural support for a translocase activity of XPB. PMID:27193682

Architecture of human translation initiation factor 3

PubMed Central

Querol-Audi, Jordi; Sun, Chaomin; Vogan, Jacob M.; Smith, Duane; Gu, Yu; Cate, Jamie; Nogales, Eva

2013-01-01

SUMMARY Eukaryotic translation initiation factor 3 (eIF3) plays a central role in protein synthesis by organizing the formation of the 43S preinitiation complex. Using genetic tag visualization by electron microscopy, we reveal the molecular organization of ten human eIF3 subunits, including an octameric core. The structure of eIF3 bears a close resemblance to that of the proteasome lid, with a conserved spatial organization of eight core subunits containing PCI and MPN domains that coordinate functional interactions in both complexes. We further show that eIF3 subunits a and c interact with initiation factors eIF1 and eIF1A, which control the stringency of start codon selection. Finally, we find that subunit j, which modulates messenger RNA interactions with the small ribosomal subunit, makes multiple independent interactions with the eIF3 octameric core. These results highlight the conserved architecture of eIF3 and how it scaffolds key factors that control translation initiation in higher eukaryotes, including humans. PMID:23623729

Defining the Status of RNA Polymerase at Promoters

PubMed Central

Core, Leighton J.; Waterfall, Joshua J.; Gilchrist, Daniel A.; Fargo, David C.; Kwak, Hojoong; Adelman, Karen; Lis, John T.

2012-01-01

Summary Recent genome-wide studies in metazoans have shown that RNA Polymerase II (Pol II) accumulates to high densities on many promoters at a rate-limited step in transcription. However, the status of this Pol II remains an area of debate. Here, we compare quantitative outputs of GRO-seq and ChIP-seq assays and demonstrate the majority of the Pol II on Drosophila promoters is transcriptionally-engaged - very little exists in a preinitiation or arrested complex. These promoter-proximal polymerases are inhibited from further elongation by detergent sensitive factors, and knockdown of negative elongation factor, NELF, reduces their levels. These results not only solidify that pausing occurs at most promoters, but demonstrate that it is the major rate-limiting step in early transcription at these promoters. Finally, the divergent elongation complexes seen at mammalian promoters are far less prevalent in Drosophila, and this specificity in orientation correlates with directional core promoter elements, which are abundant in Drosophila. PMID:23062713

Mediator Undergoes a Compositional Change during Transcriptional Activation.

PubMed

Petrenko, Natalia; Jin, Yi; Wong, Koon Ho; Struhl, Kevin

2016-11-03

Mediator is a transcriptional co-activator recruited to enhancers by DNA-binding activators, and it also interacts with RNA polymerase (Pol) II as part of the preinitiation complex (PIC). We demonstrate that a single Mediator complex associates with the enhancer and core promoter in vivo, indicating that it can physically bridge these transcriptional elements. However, the Mediator kinase module associates strongly with the enhancer, but not with the core promoter, and it dissociates from the enhancer upon depletion of the TFIIH kinase. Severing the kinase module from Mediator by removing the connecting subunit Med13 does not affect Mediator association at the core promoter but increases occupancy at enhancers. Thus, Mediator undergoes a compositional change in which the kinase module, recruited via Mediator to the enhancer, dissociates from Mediator to permit association with Pol II and the PIC. As such, Mediator acts as a dynamic bridge between the enhancer and core promoter. Copyright © 2016 Elsevier Inc. All rights reserved.

Characterization of a novel RNA-binding region of eIF4GI critical for ribosomal scanning

PubMed Central

Prévôt, Déborah; Décimo, Didier; Herbreteau, Cécile H.; Roux, Florence; Garin, Jérôme; Darlix, Jean-Luc; Ohlmann, Théophile

2003-01-01

The eukaryotic translation initiation factor eIF4GI binds several proteins and acts as a scaffold to promote preinitiation complex formation on the mRNA molecule (48S). Following mRNA attachment this complex scans along the messenger in a 5′ to 3′ direction until it locates and recognizes the initiation start codon. By using a combination of retroviral and picornaviral proteases (HIV-2 and L respectively) in the reticulocyte lysate system, we have characterized a 40 amino acid (aa) region of eIF4GI (aa 642–681) that exhibits general RNA-binding properties. Removal of this domain by proteolytic processing followed by translational assays showed virtually no inhibition of internal ribosome entry on the encephalomyocarditis virus, but resulted in drastic impairment of ribosome scanning as demonstrated by studying poliovirus and foot-and-mouth disease virus translation. Based on these findings, we propose that this 40 aa motif of eIF4GI is critical for ribosome scanning. PMID:12682023

Computational algorithm to evaluate product disassembly cost index

NASA Astrophysics Data System (ADS)

Zeid, Ibrahim; Gupta, Surendra M.

2002-02-01

Environmentally conscious manufacturing is an important paradigm in today's engineering practice. Disassembly is a crucial factor in implementing this paradigm. Disassembly allows the reuse and recycling of parts and products that reach their death after their life cycle ends. There are many questions that must be answered before a disassembly decision can be reached. The most important question is economical. The cost of disassembly versus the cost of scrapping a product is always considered. This paper develops a computational tool that allows decision-makers to calculate the disassembly cost of a product. The tool makes it simple to perform 'what if' scenarios fairly quickly. The tool is Web based and has two main parts. The front-end part is a Web page and runs on the client side in a Web browser, while the back-end part is a disassembly engine (servlet) that has disassembly knowledge and costing algorithms and runs on the server side. The tool is based on the client/server model that is pervasively utilized throughout the World Wide Web. An example is used to demonstrate the implementation and capabilities of the tool.

Transcription regulation by the Mediator complex.

PubMed

Soutourina, Julie

2018-04-01

Alterations in the regulation of gene expression are frequently associated with developmental diseases or cancer. Transcription activation is a key phenomenon in the regulation of gene expression. In all eukaryotes, mediator of RNA polymerase II transcription (Mediator), a large complex with modular organization, is generally required for transcription by RNA polymerase II, and it regulates various steps of this process. The main function of Mediator is to transduce signals from the transcription activators bound to enhancer regions to the transcription machinery, which is assembled at promoters as the preinitiation complex (PIC) to control transcription initiation. Recent functional studies of Mediator with the use of structural biology approaches and functional genomics have revealed new insights into Mediator activity and its regulation during transcription initiation, including how Mediator is recruited to transcription regulatory regions and how it interacts and cooperates with PIC components to assist in PIC assembly. Novel roles of Mediator in the control of gene expression have also been revealed by showing its connection to the nuclear pore and linking Mediator to the regulation of gene positioning in the nuclear space. Clear links between Mediator subunits and disease have also encouraged studies to explore targeting of this complex as a potential therapeutic approach in cancer and fungal infections.

Yeast eIF4A enhances recruitment of mRNAs regardless of their structural complexity

PubMed Central

Yourik, Paul; Aitken, Colin Echeverría; Zhou, Fujun; Gupta, Neha

2017-01-01

eIF4A is a DEAD-box RNA-dependent ATPase thought to unwind RNA secondary structure in the 5'-untranslated regions (UTRs) of mRNAs to promote their recruitment to the eukaryotic translation pre-initiation complex (PIC). We show that eIF4A's ATPase activity is markedly stimulated in the presence of the PIC, independently of eIF4E•eIF4G, but dependent on subunits i and g of the heteromeric eIF3 complex. Surprisingly, eIF4A accelerated the rate of recruitment of all mRNAs tested, regardless of their degree of structural complexity. Structures in the 5'-UTR and 3' of the start codon synergistically inhibit mRNA recruitment in a manner relieved by eIF4A, indicating that the factor does not act solely to melt hairpins in 5'-UTRs. Our findings that eIF4A functionally interacts with the PIC and plays important roles beyond unwinding 5'-UTR structure is consistent with a recent proposal that eIF4A modulates the conformation of the 40S ribosomal subunit to promote mRNA recruitment. PMID:29192585

Multikanban model for disassembly line with demand fluctuation

NASA Astrophysics Data System (ADS)

Udomsawat, Gun; Gupta, Surendra M.; Al-Turki, Yousef A. Y.

2004-02-01

In recent years, the continuous growth in consumer waste and dwindling natural resources has seriously threatened the environment. Realizing this, several countries have passed regulations that force manufacturers not only to manufacture environmentally conscious products, but also to take back their used products from consumers so that the components and materials recovered from the products may be reused and/or recycled. Disassembly plays an important role in product recovery. A disassembly line is perhaps the most suitable setting for disassembly of products in large quantities. Because a disassembly line has a tendency to generate excessive inventory, employing a kanban system can reduce the inventory level and let the system run more efficiently. A disassembly line is quite different from an assembly line. For example, not only can the demand arrive at the last station, it can also arrive at any of the other stations in the system. The demand for a component on the disassembly line could fluctuate widely. In fact, there are many other complicating matters that need to be considered to implement the concept of kanbans in such an environment. In this paper, we discuss the complications that are unique to a disassembly line. We discuss the complications in utilizing the conventional production control mechanisms in a disassembly line setting. We then show how to overcome them by implementing kanbans in a disassembly line setting with demand fluctuation and introduce the concept of multi-kanban mechanism. We demonstrate its effectiveness using a simulation model. An example is presented to illustrate the concept.

Dining at the periodic table: metals concentrations as they relate to recycling.

PubMed

Johnson, Jeremiah; Harper, E M; Lifset, Reid; Graedel, T E

2007-03-01

A correlation between the prices of a variety of substances and their dilutions in their initial matrices was shown in 1959 by T.K. Sherwood. The research presented here shows that the relationship holds for engineering metals today, which we termed the metals-specific Sherwood plot. The concentrations of metals in products (e.g., printed wiring boards and automobiles) and waste streams (e.g., municipal solid waste, and construction and demolition debris) were plotted with this correlation. In addition, for the products and waste streams that undergo disassembly at end-of-life, the metals concentrations of the disassembled components were also plotted. It was found that most of the metals that are currently targeted for recycling have post-disassembly concentrations that lie above the metals-specific Sherwood plot (i.e., have concentrations that are more enriched than minimum profitable ore grades). This suggests that material concentration plays a role in the viability of recycling at end-of-life. As products grow in complexity and the variety of materials used, analyses such as this one provide insight for policymakers and those interested in material sustainability into macro-level trends of material use and future recycling practices.

BAR Proteins PSTPIP1/2 Regulate Podosome Dynamics and the Resorption Activity of Osteoclasts

PubMed Central

Sztacho, Martin; Segeletz, Sandra; Sanchez-Fernandez, Maria Arantzazu; Czupalla, Cornelia; Niehage, Christian; Hoflack, Bernard

2016-01-01

Bone resorption in vertebrates relies on the ability of osteoclasts to assemble F-actin-rich podosomes that condense into podosomal belts, forming sealing zones. Sealing zones segregate bone-facing ruffled membranes from other membrane domains, and disassemble when osteoclasts migrate to new areas. How podosome/sealing zone dynamics is regulated remains unknown. We illustrate the essential role of the membrane scaffolding F-BAR-Proline-Serine-Threonine Phosphatase Interacting Proteins (PSTPIP) 1 and 2 in this process. Whereas PSTPIP2 regulates podosome assembly, PSTPIP1 regulates their disassembly. PSTPIP1 recruits, through its F-BAR domain, the protein tyrosine phosphatase non-receptor type 6 (PTPN6) that de-phosphophorylates the phosphatidylinositol 5-phosphatases SHIP1/2 bound to the SH3 domain of PSTPIP1. Depletion of any component of this complex prevents sealing zone disassembly and increases osteoclast activity. Thus, our results illustrate the importance of BAR domain proteins in podosome structure and dynamics, and identify a new PSTPIP1/PTPN6/SHIP1/2-dependent negative feedback mechanism that counterbalances Src and PI(3,4,5)P3 signalling to control osteoclast cell polarity and activity during bone resorption. PMID:27760174

Mechanistic Insights from Structural Analyses of Ran-GTPase-Driven Nuclear Export of Proteins and RNAs.

PubMed

Matsuura, Yoshiyuki

2016-05-22

Understanding how macromolecules are rapidly exchanged between the nucleus and the cytoplasm through nuclear pore complexes is a fundamental problem in biology. Exportins are Ran-GTPase-dependent nuclear transport factors that belong to the karyopherin-β family and mediate nuclear export of a plethora of proteins and RNAs, except for bulk mRNA nuclear export. Exportins bind cargo macromolecules in a Ran-GTP-dependent manner in the nucleus, forming exportin-cargo-Ran-GTP complexes (nuclear export complexes). Transient weak interactions between exportins and nucleoporins containing characteristic FG (phenylalanine-glycine) repeat motifs facilitate nuclear pore complex passage of nuclear export complexes. In the cytoplasm, nuclear export complexes are disassembled, thereby releasing the cargo. GTP hydrolysis by Ran promoted in the cytoplasm makes the disassembly reaction virtually irreversible and provides thermodynamic driving force for the overall export reaction. In the past decade, X-ray crystallography of some of the exportins in various functional states coupled with functional analyses, single-particle electron microscopy, molecular dynamics simulations, and small-angle solution X-ray scattering has provided rich insights into the mechanism of cargo binding and release and also begins to elucidate how exportins interact with the FG repeat motifs. The knowledge gained from structural analyses of nuclear export is being translated into development of clinically useful inhibitors of nuclear export to treat human diseases such as cancer and influenza. Copyright © 2015 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Paul Demkowicz; Lance Cole; Scott Ploger

The AGR-1 irradiation experiment ended on November 6, 2009, after 620 effective full power days in the Advanced Test Reactor, achieving a peak burnup of 19.6% FIMA. The test train was shipped to the Materials and Fuels Complex in March 2010 for post-irradiation examination. The first PIE activities included non-destructive examination of the test train, followed by disassembly of the test train and individual capsules and detailed inspection of the capsule contents, including the fuel compacts and the graphite fuel holders. Dimensional measurements of the compacts, graphite holders, and steel capsules shells were performed using a custom vision measurement systemmore » (for outer diameters and lengths) and conventional bore gauges (for inner diameters). Gamma spectrometry of the intact test train gave a preliminary look at the condition of the interior components. No evidence of damage to compacts or graphite components was evident from the isotopic and gross gamma scans. Neutron radiography of the intact Capsule 2 showed a high degree of detail of interior components and confirmed the observation that there was no major damage to the capsule. Disassembly of the capsules was initiated using procedures qualified during out-of-cell mockup testing. Difficulties were encountered during capsule disassembly due to irradiation-induced changes in some of the capsule components’ properties, including embrittled niobium and molybdenum parts that were susceptible to fracture and swelling of the graphite fuel holders that affected their removal from the capsule shells. This required various improvised modifications to the disassembly procedure to avoid damage to the fuel compacts. Ultimately the capsule disassembly was successful and only one compact from Capsule 4 (out of 72 total in the test train) sustained damage during the disassembly process, along with the associated graphite holder. The compacts were generally in very good condition upon removal. Only relatively minor damage or markings were visible using high resolution photographic inspection. Compact dimensional measurements indicated diametrical shrinkage of 0.9 to 1. 4%, and length shrinkage of 0.2 to 1.1%. The shrinkage was somewhat dependent on compact location within each capsule and within the test train. Compacts exhibited a maximum diametrical shrinkage at a fast neutron fluence of approximately 3×1021 n/cm2. A multivariate statistical analysis indicates that fast neutron fluence as well as compact position in the test train influence compact shrinkage.« less

Disassembling the Classroom--An Ethnographic Approach to the Materiality of Education

ERIC Educational Resources Information Center

Roehl, Tobias

2012-01-01

The ethnography of education is challenged by the materiality of the classroom. Ethnographic accounts of school lessons mostly highlight language and interaction and offer no suitable methodology for researching objects and their role in the classroom. Moreover, objects are part of complex and interwoven assemblages involving human actors,…

QIL1 mutation causes MICOS disassembly and early onset fatal mitochondrial encephalopathy with liver disease

PubMed Central

Guarani, Virginia; Jardel, Claude; Chrétien, Dominique; Lombès, Anne; Bénit, Paule; Labasse, Clémence; Lacène, Emmanuelle; Bourillon, Agnès; Imbard, Apolline; Benoist, Jean-François; Dorboz, Imen; Gilleron, Mylène; Goetzman, Eric S; Gaignard, Pauline; Slama, Abdelhamid; Elmaleh-Bergès, Monique; Romero, Norma B; Rustin, Pierre; Ogier de Baulny, Hélène; Paulo, Joao A; Harper, J Wade; Schiff, Manuel

2016-01-01

Previously, we identified QIL1 as a subunit of mitochondrial contact site (MICOS) complex and demonstrated a role for QIL1 in MICOS assembly, mitochondrial respiration, and cristae formation critical for mitochondrial architecture (Guarani et al., 2015). Here, we identify QIL1 null alleles in two siblings displaying multiple clinical symptoms of early-onset fatal mitochondrial encephalopathy with liver disease, including defects in respiratory chain function in patient muscle. QIL1 absence in patients’ fibroblasts was associated with MICOS disassembly, abnormal cristae, mild cytochrome c oxidase defect, and sensitivity to glucose withdrawal. QIL1 expression rescued cristae defects, and promoted re-accumulation of MICOS subunits to facilitate MICOS assembly. MICOS assembly and cristae morphology were not efficiently rescued by over-expression of other MICOS subunits in patient fibroblasts. Taken together, these data provide the first evidence of altered MICOS assembly linked with a human mitochondrial disease and confirm a central role for QIL1 in stable MICOS complex formation. DOI: http://dx.doi.org/10.7554/eLife.17163.001 PMID:27623147

Low cytoplasmic pH reduces ER-Golgi trafficking and induces disassembly of the Golgi apparatus.

PubMed

Soonthornsit, Jeerawat; Yamaguchi, Yoko; Tamura, Daisuke; Ishida, Ryuichi; Nakakoji, Yoko; Osako, Shiho; Yamamoto, Akitsugu; Nakamura, Nobuhiro

2014-11-01

The Golgi apparatus was dramatically disassembled when cells were incubated in a low pH medium. The cis-Golgi disassembled quickly, extended tubules and spread to the periphery of cells within 30 min. In contrast, medial- and trans-Golgi were fragmented in significantly larger structures of smaller numbers at a slower rate and remained largely in structures distinct from the cis-Golgi. Electron microscopy revealed the complete disassembly of the Golgi stack in low pH treated cells. The effect of low pH was reversible; the Golgi apparatus reassembled to form a normal ribbon-like structure within 1-2h after the addition of a control medium. The anterograde ER to Golgi transport and retrograde Golgi to ER transport were both reduced under low pH. Phospholipase A2 inhibitors (ONO, BEL) effectively suppressed the Golgi disassembly, suggesting that the phospholipase A2 was involved in the Golgi disassembly. Over-expression of Rab1, 2, 30, 33 and 41 also suppressed the Golgi disassembly under low pH, suggesting that they have protective role against Golgi disassembly. Low pH treatment reduced cytoplasmic pH, but not the luminal pH of the Golgi apparatus, strongly suggesting that reduction of the cytoplasmic pH triggered the Golgi disassembly. Because a lower cytoplasmic pH is induced in physiological or pathological conditions, disassembly of the Golgi apparatus and reduction of vesicular transport through the Golgi apparatus may play important roles in cell physiology and pathology. Furthermore, our findings indicated that low pH treatment can serve as an important tool to analyze the molecular mechanisms that support the structure and function of the Golgi apparatus. Copyright © 2014 Elsevier Inc. All rights reserved.

Comparative Proteomics Reveals Timely Transport into Cilia of Regulators or Effectors as a Mechanism Underlying Ciliary Disassembly.

PubMed

Wang, Limei; Gu, Lixiao; Meng, Dan; Wu, Qiong; Deng, Haiteng; Pan, Junmin

2017-07-07

Primary cilia are assembled and disassembled during cell cycle progression. During ciliary disassembly, ciliary axonemal microtubules (MTs) are depolymerized accompanied by extensive posttranslational protein modifications of ciliary proteins including protein phosphorylation, methylation, and ubiquitination. These events are hypothesized to involve transport of effectors or regulators into cilia at the time of ciliary disassembly from the cell body. To prove this hypothesis and identify new proteins involved in ciliary disassembly, we analyzed disassembling flagella in Chlamydomonas using comparative proteomics with TMT labeling. Ninety-one proteins were found to increase more than 1.4-fold in four replicates. The proteins of the IFT machinery not only increase but also exhibit stoichiometric changes. The other proteins that increase include signaling molecules, chaperones, and proteins involved in microtubule dynamics or stability. In particular, we have identified a ciliopathy protein C21orf2, the AAA-ATPase CDC48, that is involved in segregating polypeptides from large assemblies or cellular structures, FAP203 and FAP236, which are homologous to stabilizers of axonemal microtubules. Our data demonstrate that ciliary transport of effectors or regulators is one of the mechanisms underlying ciliary disassembly. Further characterization of the proteins identified will provide new insights into our understanding of ciliary disassembly and likely ciliopathy.

The eIF4E-binding proteins are modifiers of cytoplasmic eIF4E relocalization during the heat shock response

PubMed Central

Sukarieh, R.; Sonenberg, N.; Pelletier, J.

2009-01-01

Stress granules (SGs) arise as a consequence of cellular stress, contain stalled translation preinitiation complexes, and are associated with cell survival during environmental insults. SGs are dynamic entities with proteins relocating into and out of them during stress. Among the repertoire of proteins present in SGs is eukaryotic initiation factor 4E (eIF4E), a translation factor required for cap-dependent translation and that regulates a rate-limiting step for protein synthesis. Herein, we demonstrate that localization of eIF4E to SGs is dependent on the presence of a family of repressor proteins, eIF4E-binding proteins (4E-BPs). Our results demonstrate that 4E-BPs regulate the SG localization of eIF4E. PMID:19244480

Linear ubiquitin chains: enzymes, mechanisms and biology

PubMed Central

2017-01-01

Ubiquitination is a versatile post-translational modification that regulates a multitude of cellular processes. Its versatility is based on the ability of ubiquitin to form multiple types of polyubiquitin chains, which are recognized by specific ubiquitin receptors to induce the required cellular response. Linear ubiquitin chains are linked through Met 1 and have been established as important players of inflammatory signalling and apoptotic cell death. These chains are generated by a ubiquitin E3 ligase complex called the linear ubiquitin chain assembly complex (LUBAC) that is thus far the only E3 ligase capable of forming linear ubiquitin chains. The complex consists of three subunits, HOIP, HOIL-1L and SHARPIN, each of which have specific roles in the observed biological functions of LUBAC. Furthermore, LUBAC has been found to be associated with OTULIN and CYLD, deubiquitinases that disassemble linear chains and counterbalance the E3 ligase activity of LUBAC. Gene mutations in HOIP, HOIL-1L and OTULIN are found in human patients who suffer from autoimmune diseases, and HOIL-1L mutations are also found in myopathy patients. In this paper, we discuss the mechanisms of linear ubiquitin chain generation and disassembly by their respective enzymes and review our current understanding of their biological functions and association with human diseases. PMID:28446710

Linear ubiquitin chains: enzymes, mechanisms and biology.

PubMed

Rittinger, Katrin; Ikeda, Fumiyo

2017-04-01

Ubiquitination is a versatile post-translational modification that regulates a multitude of cellular processes. Its versatility is based on the ability of ubiquitin to form multiple types of polyubiquitin chains, which are recognized by specific ubiquitin receptors to induce the required cellular response. Linear ubiquitin chains are linked through Met 1 and have been established as important players of inflammatory signalling and apoptotic cell death. These chains are generated by a ubiquitin E3 ligase complex called the linear ubiquitin chain assembly complex (LUBAC) that is thus far the only E3 ligase capable of forming linear ubiquitin chains. The complex consists of three subunits, HOIP, HOIL-1L and SHARPIN, each of which have specific roles in the observed biological functions of LUBAC. Furthermore, LUBAC has been found to be associated with OTULIN and CYLD, deubiquitinases that disassemble linear chains and counterbalance the E3 ligase activity of LUBAC. Gene mutations in HOIP, HOIL-1L and OTULIN are found in human patients who suffer from autoimmune diseases, and HOIL-1L mutations are also found in myopathy patients. In this paper, we discuss the mechanisms of linear ubiquitin chain generation and disassembly by their respective enzymes and review our current understanding of their biological functions and association with human diseases. © 2017 The Authors.

Simultaneous detection of assembly and disassembly of multivalent HA tag and anti-HA antibody in single in-capillary assay.

PubMed

Wang, Jianhao; Qin, Yuqin; Qin, Haifang; Liu, Li; Ding, Shumin; Teng, Yiwan; Ji, Junling; Qiu, Lin; Jiang, Pengju

2016-08-01

Herein, we have developed an in-capillary assay for simultaneous detection of the assembly and disassembly of the multivalent HA tag peptide and antibody. HA tag with hexahistidine at C terminus (YPYDVPDYAG4 H6 , termed YPYDH6 ) was conjugated with quantum dots (QDs) by metal-affinity force to form a multivalent HA tag (QD-YPYDH6 ). QD-YPYDH6 and monoclonal anti-HA antibody (anti-HA) were sequentially injected into the capillary. They were mixed and assembled inside the capillary. The reaction products were online discriminated and detected by fluorescence coupled capillary electrophoresis (CE-FL). For the in-capillary assay, the binding efficiency of the multivalent HA tag and antibody on was influenced by the molar ratio and injection time. Such novel assay could even give out the self-assembly kinetic constant of QDs and YPYDH6 as KD of 34.1 μM with n (binding cooperativeness) of 2.2 by Hill equation. More importantly, the simultaneous detection of the assembly and imidazole (Im) induced disassembly of the QD-YPYDH6 -anti-HA complex was achieved in a single in-capillary assay. Our study demonstrated a new method for the online detection of antigen-antibody interactions. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Decipher the dynamic coordination between enzymatic activity and structural modulation at focal adhesions in living cells

NASA Astrophysics Data System (ADS)

Lu, Shaoying; Seong, Jihye; Wang, Yi; Chang, Shiou-Chi; Eichorst, John Paul; Ouyang, Mingxing; Li, Julie Y.-S.; Chien, Shu; Wang, Yingxiao

2014-07-01

Focal adhesions (FAs) are dynamic subcellular structures crucial for cell adhesion, migration and differentiation. It remains an enigma how enzymatic activities in these local complexes regulate their structural remodeling in live cells. Utilizing biosensors based on fluorescence resonance energy transfer (FRET), we developed a correlative FRET imaging microscopy (CFIM) approach to quantitatively analyze the subcellular coordination between the enzymatic Src activation and the structural FA disassembly. CFIM reveals that the Src kinase activity only within the microdomain of lipid rafts at the plasma membrane is coupled with FA dynamics. FA disassembly at cell periphery was linearly dependent on this raft-localized Src activity, although cells displayed heterogeneous levels of response to stimulation. Within lipid rafts, the time delay between Src activation and FA disassembly was 1.2 min in cells seeded on low fibronectin concentration ([FN]) and 4.3 min in cells on high [FN]. CFIM further showed that the level of Src-FA coupling, as well as the time delay, was regulated by cell-matrix interactions, as a tight enzyme-structure coupling occurred in FA populations mediated by integrin αvβ3, but not in those by integrin α5β1. Therefore, different FA subpopulations have distinctive regulation mechanisms between their local kinase activity and structural FA dynamics.

Application of industrial robots in automatic disassembly line of waste LCD displays

NASA Astrophysics Data System (ADS)

Wang, Sujuan

2017-11-01

In the automatic disassembly line of waste LCD displays, LCD displays are disassembled into plastic shells, metal shields, circuit boards, and LCD panels. Two industrial robots are used to cut metal shields and remove circuit boards in this automatic disassembly line. The functions of these two industrial robots, and the solutions to the critical issues of model selection, the interfaces with PLCs and the workflows were described in detail in this paper.

Structure-Function Based Molecular Relationships in Ewing's Sarcoma

PubMed Central

2015-01-01

Ewing's Sarcoma Oncogene (ews) on chromosome 22q12 is encoding a ubiquitously expressed RNA-binding protein (EWS) with unknown function that is target of tumor-specific chromosomal translocations in Ewing's sarcoma family of tumors. A model of transcription complex was proposed in which the heterodimer Rpb4/7 binds to EAD, connecting it to Core RNA Pol II. The DNA-binding domain, provided by EFP, is bound to the promoter. Rpb4/7 binds RNA, stabilizing the transcription complex. The complex Rpb4/7 can stabilize the preinitiation complexes by converting the conformation of RNA Pol II. EWS may change its conformation, so that NTD becomes accessible. Two different mechanisms of interaction between EWS and RNA Pol II are proposed: (I) an intermolecular EWS-EWS interaction between two molecules, pushing conformation from “closed” to “open” state, or (II) an intramolecular interaction inside the molecule of EWS, pushing conformation of the molecule from “closed” to “open” state. The modified forms of EWS may interact with Pol II subunits hsRpb5 and hsRpb7. The EWS and EFPs binding partners are described schematically in a model, an attempt to link the transcription with the splicing. The proposed model helps to understand the functional molecular interactions in cancer, to find new partners and ways to treat cancer. PMID:25688366

29 CFR 1926.1406 - Assembly/Disassembly-employer procedures-general requirements.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 29 Labor 8 2012-07-01 2012-07-01 false Assembly/Disassembly-employer procedures-general... CONSTRUCTION Cranes and Derricks in Construction § 1926.1406 Assembly/Disassembly—employer procedures—general requirements. (a) When using employer procedures instead of manufacturer procedures for assembly/disassembly...

29 CFR 1926.1406 - Assembly/Disassembly-employer procedures-general requirements.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 29 Labor 8 2014-07-01 2014-07-01 false Assembly/Disassembly-employer procedures-general... CONSTRUCTION Cranes and Derricks in Construction § 1926.1406 Assembly/Disassembly—employer procedures—general requirements. (a) When using employer procedures instead of manufacturer procedures for assembly/disassembly...

29 CFR 1926.1406 - Assembly/Disassembly-employer procedures-general requirements.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 29 Labor 8 2013-07-01 2013-07-01 false Assembly/Disassembly-employer procedures-general... CONSTRUCTION Cranes and Derricks in Construction § 1926.1406 Assembly/Disassembly—employer procedures—general requirements. (a) When using employer procedures instead of manufacturer procedures for assembly/disassembly...

29 CFR 1926.1406 - Assembly/Disassembly-employer procedures-general requirements.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 29 Labor 8 2011-07-01 2011-07-01 false Assembly/Disassembly-employer procedures-general... CONSTRUCTION Cranes and Derricks in Construction § 1926.1406 Assembly/Disassembly—employer procedures—general requirements. (a) When using employer procedures instead of manufacturer procedures for assembly/disassembly...

An Intelligent Agent-Controlled and Robot-Based Disassembly Assistant

NASA Astrophysics Data System (ADS)

Jungbluth, Jan; Gerke, Wolfgang; Plapper, Peter

2017-09-01

One key for successful and fluent human-robot-collaboration in disassembly processes is equipping the robot system with higher autonomy and intelligence. In this paper, we present an informed software agent that controls the robot behavior to form an intelligent robot assistant for disassembly purposes. While the disassembly process first depends on the product structure, we inform the agent using a generic approach through product models. The product model is then transformed to a directed graph and used to build, share and define a coarse disassembly plan. To refine the workflow, we formulate “the problem of loosening a connection and the distribution of the work” as a search problem. The created detailed plan consists of a sequence of actions that are used to call, parametrize and execute robot programs for the fulfillment of the assistance. The aim of this research is to equip robot systems with knowledge and skills to allow them to be autonomous in the performance of their assistance to finally improve the ergonomics of disassembly workstations.

Set1/COMPASS and Mediator are repurposed to promote epigenetic transcriptional memory.

PubMed

D'Urso, Agustina; Takahashi, Yoh-Hei; Xiong, Bin; Marone, Jessica; Coukos, Robert; Randise-Hinchliff, Carlo; Wang, Ji-Ping; Shilatifard, Ali; Brickner, Jason H

2016-06-23

In yeast and humans, previous experiences can lead to epigenetic transcriptional memory: repressed genes that exhibit mitotically heritable changes in chromatin structure and promoter recruitment of poised RNA polymerase II preinitiation complex (RNAPII PIC), which enhances future reactivation. Here, we show that INO1 memory in yeast is initiated by binding of the Sfl1 transcription factor to the cis-acting Memory Recruitment Sequence, targeting INO1 to the nuclear periphery. Memory requires a remodeled form of the Set1/COMPASS methyltransferase lacking Spp1, which dimethylates histone H3 lysine 4 (H3K4me2). H3K4me2 recruits the SET3C complex, which plays an essential role in maintaining this mark. Finally, while active INO1 is associated with Cdk8(-) Mediator, during memory, Cdk8(+) Mediator recruits poised RNAPII PIC lacking the Kin28 CTD kinase. Aspects of this mechanism are generalizable to yeast and conserved in human cells. Thus, COMPASS and Mediator are repurposed to promote epigenetic transcriptional poising by a highly conserved mechanism.

The structure of SV40 large T hexameric helicase in complex with AT-rich origin DNA

PubMed Central

Gai, Dahai; Wang, Damian; Li, Shu-Xing; Chen, Xiaojiang S

2016-01-01

DNA replication is a fundamental biological process. The initial step in eukaryotic DNA replication is the assembly of the pre-initiation complex, including the formation of two head-to-head hexameric helicases around the replication origin. How these hexameric helicases interact with their origin dsDNA remains unknown. Here, we report the co-crystal structure of the SV40 Large-T Antigen (LT) hexameric helicase bound to its origin dsDNA. The structure shows that the six subunits form a near-planar ring that interacts with the origin, so that each subunit makes unique contacts with the DNA. The origin dsDNA inside the narrower AAA+ domain channel shows partial melting due to the compression of the two phosphate backbones, forcing Watson-Crick base-pairs within the duplex to flip outward. This structure provides the first snapshot of a hexameric helicase binding to origin dsDNA, and suggests a possible mechanism of origin melting by LT during SV40 replication in eukaryotic cells. DOI: http://dx.doi.org/10.7554/eLife.18129.001 PMID:27921994

Accessory factors promote AlfA-dependent plasmid segregation by regulating filament nucleation, disassembly, and bundling

PubMed Central

Polka, Jessica K.; Kollman, Justin M.; Mullins, R. Dyche

2014-01-01

In bacteria, some plasmids are partitioned to daughter cells by assembly of actin-like proteins (ALPs). The best understood ALP, ParM, has a core set of biochemical properties that contributes to its function, including dynamic instability, spontaneous nucleation, and bidirectional elongation. AlfA, an ALP that pushes plasmids apart in Bacillus, relies on a different set of underlying properties to segregate DNA. AlfA elongates unidirectionally and is not dynamically unstable; its assembly and disassembly are regulated by a cofactor, AlfB. Free AlfB breaks up AlfA bundles and promotes filament turnover. However, when AlfB is bound to the centromeric DNA sequence, parN, it forms a segrosome complex that nucleates and stabilizes AlfA filaments. When reconstituted in vitro, this system creates polarized, motile comet tails that associate by antiparallel filament bundling to form bipolar, DNA-segregating spindles. PMID:24481252

AGR-2 Irradiated Test Train Preliminary Inspection and Disassembly First Look

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ploger, Scott; Demkowciz, Paul; Harp, Jason

2015-05-01

The AGR 2 irradiation experiment began in June 2010 and was completed in October 2013. The test train was shipped to the Materials and Fuels Complex in July 2014 for post-irradiation examination (PIE). The first PIE activities included nondestructive examination of the test train, followed by disassembly of the test train and individual capsules and detailed inspection of the capsule contents, including the fuel compacts and their graphite fuel holders. Dimensional metrology was then performed on the compacts, graphite holders, and steel capsule shells. AGR 2 disassembly and metrology were performed with the same equipment used successfully on AGR 1more » test train components. Gamma spectrometry of the intact test train gave a preliminary look at the condition of the interior components. No evidence of damage to compacts or graphite components was evident from the isotopic and gross gamma scans. Disassembly of the AGR 2 test train and its capsules was conducted rapidly and efficiently by employing techniques refined during the AGR 1 disassembly campaign. Only one major difficulty was encountered while separating the test train into capsules when thermocouples (of larger diameter than used in AGR 1) and gas lines jammed inside the through tubes of the upper capsules, which required new tooling for extraction. Disassembly of individual capsules was straightforward with only a few minor complications. On the whole, AGR 2 capsule structural components appeared less embrittled than their AGR 1 counterparts. Compacts from AGR 2 Capsules 2, 3, 5, and 6 were in very good condition upon removal. Only relatively minor damage or markings were visible using high resolution photographic inspection. Compact dimensional measurements indicated radial shrinkage between 0.8 to 1.7%, with the greatest shrinkage observed on Capsule 2 compacts that were irradiated at higher temperature. Length shrinkage ranged from 0.1 to 0.9%, with by far the lowest axial shrinkage on Capsule 3 compacts—possibly as a consequence of lower packing fraction or larger particle size. Differences in fast neutron fluence among compacts from these four capsules had no obvious effect on radial and axial shrinkage. (The AGR 2 experiment included Capsule 1 containing French compacts and Capsule 4 with compacts made at Oak Ridge National Laboratory using South African fuel particles. Information on these two batches of AGR 2 fuel compacts is confined to restricted Appendices A and B because of proprietary information limitations.)« less

Structural and functional insight into TAF1-TAF7, a subcomplex of transcription factor II D

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhattacharya, Suparna; Lou, Xiaohua; Hwang, Peter

2014-07-01

Transcription factor II D (TFIID) is a multiprotein complex that nucleates formation of the basal transcription machinery. TATA binding protein-associated factors 1 and 7 (TAF1 and TAF7), two subunits of TFIID, are integral to the regulation of eukaryotic transcription initiation and play key roles in preinitiation complex (PIC) assembly. Current models suggest that TAF7 acts as a dissociable inhibitor of TAF1 histone acetyltransferase activity and that this event ensures appropriate assembly of the RNA polymerase II-mediated PIC before transcriptional initiation. Here, we report the 3D structure of a complex of yeast TAF1 with TAF7 at 2.9 Å resolution. The structuremore » displays novel architecture and is characterized by a large predominantly hydrophobic heterodimer interface and extensive cofolding of TAF subunits. There are no obvious similarities between TAF1 and known histone acetyltransferases. Instead, the surface of the TAF1–TAF7 complex contains two prominent conserved surface pockets, one of which binds selectively to an inhibitory trimethylated histone H3 mark on Lys27 in a manner that is also regulated by phosphorylation at the neighboring H3 serine. Our findings could point toward novel roles for the TAF1–TAF7 complex in regulation of PIC assembly via reading epigenetic histone marks.« less

The Mediator complex and transcription regulation

PubMed Central

Poss, Zachary C.; Ebmeier, Christopher C.

2013-01-01

The Mediator complex is a multi-subunit assembly that appears to be required for regulating expression of most RNA polymerase II (pol II) transcripts, which include protein-coding and most non-coding RNA genes. Mediator and pol II function within the pre-initiation complex (PIC), which consists of Mediator, pol II, TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH and is approximately 4.0 MDa in size. Mediator serves as a central scaffold within the PIC and helps regulate pol II activity in ways that remain poorly understood. Mediator is also generally targeted by sequence-specific, DNA-binding transcription factors (TFs) that work to control gene expression programs in response to developmental or environmental cues. At a basic level, Mediator functions by relaying signals from TFs directly to the pol II enzyme, thereby facilitating TF-dependent regulation of gene expression. Thus, Mediator is essential for converting biological inputs (communicated by TFs) to physiological responses (via changes in gene expression). In this review, we summarize an expansive body of research on the Mediator complex, with an emphasis on yeast and mammalian complexes. We focus on the basics that underlie Mediator function, such as its structure and subunit composition, and describe its broad regulatory influence on gene expression, ranging from chromatin architecture to transcription initiation and elongation, to mRNA processing. We also describe factors that influence Mediator structure and activity, including TFs, non-coding RNAs and the CDK8 module. PMID:24088064

Archaeal TFEα/β is a hybrid of TFIIE and the RNA polymerase III subcomplex hRPC62/39

PubMed Central

Blombach, Fabian; Salvadori, Enrico; Fouqueau, Thomas; Yan, Jun; Reimann, Julia; Sheppard, Carol; Smollett, Katherine L; Albers, Sonja V; Kay, Christopher WM; Thalassinos, Konstantinos; Werner, Finn

2015-01-01

Transcription initiation of archaeal RNA polymerase (RNAP) and eukaryotic RNAPII is assisted by conserved basal transcription factors. The eukaryotic transcription factor TFIIE consists of α and β subunits. Here we have identified and characterised the function of the TFIIEβ homologue in archaea that on the primary sequence level is related to the RNAPIII subunit hRPC39. Both archaeal TFEβ and hRPC39 harbour a cubane 4Fe-4S cluster, which is crucial for heterodimerization of TFEα/β and its engagement with the RNAP clamp. TFEα/β stabilises the preinitiation complex, enhances DNA melting, and stimulates abortive and productive transcription. These activities are strictly dependent on the β subunit and the promoter sequence. Our results suggest that archaeal TFEα/β is likely to represent the evolutionary ancestor of TFIIE-like factors in extant eukaryotes. DOI: http://dx.doi.org/10.7554/eLife.08378.001 PMID:26067235

Effects of reactive oxygen species on cellular wall disassembly of banana fruit during ripening.

PubMed

Cheng, Guiping; Duan, Xuewu; Shi, John; Lu, Wangjin; Luo, Yunbo; Jiang, Weibo; Jiang, Yueming

2008-07-15

Fruit softening is generally attributed to cell wall disassembly. Experiments were conducted to investigate effects of various reactive oxygen species (ROS) on in vitro cellular wall disassembly of harvested banana fruit. The alcohol-extracted insoluble residue (AEIR) was obtained from the pulp tissues of banana fruit at various ripening stages and then used to examine the disassembly of cellular wall polysaccharides in the presence of superoxide anion (O2(-)), hydrogen peroxide (H2O2) or hydroxyl radical (OH) and their scavengers. The presence of OH accelerated significantly disassembly of cellular wall polysaccharides in terms of the increase in contents of total sugars released and uronic acid, and the decrease in molecular mass of soluble polysaccharides, using gel permeation chromatography. However, the treatment with H2O2 or O2(-) showed no significant effect on the disassembly of cellular wall polysaccharides. Furthermore, the degradation of the de-esterified AEIR was more susceptible to OH attack than the esterified AEIR. In addition, the effect of OH could be inhibited in the presence of OH scavenger. This study suggests that disassembly of cellular wall polysaccharides could be initiated by OH as the solublisation of the polysaccharides increased, which, in turn, accelerated fruit softening. Copyright © 2008 Elsevier Ltd. All rights reserved.

Protein dynamics of human RPA and RAD51 on ssDNA during assembly and disassembly of the RAD51 filament

PubMed Central

Ma, Chu Jian; Gibb, Bryan; Kwon, YoungHo; Sung, Patrick; Greene, Eric C.

2017-01-01

Homologous recombination (HR) is a crucial pathway for double-stranded DNA break (DSB) repair. During the early stages of HR, the newly generated DSB ends are processed to yield long single-stranded DNA (ssDNA) overhangs, which are quickly bound by replication protein A (RPA). RPA is then replaced by the DNA recombinase Rad51, which forms extended helical filaments on the ssDNA. The resulting nucleoprotein filament, known as the presynaptic complex, is responsible for pairing the ssDNA with homologous double-stranded DNA (dsDNA), which serves as the template to guide DSB repair. Here, we use single-molecule imaging to visualize the interplay between human RPA (hRPA) and human RAD51 during presynaptic complex assembly and disassembly. We demonstrate that ssDNA-bound hRPA can undergo facilitated exchange, enabling hRPA to undergo rapid exchange between free and ssDNA-bound states only when free hRPA is present in solution. Our results also indicate that the presence of free hRPA inhibits RAD51 filament nucleation, but has a lesser impact upon filament elongation. This finding suggests that hRPA exerts important regulatory influence over RAD51 and may in turn affect the properties of the assembled RAD51 filament. These experiments provide an important basis for further investigations into the regulation of human presynaptic complex assembly. PMID:27903895

An Early View of Real-World Patient Response to Sacubitril/Valsartan: A Retrospective Study of Patients with Heart Failure with Reduced Ejection Fraction.

PubMed

Antol, Dana Drzayich; Casebeer, Adrianne Waldman; DeClue, Richard W; Stemkowski, Stephen; Russo, Patricia A

2018-06-01

Sacubitril/valsartan has been established as an effective treatment for heart failure (HF) with reduced ejection fraction based on clinical trial data; however, little is known about its use or impact in real-world practice. This study included data from medical and pharmacy claims and medical records review for patients (n = 200) who initiated sacubitril/valsartan between August 2015 and March 2016 preceding issuance of American College of Cardiology (ACC)/American Heart Association (AHA)/Heart Failure Society of America (HFSA) focused update on new pharmacological therapy for HF (May 2016), which included recommendations for sacubitril/valsartan. A within-subject analysis compared symptoms and healthcare resource utilization before and after treatment initiation. Patients treated with sacubitril/valsartan had multiple comorbidities, and nearly all had previous treatment for HF. Most patients initiated sacubitril/valsartan at the lowest dose of 24/26 mg twice a day (BID), which remained unchanged during the observation period for half of the patients. During the first 6 weeks of treatment, few patients discontinued sacubitril/valsartan treatment (5.5%), and only 17% achieved the target dose of 97/103 mg BID after 4 months of treatment. The proportion of patients with ≥ 1 all-cause inpatient stay decreased significantly between the pre-initiation period (27.5%) and the post-initiation period (17.0%), P = 0.009. Fatigue was noted in 51.8% of patients pre-initiation and 39.5% post-initiation, P = 0.027. Shortness of breath was documented for 66.7% of patients pre-initiation and 51.8% post-initiation, P = 0.008. The findings of this real-world investigation suggest sacubitril/valsartan is associated with symptom improvements and a reduction in hospitalizations within 4 months of treatment for patients with HF and reduced ejection fraction. Novartis Pharmaceuticals Corporation.

Nanobody Binding to a Conserved Epitope Promotes Norovirus Particle Disassembly

PubMed Central

Koromyslova, Anna D.

2014-01-01

ABSTRACT Human noroviruses are icosahedral single-stranded RNA viruses. The capsid protein is divided into shell (S) and protruding (P) domains, which are connected by a flexible hinge region. There are numerous genetically and antigenically distinct noroviruses, and the dominant strains evolve every other year. Vaccine and antiviral development is hampered by the difficulties in growing human norovirus in cell culture and the continually evolving strains. Here, we show the X-ray crystal structures of human norovirus P domains in complex with two different nanobodies. One nanobody, Nano-85, was broadly reactive, while the other, Nano-25, was strain specific. We showed that both nanobodies bound to the lower region on the P domain and had nanomolar affinities. The Nano-85 binding site mainly comprised highly conserved amino acids among the genetically distinct genogroup II noroviruses. Several of the conserved residues also were recognized by a broadly reactive monoclonal antibody, which suggested this region contained a dominant epitope. Superposition of the P domain nanobody complex structures into a cryoelectron microscopy particle structure revealed that both nanobodies bound at occluded sites on the particles. The flexible hinge region, which contained ∼10 to 12 amino acids, likely permitted a certain degree of P domain movement on the particles in order to accommodate the nanobodies. Interestingly, the Nano-85 binding interaction with intact particles caused the particles to disassemble in vitro. Altogether, these results suggested that the highly conserved Nano-85 binding epitope contained a trigger mechanism for particle disassembly. Principally, this epitope represents a potential site of norovirus vulnerability. IMPORTANCE We characterized two different nanobodies (Nano-85 and Nano-25) that bind to human noroviruses. Both nanobodies bound with high affinities to the lower region of the P domain, which was occluded on intact particles. Nano-25 was specific for GII.10, whereas Nano-85 bound several different GII genotypes, including GII.4, GII.10, and GII.12. We showed that Nano-85 was able to detect norovirus virions in clinical stool specimens using a sandwich enzyme-linked immunosorbent assay. Importantly, we found that Nano-85 binding to intact particles caused the particles to disassemble. We believe that with further testing, Nano-85 not only will work as a diagnostic reagent in norovirus detection systems but also could function as a broadly reactive GII norovirus antiviral. PMID:25520510

40 CFR 1065.1109 - Post-test sampler disassembly and sample extraction.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 40 Protection of Environment 33 2014-07-01 2014-07-01 false Post-test sampler disassembly and... Semi-Volatile Organic Compounds § 1065.1109 Post-test sampler disassembly and sample extraction. This... environment as follows after the test: (1) Remove the PM filter, PUF plugs, and all the XAD-2 from the...

29 CFR 1926.1404 - Assembly/Disassembly-general requirements (applies to all assembly and disassembly operations).

Code of Federal Regulations, 2013 CFR

2013-07-01

...'s procedures must be followed. When lifting loads without using outriggers or stabilizers, the manufacturer's procedures must be met regarding truck wedges or screws. (r) Rigging. In addition to following...(o)(3) before assembly/disassembly begins. (5) Boom and jib pick points. The point(s) of attachment...

29 CFR 1926.1404 - Assembly/Disassembly-general requirements (applies to all assembly and disassembly operations).

Code of Federal Regulations, 2012 CFR

2012-07-01

...'s procedures must be followed. When lifting loads without using outriggers or stabilizers, the manufacturer's procedures must be met regarding truck wedges or screws. (r) Rigging. In addition to following...(o)(3) before assembly/disassembly begins. (5) Boom and jib pick points. The point(s) of attachment...

29 CFR 1926.1404 - Assembly/Disassembly-general requirements (applies to all assembly and disassembly operations).

Code of Federal Regulations, 2011 CFR

2011-07-01

...'s procedures must be followed. When lifting loads without using outriggers or stabilizers, the manufacturer's procedures must be met regarding truck wedges or screws. (r) Rigging. In addition to following...(o)(3) before assembly/disassembly begins. (5) Boom and jib pick points. The point(s) of attachment...

29 CFR 1926.1404 - Assembly/Disassembly-general requirements (applies to all assembly and disassembly operations).

Code of Federal Regulations, 2014 CFR

2014-07-01

...'s procedures must be followed. When lifting loads without using outriggers or stabilizers, the manufacturer's procedures must be met regarding truck wedges or screws. (r) Rigging. In addition to following...(o)(3) before assembly/disassembly begins. (5) Boom and jib pick points. The point(s) of attachment...

Recycling of electrical motors by automatic disassembly

NASA Astrophysics Data System (ADS)

Karlsson, Björn; Järrhed, Jan-Ove

2000-04-01

This paper presents a robotized workstation for end-of-life treatment of electrical motors with an electrical effect of about 1 kW. These motors can, for example, be found in washing machines and in industry. There are two main steps in the work. The first step is an inspection whereby the functionality of the motor is checked and classification either for re-use or for disassembly is done. In the second step the motors classified for disassembly are disassembled in a robotized automatic station. In the initial step measurements are performed during a start-up sequence of about 1 s. By measuring the rotation speed and the current and voltage of the three phases of the motor classification for either reuse or disassembly can be done. During the disassembly work, vision data are fused in order to classify the motors according to their type. The vision system also feeds the control system of the robot with various object co-ordinates, to facilitate correct operation of the robot. Finally, tests with a vision system and eddy-current equipment are performed to decide whether all copper has been removed from the stator.

Fault detection techniques for complex cable shield topologies

NASA Astrophysics Data System (ADS)

Coonrod, Kurt H.; Davis, Stuart L.; McLemore, Donald P.

1994-09-01

This document presents the results of a basic principles study which investigated technical approaches for developing fault detection techniques for use on cables with complex shielding topologies. The study was limited to those approaches which could realistically be implemented on a fielded cable, i.e., approaches which would require partial disassembly of a cable were not pursued. The general approach used was to start with present transfer impedance measurement techniques and modify their use to achieve the best possible measurement range. An alternative test approach, similar to a sniffer type test, was also investigated.

Identification of a small TAF complex and its role in the assembly of TAF-containing complexes.

PubMed

Demény, Màté A; Soutoglou, Evi; Nagy, Zita; Scheer, Elisabeth; Jànoshàzi, Agnes; Richardot, Magalie; Argentini, Manuela; Kessler, Pascal; Tora, Laszlo

2007-03-21

TFIID plays a role in nucleating RNA polymerase II preinitiation complex assembly on protein-coding genes. TFIID is a multisubunit complex comprised of the TATA box binding protein (TBP) and 14 TBP-associated factors (TAFs). Another class of multiprotein transcriptional regulatory complexes having histone acetyl transferase (HAT) activity, and containing TAFs, includes TFTC, STAGA and the PCAF/GCN5 complex. Looking for as yet undiscovered subunits by a proteomic approach, we had identified TAF8 and SPT7L in human TFTC preparations. Subsequently, however, we demonstrated that TAF8 was not a stable component of TFTC, but that it is present in a small TAF complex (SMAT), containing TAF8, TAF10 and SPT7L, that co-purified with TFTC. Thus, TAF8 is a subunit of both TFIID and SMAT. The latter has to be involved in a pathway of complex formation distinct from the other known TAF complexes, since these three histone fold (HF)-containing proteins (TAF8, TAF10 and SPT7L) can never be found together either in TFIID or in STAGA/TFTC HAT complexes. Here we show that TAF8 is absolutely necessary for the integration of TAF10 in a higher order TFIID core complex containing seven TAFs. TAF8 forms a heterodimer with TAF10 through its HF and proline rich domains, and also interacts with SPT7L through its C-terminal region, and the three proteins form a complex in vitro and in vivo. Thus, the TAF8-TAF10 and TAF10-SPT7L HF pairs, and also the SMAT complex, seem to be important regulators of the composition of different TFIID and/or STAGA/TFTC complexes in the nucleus and consequently may play a role in gene regulation.

Protecting the proteome: Eukaryotic cotranslational quality control pathways

PubMed Central

2014-01-01

The correct decoding of messenger RNAs (mRNAs) into proteins is an essential cellular task. The translational process is monitored by several quality control (QC) mechanisms that recognize defective translation complexes in which ribosomes are stalled on substrate mRNAs. Stalled translation complexes occur when defects in the mRNA template, the translation machinery, or the nascent polypeptide arrest the ribosome during translation elongation or termination. These QC events promote the disassembly of the stalled translation complex and the recycling and/or degradation of the individual mRNA, ribosomal, and/or nascent polypeptide components, thereby clearing the cell of improper translation products and defective components of the translation machinery. PMID:24535822

Assembly/disassembly of a complex icosahedral virus to incorporate heterologous nucleic acids

NASA Astrophysics Data System (ADS)

Pascual, Elena; Mata, Carlos P.; Carrascosa, José L.; Castón, José R.

2017-12-01

Hollow protein containers are widespread in nature, and include virus capsids as well as eukaryotic and bacterial complexes. Protein cages are studied extensively for applications in nanotechnology, nanomedicine and materials science. Their inner and outer surfaces can be modified chemically or genetically, and the internal cavity can be used to template, store and/or arrange molecular cargos. Virus capsids and virus-like particles (VLP, noninfectious particles) provide versatile platforms for nanoscale bioengineering. Study of capsid protein self-assembly into monodispersed particles, and of VLP structure and biophysics is necessary not only to understand natural processes, but also to infer how these platforms can be redesigned to furnish novel functional VLP. Here we address the assembly dynamics of infectious bursal disease virus (IBDV), a complex icosahedral virus. IBDV has a ~70 nm-diameter T  =  13 capsid with VP2 trimers as the only structural subunits. During capsid assembly, VP2 is synthesized as a precursor (pVP2) whose C terminus is cleaved. The pVP2 C terminus has an amphipathic helix that controls VP2 polymorphism. In the absence of the VP3 scaffolding protein, necessary for control of assembly, 466/456-residue pVP2 intermediates bearing this helix assemble into VLP only when expressed with an N-terminal His6 tag (the HT-VP2-466 protein). HT-VP2-466 capsids are optimal for genetic insertion of proteins (cargo space ~78 000 nm3). We established an in vitro assembly/disassembly system of HT-VP2-466-based VLP for heterologous nucleic acid packaging and/or encapsulation of drugs and other molecules. HT-VP2-466 (empty) capsids were disassembled and reassembled by dialysis against low-salt/basic pH and high-salt/acid pH buffers, respectively, thus illustrating the reversibility in vitro of IBDV capsid assembly. HT-VP2-466 VLP also packed heterologous DNA by non-specific confinement during assembly. These and previous results establish the bases for biotechnological applications based on the IBDV capsid and its ability to incorporate exogenous proteins and nucleic acids.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuuichi Tooya; Tadahiro Washiya; Kenji Koizumi

Japan Atomic Energy Agency (JAEA) has been leading feasibility study on commercialized fast reactor cycle systems in Japan. In this study, we have proposed a new disassembly technology by mechanical disassembly system that consists of a mechanical cutting step and a wrapper tube pulling step. In the mechanical disassembly system, high durability mechanical tool grinds the wrapper tube (Slit-cut (S/C) operation in circle direction), and then the wrapper tube is pulled out and removed from the fuel assembly. Then the fuel pins are cut (Crop-cut (C/C) operation at entrance nozzle side) and the entrance nozzle is removed. The fuel pinsmore » are transported to the shearing device in next process. The Fundamental tests were carried out with simulated FBR fuel pins and wrapper tube, and cutting performance and wrapper tube pulling performance has been confirmed by engineering scale. As results, we established an efficient disassembly procedure and the fundamental design of mechanical disassembly system. (authors)« less

29 CFR 1926.1407 - Power line safety (up to 350 kV)-assembly and disassembly.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 29 Labor 8 2014-07-01 2014-07-01 false Power line safety (up to 350 kV)-assembly and disassembly. 1926.1407 Section 1926.1407 Labor Regulations Relating to Labor (Continued) OCCUPATIONAL SAFETY AND... Cranes and Derricks in Construction § 1926.1407 Power line safety (up to 350 kV)—assembly and disassembly...

Septin dynamics are essential for exocytosis.

PubMed

Tokhtaeva, Elmira; Capri, Joe; Marcus, Elizabeth A; Whitelegge, Julian P; Khuzakhmetova, Venera; Bukharaeva, Ellya; Deiss-Yehiely, Nimrod; Dada, Laura A; Sachs, George; Fernandez-Salas, Ester; Vagin, Olga

2015-02-27

Septins are a family of 14 cytoskeletal proteins that dynamically form hetero-oligomers and organize membrane microdomains for protein complexes. The previously reported interactions with SNARE proteins suggested the involvement of septins in exocytosis. However, the contradictory results of up- or down-regulation of septin-5 in various cells and mouse models or septin-4 in mice suggested either an inhibitory or a stimulatory role for these septins in exocytosis. The involvement of the ubiquitously expressed septin-2 or general septin polymerization in exocytosis has not been explored to date. Here, by nano-LC with tandem MS and immunoblot analyses of the septin-2 interactome in mouse brain, we identified not only SNARE proteins but also Munc-18-1 (stabilizes assembled SNARE complexes), N-ethylmaleimide-sensitive factor (NSF) (disassembles SNARE complexes after each membrane fusion event), and the chaperones Hsc70 and synucleins (maintain functional conformation of SNARE proteins after complex disassembly). Importantly, α-soluble NSF attachment protein (SNAP), the adaptor protein that mediates NSF binding to the SNARE complex, did not interact with septin-2, indicating that septins undergo reorganization during each exocytosis cycle. Partial depletion of septin-2 by siRNA or impairment of septin dynamics by forchlorfenuron inhibited constitutive and stimulated exocytosis of secreted and transmembrane proteins in various cell types. Forchlorfenuron impaired the interaction between SNAP-25 and its chaperone Hsc70, decreasing SNAP-25 levels in cultured neuroendocrine cells, and inhibited both spontaneous and stimulated acetylcholine secretion in mouse motor neurons. The results demonstrate a stimulatory role of septin-2 and the dynamic reorganization of septin oligomers in exocytosis. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo.

PubMed

Stefan, E; Aquin, S; Berger, N; Landry, C R; Nyfeler, B; Bouvier, M; Michnick, S W

2007-10-23

The G protein-coupled receptor (GPCR) superfamily represents the most important class of pharmaceutical targets. Therefore, the characterization of receptor cascades and their ligands is a prerequisite to discovering novel drugs. Quantification of agonist-induced second messengers and downstream-coupled kinase activities is central to characterization of GPCRs or other pathways that converge on GPCR-mediated signaling. Furthermore, there is a need for simple, cell-based assays that would report on direct or indirect actions on GPCR-mediated effectors of signaling. More generally, there is a demand for sensitive assays to quantify alterations of protein complexes in vivo. We describe the development of a Renilla luciferase (Rluc)-based protein fragment complementation assay (PCA) that was designed specifically to investigate dynamic protein complexes. We demonstrate these features for GPCR-induced disassembly of protein kinase A (PKA) regulatory and catalytic subunits, a key effector of GPCR signaling. Taken together, our observations show that the PCA allows for direct and accurate measurements of live changes of absolute values of protein complex assembly and disassembly as well as cellular imaging and dynamic localization of protein complexes. Moreover, the Rluc-PCA has a sufficiently high signal-to-background ratio to identify endogenously expressed Galpha(s) protein-coupled receptors. We provide pharmacological evidence that the phosphodiesterase-4 family selectively down-regulates constitutive beta-2 adrenergic- but not vasopressin-2 receptor-mediated PKA activities. Our results show that the sensitivity of the Rluc-PCA simplifies the recording of pharmacological profiles of GPCR-based candidate drugs and could be extended to high-throughput screens to identify novel direct modulators of PKA or upstream components of GPCR signaling cascades.

Mitotic phosphorylation of SUN1 loosens its connection with the nuclear lamina while the LINC complex remains intact.

PubMed

Patel, Jennifer T; Bottrill, Andrew; Prosser, Suzanna L; Jayaraman, Sangeetha; Straatman, Kees; Fry, Andrew M; Shackleton, Sue

2014-01-01

At the onset mitosis in higher eukaryotes, the nuclear envelope (NE) undergoes dramatic deconstruction to allow separation of duplicated chromosomes. Studies have shown that during this process of nuclear envelope breakdown (NEBD), the extensive protein networks of the nuclear lamina are disassembled through phosphorylation of lamins and several inner nuclear membrane (INM) proteins. The LINC complex, composed of SUN and nesprin proteins, is involved in multiple interactions at the NE and plays vital roles in nuclear and cellular mechanics by connecting the nucleus to the cytoskeleton. Here, we show that SUN1, located in the INM, undergoes mitosis-specific phosphorylation on at least 3 sites within its nucleoplasmic N-terminus. We further identify Cdk1 as the kinase responsible for serine 48 and 333 phosphorylation, while serine 138 is phosphorylated by Plk1. In mitotic cells, SUN1 loses its interaction with N-terminal domain binding partners lamin A/C, emerin, and short nesprin-2 isoforms. Furthermore, a triple phosphomimetic SUN1 mutant displays increased solubility and reduced retention at the NE. In contrast, the central LINC complex interaction between the SUN1 C-terminus and the KASH domain of nesprin-2 is maintained during mitosis. Together, these data support a model whereby mitotic phosphorylation of SUN1 disrupts interactions with nucleoplasmic binding partners, promoting disassembly of the nuclear lamina and, potentially, its chromatin interactions. At the same time, our data add to an emerging picture that the core LINC complex plays an active role in NEBD.

Immunocytochemical localization of the major polypeptides of the nuclear pore complex-lamina fraction. Interphase and mitotic distribution

PubMed Central

1978-01-01

This laboratory has previously isolated a fraction from rat liver nuclei consisting of nuclear pore complexes associated with the proteinaceous lamina which underlies the inner nuclear membrane. Using protein eluted from sodium dodecyl sulfate (SDS) gels, we have prepared antibodies in chickens to each of the three predominant pore complex- lamina bands. Ouchterlony double diffusion analysis shows that each of these individual bands cross-reacts strongly with all three antisera. In immunofluorescence localization performed on tissue culture cells with these antibodies, we obtain a pattern of intense staining at the periphery of the interphase nucleus, with little or no cytoplasmic reaction. Electron microscope immunoperoxidase staining of rat liver nuclei with these antibodies labels exclusively the nuclear periphery. Furthermore, reaction occurs in areas which contain the lamina, but not at the pore complexes. While our isolation procedure extracts the internal contents of nuclei completely, semiquantitative Ouchterlony analysis shows that it releases negligible amounts of these lamina antigens. Considered together, our results indicate that these three bands represent major components of a peripheral nuclear lamina, and are not structural elements of an internal "nuclear protein matrix." Fluorescence microscopy shows that the perinuclear interphase localization of these lamina proteins undergoes dramatic changes during mitosis. Concomitant with nuclear envelope disassembly in prophase, these antigens assume a diffuse localization throughout the cell. This distribution persists until telophase, when the antigens become progressively and completely localized at the surface of the daughter chromosome masses. We propose that the lamina is a biological polymer which can undergo reversible disassembly during mitosis. PMID:102651

TFIIB-facilitated recruitment of preinitiation complexes by a TAF-independent mechanism.

PubMed

Hori, Roderick T; Xu, Shuping; Hu, Xianyuan; Pyo, Sung

2004-01-01

Gene activators contain activation domains that are thought to recruit limiting components of the transcription machinery to a core promoter. VP16, a viral gene activator, has served as a model for studying the mechanistic aspects of transcriptional activation from yeast to human. The VP16 activation domain can be divided into two modules--an N-terminal subdomain (VPN) and a C-terminal subdomain (VPC). This study demonstrates that VPC stimulates core promoters that are either independent or dependent on TAFs (TATA-box Binding Protein-Associated Factors). In contrast, VPN only activates the TAF-independent core promoter and this activity increases in a synergistic fashion when VPN is dimerized (VPN2). Compared to one copy of VPN (VPN1), VPN2 also displays a highly cooperative increase in binding hTFIIB. The increased TFIIB binding correlates with VPN2's increased ability to recruit a complex containing TFIID, TFIIA and TFIIB. However, VPN1 and VPN2 do not increase the assembly of a complex containing only TFIID and TFIIA. The VPN subdomain also facilitates assembly of a complex containing TBP:TFIIA:TFIIB, which lacks TAFs, and provides a mechanism that could function at TAF-independent promoters. Taken together, these results suggest the interaction between VPN and TFIIB potentially initiate a network of contacts allowing the activator to indirectly tether TFIID or TBP to DNA.

TFIIB-facilitated recruitment of preinitiation complexes by a TAF-independent mechanism

PubMed Central

Hori, Roderick T.; Xu, Shuping; Hu, Xianyuan; Pyo, Sung

2004-01-01

Gene activators contain activation domains that are thought to recruit limiting components of the transcription machinery to a core promoter. VP16, a viral gene activator, has served as a model for studying the mechanistic aspects of transcriptional activation from yeast to human. The VP16 activation domain can be divided into two modules—an N-terminal subdomain (VPN) and a C-terminal subdomain (VPC). This study demonstrates that VPC stimulates core promoters that are either independent or dependent on TAFs (TATA-box Binding Protein-Associated Factors). In contrast, VPN only activates the TAF-independent core promoter and this activity increases in a synergistic fashion when VPN is dimerized (VPN2). Compared to one copy of VPN (VPN1), VPN2 also displays a highly cooperative increase in binding hTFIIB. The increased TFIIB binding correlates with VPN2's increased ability to recruit a complex containing TFIID, TFIIA and TFIIB. However, VPN1 and VPN2 do not increase the assembly of a complex containing only TFIID and TFIIA. The VPN subdomain also facilitates assembly of a complex containing TBP:TFIIA:TFIIB, which lacks TAFs, and provides a mechanism that could function at TAF-independent promoters. Taken together, these results suggest the interaction between VPN and TFIIB potentially initiate a network of contacts allowing the activator to indirectly tether TFIID or TBP to DNA. PMID:15272087

The 'Room within a Room' Concept for Monitored Warhead Dismantlement

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tanner, Jennifer E.; Benz, Jacob M.; White, Helen

2014-12-01

Over the past 10 years, US and UK experts have engaged in a technical collaboration with the aim of improving scientific and technological abilities in support of potential future nuclear arms control and non-proliferation agreements. In 2011 a monitored dismantlement exercise provided an opportunity to develop and test potential monitoring technologies and approaches. The exercise followed a simulated nuclear object through a dismantlement process and looked to explore, with a level of realism, issues surrounding device and material monitoring, chain of custody, authentication and certification of equipment, data management and managed access. This paper focuses on the development and deploymentmore » of the ‘room-within-a-room’ system, which was designed to maintain chain of custody during disassembly operations. A key challenge for any verification regime operating within a nuclear weapon complex is to provide the monitoring party with the opportunity to gather sufficient evidence, whilst protecting sensitive or proliferative information held by the host. The requirement to address both monitoring and host party concerns led to a dual function design which: • Created a controlled boundary around the disassembly process area which could provide evidence of unauthorised diversion activities. • Shielded sensitive disassembly operations from monitoring party observation. The deployed room-within-a-room was an integrated system which combined a number of chain of custody technologies (i.e. cameras, tamper indicating panels and enclosures, seals, unique identifiers and radiation portals) and supporting deployment procedures. This paper discusses the bounding aims and constraints identified by the monitoring and host parties with respect to the disassembly phase, the design of the room-within-a-room system, lessons learned during deployment, conclusions and potential areas of future work. Overall it was agreed that the room-within-a-room approach was effective but the individual technologies used to create the system deployed during this exercise required further development.« less

Actin-filament disassembly: it takes two to shrink them fast.

PubMed

Winterhoff, Moritz; Faix, Jan

2015-06-01

Actin-filament disassembly is indispensable for replenishing the pool of polymerizable actin and allows continuous dynamic remodelling of the actin cytoskeleton. A new study now reveals that ADF/cofilin preferentially dismantles branched networks and provides new insights into the collaborative work of ADF/cofilin and Aip1 on filament disassembly at the molecular level. Copyright © 2015 Elsevier Ltd. All rights reserved.

Biodiversity loss decreases parasite diversity: theory and patterns

USGS Publications Warehouse

Lafferty, Kevin D.

2012-01-01

Past models have suggested host–parasite coextinction could lead to linear, or concave down relationships between free-living species richness and parasite richness. I explored several models for the relationship between parasite richness and biodiversity loss. Life cycle complexity, low generality of parasites and sensitivity of hosts reduced the robustness of parasite species to the loss of free-living species diversity. Food-web complexity and the ordering of extinctions altered these relationships in unpredictable ways. Each disassembly of a food web resulted in a unique relationship between parasite richness and the richness of free-living species, because the extinction trajectory of parasites was sensitive to the order of extinctions of free-living species. However, the average of many disassemblies tended to approximate an analytical model. Parasites of specialist hosts and hosts higher on food chains were more likely to go extinct in food-web models. Furthermore, correlated extinctions between hosts and parasites (e.g. if parasites share a host with a specialist predator) led to steeper declines in parasite richness with biodiversity loss. In empirical food webs with random removals of free-living species, the relationship between free-living species richness and parasite richness was, on average, quasi-linear, suggesting biodiversity loss reduces parasite diversity more than previously thought.

A Continuum Model of Actin Waves in Dictyostelium discoideum

PubMed Central

Khamviwath, Varunyu; Hu, Jifeng; Othmer, Hans G.

2013-01-01

Actin waves are complex dynamical patterns of the dendritic network of filamentous actin in eukaryotes. We developed a model of actin waves in PTEN-deficient Dictyostelium discoideum by deriving an approximation of the dynamics of discrete actin filaments and combining it with a signaling pathway that controls filament branching. This signaling pathway, together with the actin network, contains a positive feedback loop that drives the actin waves. Our model predicts the structure, composition, and dynamics of waves that are consistent with existing experimental evidence, as well as the biochemical dependence on various protein partners. Simulation suggests that actin waves are initiated when local actin network activity, caused by an independent process, exceeds a certain threshold. Moreover, diffusion of proteins that form a positive feedback loop with the actin network alone is sufficient for propagation of actin waves at the observed speed of . Decay of the wave back can be caused by scarcity of network components, and the shape of actin waves is highly dependent on the filament disassembly rate. The model allows retraction of actin waves and captures formation of new wave fronts in broken waves. Our results demonstrate that a delicate balance between a positive feedback, filament disassembly, and local availability of network components is essential for the complex dynamics of actin waves. PMID:23741312

Tripartite polyionic complex (PIC) micelles as non-viral vectors for mesenchymal stem cell siRNA transfection.

PubMed

Raisin, Sophie; Morille, Marie; Bony, Claire; Noël, Danièle; Devoisselle, Jean-Marie; Belamie, Emmanuel

2017-08-22

In the context of regenerative medicine, the use of RNA interference mechanisms has already proven its efficiency in targeting specific gene expression with the aim of enhancing, accelerating or, more generally, directing stem cell differentiation. However, achievement of good transfection levels requires the use of a gene vector. For in vivo applications, synthetic vectors are an interesting option to avoid possible issues associated with viral vectors (safety, production costs, etc.). Herein, we report on the design of tripartite polyionic complex micelles as original non-viral polymeric vectors suited for mesenchymal stem cell transfection with siRNA. Three micelle formulations were designed to exhibit pH-triggered disassembly in an acidic pH range comparable to that of endosomes. One formulation was selected as the most promising with the highest siRNA loading capacity while clearly maintaining pH-triggered disassembly properties. A thorough investigation of the internalization pathway of micelles into cells with tagged siRNA was made before showing an efficient inhibition of Runx2 expression in primary bone marrow-derived stem cells. This work evidenced PIC micelles as promising synthetic vectors that allow efficient MSC transfection and control over their behavior, from the perspective of their clinical use.

Regulating the chromatin landscape: structural and mechanistic perspectives.

PubMed

Bartholomew, Blaine

2014-01-01

A large family of chromatin remodelers that noncovalently modify chromatin is crucial in cell development and differentiation. They are often the targets of cancer, neurological disorders, and other human diseases. These complexes alter nucleosome positioning, higher-order chromatin structure, and nuclear organization. They also assemble chromatin, exchange out histone variants, and disassemble chromatin at defined locations. We review aspects of the structural organization of these complexes, the functional properties of their protein domains, and variation between complexes. We also address the mechanistic details of these complexes in mobilizing nucleosomes and altering chromatin structure. A better understanding of these issues will be vital for further analyses of subunits of these chromatin remodelers, which are being identified as targets in human diseases by NGS (next-generation sequencing).

Assembly/Disassembly of DNA-Au Nanoparticles: A Strategy of Intervention

DOE PAGES

Lim, I-Im S.; Wang, Lingyan; Chandrachud, Uma; ...

2008-01-01

This report describes the viability of a strategy for manipulating the assembly/disassembly processes of DNA-Au nanoparticles by molecular intervention. Using the temperature-induced assembly and disassembly processes of DNAs and gold nanoparticles as a model system, the introduction of a molecular recognition probe is demonstrated to lead to the intervention of the assembly/disassembly processes depending on its specific biorecognition. This process can be detected by monitoring the change in the optical properties of gold nanoparticles and their DNA assemblies. Implications of the preliminary results to exploration of the resulting nanostructures for fine-tuning of the interfacial reactivities in DNA-based bioassays and biomaterialmore » engineering are also discussed.« less

Mediator, TATA-binding Protein, and RNA Polymerase II Contribute to Low Histone Occupancy at Active Gene Promoters in Yeast*

PubMed Central

Ansari, Suraiya A.; Paul, Emily; Sommer, Sebastian; Lieleg, Corinna; He, Qiye; Daly, Alexandre Z.; Rode, Kara A.; Barber, Wesley T.; Ellis, Laura C.; LaPorta, Erika; Orzechowski, Amanda M.; Taylor, Emily; Reeb, Tanner; Wong, Jason; Korber, Philipp; Morse, Randall H.

2014-01-01

Transcription by RNA polymerase II (Pol II) in eukaryotes requires the Mediator complex, and often involves chromatin remodeling and histone eviction at active promoters. Here we address the role of Mediator in recruitment of the Swi/Snf chromatin remodeling complex and its role, along with components of the preinitiation complex (PIC), in histone eviction at inducible and constitutively active promoters in the budding yeast Saccharomyces cerevisiae. We show that recruitment of the Swi/Snf chromatin remodeling complex to the induced CHA1 promoter, as well as its association with several constitutively active promoters, depends on the Mediator complex but is independent of Mediator at the induced MET2 and MET6 genes. Although transcriptional activation and histone eviction at CHA1 depends on Swi/Snf, Swi/Snf recruitment is not sufficient for histone eviction at the induced CHA1 promoter. Loss of Swi/Snf activity does not affect histone occupancy of several constitutively active promoters; in contrast, higher histone occupancy is seen at these promoters in Mediator and PIC component mutants. We propose that an initial activator-dependent, nucleosome remodeling step allows PIC components to outcompete histones for occupancy of promoter sequences. We also observe reduced promoter association of Mediator and TATA-binding protein in a Pol II (rpb1-1) mutant, indicating mutually cooperative binding of these components of the transcription machinery and indicating that it is the PIC as a whole whose binding results in stable histone eviction. PMID:24727477

Protein dynamics of human RPA and RAD51 on ssDNA during assembly and disassembly of the RAD51 filament.

PubMed

Ma, Chu Jian; Gibb, Bryan; Kwon, YoungHo; Sung, Patrick; Greene, Eric C

2017-01-25

Homologous recombination (HR) is a crucial pathway for double-stranded DNA break (DSB) repair. During the early stages of HR, the newly generated DSB ends are processed to yield long single-stranded DNA (ssDNA) overhangs, which are quickly bound by replication protein A (RPA). RPA is then replaced by the DNA recombinase Rad51, which forms extended helical filaments on the ssDNA. The resulting nucleoprotein filament, known as the presynaptic complex, is responsible for pairing the ssDNA with homologous double-stranded DNA (dsDNA), which serves as the template to guide DSB repair. Here, we use single-molecule imaging to visualize the interplay between human RPA (hRPA) and human RAD51 during presynaptic complex assembly and disassembly. We demonstrate that ssDNA-bound hRPA can undergo facilitated exchange, enabling hRPA to undergo rapid exchange between free and ssDNA-bound states only when free hRPA is present in solution. Our results also indicate that the presence of free hRPA inhibits RAD51 filament nucleation, but has a lesser impact upon filament elongation. This finding suggests that hRPA exerts important regulatory influence over RAD51 and may in turn affect the properties of the assembled RAD51 filament. These experiments provide an important basis for further investigations into the regulation of human presynaptic complex assembly. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Regulation of Endothelial Adherens Junctions by Tyrosine Phosphorylation

PubMed Central

Adam, Alejandro Pablo

2015-01-01

Endothelial cells form a semipermeable, regulated barrier that limits the passage of fluid, small molecules, and leukocytes between the bloodstream and the surrounding tissues. The adherens junction, a major mechanism of intercellular adhesion, is comprised of transmembrane cadherins forming homotypic interactions between adjacent cells and associated cytoplasmic catenins linking the cadherins to the cytoskeleton. Inflammatory conditions promote the disassembly of the adherens junction and a loss of intercellular adhesion, creating openings or gaps in the endothelium through which small molecules diffuse and leukocytes transmigrate. Tyrosine kinase signaling has emerged as a central regulator of the inflammatory response, partly through direct phosphorylation and dephosphorylation of the adherens junction components. This review discusses the findings that support and those that argue against a direct effect of cadherin and catenin phosphorylation in the disassembly of the adherens junction. Recent findings indicate a complex interaction between kinases, phosphatases, and the adherens junction components that allow a fine regulation of the endothelial permeability to small molecules, leukocyte migration, and barrier resealing. PMID:26556953

In situ solid-state NMR and XRD studies of the ADOR process and the unusual structure of zeolite IPC-6

NASA Astrophysics Data System (ADS)

Morris, Samuel A.; Bignami, Giulia P. M.; Tian, Yuyang; Navarro, Marta; Firth, Daniel S.; Čejka, Jiří; Wheatley, Paul S.; Dawson, Daniel M.; Slawinski, Wojciech A.; Wragg, David S.; Morris, Russell E.; Ashbrook, Sharon E.

2017-10-01

The assembly-disassembly-organization-reassembly (ADOR) mechanism is a recent method for preparing inorganic framework materials and, in particular, zeolites. This flexible approach has enabled the synthesis of isoreticular families of zeolites with unprecedented continuous control over porosity, and the design and preparation of materials that would have been difficult—or even impossible—to obtain using traditional hydrothermal techniques. Applying the ADOR process to a parent zeolite with the UTL framework topology, for example, has led to six previously unknown zeolites (named IPC-n, where n = 2, 4, 6, 7, 9 and 10). To realize the full potential of the ADOR method, however, a further understanding of the complex mechanism at play is needed. Here, we probe the disassembly, organization and reassembly steps of the ADOR process through a combination of in situ solid-state NMR spectroscopy and powder X-ray diffraction experiments. We further use the insight gained to explain the formation of the unusual structure of zeolite IPC-6.

Biolayer interferometry of lipid nanodisc‐reconstituted yeast vacuolar H+‐ATPase

PubMed Central

Sharma, Stuti

2017-01-01

Abstract Vacuolar H+‐ATPase (V‐ATPase) is a large, multisubunit membrane protein complex responsible for the acidification of subcellular compartments and the extracellular space. V‐ATPase activity is regulated by reversible disassembly, resulting in cytosolic V 1‐ATPase and membrane‐integral V 0 proton channel sectors. Reversible disassembly is accompanied by transient interaction with cellular factors and assembly chaperones. Quantifying protein‐protein interactions involving membrane proteins, however, is challenging. Here we present a novel method to determine kinetic constants of membrane protein–protein interactions using biolayer interferometry (BLI). Yeast vacuoles are solubilized, vacuolar proteins are reconstituted into lipid nanodiscs with native vacuolar lipids and biotinylated membrane scaffold protein (MSP) followed by affinity purification of nanodisc‐reconstituted V‐ATPase (V 1 V 0ND). We show that V 1 V 0ND can be immobilized on streptavidin‐coated BLI sensors to quantitate binding of a pathogen derived inhibitor and to measure the kinetics of nucleotide dependent enzyme dissociation. PMID:28241399

Biolayer interferometry of lipid nanodisc-reconstituted yeast vacuolar H+ -ATPase.

PubMed

Sharma, Stuti; Wilkens, Stephan

2017-05-01

Vacuolar H + -ATPase (V-ATPase) is a large, multisubunit membrane protein complex responsible for the acidification of subcellular compartments and the extracellular space. V-ATPase activity is regulated by reversible disassembly, resulting in cytosolic V 1 -ATPase and membrane-integral V 0 proton channel sectors. Reversible disassembly is accompanied by transient interaction with cellular factors and assembly chaperones. Quantifying protein-protein interactions involving membrane proteins, however, is challenging. Here we present a novel method to determine kinetic constants of membrane protein-protein interactions using biolayer interferometry (BLI). Yeast vacuoles are solubilized, vacuolar proteins are reconstituted into lipid nanodiscs with native vacuolar lipids and biotinylated membrane scaffold protein (MSP) followed by affinity purification of nanodisc-reconstituted V-ATPase (V 1 V 0 ND). We show that V 1 V 0 ND can be immobilized on streptavidin-coated BLI sensors to quantitate binding of a pathogen derived inhibitor and to measure the kinetics of nucleotide dependent enzyme dissociation. © 2017 The Protein Society.

Identification of Neuregulin-2 as a novel stress granule component.

PubMed

Kim, Jin Ah; Jayabalan, Aravinth Kumar; Kothandan, Vinoth Kumar; Mariappan, Ramesh; Kee, Younghoon; Ohn, Takbum

2016-08-01

Stress Granules (SGs) are microscopically visible, phase dense aggregates of translationally stalled messenger ribonucleoprotein (mRNP) complexes formed in response to distinct stress conditions. It is generally considered that SG formation is induced to protect cells from conditions of stress. The precise constituents of SGs and the mechanism through which SGs are dynamically regulated in response to stress are not completely understood. Hence, it is important to identify proteins which regulate SG assembly and disassembly. In the present study, we report Neuregulin-2 (NRG2) as a novel component of SGs; furthermore, depletion of NRG2 potently inhibits SG formation. We also demonstrate that NRG2 specifically localizes to SGs under various stress conditions. Knockdown of NRG2 has no effect on stress-induced polysome disassembly, suggesting that the component does not influence early step of SG formation. It was also observed that reduced expression of NRG2 led to marginal increase in cell survival under arsenite-induced stress. [BMB Reports 2016; 49(8): 449-454].

Identification of Neuregulin-2 as a novel stress granule component

PubMed Central

Kim, Jin Ah; Jayabalan, Aravinth Kumar; Kothandan, Vinoth Kumar; Mariappan, Ramesh; Kee, Younghoon; Ohn, Takbum

2016-01-01

Stress Granules (SGs) are microscopically visible, phase dense aggregates of translationally stalled messenger ribonucleoprotein (mRNP) complexes formed in response to distinct stress conditions. It is generally considered that SG formation is induced to protect cells from conditions of stress. The precise constituents of SGs and the mechanism through which SGs are dynamically regulated in response to stress are not completely understood. Hence, it is important to identify proteins which regulate SG assembly and disassembly. In the present study, we report Neuregulin-2 (NRG2) as a novel component of SGs; furthermore, depletion of NRG2 potently inhibits SG formation. We also demonstrate that NRG2 specifically localizes to SGs under various stress conditions. Knockdown of NRG2 has no effect on stress-induced polysome disassembly, suggesting that the component does not influence early step of SG formation. It was also observed that reduced expression of NRG2 led to marginal increase in cell survival under arsenite-induced stress. [BMB Reports 2016; 49(8): 449-454] PMID:27345716

A comparison of different postures for scaffold end-frame disassembly.

PubMed

Cutlip, R; Hsiao, H; Garcia, R; Becker, E; Mayeux, B

2000-10-01

Overexertion and fall injuries comprise the largest category of nonfatal injuries among scaffold workers. This study was conducted to identify the most favourable scaffold end-frame disassembly techniques and evaluate the associated slip potential by measuring whole-body isometric strength capability and required coefficient of friction (RCOF) to reduce the incidence of injury. Forty-six male construction workers were used to study seven typical postures associated with scaffold end-frame disassembly. An analysis of variance (ANOVA) showed that the isometric forces (334.4-676.3 N) resulting from the seven postures were significantly different (p < 0.05). Three of the disassembly postures resulted in considerable biomechanical stress to workers. The symmetric front-lift method with hand locations at knuckle height would be the most favourable posture; at least 93% of the male construction worker population could handle the end frame with minimum overexertion risk. The static RCOF value resulting from this posture during the disassembly phase was less than 0.2, thus the likelihood of a slip should be low.

Direct measurement of conformational strain energy in protofilaments curling outward from disassembling microtubule tips.

PubMed

Driver, Jonathan W; Geyer, Elisabeth A; Bailey, Megan E; Rice, Luke M; Asbury, Charles L

2017-06-19

Disassembling microtubules can generate movement independently of motor enzymes, especially at kinetochores where they drive chromosome motility. A popular explanation is the 'conformational wave' model, in which protofilaments pull on the kinetochore as they curl outward from a disassembling tip. But whether protofilaments can work efficiently via this spring-like mechanism has been unclear. By modifying a previous assay to use recombinant tubulin and feedback-controlled laser trapping, we directly demonstrate the spring-like elasticity of curling protofilaments. Measuring their mechanical work output suggests they carry ~25% of the energy of GTP hydrolysis as bending strain, enabling them to drive movement with efficiency similar to conventional motors. Surprisingly, a β-tubulin mutant that dramatically slows disassembly has no effect on work output, indicating an uncoupling of disassembly speed from protofilament strain. These results show the wave mechanism can make a major contribution to kinetochore motility and establish a direct approach for measuring tubulin mechano-chemistry.

Disassembly Properties of Cementitious Finish Joints Using an Induction Heating Method

PubMed Central

Ahn, Jaecheol; Noguchi, Takafumi; Kitagaki, Ryoma

2015-01-01

Efficient maintenance and upgrading of a building during its lifecycle are difficult because a cementitious finish uses materials and parts with low disassembly properties. Additionally, the reuse and recycling processes during building demolition also present numerous problems from the perspective of environmental technology. In this study, an induction heating (IH) method was used to disassemble cementitious finish joints, which are widely used to join building members and materials. The IH rapidly and selectively heated and weakened these joints. The temperature elevation characteristics of the cementitious joint materials were measured as a function of several resistor types, including wire meshes and punching metals, which are usually used for cementitious finishing. The disassembly properties were evaluated through various tests using conductive resistors in cementitious joints such as mortar. When steel fiber, punching metal, and wire mesh were used as conductive resistors, the cementitious modifiers could be weakened within 30 s. Cementitious joints with conductive resistors also showed complete disassembly with little residual bond strength.

Direct measurement of conformational strain energy in protofilaments curling outward from disassembling microtubule tips

PubMed Central

Driver, Jonathan W; Geyer, Elisabeth A; Bailey, Megan E; Rice, Luke M; Asbury, Charles L

2017-01-01

Disassembling microtubules can generate movement independently of motor enzymes, especially at kinetochores where they drive chromosome motility. A popular explanation is the ‘conformational wave’ model, in which protofilaments pull on the kinetochore as they curl outward from a disassembling tip. But whether protofilaments can work efficiently via this spring-like mechanism has been unclear. By modifying a previous assay to use recombinant tubulin and feedback-controlled laser trapping, we directly demonstrate the spring-like elasticity of curling protofilaments. Measuring their mechanical work output suggests they carry ~25% of the energy of GTP hydrolysis as bending strain, enabling them to drive movement with efficiency similar to conventional motors. Surprisingly, a β-tubulin mutant that dramatically slows disassembly has no effect on work output, indicating an uncoupling of disassembly speed from protofilament strain. These results show the wave mechanism can make a major contribution to kinetochore motility and establish a direct approach for measuring tubulin mechano-chemistry. DOI: http://dx.doi.org/10.7554/eLife.28433.001 PMID:28628007

Damage-Free Relief-Valve Disassembly

NASA Technical Reports Server (NTRS)

Haselmaier, H.

1986-01-01

Tool safely disassembles relief valves without damage to sensitive parts. Relief-valve disassembly tool used to extract valve nozzle from its housing. Holding device on tool grops nozzle. When user strikes hammer against impact disk, holding device pulls nozzle from press fit. Previously, nozzle dislodged by striking spindle above it, but practice often damaged retaining screw. New tool removes nozzle directly. With minor modifications, tool adapted to valves from different manufacturers.

Polymer–Peptide Conjugates Disassemble Amyloid β Fibrils in a Molecular-Weight Dependent Manner

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Yang; Moore, Edwin G.; Guo, Yanshu

Amyloid aggregation and deposition are associated with many intractable human diseases. Although the inhibition of amyloid protein aggregation has been well-studied, the disaggregation and dissolution of existing amyloid fibrils is less known. Taking a fibrillar assembly of amyloid β (Aβ) peptide as the model system, in this paper we report multivalent polymer–peptide conjugates (mPPCs) that disassemble preformed Aβ fibrils into dispersible sub-100 nm structures. Atomic force microscopy and dynamic light scattering studies show that the disassembly rate of preformed Aβ fibrils is controlled by the molecular weight of mPPCs. Rate equations on fibril disappearance are deduced from a simple model,more » which indicate that the disassembly reaction is first-order in the concentration of Aβ fibrils and a pseudo-first-order reaction in the concentration of peptide moieties on mPPCs, respectively. We eliminate the possibility that the disassembly occurs by the association between mPPCs and Aβ monomer/oligomers based on circular dichroism and Thioflavin T fluorescence assays. It is mostly likely that the mPPCs disassemble Aβ fibrils through a direct interaction. Finally, the mPPCs may thus offer a general macromolecular design concept that breaks down existing amyloid fibrils in a predictable fashion.« less

Polymer–Peptide Conjugates Disassemble Amyloid β Fibrils in a Molecular-Weight Dependent Manner

DOE PAGES

Song, Yang; Moore, Edwin G.; Guo, Yanshu; ...

2017-03-14

Amyloid aggregation and deposition are associated with many intractable human diseases. Although the inhibition of amyloid protein aggregation has been well-studied, the disaggregation and dissolution of existing amyloid fibrils is less known. Taking a fibrillar assembly of amyloid β (Aβ) peptide as the model system, in this paper we report multivalent polymer–peptide conjugates (mPPCs) that disassemble preformed Aβ fibrils into dispersible sub-100 nm structures. Atomic force microscopy and dynamic light scattering studies show that the disassembly rate of preformed Aβ fibrils is controlled by the molecular weight of mPPCs. Rate equations on fibril disappearance are deduced from a simple model,more » which indicate that the disassembly reaction is first-order in the concentration of Aβ fibrils and a pseudo-first-order reaction in the concentration of peptide moieties on mPPCs, respectively. We eliminate the possibility that the disassembly occurs by the association between mPPCs and Aβ monomer/oligomers based on circular dichroism and Thioflavin T fluorescence assays. It is mostly likely that the mPPCs disassemble Aβ fibrils through a direct interaction. Finally, the mPPCs may thus offer a general macromolecular design concept that breaks down existing amyloid fibrils in a predictable fashion.« less

Electronic waste disassembly with industrial waste heat.

PubMed

Chen, Mengjun; Wang, Jianbo; Chen, Haiyian; Ogunseitan, Oladele A; Zhang, Mingxin; Zang, Hongbin; Hu, Jiukun

2013-01-01

Waste printed circuit boards (WPCBs) are resource-rich but hazardous, demanding innovative strategies for post-consumer collection, recycling, and mining for economically precious constituents. A novel technology for disassembling electronic components from WPCBs is proposed, using hot air to melt solders and to separate the components and base boards. An automatic heated-air disassembling equipment was designed to operate at a heating source temperature at a maximum of 260 °C and an inlet pressure of 0.5 MPa. A total of 13 individual WPCBs were subjected to disassembling tests at different preheat temperatures in increments of 20 °C between 80 and 160 °C, heating source temperatures ranging from 220 to 300 °C in increments of 20 °C, and incubation periods of 1, 2, 4, 6, or 8 min. For each experimental treatment, the disassembly efficiency was calculated as the ratio of electronic components released from the board to the total number of its original components. The optimal preheat temperature, heating source temperature, and incubation period to disassemble intact components were 120 °C, 260 °C, and 2 min, respectively. The disassembly rate of small surface mount components (side length ≤ 3 mm) was 40-50% lower than that of other surface mount components and pin through hole components. On the basis of these results, a reproducible and sustainable industrial ecological protocol using steam produced by industrial exhaust heat coupled to electronic-waste recycling is proposed, providing an efficient, promising, and green method for both electronic component recovery and industrial exhaust heat reutilization.

Kinetics of Surface-Driven Self-Assembly and Fatigue-Induced Disassembly of a Virus-Based Nanocoating.

PubMed

Valbuena, Alejandro; Mateu, Mauricio G

2017-02-28

Self-assembling protein layers provide a "bottom-up" approach for precisely organizing functional elements at the nanoscale over a large solid surface area. The design of protein sheets with architecture and physical properties suitable for nanotechnological applications may be greatly facilitated by a thorough understanding of the principles that underlie their self-assembly and disassembly. In a previous study, the hexagonal lattice formed by the capsid protein (CA) of human immunodeficiency virus (HIV) was self-assembled as a monomolecular layer directly onto a solid substrate, and its mechanical properties and dynamics at equilibrium were analyzed by atomic force microscopy. Here, we use atomic force microscopy to analyze the kinetics of self-assembly of the planar CA lattice on a substrate and of its disassembly, either spontaneous or induced by materials fatigue. Both self-assembly and disassembly of the CA layer are cooperative reactions that proceed until a phase equilibrium is reached. Self-assembly requires a critical protein concentration and is initiated by formation of nucleation points on the substrate, followed by lattice growth and eventual merging of CA patches into a continuous monolayer. Disassembly of the CA layer showed hysteresis and appears to proceed only after large enough defects (nucleation points) are formed in the lattice, whose number is largely increased by inducing materials fatigue that depends on mechanical load and its frequency. Implications of the kinetic results obtained for a better understanding of self-assembly and disassembly of the HIV capsid and protein-based two-dimensional nanomaterials and the design of anti-HIV drugs targeting (dis)assembly and biocompatible nanocoatings are discussed. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Efficient estimation of three-dimensional covariance and its application in the analysis of heterogeneous samples in cryo-electron microscopy

PubMed Central

Liao, Hstau Y.; Hashem, Yaser; Frank, Joachim

2015-01-01

Summary Single-particle cryogenic electron microscopy (cryo-EM) is a powerful tool for the study of macromolecular structures at high resolution. Classification allows multiple structural states to be extracted and reconstructed from the same sample. One classification approach is via the covariance matrix, which captures the correlation between every pair of voxels. Earlier approaches employ computing-intensive resampling and estimate only the eigenvectors of the matrix, which are then used in a separate fast classification step. We propose an iterative scheme to explicitly estimate the covariance matrix in its entirety. In our approach, the flexibility in choosing the solution domain allows us to examine a part of the molecule in greater detail. 3D covariance maps obtained in this way from experimental data (cryo-EM images of the eukaryotic pre-initiation complex) prove to be in excellent agreement with conclusions derived by using traditional approaches, revealing in addition the interdependencies of ligand bindings and structural changes. PMID:25982529

Efficient estimation of three-dimensional covariance and its application in the analysis of heterogeneous samples in cryo-electron microscopy.

PubMed

Liao, Hstau Y; Hashem, Yaser; Frank, Joachim

2015-06-02

Single-particle cryogenic electron microscopy (cryo-EM) is a powerful tool for the study of macromolecular structures at high resolution. Classification allows multiple structural states to be extracted and reconstructed from the same sample. One classification approach is via the covariance matrix, which captures the correlation between every pair of voxels. Earlier approaches employ computing-intensive resampling and estimate only the eigenvectors of the matrix, which are then used in a separate fast classification step. We propose an iterative scheme to explicitly estimate the covariance matrix in its entirety. In our approach, the flexibility in choosing the solution domain allows us to examine a part of the molecule in greater detail. Three-dimensional covariance maps obtained in this way from experimental data (cryo-EM images of the eukaryotic pre-initiation complex) prove to be in excellent agreement with conclusions derived by using traditional approaches, revealing in addition the interdependencies of ligand bindings and structural changes. Copyright © 2015 Elsevier Ltd. All rights reserved.

Tail and Kinase Modules Differently Regulate Core Mediator Recruitment and Function In Vivo.

PubMed

Jeronimo, Célia; Langelier, Marie-France; Bataille, Alain R; Pascal, John M; Pugh, B Franklin; Robert, François

2016-11-03

Mediator is a highly conserved transcriptional coactivator organized into four modules, namely Tail, Middle, Head, and Kinase (CKM). Previous work suggests regulatory roles for Tail and CKM, but an integrated model for these activities is lacking. Here, we analyzed the genome-wide distribution of Mediator subunits in wild-type and mutant yeast cells in which RNA polymerase II promoter escape is blocked, allowing detection of transient Mediator forms. We found that although all modules are recruited to upstream activated regions (UAS), assembly of Mediator within the pre-initiation complex is accompanied by the release of CKM. Interestingly, our data show that CKM regulates Mediator-UAS interaction rather than Mediator-promoter association. In addition, although Tail is required for Mediator recruitment to UAS, Tailless Mediator nevertheless interacts with core promoters. Collectively, our data suggest that the essential function of Mediator is mediated by Head and Middle at core promoters, while Tail and CKM play regulatory roles. Copyright © 2016 Elsevier Inc. All rights reserved.

Closure of the R Reactor Disassembly Basin at the SRS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Austin, W.E.

The Facilities Disposition Division (FDD) at the Savannah River Site is engaged in planning the deactivation/closure of three of the site's five reactor disassembly basins. Activities are currently underway at R-Reactor Disassembly Basin and will continue with the P and C disassembly basins. The basins still contain the cooling and shielding water that was present when operations ceased. Low concentrations of radionuclides are present, with tritium, Cs-137, and Sr-90 being the major contributors. Although there is no evidence that any of the basins have leaked, the 50-year-old facilities will eventually contaminate the surrounding groundwaters. The FDD is pursuing a pro-activemore » solution to close the basins in-place and prevent a release to the groundwater. In-situ ion exchange is currently underway at the R-Reactor Disassembly Basin to reduce the Cs and Sr concentrations to levels that would allow release of the treated water to previously used on-site cooling ponds or to prevent ground water impact. The closure will be accomplished under CERCLA.« less

Structure of nerve growth factor complexed with the shared neurotrophin receptor p75.

PubMed

He, Xiao-Lin; Garcia, K Christopher

2004-05-07

Neurotrophins are secreted growth factors critical for the development and maintenance of the vertebrate nervous system. Neurotrophins activate two types of cell surface receptors, the Trk receptor tyrosine kinases and the shared p75 neurotrophin receptor. We have determined the 2.4 A crystal structure of the prototypic neurotrophin, nerve growth factor (NGF), complexed with the extracellular domain of p75. Surprisingly, the complex is composed of an NGF homodimer asymmetrically bound to a single p75. p75 binds along the homodimeric interface of NGF, which disables NGF's symmetry-related second p75 binding site through an allosteric conformational change. Thus, neurotrophin signaling through p75 may occur by disassembly of p75 dimers and assembly of asymmetric 2:1 neurotrophin/p75 complexes, which could potentially engage a Trk receptor to form a trimolecular signaling complex.

19 CFR 181.132 - Disassembly.

Code of Federal Regulations, 2010 CFR

2010-04-01

... (CONTINUED) NORTH AMERICAN FREE TRADE AGREEMENT Rules of Origin § 181.132 Disassembly. (a) Treated as production. For purposes of implementing the rules of origin provisions of General Note 12, HTSUS, and...

The mediator complex in genomic and non-genomic signaling in cancer.

PubMed

Weber, Hannah; Garabedian, Michael J

2018-05-01

Mediator is a conserved, multi-subunit macromolecular machine divided structurally into head, middle, and tail modules, along with a transiently associating kinase module. Mediator functions as an integrator of transcriptional regulatory activity by interacting with DNA-bound transcription factors and with RNA polymerase II (Pol II) to both activate and repress gene expression. Mediator has been shown to affect multiple steps in transcription, including chromatin looping between enhancers and promoters, pre-initiation complex formation, transcriptional elongation, and mRNA splicing. Individual Mediator subunits participate in regulation of gene expression by the estrogen and androgen receptors and are altered in a number of endocrine cancers, including breast and prostate cancer. In addition to its role in genomic signaling, MED12 has been implicated in non-genomic signaling by interacting with and activating TGF-beta receptor 2 in the cytoplasm. Recent structural studies have revealed extensive inter-domain interactions and complex architecture of the Mediator-Pol II complex, suggesting that Mediator is capable of reorganizing its conformation and composition to fit cellular needs. We propose that alterations in Mediator subunit expression that occur in various cancers could impact the organization and function of Mediator, resulting in changes in gene expression that promote malignancy. A better understanding of the role of Mediator in cancer could reveal new approaches to the diagnosis and treatment of Mediator-dependent endocrine cancers, especially in settings of therapy resistance. Copyright © 2017 Elsevier Inc. All rights reserved.

UBF complexes with phosphatidylinositol 4,5-bisphosphate in nucleolar organizer regions regardless of ongoing RNA polymerase I activity

PubMed Central

Sobol, Margarita; Yildirim, Sukriye; Philimonenko, Vlada V; Marášek, Pavel; Castaño, Enrique; Hozák, Pavel

2013-01-01

To maintain growth and division, cells require a large-scale production of rRNAs which occurs in the nucleolus. Recently, we have shown the interaction of nucleolar phosphatidylinositol 4,5-bisphosphate (PIP2) with proteins involved in rRNA transcription and processing, namely RNA polymerase I (Pol I), UBF, and fibrillarin. Here we extend the study by investigating transcription-related localization of PIP2 in regards to transcription and processing complexes of Pol I. To achieve this, we used either physiological inhibition of transcription during mitosis or inhibition by treatment the cells with actinomycin D (AMD) or 5,6-dichloro-1β-d-ribofuranosyl-benzimidazole (DRB). We show that PIP2 is associated with Pol I subunits and UBF in a transcription-independent manner. On the other hand, PIP2/fibrillarin colocalization is dependent on the production of rRNA. These results indicate that PIP2 is required not only during rRNA production and biogenesis, as we have shown before, but also plays a structural role as an anchor for the Pol I pre-initiation complex during the cell cycle. We suggest that throughout mitosis, PIP2 together with UBF is involved in forming and maintaining the core platform of the rDNA helix structure. Thus we introduce PIP2 as a novel component of the NOR complex, which is further engaged in the renewed rRNA synthesis upon exit from mitosis. PMID:24513678

The Modifier of Transcription 1 (Mot1) ATPase and Spt16 Histone Chaperone Co-regulate Transcription through Preinitiation Complex Assembly and Nucleosome Organization.

PubMed

True, Jason D; Muldoon, Joseph J; Carver, Melissa N; Poorey, Kunal; Shetty, Savera J; Bekiranov, Stefan; Auble, David T

2016-07-15

Modifier of transcription 1 (Mot1) is a conserved and essential Swi2/Snf2 ATPase that can remove TATA-binding protein (TBP) from DNA using ATP hydrolysis and in so doing exerts global effects on transcription. Spt16 is also essential and functions globally in transcriptional regulation as a component of the facilitates chromatin transcription (FACT) histone chaperone complex. Here we demonstrate that Mot1 and Spt16 regulate a largely overlapping set of genes in Saccharomyces cerevisiae. As expected, Mot1 was found to control TBP levels at co-regulated promoters. In contrast, Spt16 did not affect TBP recruitment. On a global scale, Spt16 was required for Mot1 promoter localization, and Mot1 also affected Spt16 localization to genes. Interestingly, we found that Mot1 has an unanticipated role in establishing or maintaining the occupancy and positioning of nucleosomes at the 5' ends of genes. Spt16 has a broad role in regulating chromatin organization in gene bodies, including those nucleosomes affected by Mot1. These results suggest that the large scale overlap in Mot1 and Spt16 function arises from a combination of both their unique and shared functions in transcription complex assembly and chromatin structure regulation. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

40 CFR 82.260 - Definitions.

Code of Federal Regulations, 2010 CFR

2010-07-01

... or water; or (3) The disassembly of any halon-containing equipment for reuse of its component parts... halon-containing equipment into or on any land or water; (2) The disassembly of any halon-containing...

Regulation of mitosis by the NIMA kinase involves TINA and its newly discovered partner, An-WDR8, at spindle pole bodies

PubMed Central

Shen, Kuo-Fang; Osmani, Stephen A.

2013-01-01

The NIMA kinase is required for mitotic nuclear pore complex disassembly and potentially controls other mitotic-specific events. To investigate this possibility, we imaged NIMA–green fluorescent protein (GFP) using four-dimensional spinning disk confocal microscopy. At mitosis NIMA-GFP locates to spindle pole bodies (SPBs), which contain Cdk1/cyclin B, followed by Aurora, TINA, and the BimC kinesin. NIMA promotes NPC disassembly in a spatially regulated manner starting near SPBs. NIMA is also required for TINA, a NIMA-interacting protein, to locate to SPBs during initiation of mitosis, and TINA is then necessary for locating NIMA back to SPBs during mitotic progression. To help expand the NIMA-TINA pathway, we affinity purified TINA and found it to uniquely copurify with An-WDR8, a WD40-domain protein conserved from humans to plants. Like TINA, An-WDR8 accumulates within nuclei during G2 but disperses from nuclei before locating to mitotic SPBs. Without An-WDR8, TINA levels are greatly reduced, whereas TINA is necessary for mitotic targeting of An-WDR8. Finally, we show that TINA is required to anchor mitotic microtubules to SPBs and, in combination with An-WDR8, for successful mitosis. The findings provide new insights into SPB targeting and indicate that the mitotic microtubule-anchoring system at SPBs involves WDR8 in complex with TINA. PMID:24152731

Phosphoregulation of the C. elegans cadherin–catenin complex

PubMed Central

Callaci, Sandhya; Morrison, Kylee; Shao, Xiangqiang; Schuh, Amber L.; Wang, Yueju; Yates, John R.; Hardin, Jeff; Audhya, Anjon

2015-01-01

Adherens junctions play key roles in mediating cell–cell contacts during tissue development. In Caenorhabditis elegans embryos, the cadherin–catenin complex (CCC), composed of the classical cadherin HMR-1 and members of three catenin families, HMP-1, HMP-2 and JAC-1, is necessary for normal blastomere adhesion, gastrulation, ventral enclosure of the epidermis and embryo elongation. Disruption of CCC assembly or function results in embryonic lethality. Previous work suggests that components of the CCC are subject to phosphorylation. However, the identity of phosphorylated residues in CCC components and their contributions to CCC stability and function in a living organism remain speculative. Using mass spectrometry, we systematically identify phosphorylated residues in the essential CCC subunits HMR-1, HMP-1 and HMP-2 in vivo. We demonstrate that HMR-1/cadherin phosphorylation occurs on three sites within its β-catenin binding domain that each contributes to CCC assembly on lipid bilayers. In contrast, phosphorylation of HMP-2/β-catenin inhibits its association with HMR-1/cadherin in vitro, suggesting a role in CCC disassembly. Although HMP-1/α-catenin is also phosphorylated in vivo, phosphomimetic mutations do not affect its ability to associate with other CCC components or interact with actin in vitro. Collectively, our findings support a model in which distinct phosphorylation events contribute to rapid CCC assembly and disassembly, both of which are essential for morphogenetic rearrangements during development. PMID:26443865

Rapid Glucose Depletion Immobilizes Active Myosin-V on Stabilized Actin Cables

PubMed Central

Xu, Li; Bretscher, Anthony

2014-01-01

Summary Polarization of eukaryotic cells requires organelles and protein complexes to be transported to their proper destinations along the cytoskeleton [1]. When nutrients are abundant, budding yeast grows rapidly transporting secretory vesicles for localized growth and actively segregating organelles [2, 3]. This is mediated by myosin-Vs transporting cargos along F-actin bundles known as actin cables [4]. Actin cables are dynamic structures regulated by assembly, stabilization and disassembly [5]. Polarized growth and actin filament dynamics consume energy. For most organisms, glucose is the preferred energy source and generally represses alternative carbon source usage [6]. Thus upon abrupt glucose depletion, yeast shuts down pathways consuming large amounts of energy, including the vacuolar-ATPase [7, 8], translation [9] and phosphoinositide metabolism [10]. Here we show that glucose withdrawal rapidly (<1 min) depletes ATP levels and the yeast myosin V, Myo2, responds by relocalizing to actin cables, making it the fastest response documented. Myo2 immobilized on cables releases its secretory cargo, defining a new rigor-like state of a myosin-V in vivo. Only actively transporting Myo2 can be converted to the rigor-like state. Glucose depletion has differential effects on the actin cytoskeleton resulting in disassembly of actin patches with concomitant inhibition of endocytosis, and strong stabilization of actin cables, thereby revealing a selective and previously unappreciated ATP requirement for actin cable disassembly. A similar response is seen in HeLa cells to ATP depletion. These findings reveal a new fast-acting energy conservation strategy halting growth by immobilizing myosin-V in a newly described state on selectively stabilized actin cables. PMID:25308080

Design and analysis of sustainable computer mouse using design for disassembly methodology

NASA Astrophysics Data System (ADS)

Roni Sahroni, Taufik; Fitri Sukarman, Ahmad; Agung Mahardini, Karunia

2017-12-01

This paper presents the design and analysis of computer mouse using Design for Disassembly methodology. Basically, the existing computer mouse model consist a number of unnecessary part that cause the assembly and disassembly time in production. The objective of this project is to design a new computer mouse based on Design for Disassembly (DFD) methodology. The main methodology of this paper was proposed from sketch generation, concept selection, and concept scoring. Based on the design screening, design concept B was selected for further analysis. New design of computer mouse is proposed using fastening system. Furthermore, three materials of ABS, Polycarbonate, and PE high density were prepared to determine the environmental impact category. Sustainable analysis was conducted using software SolidWorks. As a result, PE High Density gives the lowers amount in the environmental category with great maximum stress value.

Mediator, TATA-binding protein, and RNA polymerase II contribute to low histone occupancy at active gene promoters in yeast.

PubMed

Ansari, Suraiya A; Paul, Emily; Sommer, Sebastian; Lieleg, Corinna; He, Qiye; Daly, Alexandre Z; Rode, Kara A; Barber, Wesley T; Ellis, Laura C; LaPorta, Erika; Orzechowski, Amanda M; Taylor, Emily; Reeb, Tanner; Wong, Jason; Korber, Philipp; Morse, Randall H

2014-05-23

Transcription by RNA polymerase II (Pol II) in eukaryotes requires the Mediator complex, and often involves chromatin remodeling and histone eviction at active promoters. Here we address the role of Mediator in recruitment of the Swi/Snf chromatin remodeling complex and its role, along with components of the preinitiation complex (PIC), in histone eviction at inducible and constitutively active promoters in the budding yeast Saccharomyces cerevisiae. We show that recruitment of the Swi/Snf chromatin remodeling complex to the induced CHA1 promoter, as well as its association with several constitutively active promoters, depends on the Mediator complex but is independent of Mediator at the induced MET2 and MET6 genes. Although transcriptional activation and histone eviction at CHA1 depends on Swi/Snf, Swi/Snf recruitment is not sufficient for histone eviction at the induced CHA1 promoter. Loss of Swi/Snf activity does not affect histone occupancy of several constitutively active promoters; in contrast, higher histone occupancy is seen at these promoters in Mediator and PIC component mutants. We propose that an initial activator-dependent, nucleosome remodeling step allows PIC components to outcompete histones for occupancy of promoter sequences. We also observe reduced promoter association of Mediator and TATA-binding protein in a Pol II (rpb1-1) mutant, indicating mutually cooperative binding of these components of the transcription machinery and indicating that it is the PIC as a whole whose binding results in stable histone eviction. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures

PubMed Central

Schaffter, Samuel W; Green, Leopold N; Schneider, Joanna; Subramanian, Hari K K; Schulman, Rebecca

2018-01-01

Abstract The use of proteins that bind and catalyze reactions with DNA alongside DNA nanostructures has broadened the functionality of DNA devices. DNA binding proteins have been used to specifically pattern and tune structural properties of DNA nanostructures and polymerases have been employed to directly and indirectly drive structural changes in DNA structures and devices. Despite these advances, undesired and poorly understood interactions between DNA nanostructures and proteins that bind DNA continue to negatively affect the performance and stability of DNA devices used in conjunction with enzymes. A better understanding of these undesired interactions will enable the construction of robust DNA nanostructure-enzyme hybrid systems. Here, we investigate the undesired disassembly of DNA nanotubes in the presence of viral RNA polymerases (RNAPs) under conditions used for in vitro transcription. We show that nanotubes and individual nanotube monomers (tiles) are non-specifically transcribed by T7 RNAP, and that RNA transcripts produced during non-specific transcription disassemble the nanotubes. Disassembly requires a single-stranded overhang on the nanotube tiles where transcripts can bind and initiate disassembly through strand displacement, suggesting that single-stranded domains on other DNA nanostructures could cause unexpected interactions in the presence of viral RNA polymerases. PMID:29718412

Disassembly factories for electrical and electronic products to recover resources in product and material cycles.

PubMed

Basdere, Bahadir; Seliger, Guenther

2003-12-01

Cycle economy as a paradigm for industry in the 21st century depends on the economical and ecological treatment of limited resources. The objective is to achieve more use with fewer resources to increase the use-productivity of these resources. The European Union, aware of the adverse environmental impacts associated with electrical and electronic consumer goods in particular, has passed legislation regulating their appropriate end-of-life treatment. Adaptation processes, including essential disassembly and re-assembly operations, contribute significantly toward the economical fulfillment of these new legal requirements. Typically, the disassembly of used products is characterized by a high rate of manual operations, wide variety of product types, and unknown product properties. To cope with such demands, life cycle units or product accompanying information systems, are being developed and used for acquiring data about a specific product throughout its life cycle to aid in determining the level of product deterioration. Modular disassembly processes and tools have been developed and realized to enable the handling of multiple productvariants. They are being implemented in prototypical hybrid disassembly systemsfor large- and small-size electrical and electronic consumer goods.

Deactivation of the P, C, and R Reactor Disassembly Basins at the SRS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pickett, J.B.

The Facilities Disposition Division (FDD) at the Savannah River Site is engaged in planning the deactivation/closure of three of the site's five reactor disassembly basins. Activities are currently underway at 105-R Disassembly Basin and will continue with the 105-P and 105-C disassembly basins. The basins still contain the cooling and shielding water that was present when operations ceased. Low concentrations of radionuclides are present, with tritium, Cs-137, and Sr-90 being the major contributors. Although there is no evidence that any of the basins have leaked, the 50-year-old facilities will eventually contaminate the surrounding groundwaters. The FDD is pursuing a pro-activemore » solution to close the basins in-place and prevent a release to the groundwater. In-situ ion-exchange is currently underway at the R-Reactor Disassembly Basin to reduce the Cs and Sr concentrations to levels that would allow release of the treated water to previously used on-site cooling ponds. A NEPA Environmental Assessment (EA) is being prepared to propose the preferred closure alternative for each of the three basins. The EA will be the primary mechanism to inform the public and gain stakeholder and regulatory approval.« less

T7 RNA polymerase non-specifically transcribes and induces disassembly of DNA nanostructures.

PubMed

Schaffter, Samuel W; Green, Leopold N; Schneider, Joanna; Subramanian, Hari K K; Schulman, Rebecca; Franco, Elisa

2018-06-01

The use of proteins that bind and catalyze reactions with DNA alongside DNA nanostructures has broadened the functionality of DNA devices. DNA binding proteins have been used to specifically pattern and tune structural properties of DNA nanostructures and polymerases have been employed to directly and indirectly drive structural changes in DNA structures and devices. Despite these advances, undesired and poorly understood interactions between DNA nanostructures and proteins that bind DNA continue to negatively affect the performance and stability of DNA devices used in conjunction with enzymes. A better understanding of these undesired interactions will enable the construction of robust DNA nanostructure-enzyme hybrid systems. Here, we investigate the undesired disassembly of DNA nanotubes in the presence of viral RNA polymerases (RNAPs) under conditions used for in vitro transcription. We show that nanotubes and individual nanotube monomers (tiles) are non-specifically transcribed by T7 RNAP, and that RNA transcripts produced during non-specific transcription disassemble the nanotubes. Disassembly requires a single-stranded overhang on the nanotube tiles where transcripts can bind and initiate disassembly through strand displacement, suggesting that single-stranded domains on other DNA nanostructures could cause unexpected interactions in the presence of viral RNA polymerases.

Polygons and adhesion plaques and the disassembly and assembly of myofibrils in cardiac myocytes.

PubMed

Lin, Z X; Holtzer, S; Schultheiss, T; Murray, J; Masaki, T; Fischman, D A; Holtzer, H

1989-06-01

Successive stages in the disassembly of myofibrils and the subsequent assembly of new myofibrils have been studied in cultures of dissociated chick cardiac myocytes. The myofibrils in trypsinized and dispersed myocytes are sequentially disassembled during the first 3 d of culture. They split longitudinally and then assemble into transitory polygons. Multiples of single sarcomeres, the cardiac polygons, are analogous to the transitory polygonal configurations assumed by stress fibers in spreading fibroblasts. They differ from their counterparts in fibroblasts in that they consist of muscle alpha-actinin vertices and muscle myosin heavy chain struts, rather than of the nonmuscle contractile protein isoforms of stress fiber polygons. EM sections reveal the vertices and struts in cardiac polygons to be typical Z and A bands. Most cardiac polygons are eliminated by day 5 of culture. Concurrent with the disassembly and elimination of the original myofibrils new myofibrils are rapidly assembled elsewhere in the same myocyte. Without exception both distal tips of each nascent myofibril terminate in adhesion plaques. The morphology and composition of the adhesion plaques capping each end of each myofibril are similar to those of the termini of stress fibers in fibroblasts. However, whereas the adhesion complexes involving stress fibers in fibroblasts consist of vinculin/nonmuscle alpha-actinin/beta- and gamma-actins, the analogous structures in myocytes involving myofibrils consist of vinculin/muscle alpha-actinin/alpha-actin. The addition of 1.7-2.0 microns sarcomeres to the distal tips of an elongating myofibril, irrespective of whether the myofibril consists of 1, 10, or several hundred tandem sarcomeres, occurs while the myofibril appears to remain linked to its respective adhesion plaques. The adhesion plaques in vitro are the equivalent of the in vivo intercalated discs, both in terms of their molecular composition and with respect to their functioning as initiating sites for the assembly of new sarcomeres. How 1.7-2.0 microns nascent sarcomeres can be added distally during elongation while the tips of the myofibrils remain inserted into submembranous adhesion plaques is unknown.

Polygons and adhesion plaques and the disassembly and assembly of myofibrils in cardiac myocytes

PubMed Central

1989-01-01

Successive stages in the disassembly of myofibrils and the subsequent assembly of new myofibrils have been studied in cultures of dissociated chick cardiac myocytes. The myofibrils in trypsinized and dispersed myocytes are sequentially disassembled during the first 3 d of culture. They split longitudinally and then assemble into transitory polygons. Multiples of single sarcomeres, the cardiac polygons, are analogous to the transitory polygonal configurations assumed by stress fibers in spreading fibroblasts. They differ from their counterparts in fibroblasts in that they consist of muscle alpha-actinin vertices and muscle myosin heavy chain struts, rather than of the nonmuscle contractile protein isoforms of stress fiber polygons. EM sections reveal the vertices and struts in cardiac polygons to be typical Z and A bands. Most cardiac polygons are eliminated by day 5 of culture. Concurrent with the disassembly and elimination of the original myofibrils new myofibrils are rapidly assembled elsewhere in the same myocyte. Without exception both distal tips of each nascent myofibril terminate in adhesion plaques. The morphology and composition of the adhesion plaques capping each end of each myofibril are similar to those of the termini of stress fibers in fibroblasts. However, whereas the adhesion complexes involving stress fibers in fibroblasts consist of vinculin/nonmuscle alpha-actinin/beta- and gamma-actins, the analogous structures in myocytes involving myofibrils consist of vinculin/muscle alpha-actinin/alpha-actin. The addition of 1.7-2.0 microns sarcomeres to the distal tips of an elongating myofibril, irrespective of whether the myofibril consists of 1, 10, or several hundred tandem sarcomeres, occurs while the myofibril appears to remain linked to its respective adhesion plaques. The adhesion plaques in vitro are the equivalent of the in vivo intercalated discs, both in terms of their molecular composition and with respect to their functioning as initiating sites for the assembly of new sarcomeres. How 1.7-2.0 microns nascent sarcomeres can be added distally during elongation while the tips of the myofibrils remain inserted into submembranous adhesion plaques is unknown. PMID:2472405

Human U2 snRNA Genes Exhibit a Persistently Open Transcriptional State and Promoter Disassembly at Metaphase▿

PubMed Central

Pavelitz, Thomas; Bailey, Arnold D.; Elco, Christopher P.; Weiner, Alan M.

2008-01-01

In mammals, small multigene families generate spliceosomal U snRNAs that are nearly as abundant as rRNA. Using the tandemly repeated human U2 genes as a model, we show by footprinting with DNase I and permanganate that nearly all sequences between the enhancer-like distal sequence element and the initiation site are protected during interphase whereas the upstream half of the U2 snRNA coding region is exposed. We also show by chromatin immunoprecipitation that the SNAPc complex, which binds the TATA-like proximal sequence element, is removed at metaphase but remains bound under conditions that induce locus-specific metaphase fragility of the U2 genes, such as loss of CSB, BRCA1, or BRCA2 function, treatment with actinomycin D, or overexpression of the tetrameric p53 C terminus. We propose that the U2 snRNA promoter establishes a persistently open state to facilitate rapid reinitiation and perhaps also to bypass TFIIH-dependent promoter melting; this open state would then be disassembled to allow metaphase chromatin condensation. PMID:18378697

TRIP13 is a protein-remodeling AAA+ ATPase that catalyzes MAD2 conformation switching

DOE PAGES

Ye, Qiaozhen; Rosenberg, Scott C.; Moeller, Arne; ...

2015-04-28

The AAA+ family ATPase TRIP13 is a key regulator of meiotic recombination and the spindle assembly checkpoint, acting on signaling proteins of the conserved HORMA domain family. Here we present the structure of the Caenorhabditis elegans TRIP13 ortholog PCH-2, revealing a new family of AAA+ ATPase protein remodelers. PCH-2 possesses a substrate-recognition domain related to those of the protein remodelers NSF and p97, while its overall hexameric architecture and likely structural mechanism bear close similarities to the bacterial protein unfoldase ClpX. We find that TRIP13, aided by the adapter protein p31(comet), converts the HORMA-family spindle checkpoint protein MAD2 from amore » signaling-active ‘closed’ conformer to an inactive ‘open’ conformer. We propose that TRIP13 and p31(comet) collaborate to inactivate the spindle assembly checkpoint through MAD2 conformational conversion and disassembly of mitotic checkpoint complexes. A parallel HORMA protein disassembly activity likely underlies TRIP13's critical regulatory functions in meiotic chromosome structure and recombination.« less

BiP and Multiple DNAJ Molecular Chaperones in the Endoplasmic Reticulum Are Required for Efficient Simian Virus 40 Infection

PubMed Central

Goodwin, Edward C.; Lipovsky, Alex; Inoue, Takamasa; Magaldi, Thomas G.; Edwards, Anne P. B.; Van Goor, Kristin E. Y.; Paton, Adrienne W.; Paton, James C.; Atwood, Walter J.; Tsai, Billy; DiMaio, Daniel

2011-01-01

ABSTRACT Simian virus 40 (SV40) is a nonenveloped DNA virus that traffics through the endoplasmic reticulum (ER) en route to the nucleus, but the mechanisms of capsid disassembly and ER exit are poorly understood. We conducted an unbiased RNA interference screen to identify cellular genes required for SV40 infection. SV40 infection was specifically inhibited by up to 50-fold by knockdown of four different DNAJ molecular cochaperones or by inhibition of BiP, the Hsp70 partner of DNAJB11. These proteins were not required for the initiation of capsid disassembly, but knockdown markedly inhibited SV40 exit from the ER. In addition, BiP formed a complex with SV40 capsids in the ER in a DNAJB11-dependent fashion. These experiments identify five new cellular proteins required for SV40 infection and suggest that the binding of BiP to the capsid is required for ER exit. Further studies of these proteins will provide insight into the molecular mechanisms of polyomavirus infection and ER function. PMID:21673190

Disassembly properties and material characterisation of household small waste electric and electronic equipment.

PubMed

Bovea, María D; Pérez-Belis, Victoria; Ibáñez-Forés, Valeria; Quemades-Beltrán, Pilar

2016-07-01

This paper is focused on characterising small waste electric and electronic equipment, specifically small household appliances, from two different points of views: disassembly properties and material identification. The sample for this characterisation was obtained from a selective collection campaign organised in Castellón de la Plana (Spain). A total amount of 833.7kg (749 units) of small waste electric and electronic equipment was collected, of which 23.3% by weight and 22.4% by units belonged to the subcategory household equipment. This subcategory, composed of appliances such as vacuum cleaners, toasters, sandwich makers, hand blenders, juicers, coffee makers, hairdryers, scales, irons and heaters, was first disassembled in order to analyse different aspects of the disassembly process for each equipment type: type of joints, ease of identification of materials, ease of access to joints for extracting components, ease of separation of components from the whole, uniformity of tools needed for the disassembly process and possibility of reassembly after disassembly. Results show that the most common joints used in these equipment types are snap-fits and screws, although some permanent joints have also been identified. Next, the material composition of each component of each appliance belonging to each equipment type was identified visually and with additional mechanical trials and testing. It can be observed that plastic and electric/electronic components are present in all the equipment types analysed and are also the material fractions that appear with higher percentages in the material composition: 41.1wt% and 39.1wt% for the plastic fraction and electric/electronic components, respectively. The most common plastics are: polypropylene (PP), acrylonitrile butadiene styrene (ABS) and polycarbonate (PC), while the most common electric/electronic components are: cable, plug and printed circuit boards. Results also show that disassembly properties and material characterisation vary widely from one equipment type to another. Copyright © 2016 Elsevier Ltd. All rights reserved.

Nucleoside-2',3'/3',5'-bis(thio)phosphate antioxidants are also capable of disassembly of amyloid beta42-Zn(ii)/Cu(ii) aggregates via Zn(ii)/Cu(ii)-chelation.

PubMed

Hevroni, Bosmat Levi; Major, Dan Thomas; Dixit, Mudit; Mhashal, Anil Ranu; Das, Susanta; Fischer, Bilha

2016-05-18

Currently, there is an urgent need for biocompatible metal-ion chelators capable of antioxidant activity and disassembly of amyloid beta (Aβ)-aggregates as potential therapeutics for Alzheimer's disease (AD). We recently demonstrated the promising antioxidant activity of adenine/guanine 2',3' or 3',5'-bis(thio)phosphate analogues, 2'-dA/G3'5'PO/S and A2'3'PO/S, and their affinity to Zn(ii)-ions. These findings encouraged us to evaluate them as agents for the dissolution of Aβ42-Zn(ii)/Cu(ii) aggregates. Specifically, we explored their ability to bind Cu(ii)/Zn(ii)-ions, the geometry and stoichiometry of these complexes, Cu(ii)/Zn(ii)-binding-sites and binding mode, and the ability of these analogues to dissolve Aβ42-Zn(ii)/Cu(ii) aggregates, as well as their effect on the secondary structure of those aggregates. Finally, we identified the most promising agents for dissolution of Aβ42-Zn(ii)/Cu(ii) aggregates. Specifically, we observed the formation of a 1 : 1 complex between 2'-dG3'5'PO and Cu(ii), involving O4 ligands. Zn(ii) was coordinated by both thiophosphate groups of 2'-dA3'5'PS and A2'3'PS involving O2S2 ligands in a 1 : 1 stoichiometry. A2'3'PS dissolves Aβ42-Zn(ii) and Aβ42-Cu(ii) aggregates as effectively as, and 2.5-fold more effectively than EDTA, respectively. Furthermore, 2'-dG3'5'PS and A2'3'PS reverted the Aβ42-M(ii) structure, back to that of the free Aβ42. Finally, cryo-TEM and TEM images confirmed the disassembly of Aβ42 and Aβ42-M(ii) aggregates by A2'3'PS. Hence, 2'-dG3'5'PS and A2'3'PS may serve as promising scaffolds for new AD therapeutics, acting as both effective antioxidants and agents for solubilization of Aβ42-Cu(ii)/Zn(ii) aggregates.

Impact of physicochemical parameters on in vitro assembly and disassembly kinetics of recombinant triple-layered rotavirus-like particles.

PubMed

Mellado, Maria Candida M; Mena, Jimmy A; Lopes, António; Ramírez, Octavio T; Carrondo, Manuel J T; Palomares, Laura A; Alves, Paula M

2009-11-01

Virus-like particles constitute potentially relevant vaccine candidates. Nevertheless, their behavior in vitro and assembly process needs to be understood in order to improve their yield and quality. In this study we aimed at addressing these issues and for that purpose triple- and double-layered rotavirus-like particles (TLP 2/6/7 and DLP 2/6, respectively) size and zeta potential were measured using dynamic light scattering at different physicochemical conditions, namely pH, ionic strength, and temperature. Both TLP and DLP were stable within a pH range of 3-7 and at 5-25 degrees C. Aggregation occurred at 35-45 degrees C and their disassembly became evident at 65 degrees C. The isoelectric points of TLP and DLP were 3.0 and 3.8, respectively. In vitro kinetics of TLP disassembly was monitored. Ionic strength, temperature, and the chelating agent employed determined disassembly kinetics. Glycerol (10%) stabilized TLP by preventing its disassembly. Disassembled TLP was able to reassemble by dialysis at high calcium conditions. VP7 monomers were added to DLP in the presence of calcium to follow in vitro TLP assembly kinetics; its assembly rate being mostly affected by pH. Finally, DLP and TLP were found to coexist under certain conditions as determined from all reaction products analyzed by capillary electrophoresis. Overall, these results contribute to the design of new strategies for the improvement of TLP yield and quality by reducing the VP7 detachment from TLP. Copyright 2009 Wiley Periodicals, Inc.

12. INTERIOR VIEW TO THE SOUTH OF COOLING VATS AT ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

12. INTERIOR VIEW TO THE SOUTH OF COOLING VATS AT THE WEST END OF ROOM 102, THE DISASSEMBLY BAY. - Nevada Test Site, Pluto Facility, Disassembly Building, Area 26, Wahmonie Flats, Cane Spring Road, Mercury, Nye County, NV

Correlation between femoral offset loss and dynamic hip screw cut-out complications after pertrochanteric fractures: a case-control study.

PubMed

Boukebous, Baptiste; Guillon, Pascal; Vandenbussche, Eric; Rousseau, Marc Antoine

2018-04-27

Screw-plates disassembly incidence after pertrochanteric fracture (PF) amounts to 1 and 16% among the elderly population. The main occurrence is early cervical screw cut-out. The population at highest risk of disassembly remains difficult to identify. The correlation between femoral offset loss and disassembly occurrence has never been surveyed. A radiological prognosis score for screw plate disassembly was defined to reflect trochanteric impaction (TI); it was based on a femoral offset ratio. Our single-centre retrospective case-control study surveyed patients suffering from Dynamic Hip Screw (DHS, Synthes ® ) disassembly following osteosynthesis of non-pathological osteoporotic PF between 2004 and 2014. All cases were categorised by age and gender and paired to three patients in the control group. The primary endpoint was TI measurement, corresponding to offset loss on the operated hip compared to healthy hip offset and expressed as a percentage. The measurement was done on an immediate postoperative X-ray. The secondary endpoints were tip apex distance (TAD) measurement, Ender and AO classifications, as well as postoperative weight-bearing prescription. Twenty-three cases and 69 controls were surveyed. The case group's average age was 87; 70% of the cases were women. The main disassembly occurrence delay was after 27 days. Average TI was 26% within the patients global group and 12% within the control group (p < 10 -5 ). Over a 21% impaction percentage, disassembly occurrence represents a greater risk: OR = 21.95% CI [5.4-104.3], p < 10 -5 . Ender 3 type fractures were the most frequent indication for surgery within the case group. Average TAD was 20 mm within the case group, and 17 mm within the control group (p = 0.03). The weight-bearing prescription rate was 52% within the control group and 21% within the case group (p = 0.014). 14.5% of the control group had a TI > 21%. Using the offset ratio tool, TI measurement was associated with a greater risk of DHS disassembly when it was higher than 21%. The exclusive use of a DHS device does not seem optimal for a TI > 21%. Weight-bearing may be prescribed for all the patients with a TI < 21%, provided good implant positioning is secured.

α-SNAP Interferes with the Zippering of the SNARE Protein Membrane Fusion Machinery

PubMed Central

Park, Yongsoo; Vennekate, Wensi; Yavuz, Halenur; Preobraschenski, Julia; Hernandez, Javier M.; Riedel, Dietmar; Walla, Peter Jomo; Jahn, Reinhard

2014-01-01

Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Before fusion, SNARE proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase N-ethylmaleimide-sensitive factor that requires the cofactor α-SNAP to first bind to the assembled SNARE complex. Using chromaffin granules and liposomes we now show that α-SNAP on its own interferes with the zippering of membrane-anchored SNARE complexes midway through the zippering reaction, arresting SNAREs in a partially assembled trans-complex and preventing fusion. Intriguingly, the interference does not result in an inhibitory effect on synaptic vesicles, suggesting that membrane properties also influence the final outcome of α-SNAP interference with SNARE zippering. We suggest that binding of α-SNAP to the SNARE complex affects the ability of the SNARE complex to harness energy or transmit force to the membrane. PMID:24778182

Metrics and experimental data for assessing unbalanced disassembly lines.

DOT National Transportation Integrated Search

2011-01-01

Disassembly lines are inherently multi-criteria, with balance having the possibility of : being one of the lower priorities. This is due to the fact that other criteria for example, : removing valuable or hazardous materials early on in the proce...

Highly Selective Nuclide Removal from the R-Reactor Disassembly Basin at the SRS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pickett, J. B.; Austin, W. E.; Dukes, H. H.

This paper describes the results of a deployment of highly selective ion-exchange resin technologies for the in-situ removal of Cs-137 and Sr-90 from the Savannah River Site (SRS) R-Reactor Disassembly Basin. The deployment was supported by the DOE Office of Science and Technology's (OST, EM-50) National Engineering Technology Laboratory (NETL), as a part of an Accelerated Site Technology Deployment (ASTD) project. The Facilities Decontamination and Decommissioning (FDD) Program at the SRS conducted this deployment as a part of an overall program to deactivate three of the site's five reactor disassembly basins.

Highly Selective Nuclide Removal from the R-Reactor Disassembly Basin at SRS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pickett, J.B.

This paper describes the results of a deployment of highly selective ion-exchange resin technologies for the in-situ removal of Cs-137 and Sr-90 from the Savannah River Site (SRS) R-Reactor Disassembly Basin. The deployment was supported by the DOE Office of Science and Technology's (OST, EM-50) National Engineering Technology Laboratory (NETL), as a part of an Accelerated Site Technology Deployment (ASTD) project. The Facilities Decontamination and Decommissioning (FDD) Program at the SRS conducted this deployment as a part of an overall program to deactivate three of the site's five reactor disassembly basins

Deactivation of the P, C, and R Reactor Disassembly Basins at the SRS

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pickett, J.B.

The Facilities Disposition Division (FDD) at the Savannah River Site is engaged in planning the deactivation/closure of three of the site's five reactor disassembly basins. Activities are currently underway at 105-R Disassembly Basin and will continue with the 105-P and 105-C disassembly basins. The basins still contain the cooling and shielding water that was present when operations ceased. Low concentrations of radionuclides are present, with tritium, Cs-137, and Sr-90 being the major contributors. Although there is no evidence that any of the basins have leaked, the 50-year-old facilities will eventually contaminate the surrounding groundwaters. The FDD is pursuing a pro-activemore » solution to close the basins in-place and prevent a release to the groundwater. In-situ ion-exchange is currently underway at the R-Reactor Disassembly Basin to reduce the Cs and Sr concentrations to levels that would allow release of the treated water to previously used on-site cooling ponds. A NEPA Environm ental Assessment (EA) is being prepared to propose the preferred closure alternative for each of the three basins. The EA will be the primary mechanism to inform the public and gain stakeholder and regulatory approval.« less

Desmosome Assembly and Disassembly Are Membrane Raft-Dependent

PubMed Central

Faundez, Victor; Koval, Michael; Mattheyses, Alexa L.; Kowalczyk, Andrew P.

2014-01-01

Strong intercellular adhesion is critical for tissues that experience mechanical stress, such as the skin and heart. Desmosomes provide adhesive strength to tissues by anchoring desmosomal cadherins of neighboring cells to the intermediate filament cytoskeleton. Alterations in assembly and disassembly compromise desmosome function and may contribute to human diseases, such as the autoimmune skin blistering disease pemphigus vulgaris (PV). We previously demonstrated that PV auto-antibodies directed against the desmosomal cadherin desmoglein 3 (Dsg3) cause loss of adhesion by triggering membrane raft-mediated Dsg3 endocytosis. We hypothesized that raft membrane microdomains play a broader role in desmosome homeostasis by regulating the dynamics of desmosome assembly and disassembly. In human keratinocytes, Dsg3 is raft associated as determined by biochemical and super resolution immunofluorescence microscopy methods. Cholesterol depletion, which disrupts rafts, prevented desmosome assembly and adhesion, thus functionally linking rafts to desmosome formation. Interestingly, Dsg3 did not associate with rafts in cells lacking desmosomal proteins. Additionally, PV IgG-induced desmosome disassembly occurred by redistribution of Dsg3 into raft-containing endocytic membrane domains, resulting in cholesterol-dependent loss of adhesion. These findings demonstrate that membrane rafts are required for desmosome assembly and disassembly dynamics, suggesting therapeutic potential for raft targeting agents in desmosomal diseases such as PV. PMID:24498201

Multi-kanban mechanism for appliance disassembly

NASA Astrophysics Data System (ADS)

Udomsawat, Gun; Gupta, Surendra M.

2005-11-01

The use of household appliances continues to rise every year. A significant number of End-Of-Life (EOL) appliances are generated because of the introduction of newer models that are more attractive, efficient and affordable. Others are, of course, generated when they become non-functional. Many regulations encourage recycling of EOL appliances to reduce the amount of waste sent to landfills. In addition, EOL appliances offer the appliance manufacturing and remanufacturing industries a source of less expensive raw materials and components. For this reason product recovery has become a subject of interest during the past decade. In this paper, we study the disassembly line for appliance disassembly. We discuss and incorporate some of the complications that are inherent in disassembly line including product arrival, demand arrival, inventory fluctuation and production control mechanisms. We show how to overcome such complications by implementing a multi-kanban system in the appliance disassembly line setting. The multi-kanban system (MKS) relies on dynamic routing of kanbans according to the state of the system. We investigate the multi-kanban mechanism using simulation and explore the effect of product mix on performance of the traditional push system (TPS) and MKS in terms of controlling the system's inventory while attempting to achieve a decent customer service level.

Disassembling Iron Availability to Phytoplankton

PubMed Central

Shaked, Yeala; Lis, Hagar

2012-01-01

The bioavailability of iron to microorganisms and its underlying mechanisms have far reaching repercussions to many natural systems and diverse fields of research, including ocean biogeochemistry, carbon cycling and climate, harmful algal blooms, soil and plant research, bioremediation, pathogenesis, and medicine. Within the framework of ocean sciences, short supply and restricted bioavailability of Fe to phytoplankton is thought to limit primary production and curtail atmospheric CO2 drawdown in vast ocean regions. Yet a clear-cut definition of bioavailability remains elusive, with elements of iron speciation and kinetics, phytoplankton physiology, light, temperature, and microbial interactions, to name a few, all intricately intertwined into this concept. Here, in a synthesis of published and new data, we attempt to disassemble the complex concept of iron bioavailability to phytoplankton by individually exploring some of its facets. We distinguish between the fundamentals of bioavailability – the acquisition of Fe-substrate by phytoplankton – and added levels of complexity involving interactions among organisms, iron, and ecosystem processes. We first examine how phytoplankton acquire free and organically bound iron, drawing attention to the pervasiveness of the reductive uptake pathway in both prokaryotic and eukaryotic autotrophs. Turning to acquisition rates, we propose to view the availability of various Fe-substrates to phytoplankton as a spectrum rather than an absolute “all or nothing.” We then demonstrate the use of uptake rate constants to make comparisons across different studies, organisms, Fe-compounds, and environments, and for gaging the contribution of various Fe-substrates to phytoplankton growth in situ. Last, we describe the influence of aquatic microorganisms on iron chemistry and fate by way of organic complexation and bio-mediated redox transformations and examine the bioavailability of these bio-modified Fe species. PMID:22529839

The Chloroplast Division Protein ARC6 Acts to Inhibit Disassembly of GDP-bound FtsZ2.

PubMed

Sung, Min Woo; Shaik, Rahamthulla; TerBush, Allan D; Osteryoung, Katherine W; Vitha, Stanislav; Holzenburg, Andreas

2018-05-16

Chloroplasts host photosynthesis and fulfill other metabolic functions that are essential to plant life. They have to divide by binary fission to maintain their numbers throughout cycles of cell division. Chloroplast division is achieved by a complex ring-shaped division machinery located on both the inner (stromal) and the outer (cytosolic) side of the chloroplast envelope. The inner division ring (termed the Z ring) is formed by the assembly of tubulin-like FtsZ1 and FtsZ2 proteins. ARC6 is a key chloroplast division protein that interacts with the Z ring. ARC6 spans the inner envelope membrane, is known to stabilize or maintain the Z ring, and anchors the Z ring to the inner membrane through interaction with FtsZ2. The underlying mechanism of Z-ring stabilization is not well understood. Here, biochemical and structural characterization of ARC6 was conducted using light scattering, sedimentation, and light and transmission electron microscopy. The recombinant protein was purified as a dimer. The results indicated that a truncated form of ARC6 (tARC6), representing the stromal portion of ARC6, affects FtsZ2 assembly without forming higher-order structures, and exerts its effect via FtsZ2 dynamics. tARC6 prevented GDP-induced FtsZ2 disassembly and caused a significant net increase in FtsZ2 assembly when GDP was present. Single particle analysis and 3D reconstruction were performed to elucidate the structural basis of ARC6 activity. Together, the data reveal that a dimeric form of tARC6 binds to FtsZ2 filaments and does not increase FtsZ polymerization rates but rather inhibits GDP-associated FtsZ2 disassembly. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

A disassembly-free method for evaluation of spiral bevel gear assembly

NASA Astrophysics Data System (ADS)

Jedliński, Łukasz; Jonak, Józef

2017-05-01

The paper presents a novel method for evaluation of assembly of spiral bevel gears. The examination of the approaches to the problem of gear control diagnostics without disassembly has revealed that residual processes in the form of vibrations (or noise) are currently the most suitable to this end. According to the literature, contact pattern is a complex parameter for describing gear position. Therefore, the task is to determine the correlation between contact pattern and gear vibrations. Although the vibration signal contains a great deal of information, it also has a complex spectral structure and contains interferences. For this reason, the proposed method has three variants which determine the effect of preliminary processing of the signal on the results. In Variant 2, stage 1, the vibration signal is subjected to multichannel denoising using a wavelet transform (WT), and in Variant 3 - to a combination of WT and principal component analysis (PCA). This denoising procedure does not occur in Variant 1. Next, we determine the features of the vibration signal in order to focus on information which is crucial regarding the objective of the study. Given the lack of unequivocal premises enabling selection of optimum features, we calculate twenty features, rank them and finally select the appropriate ones using an algorithm. Diagnostic rules were created using artificial neural networks. We investigated the suitability of three network types: multilayer perceptron (MLP), radial basis function (RBF) and support vector machine (SVM).

Surfactant-free purification of membrane protein complexes from bacteria: application to the staphylococcal penicillin-binding protein complex PBP2/PBP2a

NASA Astrophysics Data System (ADS)

Paulin, Sarah; Jamshad, Mohammed; Dafforn, Timothy R.; Garcia-Lara, Jorge; Foster, Simon J.; Galley, Nicola F.; Roper, David I.; Rosado, Helena; Taylor, Peter W.

2014-07-01

Surfactant-mediated removal of proteins from biomembranes invariably results in partial or complete loss of function and disassembly of multi-protein complexes. We determined the capacity of styrene-co-maleic acid (SMA) co-polymer to remove components of the cell division machinery from the membrane of drug-resistant staphylococcal cells. SMA-lipid nanoparticles solubilized FtsZ-PBP2-PBP2a complexes from intact cells, demonstrating the close physical proximity of these proteins within the lipid bilayer. Exposure of bacteria to (-)-epicatechin gallate, a polyphenolic agent that abolishes β-lactam resistance in staphylococci, disrupted the association between PBP2 and PBP2a. Thus, SMA purification provides a means to remove native integral membrane protein assemblages with minimal physical disruption and shows promise as a tool for the interrogation of molecular aspects of bacterial membrane protein structure and function.

Multivalency of NDC80 in the outer kinetochore is essential to track shortening microtubules and generate forces

PubMed Central

2018-01-01

Presence of multiple copies of the microtubule-binding NDC80 complex is an evolutionary conserved feature of kinetochores, points of attachment of chromosomes to spindle microtubules. This may enable multivalent attachments to microtubules, with implications that remain unexplored. Using recombinant human kinetochore components, we show that while single NDC80 complexes do not track depolymerizing microtubules, reconstituted particles containing the NDC80 receptor CENP-T bound to three or more NDC80 complexes do so effectively, as expected for a kinetochore force coupler. To study multivalency systematically, we engineered modules allowing incremental addition of NDC80 complexes. The modules’ residence time on microtubules increased exponentially with the number of NDC80 complexes. Modules with two or more complexes tracked depolymerizing microtubules with increasing efficiencies, and stalled and rescued microtubule depolymerization in a force-dependent manner when conjugated to cargo. Our observations indicate that NDC80, rather than through biased diffusion, tracks depolymerizing microtubules by harnessing force generated during microtubule disassembly. PMID:29629870

Discrete Address Beacon System (DABS) Software System Reliability Modeling and Prediction.

DTIC Science & Technology

1981-06-01

Service ( ATARS ) module because of its interim status. Reliability prediction models for software modules were derived and then verified by matching...System (A’iCR3BS) and thus can be introduced gradually and economically without ma jor olper- ational or procedural change. Since DABS uses monopulse...lineanaly- sis tools or are ured during maintenance or pre-initialization were not modeled because they are not part of the mission software. The ATARS

Altered visual focus on sensorimotor control in people with chronic ankle instability.

PubMed

Terada, Masafumi; Ball, Lindsay M; Pietrosimone, Brian G; Gribble, Phillip A

2016-01-01

The purpose of this investigation was to examine the effects of the combination of chronic ankle instability (CAI) and altered visual focus on strategies for dynamic stability during a drop-jump task. Nineteen participants with self-reported CAI and 19 healthy participants performed a drop-jump task in looking-up and looking-down conditions. For the looking-up condition, participants looked up and read a random number that flashed on a computer monitor. For the looking-down condition, participants focused their vision on the force plate. Sagittal- and frontal-plane kinematics in the hip, knee and ankle were calculated at the time points of 100 ms pre-initial foot contact to ground and at IC. The resultant vector time to stabilisation was calculated with ground reaction force data. The CAI group demonstrated less hip flexion at the point of 100 ms pre-initial contact (P < 0.01), and less hip flexion (P = 0.03) and knee flexion at initial contact (P = 0.047) compared to controls. No differences in kinematics or dynamic stability were observed in either looking-up or looking-down conditions (P > 0.05). Altered visual focus did not influence movement patterns during the drop-jump task, but the presence of CAI did. The current data suggests that centrally mediated changes associated with CAI may lead to global alterations in the sensorimotor control.

Apparatus and methods for installing, removing and adjusting an inner turbine shell section relative to an outer turbine shell section

DOEpatents

Leach, David; Bergendahl, Peter Allen; Waldo, Stuart Forrest; Smith, Robert Leroy; Phelps, Robert Kim

2001-01-01

A turbine includes upper and lower inner shell sections mounting the nozzles and shrouds and which inner shell is supported by pins secured to a surrounding outer shell. To disassemble the turbine for access to the inner shell sections and rotor, an alignment fixture is secured to the lower outer shell section and has pins engaging the inner shell section. To disassemble the turbine, the inner shell weight is transferred to the lower outer shell section via the alignment fixture and cradle pins. Roller assemblies are inserted through access openings vacated by support pins to permit rotation of the lower inner shell section out of and into the lower outer shell section during disassembly and assembly. The alignment fixture includes adjusting rods for adjusting the inner shell axially, vertically, laterally and about a lateral axis. A roller over-cage is provided to rotate the inner shell and a dummy shell to facilitate assembly and disassembly in the field.

Regulatory assembly of the vacuolar proton pump VoV1-ATPase in yeast cells by FLIM-FRET

NASA Astrophysics Data System (ADS)

Ernst, Stefan; Batisse, Claire; Zarrabi, Nawid; Böttcher, Bettina; Börsch, Michael

2010-02-01

We investigate the reversible disassembly of VOV1-ATPase in life yeast cells by time resolved confocal FRET imaging. VOV1-ATPase in the vacuolar membrane pumps protons from the cytosol into the vacuole. VOV1-ATPase is a rotary biological nanomotor driven by ATP hydrolysis. The emerging proton gradient is used for secondary transport processes as well as for pH and Ca2+ homoeostasis in the cell. The activity of the VOV1-ATPase is regulated through assembly / disassembly processes. During starvation the two parts of VOV1-ATPase start to disassemble. This process is reversed after addition of glucose. The exact mechanisms are unknown. To follow the disassembly / reassembly in vivo we tagged two subunits C and E with different fluorescent proteins. Cellular distributions of C and E were monitored using a duty cycle-optimized alternating laser excitation scheme (DCO-ALEX) for time resolved confocal FRET-FLIM measurements.

Myosin-dependent remodeling of adherens junctions protects junctions from Snail-dependent disassembly

PubMed Central

Weng, Mo

2016-01-01

Although Snail is essential for disassembly of adherens junctions during epithelial–mesenchymal transitions (EMTs), loss of adherens junctions in Drosophila melanogaster gastrula is delayed until mesoderm is internalized, despite the early expression of Snail in that primordium. By combining live imaging and quantitative image analysis, we track the behavior of E-cadherin–rich junction clusters, demonstrating that in the early stages of gastrulation most subapical clusters in mesoderm not only persist, but move apically and enhance in density and total intensity. All three phenomena depend on myosin II and are temporally correlated with the pulses of actomyosin accumulation that drive initial cell shape changes during gastrulation. When contractile myosin is absent, the normal Snail expression in mesoderm, or ectopic Snail expression in ectoderm, is sufficient to drive early disassembly of junctions. In both cases, junctional disassembly can be blocked by simultaneous induction of myosin contractility. Our findings provide in vivo evidence for mechanosensitivity of cell–cell junctions and imply that myosin-mediated tension can prevent Snail-driven EMT. PMID:26754645

Foraging traits modulate stingless bee community disassembly under forest loss.

PubMed

Lichtenberg, Elinor M; Mendenhall, Chase D; Brosi, Berry

2017-10-01

Anthropogenic land use change is an important driver of impacts to biological communities and the ecosystem services they provide. Pollination is one ecosystem service that may be threatened by community disassembly. Relatively little is known about changes in bee community composition in the tropics, where pollination limitation is most severe and land use change is rapid. Understanding how anthropogenic changes alter community composition and functioning has been hampered by high variability in responses of individual species. Trait-based approaches, however, are emerging as a potential method for understanding responses of ecologically similar species to global change. We studied how communities of tropical, eusocial stingless bees (Apidae: Meliponini) disassemble when forest is lost. These bees are vital tropical pollinators that exhibit high trait diversity, but are under considerable threat from human activities. We compared functional traits of stingless bee species found in pastures surrounded by differing amounts of forest in an extensively deforested landscape in southern Costa Rica. Our results suggest that foraging traits modulate competitive interactions that underlie community disassembly patterns. In contrast to both theoretical predictions and temperate bee communities, we found that stingless bee species with the widest diet breadths were less likely to persist in sites with less forest. These wide-diet-breadth species also tend to be solitary foragers, and are competitively subordinate to group-foraging stingless bee species. Thus, displacement by dominant, group-foraging species may make subordinate species more dependent on the larger or more diversified resource pool that natural habitats offer. We also found that traits that may reduce reliance on trees-nesting in the ground or inside nests of other species-correlated with persistence in highly deforested landscapes. The functional trait perspective we employed enabled capturing community processes in analyses and suggests that land use change may disassemble bee communities via different mechanisms in temperate and tropical areas. Our results further suggest that community processes, such as competition, can be important regulators of community disassembly under land use change. A better understanding of community disassembly processes is critical for conserving and restoring pollinator communities and the ecosystem services and functions they provide. © 2017 The Authors. Journal of Animal Ecology © 2017 British Ecological Society.

LDB1-mediated enhancer looping can be established independent of mediator and cohesin.

PubMed

Krivega, Ivan; Dean, Ann

2017-08-21

Mechanistic studies in erythroid cells indicate that LDB1, as part of a GATA1/TAL1/LMO2 complex, brings erythroid-expressed genes into proximity with enhancers for transcription activation. The role of co-activators in establishing this long-range interaction is poorly understood. Here we tested the contributions of the RNA Pol II pre-initiation complex (PIC), mediator and cohesin to establishment of locus control region (LCR)/β-globin proximity. CRISPR/Cas9 editing of the β-globin promoter to eliminate the RNA Pol II PIC by deleting the TATA-box resulted in loss of transcription, but enhancer-promoter interaction was unaffected. Additional deletion of the promoter GATA1 site eliminated LDB1 complex and mediator occupancy and resulted in loss of LCR/β-globin proximity. To separate the roles of LDB1 and mediator in LCR looping, we expressed a looping-competent but transcription-activation deficient form of LDB1 in LDB1 knock down cells: LCR/β-globin proximity was restored without mediator core occupancy. Further, Cas9-directed tethering of mutant LDB1 to the β-globin promoter forced LCR loop formation in the absence of mediator or cohesin occupancy. Moreover, ENCODE data and our chromatin immunoprecipitation results indicate that cohesin is almost completely absent from validated and predicted LDB1-regulated erythroid enhancer-gene pairs. Thus, lineage specific factors largely mediate enhancer-promoter looping in erythroid cells independent of mediator and cohesin. Published by Oxford University Press on behalf of Nucleic Acids Research 2017.

Distinct mutations in yeast TAF(II)25 differentially affect the composition of TFIID and SAGA complexes as well as global gene expression patterns.

PubMed

Kirschner, Doris B; vom Baur, Elmar; Thibault, Christelle; Sanders, Steven L; Gangloff, Yann-Gaël; Davidson, Irwin; Weil, P Anthony; Tora, Làszlò

2002-05-01

The RNA polymerase II transcription factor TFIID, composed of the TATA-binding protein (TBP) and TBP-associated factors (TAF(II)s), nucleates preinitiation complex formation at protein-coding gene promoters. SAGA, a second TAF(II)-containing multiprotein complex, is involved in transcription regulation in Saccharomyces cerevisiae. One of the essential protein components common to SAGA and TFIID is yTAF(II)25. We define a minimal evolutionarily conserved 91-amino-acid region of TAF(II)25 containing a histone fold domain that is necessary and sufficient for growth in vivo. Different temperature-sensitive mutations of yTAF(II)25 or chimeras with the human homologue TAF(II)30 arrested cell growth at either the G(1) or G(2)/M cell cycle phase and displayed distinct phenotypic changes and gene expression patterns. Immunoprecipitation studies revealed that TAF(II)25 mutation-dependent gene expression and phenotypic changes correlated at least partially with the integrity of SAGA and TFIID. Genome-wide expression analysis revealed that the five TAF(II)25 temperature-sensitive mutant alleles individually affect the expression of between 18 and 33% of genes, whereas taken together they affect 64% of all class II genes. Thus, different yTAF(II)25 mutations induce distinct phenotypes and affect the regulation of different subsets of genes, demonstrating that no individual TAF(II) mutant allele reflects the full range of its normal functions.

Structural dissection of an interaction between transcription initiation and termination factors implicated in promoter-terminator cross-talk.

PubMed

Bratkowski, Matthew; Unarta, Ilona Christy; Zhu, Lizhe; Shubbar, Murtada; Huang, Xuhui; Liu, Xin

2018-02-02

Functional cross-talk between the promoter and terminator of a gene has long been noted. Promoters and terminators are juxtaposed to form gene loops in several organisms, and gene looping is thought to be involved in transcriptional regulation. The general transcription factor IIB (TFIIB) and the C-terminal domain phosphatase Ssu72, essential factors of the transcription preinitiation complex and the mRNA processing and polyadenylation complex, respectively, are important for gene loop formation. TFIIB and Ssu72 interact both genetically and physically, but the molecular basis of this interaction is not known. Here we present a crystal structure of the core domain of TFIIB in two new conformations that differ in the relative distance and orientation of the two cyclin-like domains. The observed extraordinary conformational plasticity may underlie the binding of TFIIB to multiple transcription factors and promoter DNAs that occurs in distinct stages of transcription, including initiation, reinitiation, and gene looping. We mapped the binding interface of the TFIIB-Ssu72 complex using a series of systematic, structure-guided in vitro binding and site-specific photocross-linking assays. Our results indicate that Ssu72 competes with acidic activators for TFIIB binding and that Ssu72 disrupts an intramolecular TFIIB complex known to impede transcription initiation. We also show that the TFIIB-binding site on Ssu72 overlaps with the binding site of symplekin, a component of the mRNA processing and polyadenylation complex. We propose a hand-off model in which Ssu72 mediates a conformational transition in TFIIB, accounting for the role of Ssu72 in transcription reinitiation, gene looping, and promoter-terminator cross-talk. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

TFIID TAF6-TAF9 Complex Formation Involves the HEAT Repeat-containing C-terminal Domain of TAF6 and Is Modulated by TAF5 Protein*

PubMed Central

Scheer, Elisabeth; Delbac, Frédéric; Tora, Laszlo; Moras, Dino; Romier, Christophe

2012-01-01

The general transcription factor TFIID recognizes specifically the core promoter of genes transcribed by eukaryotic RNA polymerase II, nucleating the assembly of the preinitiation complex at the transcription start site. However, the understanding in molecular terms of TFIID assembly and function remains poorly understood. Histone fold motifs have been shown to be extremely important for the heterodimerization of many TFIID subunits. However, these subunits display several evolutionary conserved noncanonical features when compared with histones, including additional regions whose role is unknown. Here we show that the conserved additional C-terminal region of TFIID subunit TAF6 can be divided into two domains: a small middle domain (TAF6M) and a large C-terminal domain (TAF6C). Our crystal structure of the TAF6C domain from Antonospora locustae at 1.9 Å resolution reveals the presence of five conserved HEAT repeats. Based on these data, we designed several mutants that were introduced into full-length human TAF6. Surprisingly, the mutants affect the interaction between TAF6 and TAF9, suggesting that the formation of the complex between these two TFIID subunits do not only depend on their histone fold motifs. In addition, the same mutants affect even more strongly the interaction between TAF6 and TAF9 in the context of a TAF5-TAF6-TAF9 complex. Expression of these mutants in HeLa cells reveals that most of them are unstable, suggesting their poor incorporation within endogenous TFIID. Taken together, our results suggest that the conserved additional domains in histone fold-containing subunits of TFIID and of co-activator SAGA are important for the assembly of these complexes. PMID:22696218

Capillarity-induced disassembly of virions in carbon nanotubes

NASA Astrophysics Data System (ADS)

Fan, Xiaobin; Barclay, J. Elaine; Peng, Wenchao; Li, Yang; Li, Xianyu; Zhang, Guoliang; Evans, David J.; Zhang, Fengbao

2008-04-01

Studying the transport and fate of viruses through nanochannels is of great importance. By using the nanochannel of a carbon nanotube (CNT) as an ideal model, we evaluated the possibility of capillarity-induced viral transport through a closely fitting nanochannel and explored the mechanisms involved. It is shown both experimentally and theoretically that Cowpea mosaic virus can enter CNTs by capillarity. However, when introduced into a nanotube the protein capsid may disassemble. During the initial capillary filling stage, anomalous needle-shaped high pressure exists in the centre of the nanotube's entrance. This high pressure, combining with the significant negative pressure within the nanotube, may account for the disassembly of the virions.

Genomic localization of the human gene encoding Dr1, a negative modulator of transcription of class II and class III genes.

PubMed

Purrello, M; Di Pietro, C; Rapisarda, A; Viola, A; Corsaro, C; Motta, S; Grzeschik, K H; Sichel, G

1996-01-01

Dr1 is a nuclear protein of 19 kDa that exists in the nucleoplasm as a homotetramer. By binding to TBP (the DNA-binding subunit of TFIID, and also a subunit of SL1 and TFIIIB), the protein blocks class II and class III preinitiation complex assembly, thus repressing the activity of the corresponding promoters. Since transcription of class I genes is unaffected by Dr1. it has been proposed that the protein may coordinate the expression of class I, class II and class III genes. By somatic cell genetics and fluorescence in situ hybridization, we have localized the gene (DR1), present in the genome of higher eukaryotes as a single copy, to human chromosome region 1p21-->p13. The nucleotide sequence conservation of the coding segment of the gene, as determined by Noah's ark blot analysis, and its ubiquitous transcription suggest that Dr1 has an important biological role, which could be related to the negative control of cell proliferation.

Essential role of the TFIID subunit TAF4 in murine embryogenesis and embryonic stem cell differentiation

PubMed Central

Langer, Diana; Martianov, Igor; Alpern, Daniel; Rhinn, Muriel; Keime, Céline; Dollé, Pascal; Mengus, Gabrielle; Davidson, Irwin

2016-01-01

TAF4 (TATA-binding protein-associated factor 4) and its paralogue TAF4b are components of the TFIID core module. We inactivated the murine Taf4a gene to address Taf4 function during embryogenesis. Here we show that Taf4a−/− embryos survive until E9.5 where primary germ layers and many embryonic structures are identified showing Taf4 is dispensable for their specification. In contrast, Taf4 is required for correct patterning of the trunk and anterior structures, ventral morphogenesis and proper heart positioning. Overlapping expression of Taf4a and Taf4b during embryogenesis suggests their redundancy at early stages. In agreement with this, Taf4a−/− embryonic stem cells (ESCs) are viable and comprise Taf4b-containing TFIID. Nevertheless, Taf4a−/− ESCs do not complete differentiation into glutamatergic neurons and cardiomyocytes in vitro due to impaired preinitiation complex formation at the promoters of critical differentiation genes. We define an essential role of a core TFIID TAF in differentiation events during mammalian embryogenesis. PMID:27026076

Essential role of the TFIID subunit TAF4 in murine embryogenesis and embryonic stem cell differentiation.

PubMed

Langer, Diana; Martianov, Igor; Alpern, Daniel; Rhinn, Muriel; Keime, Céline; Dollé, Pascal; Mengus, Gabrielle; Davidson, Irwin

2016-03-30

TAF4 (TATA-binding protein-associated factor 4) and its paralogue TAF4b are components of the TFIID core module. We inactivated the murine Taf4a gene to address Taf4 function during embryogenesis. Here we show that Taf4a(-/-) embryos survive until E9.5 where primary germ layers and many embryonic structures are identified showing Taf4 is dispensable for their specification. In contrast, Taf4 is required for correct patterning of the trunk and anterior structures, ventral morphogenesis and proper heart positioning. Overlapping expression of Taf4a and Taf4b during embryogenesis suggests their redundancy at early stages. In agreement with this, Taf4a(-/-) embryonic stem cells (ESCs) are viable and comprise Taf4b-containing TFIID. Nevertheless, Taf4a(-/-) ESCs do not complete differentiation into glutamatergic neurons and cardiomyocytes in vitro due to impaired preinitiation complex formation at the promoters of critical differentiation genes. We define an essential role of a core TFIID TAF in differentiation events during mammalian embryogenesis.

The histone chaperone TAF-I/SET/INHAT is required for transcription in vitro of chromatin templates.

PubMed

Gamble, Matthew J; Erdjument-Bromage, Hediye; Tempst, Paul; Freedman, Leonard P; Fisher, Robert P

2005-01-01

To uncover factors required for transcription by RNA polymerase II on chromatin, we fractionated a mammalian cell nuclear extract. We identified the histone chaperone TAF-I (also known as INHAT [inhibitor of histone acetyltransferase]), which was previously proposed to repress transcription, as a potent activator of chromatin transcription responsive to the vitamin D3 receptor or to Gal4-VP16. TAF-I associates with chromatin in vitro and can substitute for the related protein NAP-1 in assembling chromatin onto cloned DNA templates in cooperation with the remodeling enzyme ATP-dependent chromatin assembly factor (ACF). The chromatin assembly and transcriptional activation functions are distinct, however, and can be dissociated temporally. Efficient transcription of chromatin assembled with TAF-I still requires the presence of TAF-I during the polymerization reaction. Conversely, TAF-I cannot stimulate transcript elongation when added after the other factors necessary for assembly of a preinitiation complex on naked DNA. Thus, TAF-I is required to facilitate transcription at a step after chromatin assembly but before transcript elongation.

Global transcriptional repression in C. elegans germline precursors by regulated sequestration of TAF-4.

PubMed

Guven-Ozkan, Tugba; Nishi, Yuichi; Robertson, Scott M; Lin, Rueyling

2008-10-03

In C. elegans, four asymmetric divisions, beginning with the zygote (P0), generate transcriptionally repressed germline blastomeres (P1-P4) and somatic sisters that become transcriptionally active. The protein PIE-1 represses transcription in the later germline blastomeres but not in the earlier germline blastomeres P0 and P1. We show here that OMA-1 and OMA-2, previously shown to regulate oocyte maturation, repress transcription in P0 and P1 by binding to and sequestering in the cytoplasm TAF-4, a component critical for assembly of TFIID and the pol II preinitiation complex. OMA-1/2 binding to TAF-4 is developmentally regulated, requiring phosphorylation by the DYRK kinase MBK-2, which is activated at meiosis II after fertilization. OMA-1/2 are normally degraded after the first mitosis, but ectopic expression of wild-type OMA-1 is sufficient to repress transcription in both somatic and later germline blastomeres. We propose that phosphorylation by MBK-2 serves as a developmental switch, converting OMA-1/2 from oocyte to embryo regulators.

Global transcriptional repression in C. elegans germline precursors by regulated sequestration of TFIID component TAF-4

PubMed Central

Guven-Ozkan, Tugba; Nishi, Yuichi; Robertson, Scott M.; Lin, Rueyling

2008-01-01

In C. elegans, four asymmetric divisions, beginning with the zygote (P0), generate transcriptionally repressed germline blastomeres (P1–P4) and somatic sisters that become transcriptionally active. The protein PIE-1 represses transcription in the later germline blastomeres, but not in the earlier germline blastomeres P0 and P1. We show here that OMA-1 and OMA-2, previously shown to regulate oocyte maturation, repress transcription in P0 and P1 by binding to and sequestering in the cytoplasm TAF-4, a component critical for assembly of TFIID and the pol II preinitiation complex. OMA-1/2 binding to TAF-4 is developmentally regulated, requiring phosphorylation by the DYRK kinase MBK-2, which is activated at meiosis II following fertilization. OMA-1/2 are normally degraded after the first mitosis, but ectopic expression of wildtype OMA-1 is sufficient to repress transcription in both somatic and later germline blastomeres. We propose that phosphorylation by MBK-2 serves as a developmental switch, converting OMA-1/2 from oocyte to embryo regulators. PMID:18854162

Inhibition of ribosome recruitment induces stress granule formation independently of eukaryotic initiation factor 2alpha phosphorylation.

PubMed

Mazroui, Rachid; Sukarieh, Rami; Bordeleau, Marie-Eve; Kaufman, Randal J; Northcote, Peter; Tanaka, Junichi; Gallouzi, Imed; Pelletier, Jerry

2006-10-01

Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2alpha phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2alpha phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2alpha to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2alpha phosphorylation-dependent and -independent pathways that target translation initiation.

Inhibition of Ribosome Recruitment Induces Stress Granule Formation Independently of Eukaryotic Initiation Factor 2α Phosphorylation

PubMed Central

Mazroui, Rachid; Sukarieh, Rami; Bordeleau, Marie-Eve; Kaufman, Randal J.; Northcote, Peter; Tanaka, Junichi; Gallouzi, Imed

2006-01-01

Cytoplasmic aggregates known as stress granules (SGs) arise as a consequence of cellular stress and contain stalled translation preinitiation complexes. These foci are thought to serve as sites of mRNA storage or triage during the cell stress response. SG formation has been shown to require induction of eukaryotic initiation factor (eIF)2α phosphorylation. Herein, we investigate the potential role of other initiation factors in this process and demonstrate that interfering with eIF4A activity, an RNA helicase required for the ribosome recruitment phase of translation initiation, induces SG formation and that this event is not dependent on eIF2α phosphorylation. We also show that inhibition of eIF4A activity does not impair the ability of eIF2α to be phosphorylated under stress conditions. Furthermore, we observed SG assembly upon inhibition of cap-dependent translation after poliovirus infection. We propose that SG modeling can occur via both eIF2α phosphorylation-dependent and -independent pathways that target translation initiation. PMID:16870703

Assembly via disassembly: A case in machine perceptual development

NASA Technical Reports Server (NTRS)

Bajcsy, Ruzena K.; Tsikos, Constantine J.

1989-01-01

First results in the effort of learning about representations of objects is presented. The questions attempted to be answered are: What is innate and what must be derived from the environment. The problem is casted in the framework of disassembly of an object into two parts.

pH-Sensitive Reversible Programmed Targeting Strategy by the Self-Assembly/Disassembly of Gold Nanoparticles.

PubMed

Ma, Jinlong; Hu, Zhenpeng; Wang, Wei; Wang, Xinyu; Wu, Qiang; Yuan, Zhi

2017-05-24

A reversible programmed targeting strategy could achieve high tumor accumulation due to its long blood circulation time and high cellular internalization. Here, targeting ligand-modified poly(ethylene glycol) (PEG-ligand), dibutylamines (Bu), and pyrrolidinamines (Py) were introduced on the surface of gold nanoparticles (Au NPs) for reversible shielding/deshielding of the targeting ligands by pH-responsive self-assembly. Hydrophobic interaction and steric repulsion are the main driving forces for the self-assembly/disassembly of Au NPs. The precise self-assembly (pH ≥ 7.2) and disassembly (pH ≤ 6.8) of Au NPs with different ligands could be achieved by fine-tuning the modifying molar ratio of Bu and Py (R m ), which followed the formula R m = 1/(-0.0013X 2 + 0.0323X + 1), in which X is the logarithm of the partition coefficient of the targeting ligand. The assembled/disassembled behavior of Au NPs at pH 7.2 and 6.8 was confirmed by transmission electron microscopy and dynamic light scattering. Enzyme-linked immunosorbent assays and cellular uptake studies showed that the ligands could be buried inside the assembly and exposed when disassembled. More importantly, this process was reversible, which provides the possibility of prolonging blood circulation by shielding ligands associated with the NPs that were effused from tumor tissue.

Photodissociation of Non-Covalent Peptide-Crown Ether Complexes

PubMed Central

Wilson, Jeffrey J.; Kirkovits, Gregory J.; Sessler, Jonathan L.; Brodbelt, Jennifer S.

2008-01-01

Highly chromogenic 18-crown-6-dipyrrolylquinoxaline coordinates primary amines of peptides, forming non-covalent complexes that can be transferred to the gas phase by electrospray ionization. The appended chromogenic crown ether facilitates efficient energy transfer to the peptide upon ultraviolet irradiation in the gas phase, resulting in diagnostic peptide fragmentation. Collisional activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) of these non-covalent complexes results only in their disassembly with the charge retained on either the peptide or crown ether, yielding no sequence ions. Upon UV photon absorption the intermolecular energy transfer is facilitated by the fast activation time scale of UVPD (< 10 ns) and by the collectively strong hydrogen bonding between the crown ether and peptide, thus allowing effective transfer of energy to the peptide moiety prior to disruption of the intermolecular hydrogen bonds. PMID:18077179

Nano-functionalized filamentous fungus hyphae with fast reversible macroscopic assembly & disassembly features.

PubMed

Wang, Haiying; Li, Xiaorui; Chai, Liyuan; Zhang, Liyuan

2015-05-18

A uniform decoration of hyphae by polyaniline nanoparticles (PANI NPs) was achieved here. This novel hybrid structure can be effectively assembled into a film by filtration and disassembled in water by shaking. This reversible process is very fast, which promises applications in nanomaterials including adsorption.

ARC: A compact, high-field, disassemblable fusion nuclear science facility and demonstration power plant

NASA Astrophysics Data System (ADS)

Sorbom, Brandon; Ball, Justin; Palmer, Timothy; Mangiarotti, Franco; Sierchio, Jennifer; Bonoli, Paul; Kasten, Cale; Sutherland, Derek; Barnard, Harold; Haakonsen, Christian; Goh, Jon; Sung, Choongki; Whyte, Dennis

2014-10-01

The Affordable, Robust, Compact (ARC) reactor conceptual design aims to reduce the size, cost, and complexity of a combined Fusion Nuclear Science Facility (FNSF) and demonstration fusion pilot power plant. ARC is a 270 MWe tokamak reactor with a major radius of 3.3 m, a minor radius of 1.1 m, and an on-axis magnetic field of 9.2 T. ARC has Rare Earth Barium Copper Oxide (REBCO) superconducting toroidal field coils with joints to allow disassembly, allowing for removal and replacement of the vacuum vessel as a single component. Inboard-launched current drive of 25 MW LHRF power and 13.6 MW ICRF power is used to provide a robust, steady state core plasma far from disruptive limits. ARC uses an all-liquid blanket, consisting of low pressure, slowly flowing Fluorine Lithium Beryllium (FLiBe) molten salt. The liquid blanket acts as a working fluid, coolant, and tritium breeder, and minimizes the solid material that can become activated. The large temperature range over which FLiBe is liquid permits blanket operation at 800-900 K with single phase fluid cooling and allows use of a high-efficiency Brayton cycle for electricity production in the secondary coolant loop.

The yeast actin cytoskeleton.

PubMed

Mishra, Mithilesh; Huang, Junqi; Balasubramanian, Mohan K

2014-03-01

The actin cytoskeleton is a complex network of dynamic polymers, which plays an important role in various fundamental cellular processes, including maintenance of cell shape, polarity, cell division, cell migration, endocytosis, vesicular trafficking, and mechanosensation. Precise spatiotemporal assembly and disassembly of actin structures is regulated by the coordinated activity of about 100 highly conserved accessory proteins, which nucleate, elongate, cross-link, and sever actin filaments. Both in vivo studies in a wide range of organisms from yeast to metazoans and in vitro studies of purified proteins have helped shape the current understanding of actin dynamics and function. Molecular genetics, genome-wide functional analysis, sophisticated real-time imaging, and ultrastructural studies in concert with biochemical analysis have made yeast an attractive model to understand the actin cytoskeleton, its molecular dynamics, and physiological function. Studies of the yeast actin cytoskeleton have contributed substantially in defining the universal mechanism regulating actin assembly and disassembly in eukaryotes. Here, we review some of the important insights generated by the study of actin cytoskeleton in two important yeast models the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

The NSL Complex Regulates Housekeeping Genes in Drosophila

PubMed Central

Raja, Sunil Jayaramaiah; Holz, Herbert; Luscombe, Nicholas M.; Manke, Thomas; Akhtar, Asifa

2012-01-01

MOF is the major histone H4 lysine 16-specific (H4K16) acetyltransferase in mammals and Drosophila. In flies, it is involved in the regulation of X-chromosomal and autosomal genes as part of the MSL and the NSL complexes, respectively. While the function of the MSL complex as a dosage compensation regulator is fairly well understood, the role of the NSL complex in gene regulation is still poorly characterized. Here we report a comprehensive ChIP–seq analysis of four NSL complex members (NSL1, NSL3, MBD-R2, and MCRS2) throughout the Drosophila melanogaster genome. Strikingly, the majority (85.5%) of NSL-bound genes are constitutively expressed across different cell types. We find that an increased abundance of the histone modifications H4K16ac, H3K4me2, H3K4me3, and H3K9ac in gene promoter regions is characteristic of NSL-targeted genes. Furthermore, we show that these genes have a well-defined nucleosome free region and broad transcription initiation patterns. Finally, by performing ChIP–seq analyses of RNA polymerase II (Pol II) in NSL1- and NSL3-depleted cells, we demonstrate that both NSL proteins are required for efficient recruitment of Pol II to NSL target gene promoters. The observed Pol II reduction coincides with compromised binding of TBP and TFIIB to target promoters, indicating that the NSL complex is required for optimal recruitment of the pre-initiation complex on target genes. Moreover, genes that undergo the most dramatic loss of Pol II upon NSL knockdowns tend to be enriched in DNA Replication–related Element (DRE). Taken together, our findings show that the MOF-containing NSL complex acts as a major regulator of housekeeping genes in flies by modulating initiation of Pol II transcription. PMID:22723752

Mto2 multisite phosphorylation inactivates non-spindle microtubule nucleation complexes during mitosis

PubMed Central

Borek, Weronika E.; Groocock, Lynda M.; Samejima, Itaru; Zou, Juan; de Lima Alves, Flavia; Rappsilber, Juri; Sawin, Kenneth E.

2015-01-01

Microtubule nucleation is highly regulated during the eukaryotic cell cycle, but the underlying molecular mechanisms are largely unknown. During mitosis in fission yeast Schizosaccharomyces pombe, cytoplasmic microtubule nucleation ceases simultaneously with intranuclear mitotic spindle assembly. Cytoplasmic nucleation depends on the Mto1/2 complex, which binds and activates the γ-tubulin complex and also recruits the γ-tubulin complex to both centrosomal (spindle pole body) and non-centrosomal sites. Here we show that the Mto1/2 complex disassembles during mitosis, coincident with hyperphosphorylation of Mto2 protein. By mapping and mutating multiple Mto2 phosphorylation sites, we generate mto2-phosphomutant strains with enhanced Mto1/2 complex stability, interaction with the γ-tubulin complex and microtubule nucleation activity. A mutant with 24 phosphorylation sites mutated to alanine, mto2[24A], retains interphase-like behaviour even in mitotic cells. This provides a molecular-level understanding of how phosphorylation ‘switches off' microtubule nucleation complexes during the cell cycle and, more broadly, illuminates mechanisms regulating non-centrosomal microtubule nucleation. PMID:26243668

View looking down to the Oscar S. Straus Memorial Fountain. ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

View looking down to the Oscar S. Straus Memorial Fountain. The monument was authorized by Congress in 1927 and dedicated in 1947. It consists of the fountain and two groups of statues, Religious Freedom and Reason. It was disassembled in 1991 and reinstalled after the construction of the Ronald Reagan Building and the International Trade Center. The rededication took place in 1998 and the fountain is located near the west entrance of the building complex. - Ronald Reagan Building and International Trade Center, 1300 Pennsylvania Avenue, NW, Washington, District of Columbia, DC

Elongation Factor Ts Directly Facilitates the Formation and Disassembly of the Escherichia coli Elongation Factor Tu·GTP·Aminoacyl-tRNA Ternary Complex*

PubMed Central

Burnett, Benjamin J.; Altman, Roger B.; Ferrao, Ryan; Alejo, Jose L.; Kaur, Navdep; Kanji, Joshua; Blanchard, Scott C.

2013-01-01

Aminoacyl-tRNA enters the translating ribosome in a ternary complex with elongation factor Tu (EF-Tu) and GTP. Here, we describe bulk steady state and pre-steady state fluorescence methods that enabled us to quantitatively explore the kinetic features of Escherichia coli ternary complex formation and decay. The data obtained suggest that both processes are controlled by a nucleotide-dependent, rate-determining conformational change in EF-Tu. Unexpectedly, we found that this conformational change is accelerated by elongation factor Ts (EF-Ts), the guanosine nucleotide exchange factor for EF-Tu. Notably, EF-Ts attenuates the affinity of EF-Tu for GTP and destabilizes ternary complex in the presence of non-hydrolyzable GTP analogs. These results suggest that EF-Ts serves an unanticipated role in the cell of actively regulating the abundance and stability of ternary complex in a manner that contributes to rapid and faithful protein synthesis. PMID:23539628

Role of the Escherichia coli grpE heat shock protein in the initiation of bacteriophage lambda DNA replication.

PubMed

Osipiuk, J; Zylicz, M

1991-01-01

Initiation of replication of lambda DNA requires assembly of the proper nucleoprotein complex consisting of the lambda origin of replication-lambda O-lambda P-dnaB proteins. The dnaJ, dnaK and grpE heat shock proteins destabilize the lambda P-dnaB interaction in this complex permitting dnaB helicase to unwind lambda DNA near ori lambda sequence. First step of this disassembling reaction is the binding of dnaK protein to lambda P protein. In this report we examined the influence of dnaJ and grpE proteins on stability of the lambda P-dnaK complex. Our results show that grpE alone dissociates this complex, but both grpE and dnaJ together do not. These results suggest that, in the presence of grpE protein, dnaK protein has a higher affinity for lambda P protein complexed with dnaJ protein than in the situation where grpE protein is not used.

Dissipative particle dynamics simulation on the self-assembly and disassembly of pH-sensitive polymeric micelle with coating repair agent

NASA Astrophysics Data System (ADS)

Wang, Xiumin; Gao, Jianbang; Wang, Zhikun; Xu, Jianchang; Li, Chunling; Sun, Shuangqing; Hu, Songqing

2017-10-01

Dissipative particle dynamics (DPD) simulations were applied to investigate the coating repair agent dicyclopentadience (DCPD) in pH-sensitive micelles. The results show micelles self-assembled from triblock copolymers with strong hydrophobic interaction are not conducive to loading DCPD, and only micelles with weak interaction parameter can encapsulate DCPD well. After protonation, the structure of micelle was disassembled and DCPD beads have a stronger ability to shrink polymer chains and exposed to water. This work provides mesoscopic insight into self-assembly and disassembly of desired agent-loaded micelle, and might be useful for the design of new materials for agent delivery.

THE MAN&RSQUO;S JACKET DESIGN FOR DISASSEMBLY: AN IMPLEMENTATION OF C2CAD FRAMEWORK

EPA Science Inventory

The C2CAD model served as the basis in the man’s jacket design and production. In man’s jackets, both natural and synthetic materials are commonly used for fabrics, threads, and buttons. To promote disassembly and value retention, we minimized material diversity an...

Molecular disassembly of rice and lotus starches during thermal processing and its effect on starch digestibility.

PubMed

Wang, Shujun; Sun, Yue; Wang, Jinrong; Wang, Shuo; Copeland, Les

2016-02-01

The molecular disassembly of starch during thermal processing is a major determinant for the susceptibility of starch to enzymatic digestion. In the present study, the effects of thermal processing on the disassembly of the granular structure and the in vitro enzymatic digestibility of rice and lotus starches were investigated. After heating at 50 °C, rice and lotus starches did not show significant changes in granular morphology, long-range crystallinity and short-range molecular order. As the temperature increased to 60 °C, rice starch underwent a partial gelatinization followed by an incomplete disruption of granular morphology, crystallites and molecular order. In contrast, lotus starch was almost completely gelatinized at 60 °C. At 70 °C or higher, both starches were fully gelatinized with complete disruption of the micro and macro structures. Our results show that gelatinization greatly increased the in vitro enzymatic digestibility of both starches, but that the degree of disassembly of the starch structure during thermal processing was not a major determinant of the digestibility of gelatinized starch.

Microtubule dissassembly in vivo: intercalary destabilization and breakdown of microtubules in the heliozoan Actinocoryne contractilis

PubMed Central

1992-01-01

In the marine heliozoan Actinocoryne contractilis, uninterrupted rods of microtubules stiffen the axopodia and the stalk. Stimulation in sea water elicits an extremely fast contraction (millisecond range) accompanied by almost complete Mt dissociation. Using high-speed cinematography and light transmittance measurements, we have studied the process of Mt disassembly in real time. In sea water, Mt disassembly follows an exponential decrease (mean half time of 4 ms) or proceeds by short steps. Cell contraction and Mt disassembly have been inhibited or slowed down through the use of artificial media. Although kinetics are slower (mean half time of 3 s), the curves of the length change against time look similar. The rapid as well as the slower process are accompanied by the formation of breakpoints on the stalk, from which disassembly proceeds. In specimens fixed during the slowed contraction, the presence across the Mt rods, of a single or multiple destabilization band that may consist of granular material and polymorphic forms of tubulin supports the hypothesis of "intercalary destabilization and breakdown" of axonemal Mts. PMID:1639845

α-SNAP interferes with the zippering of the SNARE protein membrane fusion machinery.

PubMed

Park, Yongsoo; Vennekate, Wensi; Yavuz, Halenur; Preobraschenski, Julia; Hernandez, Javier M; Riedel, Dietmar; Walla, Peter Jomo; Jahn, Reinhard

2014-06-06

Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Before fusion, SNARE proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase N-ethylmaleimide-sensitive factor that requires the cofactor α-SNAP to first bind to the assembled SNARE complex. Using chromaffin granules and liposomes we now show that α-SNAP on its own interferes with the zippering of membrane-anchored SNARE complexes midway through the zippering reaction, arresting SNAREs in a partially assembled trans-complex and preventing fusion. Intriguingly, the interference does not result in an inhibitory effect on synaptic vesicles, suggesting that membrane properties also influence the final outcome of α-SNAP interference with SNARE zippering. We suggest that binding of α-SNAP to the SNARE complex affects the ability of the SNARE complex to harness energy or transmit force to the membrane. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Disassembly and physical separation of electric/electronic components layered in printed circuit boards (PCB).

PubMed

Lee, Jaeryeong; Kim, Youngjin; Lee, Jae-chun

2012-11-30

Although printed circuit boards (PCBs) contain various elements, only the major elements (i.e., those with content levels in wt% or over grade) of and precious metals (e.g., Ag, Au, and platinum groups) contained within PCBs can be recycled. To recover other elements from PCBs, the PCBs should be properly disassembled as the first step of the recycling process. The recovery of these other elements would be beneficial for efforts to conserve scarce resources, reuse electric/electronic components (EECs), and eliminate environmental problems. This paper examines the disassembly of EECs from wasted PCBs (WPCBs) and the physical separation of these EECs using a self-designed disassembling apparatus and a 3-step separation process of sieving, magnetic separation, and dense medium separation. The disassembling efficiencies were evaluated by using the ratio of grinding area (E(area)) and the weight ratio of the detached EECs (E(weight)). In the disassembly treatment, these efficiencies were improved with an increase of grinder speed and grinder height. 97.7% (E(area)) and 98% (E(weight)) could be accomplished ultimately by 3 repetitive treatments at a grinder speed of 5500 rpm and a grinder height of 1.5mm. Through a series of physical separations, most groups of the EECs (except for the diode, transistor, and IC chip groups) could be sorted at a relatively high separation efficiency of about 75% or more. To evaluate the separation efficiency with regard to the elemental composition, the distribution ratio (R(dis)) and the concentration ratio (R(conc)) were used. 15 elements could be separated with the highest R(dis) and R(conc) in the same separated division. This result implies that the recyclability of the elements is highly feasible, even though the initial content in EECs is lower than several tens of mg/kg. Copyright © 2012 Elsevier B.V. All rights reserved.

The unique self-assembly/disassembly property of Archaeoglobus fulgidus ferritin and its implications on molecular release from the protein cage.

PubMed

Sana, Barindra; Johnson, Eric; Lim, Sierin

2015-12-01

In conventional in vitro encapsulation of molecular cargo, the multi-subunit ferritin protein cages are disassembled in extremely acidic pH and re-assembled in the presence of highly concentrated cargo materials, which results in poor yields due to the low-pH treatment. In contrast, Archaeoglobus fulgidus open-pore ferritin (AfFtn) and its closed-pore mutant (AfFtn-AA) are present as dimeric species in neutral buffers that self-assemble into cage-like structure upon addition of metal ions. To understand the iron-mediated self-assembly and ascorbate-mediated disassembly properties, we studied the iron binding and release profile of the AfFtn and AfFtn-AA, and the corresponding oligomerization of their subunits. Fe(2+) binding and conversion to Fe(3+) triggered the self-assembly of cage-like structures from dimeric species of AfFtn and AfFtn-AA subunits, while disassembly was induced by dissolving the iron core with reducing agents. The closed-pore AfFtn-AA has identical iron binding kinetics but lower iron release rates when compared to AfFtn. While the iron binding rate is proportional to Fe(2+) concentration, the iron release rate can be controlled by varying ascorbate concentrations. The AfFtn and AfFtn-AA cages formed by iron mineralization could be disassembled by dissolving the iron core. The open-pores of AfFtn contribute to enhanced reductive iron release while the small channels located at the 3-fold symmetry axis (3-fold channels) are used for iron uptake. The iron-mediated self-assembly/disassembly property of AfFtn offers a new set of molecular trigger for formation and dissociation of the protein cage, which can potentially regulate uptake and release of molecular cargo from protein cages. Copyright © 2015 Elsevier B.V. All rights reserved.

Multi-kanban mechanism for personal computer disassembly

NASA Astrophysics Data System (ADS)

Udomsawat, Gun; Gupta, Surendra M.; Kamarthi, Sagar V.

2004-12-01

The use of personal computers (PCs) continues to increase every year. According to a 1999 figure, 50 percent of all US households owned PCs, a figure that continues to rise every year. With continuous development of sophisticated software, PCs are becoming increasingly powerful. In addition, the price of a PC continues to steadily decline. Furthermore, the typical life of a PC in the workplace is approximately two to three years while in the home it is three to five years. As these PCs become obsolete, they are replaced and the old PCs are disposed of. It is estimated that between 14 and 20 million PCs are retired annually in the US. While 20 to 30% of the units may be resold, the others are discarded. These discards represent a significant potential source of lead for the waste stream. In some communities, waste cathode ray tubes (CRTs) represent the second largest source of lead in the waste stream after vehicular lead acid batteries. PCs are, therefore, not suitable for dumping in landfills. Besides, several components of a PC can be reused and then there are other valuable materials that can also be harvested. And with the advent of product stewardship, product recovery is the best solution for manufacturers. Disassembly line is perhaps the most suitable set up for disassembling PCs. However, planning and scheduling of disassembly on a disassembly line is complicated. In this paper, we discuss some of the complications including product arrival, demand arrival, inventory fluctuation and production control mechanisms. We then show how to overcome them by implementing a multi-kanban mechanism in the PC disassembly line setting. The multi-kanban mechanism relies on dynamic routing of kanbans according to the state of the system. We investigate the multi-kanban mechanism using simulation and demonstrate that this mechanism is superior to the traditional push system in terms of controlling the system"s inventory while maintaining a decent customer service level.

SCF(FBXW7α) modulates the intra-S-phase DNA-damage checkpoint by regulating Polo like kinase-1 stability.

PubMed

Giráldez, Servando; Herrero-Ruiz, Joaquín; Mora-Santos, Mar; Japón, Miguel Á; Tortolero, Maria; Romero, Francisco

2014-06-30

The intra-S-checkpoint is essential to control cell progression through S phase under normal conditions and in response to replication stress. When DNA lesions are detected, replication fork progression is blocked allowing time for repair to avoid genomic instability and the risk of cancer. DNA replication initiates at many origins of replication in eukaryotic cells, where a series of proteins form pre-replicative complexes (pre-RCs) that are activated to become pre-initiation complexes and ensure a single round of replication in each cell cycle. PLK1 plays an important role in the regulation of DNA replication, contributing to the regulation of pre-RCs formation by phosphorylating several proteins, under both normal and stress conditions. Here we report that PLK1 is ubiquitinated and degraded by SCFFBXW7α/proteasome. Moreover, we identified a new Cdc4 phosphodegron in PLK1, conserved from yeast to humans, whose mutation prevents PLK1 destruction. We established that endogenous SCFFBXW7α degrades PLK1 in the G1 and S phases of an unperturbed cell cycle and in S phase following UV irradiation. Furthermore, we showed that FBXW7α overexpression or UV irradiation prevented the loading of proteins onto chromatin to form pre-RCs and, accordingly, reduced cell proliferation. We conclude that PLK1 degradation mediated by SCFFBXW7α modulates the intra-S-phase checkpoint.

SCFFBXW7α modulates the intra-S-phase DNA-damage checkpoint by regulating Polo like kinase-1 stability

PubMed Central

Giráldez, Servando; Herrero-Ruiz, Joaquín; Mora-Santos, Mar; Japón, Miguel Á.; Tortolero, Maria; Romero, Francisco

2014-01-01

The intra-S-checkpoint is essential to control cell progression through S phase under normal conditions and in response to replication stress. When DNA lesions are detected, replication fork progression is blocked allowing time for repair to avoid genomic instability and the risk of cancer. DNA replication initiates at many origins of replication in eukaryotic cells, where a series of proteins form pre-replicative complexes (pre-RCs) that are activated to become pre-initiation complexes and ensure a single round of replication in each cell cycle. PLK1 plays an important role in the regulation of DNA replication, contributing to the regulation of pre-RCs formation by phosphorylating several proteins, under both normal and stress conditions. Here we report that PLK1 is ubiquitinated and degraded by SCFFBXW7α/proteasome. Moreover, we identified a new Cdc4 phosphodegron in PLK1, conserved from yeast to humans, whose mutation prevents PLK1 destruction. We established that endogenous SCFFBXW7α degrades PLK1 in the G1 and S phases of an unperturbed cell cycle and in S phase following UV irradiation. Furthermore, we showed that FBXW7α overexpression or UV irradiation prevented the loading of proteins onto chromatin to form pre-RCs and, accordingly, reduced cell proliferation. We conclude that PLK1 degradation mediated by SCFFBXW7α modulates the intra-S-phase checkpoint. PMID:24970797

PIC Activation through Functional Interplay between Mediator and TFIIH.

PubMed

Malik, Sohail; Molina, Henrik; Xue, Zhu

2017-01-06

The multiprotein Mediator coactivator complex functions in large part by controlling the formation and function of the promoter-bound preinitiation complex (PIC), which consists of RNA polymerase II and general transcription factors. However, precisely how Mediator impacts the PIC, especially post-recruitment, has remained unclear. Here, we have studied Mediator effects on basal transcription in an in vitro transcription system reconstituted from purified components. Our results reveal a close functional interplay between Mediator and TFIIH in the early stages of PIC development. We find that under conditions when TFIIH is not normally required for transcription, Mediator actually represses transcription. TFIIH, whose recruitment to the PIC is known to be facilitated by the Mediator, then acts to relieve Mediator-induced repression to generate an active form of the PIC. Gel mobility shift analyses of PICs and characterization of TFIIH preparations carrying mutant XPB translocase subunit further indicate that this relief of repression is achieved through expending energy via ATP hydrolysis, suggesting that it is coupled to TFIIH's established promoter melting activity. Our interpretation of these results is that Mediator functions as an assembly factor that facilitates PIC maturation through its various stages. Whereas the overall effect of the Mediator is to stimulate basal transcription, its initial engagement with the PIC generates a transcriptionally inert PIC intermediate, which necessitates energy expenditure to complete the process. Copyright © 2016 Elsevier Ltd. All rights reserved.

Suppression of Mediator is regulated by Cdk8-dependent Grr1 turnover of the Med3 coactivator.

PubMed

Gonzalez, Deyarina; Hamidi, Nurul; Del Sol, Ricardo; Benschop, Joris J; Nancy, Thomas; Li, Chao; Francis, Lewis; Tzouros, Manuel; Krijgsveld, Jeroen; Holstege, Frank C P; Conlan, R Steven

2014-02-18

Mediator, an evolutionary conserved large multisubunit protein complex with a central role in regulating RNA polymerase II-transcribed genes, serves as a molecular switchboard at the interface between DNA binding transcription factors and the general transcription machinery. Mediator subunits include the Cdk8 module, which has both positive and negative effects on activator-dependent transcription through the activity of the cyclin-dependent kinase Cdk8, and the tail module, which is required for positive and negative regulation of transcription, correct preinitiation complex formation in basal and activated transcription, and Mediator recruitment. Currently, the molecular mechanisms governing Mediator function remain largely undefined. Here we demonstrate an autoregulatory mechanism used by Mediator to repress transcription through the activity of distinct components of different modules. We show that the function of the tail module component Med3, which is required for transcription activation, is suppressed by the kinase activity of the Cdk8 module. Med3 interacts with, and is phosphorylated by, Cdk8; site-specific phosphorylation triggers interaction with and degradation by the Grr1 ubiquitin ligase, thereby preventing transcription activation. This active repression mechanism involving Grr1-dependent ubiquitination of Med3 offers a rationale for the substoichiometric levels of the tail module that are found in purified Mediator and the corresponding increase in tail components seen in cdk8 mutants.

Evidence that Hsc70 Is Associated with Cucumber Necrosis Virus Particles and Plays a Role in Particle Disassembly

PubMed Central

Alam, Syed Benazir

2016-01-01

ABSTRACT Uncoating of a virus particle to expose its nucleic acid is a critical aspect of the viral multiplication cycle, as it is essential for the establishment of infection. In the present study, we investigated the role of plant HSP70 homologs in the uncoating process of Cucumber necrosis virus (CNV), a nonenveloped positive-sense single-stranded RNA [(+)ssRNA] virus having a T=3 icosahedral capsid. We have found through Western blot analysis and mass spectrometry that the HSP70 homolog Hsc70-2 copurifies with CNV particles. Virus overlay and immunogold labeling assays suggest that Hsc70-2 is physically bound to virions. Furthermore, trypsin digestion profiles suggest that the bound Hsc70-2 is partially protected by the virus, indicating an intimate association with particles. In investigating a possible role of Hsc70-2 in particle disassembly, we showed that particles incubated with Hsp70/Hsc70 antibody produce fewer local lesions than those incubated with prebleed control antibody on Chenopodium quinoa. In conjunction, CNV virions purified using CsCl and having undetectable amounts of Hsc70-2 produce fewer local lesions. We also have found that plants with elevated levels of HSP70/Hsc70 produce higher numbers of local lesions following CNV inoculation. Finally, incubation of recombinant Nicotiana benthamiana Hsc70-2 with virus particles in vitro leads to conformational changes or partial disassembly of capsids as determined by transmission electron microscopy, and particles are more sensitive to chymotrypsin digestion. This is the first report suggesting that a cellular Hsc70 chaperone is involved in disassembly of a plant virus. IMPORTANCE Virus particles must disassemble and release their nucleic acid in order to establish infection in a cell. Despite the importance of disassembly in the ability of a virus to infect its host, little is known about this process, especially in the case of nonenveloped spherical RNA viruses. Previous work has shown that host HSP70 homologs play multiple roles in the CNV infection cycle. We therefore examined the potential role of these cellular components in the CNV disassembly process. We show that the HSP70 family member Hsc70-2 is physically associated with CNV virions and that HSP70 antibody reduces the ability of CNV to establish infection. Statistically significantly fewer lesions are produced when virions having undetectable HSc70-2 are used as an inoculum. Finally incubation of Hsc70-2 with CNV particles results in conformational changes in particles. Taken together, our data point to an important role of the host factor Hsc70-2 in CNV disassembly. PMID:27807229

Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends

PubMed Central

Volkov, Vladimir A.; Zaytsev, Anatoly V.; Grishchuk, Ekaterina L.

2014-01-01

Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are thought to play a vital role in the microtubule-dependent chromosome motions during cell division, but the relevant proteins and their exact roles are ill-defined. Thus, there is a growing need to develop assays with which to study such motility in vitro using purified components and defined biochemical milieu. Microtubules, however, are inherently unstable polymers; their switching between growth and shortening is stochastic and difficult to control. The protocols we describe here take advantage of the segmented microtubules that are made with the photoablatable stabilizing caps. Depolymerization of such segmented microtubules can be triggered with high temporal and spatial resolution, thereby assisting studies of motility at the disassembling microtubule ends. This technique can be used to carry out a quantitative analysis of the number of molecules in the fluorescently-labeled protein complexes, which move processively with dynamic microtubule ends. To optimize a signal-to-noise ratio in this and other quantitative fluorescent assays, coverslips should be treated to reduce nonspecific absorption of soluble fluorescently-labeled proteins. Detailed protocols are provided to take into account the unevenness of fluorescent illumination, and determine the intensity of a single fluorophore using equidistant Gaussian fit. Finally, we describe the use of segmented microtubules to study microtubule-dependent motions of the protein-coated microbeads, providing insights into the ability of different motor and nonmotor proteins to couple microtubule depolymerization to processive cargo motion. PMID:24686554

One-step fabrication of multifunctional micromotors

NASA Astrophysics Data System (ADS)

Gao, Wenlong; Liu, Mei; Liu, Limei; Zhang, Hui; Dong, Bin; Li, Christopher Y.

2015-08-01

Although artificial micromotors have undergone tremendous progress in recent years, their fabrication normally requires complex steps or expensive equipment. In this paper, we report a facile one-step method based on an emulsion solvent evaporation process to fabricate multifunctional micromotors. By simultaneously incorporating various components into an oil-in-water droplet, upon emulsification and solidification, a sphere-shaped, asymmetric, and multifunctional micromotor is formed. Some of the attractive functions of this model micromotor include autonomous movement in high ionic strength solution, remote control, enzymatic disassembly and sustained release. This one-step, versatile fabrication method can be easily scaled up and therefore may have great potential in mass production of multifunctional micromotors for a wide range of practical applications.Although artificial micromotors have undergone tremendous progress in recent years, their fabrication normally requires complex steps or expensive equipment. In this paper, we report a facile one-step method based on an emulsion solvent evaporation process to fabricate multifunctional micromotors. By simultaneously incorporating various components into an oil-in-water droplet, upon emulsification and solidification, a sphere-shaped, asymmetric, and multifunctional micromotor is formed. Some of the attractive functions of this model micromotor include autonomous movement in high ionic strength solution, remote control, enzymatic disassembly and sustained release. This one-step, versatile fabrication method can be easily scaled up and therefore may have great potential in mass production of multifunctional micromotors for a wide range of practical applications. Electronic supplementary information (ESI) available: Videos S1-S4 and Fig. S1-S3. See DOI: 10.1039/c5nr03574k

Teaching Assembly for Disassembly; An Under-Graduate Module Experience

ERIC Educational Resources Information Center

Alexandri, Eleftheria

2014-01-01

This paper is about the experience of teaching Assembly for Disassembly to fourth year architect students within the module of sustainable design. When designing a sustainable building one should take into consideration the fact that the building is going to be demolished in some years; thus the materials should be assembled in such a way so that…

Easily disassembled electrical connector for high voltage, high frequency connections

DOEpatents

Milner, Joseph R.

1994-01-01

An easily accessible electrical connector capable of rapid assembly and disassembly wherein a wide metal conductor sheet may be evenly contacted over the entire width of the conductor sheet by opposing surfaces on the connector which provide an even clamping pressure against opposite surfaces of the metal conductor sheet using a single threaded actuating screw.

A Summary Report on the NPH Evaluation of 105-L Disassembly Basin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Joshi, J.R.

2002-04-30

The L Area Disassembly Basin (LDB) is evaluated for the natural phenomena hazards (NPH) effects due to earthquake, wind, and tornado in accordance with DOE Order 420.1 and DOE-STD-1020. The deterministic analysis is performed for a Performance Category 3 (PC3) level of loads. Savannah River Site (SRS) specific NPH loads and design criteria are obtained from Engineering Standard 01060. It is demonstrated that the demand to capacity (D/C) ratios for primary and significant structural elements are acceptable (equal to or less than 1.0). Thus, 105-L Disassembly Basin building structure is qualified for the PC3 NPH effects in accordance with DOEmore » Order 420.1.« less

Phosphorylation-mediated RNA/peptide complex coacervation as a model for intracellular liquid organelles

NASA Astrophysics Data System (ADS)

Aumiller, William M.; Keating, Christine D.

2016-02-01

Biological cells are highly organized, with numerous subcellular compartments. Phosphorylation has been hypothesized as a means to control the assembly/disassembly of liquid-like RNA- and protein-rich intracellular bodies, or liquid organelles, that lack delimiting membranes. Here, we demonstrate that charge-mediated phase separation, or complex coacervation, of RNAs with cationic peptides can generate simple model liquid organelles capable of reversibly compartmentalizing biomolecules. Formation and dissolution of these liquid bodies was controlled by changes in peptide phosphorylation state using a kinase/phosphatase enzyme pair. The droplet-generating phase transition responded to modification of even a single serine residue. Electrostatic interactions between the short cationic peptides and the much longer polyanionic RNAs drove phase separation. Coacervates were also formed on silica beads, a primitive model for localization at specific intracellular sites. This work supports phosphoregulation of complex coacervation as a viable mechanism for dynamic intracellular compartmentalization in membraneless organelles.

Membranes linked by trans-SNARE complexes require lipids prone to non-bilayer structure for progression to fusion.

PubMed

Zick, Michael; Stroupe, Christopher; Orr, Amy; Douville, Deborah; Wickner, William T

2014-01-01

Like other intracellular fusion events, the homotypic fusion of yeast vacuoles requires a Rab GTPase, a large Rab effector complex, SNARE proteins which can form a 4-helical bundle, and the SNARE disassembly chaperones Sec17p and Sec18p. In addition to these proteins, specific vacuole lipids are required for efficient fusion in vivo and with the purified organelle. Reconstitution of vacuole fusion with all purified components reveals that high SNARE levels can mask the requirement for a complex mixture of vacuole lipids. At lower, more physiological SNARE levels, neutral lipids with small headgroups that tend to form non-bilayer structures (phosphatidylethanolamine, diacylglycerol, and ergosterol) are essential. Membranes without these three lipids can dock and complete trans-SNARE pairing but cannot rearrange their lipids for fusion. DOI: http://dx.doi.org/10.7554/eLife.01879.001.

The nuclear retention signal of HPV16 L2 protein is essential for incoming viral genome to transverse the trans-Golgi network

PubMed Central

DiGiuseppe, Stephen; Bienkowska-Haba, Malgorzata; Hilbig, Lydia; Sapp, Martin

2014-01-01

The Human papillomavirus (HPV) capsid is composed of the major and minor capsid proteins, L1 and L2, respectively. Infectious entry requires a complex series of conformational changes in both proteins that lead to uptake and allow uncoating to occur. During entry, the capsid is disassembled and host cyclophilins dissociate L1 protein from the L2/DNA complex. Herein, we describe a mutant HPV16 L2 protein (HPV16 L2-R302/5A) that traffics pseudogenome to the trans-Golgi network (TGN) but fails to egress. Our data provide further evidence that HPV16 traffics through the TGN and demonstrates that L2 is essential for TGN egress. Furthermore, we show that cyclophilin activity is required for the L2/DNA complex to be transported to the TGN which is accompanied by a reduced L1 protein levels. PMID:24928042

Conformational changes in the AAA ATPase p97–p47 adaptor complex

PubMed Central

Beuron, Fabienne; Dreveny, Ingrid; Yuan, Xuemei; Pye, Valerie E; Mckeown, Ciaran; Briggs, Louise C; Cliff, Matthew J; Kaneko, Yayoi; Wallis, Russell; Isaacson, Rivka L; Ladbury, John E; Matthews, Steve J; Kondo, Hisao; Zhang, Xiaodong; Freemont, Paul S

2006-01-01

The AAA+ATPase p97/VCP, helped by adaptor proteins, exerts its essential role in cellular events such as endoplasmic reticulum-associated protein degradation or the reassembly of Golgi, ER and the nuclear envelope after mitosis. Here, we report the three-dimensional cryo-electron microscopy structures at ∼20 Å resolution in two nucleotide states of the endogenous hexameric p97 in complex with a recombinant p47 trimer, one of the major p97 adaptor proteins involved in membrane fusion. Depending on the nucleotide state, we observe the p47 trimer to be in two distinct arrangements on top of the p97 hexamer. By combining the EM data with NMR and other biophysical measurements, we propose a model of ATP-dependent p97(N) domain motions that lead to a rearrangement of p47 domains, which could result in the disassembly of target protein complexes. PMID:16601695

Hsc70 chaperone activity is required for the cytosolic slow axonal transport of synapsin

PubMed Central

Ganguly, Archan; Han, Xuemei; Das, Utpal; Caillol, Ghislaine

2017-01-01

Soluble cytosolic proteins vital to axonal and presynaptic function are synthesized in the neuronal soma and conveyed via slow axonal transport. Our previous studies suggest that the overall slow transport of synapsin is mediated by dynamic assembly/disassembly of cargo complexes followed by short-range vectorial transit (the “dynamic recruitment” model). However, neither the composition of these complexes nor the mechanistic basis for the dynamic behavior is understood. In this study, we first examined putative cargo complexes associated with synapsin using coimmunoprecipitation and multidimensional protein identification technology mass spectrometry (MS). MS data indicate that synapsin is part of a multiprotein complex enriched in chaperones/cochaperones including Hsc70. Axonal synapsin–Hsc70 coclusters are also visualized by two-color superresolution microscopy. Inhibition of Hsc70 ATPase activity blocked the slow transport of synapsin, disrupted axonal synapsin organization, and attenuated Hsc70–synapsin associations, advocating a model where Hsc70 activity dynamically clusters cytosolic proteins into cargo complexes, allowing transport. Collectively, our study offers insight into the molecular organization of cytosolic transport complexes and identifies a novel regulator of slow transport. PMID:28559423

A novel TBP-TAF complex on RNA polymerase II-transcribed snRNA genes.

PubMed

Zaborowska, Justyna; Taylor, Alice; Roeder, Robert G; Murphy, Shona

2012-01-01

Initiation of transcription of most human genes transcribed by RNA polymerase II (RNAP II) requires the formation of a preinitiation complex comprising TFIIA, B, D, E, F, H and RNAP II. The general transcription factor TFIID is composed of the TATA-binding protein and up to 13 TBP-associated factors. During transcription of snRNA genes, RNAP II does not appear to make the transition to long-range productive elongation, as happens during transcription of protein-coding genes. In addition, recognition of the snRNA gene-type specific 3' box RNA processing element requires initiation from an snRNA gene promoter. These characteristics may, at least in part, be driven by factors recruited to the promoter. For example, differences in the complement of TAFs might result in differential recruitment of elongation and RNA processing factors. As precedent, it already has been shown that the promoters of some protein-coding genes do not recruit all the TAFs found in TFIID. Although TAF5 has been shown to be associated with RNAP II-transcribed snRNA genes, the full complement of TAFs associated with these genes has remained unclear. Here we show, using a ChIP and siRNA-mediated approach, that the TBP/TAF complex on snRNA genes differs from that found on protein-coding genes. Interestingly, the largest TAF, TAF1, and the core TAFs, TAF10 and TAF4, are not detected on snRNA genes. We propose that this snRNA gene-specific TAF subset plays a key role in gene type-specific control of expression.

Parvoviruses Cause Nuclear Envelope Breakdown by Activating Key Enzymes of Mitosis

PubMed Central

Porwal, Manvi; Cohen, Sarah; Snoussi, Kenza; Popa-Wagner, Ruth; Anderson, Fenja; Dugot-Senant, Nathalie; Wodrich, Harald; Dinsart, Christiane; Kleinschmidt, Jürgen A.; Panté, Nelly; Kann, Michael

2013-01-01

Disassembly of the nuclear lamina is essential in mitosis and apoptosis requiring multiple coordinated enzymatic activities in nucleus and cytoplasm. Activation and coordination of the different activities is poorly understood and moreover complicated as some factors translocate between cytoplasm and nucleus in preparatory phases. Here we used the ability of parvoviruses to induce nuclear membrane breakdown to understand the triggers of key mitotic enzymes. Nuclear envelope disintegration was shown upon infection, microinjection but also upon their application to permeabilized cells. The latter technique also showed that nuclear envelope disintegration was independent upon soluble cytoplasmic factors. Using time-lapse microscopy, we observed that nuclear disassembly exhibited mitosis-like kinetics and occurred suddenly, implying a catastrophic event irrespective of cell- or type of parvovirus used. Analyzing the order of the processes allowed us to propose a model starting with direct binding of parvoviruses to distinct proteins of the nuclear pore causing structural rearrangement of the parvoviruses. The resulting exposure of domains comprising amphipathic helices was required for nuclear envelope disintegration, which comprised disruption of inner and outer nuclear membrane as shown by electron microscopy. Consistent with Ca++ efflux from the lumen between inner and outer nuclear membrane we found that Ca++ was essential for nuclear disassembly by activating PKC. PKC activation then triggered activation of cdk-2, which became further activated by caspase-3. Collectively our study shows a unique interaction of a virus with the nuclear envelope, provides evidence that a nuclear pool of executing enzymes is sufficient for nuclear disassembly in quiescent cells, and demonstrates that nuclear disassembly can be uncoupled from initial phases of mitosis. PMID:24204256

Easily disassembled electrical connector for high voltage, high frequency connections

DOEpatents

Milner, J.R.

1994-05-10

An easily accessible electrical connector capable of rapid assembly and disassembly is described wherein a wide metal conductor sheet may be evenly contacted over the entire width of the conductor sheet by opposing surfaces on the connector which provide an even clamping pressure against opposite surfaces of the metal conductor sheet using a single threaded actuating screw. 13 figures.

A sequential mechanism for clathrin cage disassembly by 70-kDa heat-shock cognate protein (Hsc70) and auxilin

PubMed Central

Rothnie, Alice; Clarke, Anthony R.; Kuzmic, Petr; Cameron, Angus; Smith, Corinne J.

2011-01-01

An essential stage in endocytic coated vesicle recycling is the dissociation of clathrin from the vesicle coat by the molecular chaperone, 70-kDa heat-shock cognate protein (Hsc70), and the J-domain-containing protein, auxilin, in an ATP-dependent process. We present a detailed mechanistic analysis of clathrin disassembly catalyzed by Hsc70 and auxilin, using loss of perpendicular light scattering to monitor the process. We report that a single auxilin per clathrin triskelion is required for maximal rate of disassembly, that ATP is hydrolyzed at the same rate that disassembly occurs, and that three ATP molecules are hydrolyzed per clathrin triskelion released. Stopped-flow measurements revealed a lag phase in which the scattering intensity increased owing to association of Hsc70 with clathrin cages followed by serial rounds of ATP hydrolysis prior to triskelion removal. Global fit of stopped-flow data to several physically plausible mechanisms showed the best fit to a model in which sequential hydrolysis of three separate ATP molecules is required for the eventual release of a triskelion from the clathrin–auxilin cage. PMID:21482805

Superelastic Ball Bearings: Materials and Design to Avoid Mounting and Dismounting Brinell Damage in an Inaccessible Press-Fit Application-. I ; Design Approach

NASA Technical Reports Server (NTRS)

Dellacorte, Christopher; Howard, S. Adam

2015-01-01

Ball bearings require proper fit and installation into machinery structures (onto shafts and into bearing housings) to ensure optimal performance. For some applications, both the inner and outer race must be mounted with an interference fit and care must be taken during assembly and disassembly to avoid placing heavy static loads between the balls and races otherwise Brinell dent type damage can occur. In this paper, a highly dent resistant superelastic alloy, 60NiTi, is considered for rolling element bearing applications that encounter excessive static axial loading during assembly or disassembly. A small (R8) ball bearing is designed for an application in which access to the bearing races to apply disassembly tools is precluded. First Principles analyses show that by careful selection of materials, raceway curvature and land geometry, a bearing can be designed that allows blind assembly and disassembly without incurring raceway damage due to ball denting. Though such blind assembly applications are uncommon, the availability of bearings with unusually high static load capability may enable more such applications with additional benefits, especially for miniature bearings.

Molecular architecture of the human Mediator-RNA polymerase II-TFIIF assembly.

PubMed

Bernecky, Carrie; Grob, Patricia; Ebmeier, Christopher C; Nogales, Eva; Taatjes, Dylan J

2011-03-01

The macromolecular assembly required to initiate transcription of protein-coding genes, known as the Pre-Initiation Complex (PIC), consists of multiple protein complexes and is approximately 3.5 MDa in size. At the heart of this assembly is the Mediator complex, which helps regulate PIC activity and interacts with the RNA polymerase II (pol II) enzyme. The structure of the human Mediator-pol II interface is not well-characterized, whereas attempts to structurally define the Mediator-pol II interaction in yeast have relied on incomplete assemblies of Mediator and/or pol II and have yielded inconsistent interpretations. We have assembled the complete, 1.9 MDa human Mediator-pol II-TFIIF complex from purified components and have characterized its structural organization using cryo-electron microscopy and single-particle reconstruction techniques. The orientation of pol II within this assembly was determined by crystal structure docking and further validated with projection matching experiments, allowing the structural organization of the entire human PIC to be envisioned. Significantly, pol II orientation within the Mediator-pol II-TFIIF assembly can be reconciled with past studies that determined the location of other PIC components relative to pol II itself. Pol II surfaces required for interacting with TFIIB, TFIIE, and promoter DNA (i.e., the pol II cleft) are exposed within the Mediator-pol II-TFIIF structure; RNA exit is unhindered along the RPB4/7 subunits; upstream and downstream DNA is accessible for binding additional factors; and no major structural re-organization is necessary to accommodate the large, multi-subunit TFIIH or TFIID complexes. The data also reveal how pol II binding excludes Mediator-CDK8 subcomplex interactions and provide a structural basis for Mediator-dependent control of PIC assembly and function. Finally, parallel structural analysis of Mediator-pol II complexes lacking TFIIF reveal that TFIIF plays a key role in stabilizing pol II orientation within the assembly.

Molecular Architecture of the Human Mediator–RNA Polymerase II–TFIIF Assembly

PubMed Central

Bernecky, Carrie; Grob, Patricia; Ebmeier, Christopher C.; Nogales, Eva; Taatjes, Dylan J.

2011-01-01

The macromolecular assembly required to initiate transcription of protein-coding genes, known as the Pre-Initiation Complex (PIC), consists of multiple protein complexes and is approximately 3.5 MDa in size. At the heart of this assembly is the Mediator complex, which helps regulate PIC activity and interacts with the RNA polymerase II (pol II) enzyme. The structure of the human Mediator–pol II interface is not well-characterized, whereas attempts to structurally define the Mediator–pol II interaction in yeast have relied on incomplete assemblies of Mediator and/or pol II and have yielded inconsistent interpretations. We have assembled the complete, 1.9 MDa human Mediator–pol II–TFIIF complex from purified components and have characterized its structural organization using cryo-electron microscopy and single-particle reconstruction techniques. The orientation of pol II within this assembly was determined by crystal structure docking and further validated with projection matching experiments, allowing the structural organization of the entire human PIC to be envisioned. Significantly, pol II orientation within the Mediator–pol II–TFIIF assembly can be reconciled with past studies that determined the location of other PIC components relative to pol II itself. Pol II surfaces required for interacting with TFIIB, TFIIE, and promoter DNA (i.e., the pol II cleft) are exposed within the Mediator–pol II–TFIIF structure; RNA exit is unhindered along the RPB4/7 subunits; upstream and downstream DNA is accessible for binding additional factors; and no major structural re-organization is necessary to accommodate the large, multi-subunit TFIIH or TFIID complexes. The data also reveal how pol II binding excludes Mediator–CDK8 subcomplex interactions and provide a structural basis for Mediator-dependent control of PIC assembly and function. Finally, parallel structural analysis of Mediator–pol II complexes lacking TFIIF reveal that TFIIF plays a key role in stabilizing pol II orientation within the assembly. PMID:21468301

Reprocessing anesthesia instruments and devices.

PubMed

Ball, K

2000-02-01

Reprocessing anesthesia instruments and devices can often present a challenge for anesthesia providers because anesthesia devices have become more complex, cross-contamination with disease-forming pathogens can occur, and the importance of appropriate reprocessing may not be fully understood. Based on accepted practice recommendations, regulations, and research, reprocessing must be performed by skilled individuals who understand asepsis, cleaning, disinfection, and sterilization principles. This article describes the art of reprocessing and includes highlighted information on recommended practices, Spaulding's classifications, personal protective attire, precleaning, leak testing of flexible endoscopes, device disassembly, cleaning supplies and solutions, cleaning methods, rinsing, reassembly of the device, inspection, disinfection, and sterilization.

African Swine Fever Virus Gets Undressed: New Insights on the Entry Pathway.

PubMed

Andrés, Germán

2017-02-15

African swine fever virus (ASFV) is a large, multienveloped DNA virus composed of a genome-containing core successively wrapped by an inner lipid envelope, an icosahedral protein capsid, and an outer lipid envelope. In keeping with this structural complexity, recent studies have revealed an intricate entry program. This Gem highlights how ASFV uses two alternative pathways, macropinocytosis and clathrin-mediated endocytosis, to enter into the host macrophage and how the endocytosed particles undergo a stepwise, low pH-driven disassembly leading to inner envelope fusion and core delivery in the cytoplasm. Copyright © 2017 American Society for Microbiology.

Versatile microrobotics using simple modular subunits

NASA Astrophysics Data System (ADS)

Cheang, U. Kei; Meshkati, Farshad; Kim, Hoyeon; Lee, Kyoungwoo; Fu, Henry Chien; Kim, Min Jun

2016-07-01

The realization of reconfigurable modular microrobots could aid drug delivery and microsurgery by allowing a single system to navigate diverse environments and perform multiple tasks. So far, microrobotic systems are limited by insufficient versatility; for instance, helical shapes commonly used for magnetic swimmers cannot effectively assemble and disassemble into different size and shapes. Here by using microswimmers with simple geometries constructed of spherical particles, we show how magnetohydrodynamics can be used to assemble and disassemble modular microrobots with different physical characteristics. We develop a mechanistic physical model that we use to improve assembly strategies. Furthermore, we experimentally demonstrate the feasibility of dynamically changing the physical properties of microswimmers through assembly and disassembly in a controlled fluidic environment. Finally, we show that different configurations have different swimming properties by examining swimming speed dependence on configuration size.

Versatile microrobotics using simple modular subunits

PubMed Central

Cheang, U Kei; Meshkati, Farshad; Kim, Hoyeon; Lee, Kyoungwoo; Fu, Henry Chien; Kim, Min Jun

2016-01-01

The realization of reconfigurable modular microrobots could aid drug delivery and microsurgery by allowing a single system to navigate diverse environments and perform multiple tasks. So far, microrobotic systems are limited by insufficient versatility; for instance, helical shapes commonly used for magnetic swimmers cannot effectively assemble and disassemble into different size and shapes. Here by using microswimmers with simple geometries constructed of spherical particles, we show how magnetohydrodynamics can be used to assemble and disassemble modular microrobots with different physical characteristics. We develop a mechanistic physical model that we use to improve assembly strategies. Furthermore, we experimentally demonstrate the feasibility of dynamically changing the physical properties of microswimmers through assembly and disassembly in a controlled fluidic environment. Finally, we show that different configurations have different swimming properties by examining swimming speed dependence on configuration size. PMID:27464852

Lignin Hydrogenolysis: Improving Lignin Disassembly through Formaldehyde Stabilization

PubMed Central

2017-01-01

Abstract Lignocellulosic biomass is available in large quantities and constitutes an attractive feedstock for the sustainable production of bulk and fine chemicals. Although methods have been established for the conversion of its cellulosic fractions, valorization of lignin has proven to be challenging. The difficulty in disassembling lignin originates from its heterogeneous structure and its propensity to undergo skeletal rearrangements and condensation reactions during biorefinery fractionation or biomass pretreatment processes. A strategy for hindering the generation of these resistive interunit linkages during biomass pretreatment has now been devised using formaldehyde as a stabilizing agent. The developed method when combined with Ru/C‐catalyzed hydrogenolysis allows for efficient disassembly of all three biomass fractions: (cellulose, hemicellulose, and lignin) and suggests that lignin upgrading can be integrated into prevailing biorefinery schemes. PMID:28394095

Mechanical recycling of waste electric and electronic equipment: a review.

PubMed

Cui, Jirang; Forssberg, Eric

2003-05-30

The production of electric and electronic equipment (EEE) is one of the fastest growing areas. This development has resulted in an increase of waste electric and electronic equipment (WEEE). In view of the environmental problems involved in the management of WEEE, many counties and organizations have drafted national legislation to improve the reuse, recycling and other forms of recovery of such wastes so as to reduce disposal. Recycling of WEEE is an important subject not only from the point of waste treatment but also from the recovery of valuable materials.WEEE is diverse and complex, in terms of materials and components makeup as well as the original equipment's manufacturing processes. Characterization of this waste stream is of paramount importance for developing a cost-effective and environmentally friendly recycling system. In this paper, the physical and particle properties of WEEE are presented. Selective disassembly, targeting on singling out hazardous and/or valuable components, is an indispensable process in the practice of recycling of WEEE. Disassembly process planning and innovation of disassembly facilities are most active research areas. Mechanical/physical processing, based on the characterization of WEEE, provides an alternative means of recovering valuable materials. Mechanical processes, such as screening, shape separation, magnetic separation, Eddy current separation, electrostatic separation, and jigging have been widely utilized in recycling industry. However, recycling of WEEE is only beginning. For maximum separation of materials, WEEE should be shredded to small, even fine particles, generally below 5 or 10mm. Therefore, a discussion of mechanical separation processes for fine particles is highlighted in this paper. Consumer electronic equipment (brown goods), such as television sets, video recorders, are most common. It is very costly to perform manual dismantling of those products, due to the fact that brown goods contain very low-grade precious metals and copper. It is expected that a mechanical recycling process will be developed for the upgrading of low metal content scraps.

Fungus hyphae-supported alumina: An efficient and reclaimable adsorbent for fluoride removal from water.

PubMed

Yang, Weichun; Tian, Shunqi; Tang, Qiongzhi; Chai, Liyuan; Wang, Haiying

2017-06-15

A reclaimable adsorbent of fungus hyphae-supported alumina (FHSA) bio-nanocomposites was developed, characterized and applied in fluoride removal from water. This adsorbent can be fast assembled and disassemble reversibly, promising efficient reclamation and high accessible surface area for fluoride adsorption. Adsorption experiments demonstrate that the FHSA performed well over a considerable wide pH range of 3-10 with high fluoride removal efficiencies (>66.3%). The adsorption capacity was 105.60mgg -1 for FHSA, much higher than that for the alumina nanoparticles (50.55mgg -1 ) and pure fungus hyphae (22.47mgg -1 ). The adsorption capacity calculated by the pure content of alumina in the FHSA is 340.27mgg -1 of alumina. Kinetics data reveal that the fluoride adsorption process on the FHSA was fast, nearly 90% fluoride adsorption can be achieved within 40min. The fluoride adsorption on the FHSA is mainly due to the surface complexes formation of fluoride with AlOH and the attraction between protonated NH 2 and fluoride through hydrogen bonding. Findings demonstrate that the FHSA has potential applicability in fluoride removal due to its strong fluoride adsorbility and the easy reclamation by its fast reversible assembly and disassembly feature. Copyright © 2017 Elsevier Inc. All rights reserved.

Analysis of interphase node proteins in fission yeast by quantitative and superresolution fluorescence microscopy

PubMed Central

Akamatsu, Matthew; Lin, Yu; Bewersdorf, Joerg; Pollard, Thomas D.

2017-01-01

We used quantitative confocal microscopy and FPALM superresolution microscopy of live fission yeast to investigate the structures and assembly of two types of interphase nodes—multiprotein complexes associated with the plasma membrane that merge together and mature into the precursors of the cytokinetic contractile ring. During the long G2 phase of the cell cycle, seven different interphase node proteins maintain constant concentrations as they accumulate in proportion to cell volume. During mitosis, the total numbers of type 1 node proteins (cell cycle kinases Cdr1p, Cdr2p, Wee1p, and anillin Mid1p) are constant even when the nodes disassemble. Quantitative measurements provide strong evidence that both types of nodes have defined sizes and numbers of constituent proteins, as observed for cytokinesis nodes. Type 1 nodes assemble in two phases—a burst at the end of mitosis, followed by steady increase during interphase to double the initial number. Type 2 nodes containing Blt1p, Rho-GEF Gef2p, and kinesin Klp8p remain intact throughout the cell cycle and are constituents of the contractile ring. They are released from the contractile ring as it disassembles and then associate with type 1 nodes around the equator of the cell during interphase. PMID:28539404

32. INTERIOR VIEW TO THE NORTH OF THE FIRST FLOOR ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

32. INTERIOR VIEW TO THE NORTH OF THE FIRST FLOOR EAST CORRIDOR AND VIEWING GALLERY TO THE DISASSEMBLY BAY. A VIEWING AND WORK STATION FOR THE EAST SIDE OF THE UPPER LEVEL OF THE DISASSEMBLY BAY IS ON THE WEST SIDE OF THE CORRIDOR. - Nevada Test Site, Reactor Maintenance Assembly & Dissassembly Facility, Area 25, Jackass Flats, Junction of Roads F & G, Mercury, Nye County, NV

Adsorption of virus-like particles on ion exchange surface: Conformational changes at different pH detected by dual polarization interferometry.

PubMed

Yang, Yanli; Mengran Yu; Zhang, Songping; Ma, Guanghui; Su, Zhiguo

2015-08-21

Disassembling of virus-like particles (VLPs) like hepatitis B virus surface antigen (HB-VLPs) during chromatographic process has been identified as a major cause of loss of antigen activity. In this study, dual polarization interferometry (DPI) measurement, together with chromatography experiments, were performed to study the adsorption and conformational change of HB-VLPs on ion exchange surface at three different pHs. Changes in pH values of buffer solution showed only minimal effect on the HB-VLPs assembly and antigen activity, while significantly different degree of HB-VLPs disassembling was observed after ion exchange chromatography (IEC) at different pHs, indicating the conformational change of HB-VLPs caused mainly by its interactions with the adsorbent surface. By creating an ion exchange surface on chip surface, the conformational changes of HB-VLPs during adsorption to the surface were monitored in real time by DPI for the first time. As pH increased from 7.0 to 9.0, strong electrostatic interactions between oppositely charged HB-VLPs and the ion exchange surface make the HB-VLPs spread thinly or even adsorbed in disassembled formation on the surface as revealed by significant decrease in thickness of the adsorbed layer measured by DPI. Such findings were consistent with the results of IEC experiments operated at different pHs, that more disassembled HB-VLPs were detected in the eluted proteins at pH 9.0. At low pH like pH 5.0, however, possible bi-layer adsorption was involved as evidenced by an adsorbed layer thickness higher than average diameter of the HB-VLPs. The "lateral" protein-protein interactions might be unfavorable and would make additional contribution to the disassembling of HB-VLPs besides the primary mechanism related to the protein-surface interactions; therefore, the lowest antigen activity was observed after IEC at pH 5.0. Such real-time information on conformational change of VLPs is helpful for better understanding the real mechanism for the disassembling of VLPs on the solid-liquid interface. Copyright © 2015 Elsevier B.V. All rights reserved.

Direct TFIIA-TFIID Protein Contacts Drive Budding Yeast Ribosomal Protein Gene Transcription*

PubMed Central

Layer, Justin H.; Weil, P. Anthony

2013-01-01

We have previously shown that yeast TFIID provides coactivator function on the promoters of ribosomal protein-encoding genes (RPGs) by making direct contact with the transactivator repressor activator protein 1 (Rap1). Further, our structural studies of assemblies generated with purified Rap1, TFIID, and TFIIA on RPG enhancer-promoter DNA indicate that Rap1-TFIID interaction induces dramatic conformational rearrangements of enhancer-promoter DNA and TFIID-bound TFIIA. These data indicate a previously unknown yet critical role for yeast TFIIA in the integration of activator-TFIID contacts with promoter conformation and downstream preinitiation complex formation and/or function. Here we describe the use of systematic mutagenesis to define how specific TFIIA contacts contribute to these processes. We have verified that TFIIA is required for RPG transcription in vivo and in vitro, consistent with the existence of a critical Rap1-TFIIA-TFIID interaction network. We also identified essential points of contact for TFIIA and Rap1 within the Rap1 binding domain of the Taf4 subunit of TFIID. These data suggest a mechanism for how interactions between TFIID, TFIIA, and Rap1 contribute to the high rate of transcription initiation seen on RPGs in vivo. PMID:23814059

Translation suppression promotes stress granule formation and cell survival in response to cold shock

PubMed Central

Hofmann, Sarah; Cherkasova, Valeria; Bankhead, Peter; Bukau, Bernd; Stoecklin, Georg

2012-01-01

Cells respond to different types of stress by inhibition of protein synthesis and subsequent assembly of stress granules (SGs), cytoplasmic aggregates that contain stalled translation preinitiation complexes. Global translation is regulated through the translation initiation factor eukaryotic initiation factor 2α (eIF2α) and the mTOR pathway. Here we identify cold shock as a novel trigger of SG assembly in yeast and mammals. Whereas cold shock–induced SGs take hours to form, they dissolve within minutes when cells are returned to optimal growth temperatures. Cold shock causes eIF2α phosphorylation through the kinase PERK in mammalian cells, yet this pathway is not alone responsible for translation arrest and SG formation. In addition, cold shock leads to reduced mitochondrial function, energy depletion, concomitant activation of AMP-activated protein kinase (AMPK), and inhibition of mTOR signaling. Compound C, a pharmacological inhibitor of AMPK, prevents the formation of SGs and strongly reduces cellular survival in a translation-dependent manner. Our results demonstrate that cells actively suppress protein synthesis by parallel pathways, which induce SG formation and ensure cellular survival during hypothermia. PMID:22875991

Promoter selection in human mitochondria involves binding of a transcription factor to orientation-independent upstream regulatory elements.

PubMed

Fisher, R P; Topper, J N; Clayton, D A

1987-07-17

Selective transcription of human mitochondrial DNA requires a transcription factor (mtTF) in addition to an essentially nonselective RNA polymerase. Partially purified mtTF is able to sequester promoter-containing DNA in preinitiation complexes in the absence of mitochondrial RNA polymerase, suggesting a DNA-binding mechanism for factor activity. Functional domains, required for positive transcriptional regulation by mtTF, are identified within both major promoters of human mtDNA through transcription of mutant promoter templates in a reconstituted in vitro system. These domains are essentially coextensive with DNA sequences protected from nuclease digestion by mtTF-binding. Comparison of the sequences of the two mtTF-responsive elements reveals significant homology only when one sequence is inverted; the binding sites are in opposite orientations with respect to the predominant direction of transcription. Thus mtTF may function bidirectionally, requiring additional protein-DNA interactions to dictate transcriptional polarity. The mtTF-responsive elements are arrayed as direct repeats, separated by approximately 80 bp within the displacement-loop region of human mitochondrial DNA; this arrangement may reflect duplication of an ancestral bidirectional promoter, giving rise to separate, unidirectional promoters for each strand.

Structural insight into the TFIIE–TFIIH interaction: TFIIE and p53 share the binding region on TFIIH

PubMed Central

Okuda, Masahiko; Tanaka, Aki; Satoh, Manami; Mizuta, Shoko; Takazawa, Manabu; Ohkuma, Yoshiaki; Nishimura, Yoshifumi

2008-01-01

RNA polymerase II and general transcription factors (GTFs) assemble on a promoter to form a transcription preinitiation complex (PIC). Among the GTFs, TFIIE recruits TFIIH to complete the PIC formation and regulates enzymatic activities of TFIIH. However, the mode of binding between TFIIE and TFIIH is poorly understood. Here, we demonstrate the specific binding of the C-terminal acidic domain (AC-D) of the human TFIIEα subunit to the pleckstrin homology domain (PH-D) of the human TFIIH p62 subunit and describe the solution structures of the free and PH-D-bound forms of AC-D. Although the flexible N-terminal acidic tail from AC-D wraps around PH-D, the core domain of AC-D also interacts with PH-D. AC-D employs an entirely novel binding mode, which differs from the amphipathic helix method used by many transcriptional activators. So the binding surface between PH-D and AC-D is much broader than the specific binding surface between PH-D and the p53 acidic fragments. From our in vitro studies, we demonstrate that this interaction could be a switch to replace p53 with TFIIE on TFIIH in transcription. PMID:18354501

Supramolecular Disassembly of Facially Amphiphilic Dendrimer Assemblies in Response to Physical, Chemical, and Biological Stimuli

PubMed Central

2015-01-01

Conspectus Supramolecular assemblies formed from spontaneous self-assembly of amphiphilic macromolecules are explored as biomimetic architectures and for applications in areas such as sensing, drug delivery, and diagnostics. Macromolecular assemblies are usually preferred, compared with their simpler small molecule counterparts, due to their low critical aggregate concentrations (CAC) and high thermodynamic stability. This Account focuses on the structural and functional aspects of assemblies formed from dendrimers, specifically facially amphiphilic dendrons that form micelle or inverse micelle type supramolecular assemblies depending on the nature of the solvent medium. The micelle type assemblies formed from facially amphiphilic dendrons sequester hydrophobic guest molecules in their interiors. The stability of these assemblies is dependent on the relative compatibility of the hydrophilic and hydrophobic functionalities with water, often referred to as hydrophilic–lipophilic balance (HLB). Disruption of the HLB, using an external stimulus, could lead to disassembly of the aggregates, which can then be utilized to cause an actuation event, such as guest molecule release. Studying these possibilities has led to (i) a robust and general strategy for stimulus-induced disassembly and molecular release and (ii) the introduction of a new approach to protein-responsive supramolecular disassembly. The latter strategy provides a particularly novel avenue for impacting biomedical applications. Most of the stimuli-sensitive supramolecular assemblies have been designed to be responsive to factors such pH, temperature, and redox conditions. The reason for this interest stems from the fact that certain disease microenvironments have aberrations in these factors. However, these variations are the secondary imbalances in biology. Imbalances in protein activity are the primary reasons for most, if not all, human pathology. There have been no robust strategies in stimulus-responsive assemblies that respond to these variations. The facially amphiphilic dendrimers provide a unique opportunity to explore this possibility. Similarly, the propensity of these molecules to form inverse micelles in apolar solvents and thus bind polar guest molecules, combined with the fact that these assemblies do not thermodynamically equilibrate in biphasic mixtures, was used to predictably simplify peptide mixtures. The structure–property relationships developed from these studies have led to a selective and highly sensitive detection of peptides in complex mixtures. Selectivity in peptide extraction was achieved using charge complementarity between the peptides and the hydrophilic components present in inverse micellar interiors. These findings will have implications in areas such as proteomics and biomarker detection. PMID:24937682

The Amphipathic Helix of Adenovirus Capsid Protein VI Contributes to Penton Release and Postentry Sorting

PubMed Central

Martinez, Ruben; Schellenberger, Pascale; Vasishtan, Daven; Aknin, Cindy; Austin, Sisley; Dacheux, Denis; Rayne, Fabienne; Siebert, Alistair; Ruzsics, Zsolt; Gruenewald, Kay

2014-01-01

ABSTRACT Nuclear delivery of the adenoviral genome requires that the capsid cross the limiting membrane of the endocytic compartment and traverse the cytosol to reach the nucleus. This endosomal escape is initiated upon internalization and involves a highly coordinated process of partial disassembly of the entering capsid to release the membrane lytic internal capsid protein VI. Using wild-type and protein VI-mutated human adenovirus serotype 5 (HAdV-C5), we show that capsid stability and membrane rupture are major determinants of entry-related sorting of incoming adenovirus virions. Furthermore, by using electron cryomicroscopy, as well as penton- and protein VI-specific antibodies, we show that the amphipathic helix of protein VI contributes to capsid stability by preventing premature disassembly and deployment of pentons and protein VI. Thus, the helix has a dual function in maintaining the metastable state of the capsid by preventing premature disassembly and mediating efficient membrane lysis to evade lysosomal targeting. Based on these findings and structural data from cryo-electron microscopy, we suggest a refined disassembly mechanism upon entry. IMPORTANCE In this study, we show the intricate connection of adenovirus particle stability and the entry-dependent release of the membrane-lytic capsid protein VI required for endosomal escape. We show that the amphipathic helix of the adenovirus internal protein VI is required to stabilize pentons in the particle while coinciding with penton release upon entry and that release of protein VI mediates membrane lysis, thereby preventing lysosomal sorting. We suggest that this dual functionality of protein VI ensures an optimal disassembly process by balancing the metastable state of the mature adenovirus particle. PMID:25473051

Robustness analysis of non-ordinary Petri nets for flexible assembly/disassembly processes based on structural decomposition

NASA Astrophysics Data System (ADS)

Hsieh, Fu-Shiung

2011-03-01

Design of robust supervisory controllers for manufacturing systems with unreliable resources has received significant attention recently. Robustness analysis provides an alternative way to analyse a perturbed system to quickly respond to resource failures. Although we have analysed the robustness properties of several subclasses of ordinary Petri nets (PNs), analysis for non-ordinary PNs has not been done. Non-ordinary PNs have weighted arcs and have the advantage to compactly model operations requiring multiple parts or resources. In this article, we consider a class of flexible assembly/disassembly manufacturing systems and propose a non-ordinary flexible assembly/disassembly Petri net (NFADPN) model for this class of systems. As the class of flexible assembly/disassembly manufacturing systems can be regarded as the integration and interactions of a set of assembly/disassembly subprocesses, a bottom-up approach is adopted in this article to construct the NFADPN models. Due to the routing flexibility in NFADPN, there may exist different ways to accomplish the tasks. To characterise different ways to accomplish the tasks, we propose the concept of completely connected subprocesses. As long as there exists a set of completely connected subprocesses for certain type of products, the production of that type of products can still be maintained without requiring the whole NFADPN to be live. To take advantage of the alternative routes without enforcing liveness for the whole system, we generalise the concept of persistent production proposed to NFADPN. We propose a condition for persistent production based on the concept of completely connected subprocesses. We extend robustness analysis to NFADPN by exploiting its structure. We identify several patterns of resource failures and characterise the conditions to maintain operation in the presence of resource failures.

RNA binding to APOBEC3G induces the disassembly of functional deaminase complexes by displacing single-stranded DNA substrates

PubMed Central

Polevoda, Bogdan; McDougall, William M.; Tun, Bradley N.; Cheung, Michael; Salter, Jason D.; Friedman, Alan E.; Smith, Harold C.

2015-01-01

APOBEC3G (A3G) DNA deaminase activity requires a holoenzyme complex whose assembly on nascent viral reverse transcripts initiates with A3G dimers binding to ssDNA followed by formation of higher-order A3G homo oligomers. Catalytic activity is inhibited when A3G binds to RNA. Our prior studies suggested that RNA inhibited A3G binding to ssDNA. In this report, near equilibrium binding and gel shift analyses showed that A3G assembly and disassembly on ssDNA was an ordered process involving A3G dimers and multimers thereof. Although, fluorescence anisotropy showed that A3G had similar nanomolar affinity for RNA and ssDNA, RNA stochastically dissociated A3G dimers and higher-order oligomers from ssDNA, suggesting a different modality for RNA binding. Mass spectrometry mapping of A3G peptides cross-linked to nucleic acid suggested ssDNA only bound to three peptides, amino acids (aa) 181–194 in the N-terminus and aa 314–320 and 345–374 in the C-terminus that were part of a continuous exposed surface. RNA bound to these peptides and uniquely associated with three additional peptides in the N- terminus, aa 15–29, 41–52 and 83–99, that formed a continuous surface area adjacent to the ssDNA binding surface. The data predict a mechanistic model of RNA inhibition of ssDNA binding to A3G in which competitive and allosteric interactions determine RNA-bound versus ssDNA-bound conformational states. PMID:26424853

Preliminary topical report on comparison reactor disassembly calculations

DOE Office of Scientific and Technical Information (OSTI.GOV)

McLaughlin, T.P.

1975-11-01

Preliminary results of comparison disassembly calculations for a representative LMFBR model (2100-l voided core) and arbitrary accident conditions are described. The analytical methods employed were the computer programs: FX2- POOL, PAD, and VENUS-II. The calculated fission energy depositions are in good agreement, as are measures of the destructive potential of the excursions, kinetic energy, and work. However, in some cases the resulting fuel temperatures are substantially divergent. Differences in the fission energy deposition appear to be attributable to residual inconsistencies in specifying the comparison cases. In contrast, temperature discrepancies probably stem from basic differences in the energy partition models inherentmore » in the codes. Although explanations of the discrepancies are being pursued, the preliminary results indicate that all three computational methods provide a consistent, global characterization of the contrived disassembly accident. (auth)« less

Mechanisms of DNA replication termination.

PubMed

Dewar, James M; Walter, Johannes C

2017-08-01

Genome duplication is carried out by pairs of replication forks that assemble at origins of replication and then move in opposite directions. DNA replication ends when converging replication forks meet. During this process, which is known as replication termination, DNA synthesis is completed, the replication machinery is disassembled and daughter molecules are resolved. In this Review, we outline the steps that are likely to be common to replication termination in most organisms, namely, fork convergence, synthesis completion, replisome disassembly and decatenation. We briefly review the mechanism of termination in the bacterium Escherichia coli and in simian virus 40 (SV40) and also focus on recent advances in eukaryotic replication termination. In particular, we discuss the recently discovered E3 ubiquitin ligases that control replisome disassembly in yeast and higher eukaryotes, and how their activity is regulated to avoid genome instability.

Human Ska complex and Ndc80 complex interact to form a load-bearing assembly that strengthens kinetochore–microtubule attachments

PubMed Central

Zelter, Alex; Riffle, Michael; MacCoss, Michael J.; Asbury, Charles L.; Davis, Trisha N.

2018-01-01

Accurate segregation of chromosomes relies on the force-bearing capabilities of the kinetochore to robustly attach chromosomes to dynamic microtubule tips. The human Ska complex and Ndc80 complex are outer-kinetochore components that bind microtubules and are required to fully stabilize kinetochore–microtubule attachments in vivo. While purified Ska complex tracks with disassembling microtubule tips, it remains unclear whether the Ska complex–microtubule interaction is sufficiently strong to make a significant contribution to kinetochore–microtubule coupling. Alternatively, Ska complex might affect kinetochore coupling indirectly, through recruitment of phosphoregulatory factors. Using optical tweezers, we show that the Ska complex itself bears load on microtubule tips, strengthens Ndc80 complex-based tip attachments, and increases the switching dynamics of the attached microtubule tips. Cross-linking mass spectrometry suggests the Ska complex directly binds Ndc80 complex through interactions between the Ska3 unstructured C-terminal region and the coiled-coil regions of each Ndc80 complex subunit. Deletion of the Ska complex microtubule-binding domain or the Ska3 C terminus prevents Ska complex from strengthening Ndc80 complex-based attachments. Together, our results indicate that the Ska complex can directly strengthen the kinetochore–microtubule interface and regulate microtubule tip dynamics by forming an additional connection between the Ndc80 complex and the microtubule. PMID:29487209

An Efficient Procedure for Removal and Inactivation of Alpha-Synuclein Assemblies from Laboratory Materials.

PubMed

Bousset, Luc; Brundin, Patrik; Böckmann, Anja; Meier, Beat; Melki, Ronald

2016-01-01

Preformed α-synuclein fibrils seed the aggregation of soluble α-synuclein in cultured cells and in vivo. This, and other findings, has kindled the idea that α-synuclein fibrils possess prion-like properties. As α-synuclein fibrils should not be considered as innocuous, there is a need for decontamination and inactivation procedures for laboratory benches and non-disposable laboratory material. We assessed the effectiveness of different procedures designed to disassemble α-synuclein fibrils and reduce their infectivity. We examined different commercially available detergents to remove α-synuclein assemblies adsorbed on materials that are not disposable and that are most found in laboratories (e.g. plastic, glass, aluminum or stainless steel surfaces). We show that methods designed to decrease PrP prion infectivity neither effectively remove α-synuclein assemblies adsorbed to different materials commonly used in the laboratory nor disassemble the fibrillar form of the protein with efficiency. In contrast, both commercial detergents and SDS detached α-synuclein assemblies from contaminated surfaces and disassembled the fibrils. We describe three cleaning procedures that effectively remove and disassemble α-synuclein seeds. The methods rely on the use of detergents that are compatible with most non-disposable tools in a laboratory. The procedures are easy to implement and significantly decrease any potential risks associated to handling α-synuclein assemblies.

MCM5: a new actor in the link between DNA replication and Meier-Gorlin syndrome.

PubMed

Vetro, Annalisa; Savasta, Salvatore; Russo Raucci, Annalisa; Cerqua, Cristina; Sartori, Geppo; Limongelli, Ivan; Forlino, Antonella; Maruelli, Silvia; Perucca, Paola; Vergani, Debora; Mazzini, Giuliano; Mattevi, Andrea; Stivala, Lucia Anna; Salviati, Leonardo; Zuffardi, Orsetta

2017-05-01

Meier-Gorlin syndrome (MGORS) is a rare disorder characterized by primordial dwarfism, microtia, and patellar aplasia/hypoplasia. Recessive mutations in ORC1, ORC4, ORC6, CDT1, CDC6, and CDC45, encoding members of the pre-replication (pre-RC) and pre-initiation (pre-IC) complexes, and heterozygous mutations in GMNN, a regulator of cell-cycle progression and DNA replication, have already been associated with this condition. We performed whole-exome sequencing (WES) in a patient with a clinical diagnosis of MGORS and identified biallelic variants in MCM5. This gene encodes a subunit of the replicative helicase complex, which represents a component of the pre-RC. Both variants, a missense substitution within a conserved domain critical for the helicase activity, and a single base deletion causing a frameshift and a premature stop codon, were predicted to be detrimental for the MCM5 function. Although variants of MCM5 have never been reported in specific human diseases, defect of this gene in zebrafish causes a phenotype of growth restriction overlapping the one associated with orc1 depletion. Complementation experiments in yeast showed that the plasmid carrying the missense variant was unable to rescue the lethal phenotype caused by mcm5 deletion. Moreover cell-cycle progression was delayed in patient's cells, as already shown for mutations in the ORC1 gene. Altogether our findings support the role of MCM5 as a novel gene involved in MGORS, further emphasizing that this condition is caused by impaired DNA replication.

Identification of EhTIF-IA: The putative E. histolytica orthologue of the human ribosomal RNA transcription initiation factor-IA.

PubMed

Srivastava, Ankita; Bhattacharya, Alok; Bhattacharya, Sudha; Jhingan, Gagan Deep

2016-03-01

Initiation of rDNA transcription requires the assembly of a specific multi-protein complex at the rDNA promoter containing the RNA Pol I with auxiliary factors. One of these factors is known as Rrn3P in yeast and Transcription Initiation Factor IA (TIF-IA) in mammals. Rrn3p/TIF-IA serves as a bridge between RNA Pol I and the pre-initiation complex at the promoter. It is phosphorylated at multiple sites and is involved in regulation of rDNA transcription in a growth-dependent manner. In the early branching parasitic protist Entamoeba histolytica, the rRNA genes are present exclusively on circular extra chromosomal plasmids. The protein factors involved in regulation of rDNA transcription in E. histolytica are not known. We have identified the E. histolytica equivalent of TIF-1A (EhTIF-IA) by homology search within the database and was further cloned and expressed. Immuno-localization studies showed that EhTIF-IA co-localized partially with fibrillarin in the peripherally localized nucleolus. EhTIF-IA was shown to interact with the RNA Pol I-specific subunit RPA12 both in vivo and in vitro. Mass spectroscopy data identified RNA Pol I-specific subunits and other nucleolar proteins to be the interacting partners of EhTIF-IA. Our study demonstrates for the first time a conserved putative RNA Pol I transcription factor TIF-IA in E. histolytica.

Mechanism of Mediator recruitment by tandem Gcn4 activation domains and three Gal11 activator-binding domains.

PubMed

Herbig, Eric; Warfield, Linda; Fish, Lisa; Fishburn, James; Knutson, Bruce A; Moorefield, Beth; Pacheco, Derek; Hahn, Steven

2010-05-01

Targets of the tandem Gcn4 acidic activation domains in transcription preinitiation complexes were identified by site-specific cross-linking. The individual Gcn4 activation domains cross-link to three common targets, Gal11/Med15, Taf12, and Tra1, which are subunits of four conserved coactivator complexes, Mediator, SAGA, TFIID, and NuA4. The Gcn4 N-terminal activation domain also cross-links to the Mediator subunit Sin4/Med16. The contribution of the two Gcn4 activation domains to transcription was gene specific and varied from synergistic to less than additive. Gcn4-dependent genes had a requirement for Gal11 ranging from 10-fold dependence to complete Gal11 independence, while the Gcn4-Taf12 interaction did not significantly contribute to the expression of any gene studied. Complementary methods identified three conserved Gal11 activator-binding domains that bind each Gcn4 activation domain with micromolar affinity. These Gal11 activator-binding domains contribute additively to transcription activation and Mediator recruitment at Gcn4- and Gal11-dependent genes. Although we found that the conserved Gal11 KIX domain contributes to Gal11 function, we found no evidence of specific Gcn4-KIX interaction and conclude that the Gal11 KIX domain does not function by specific interaction with Gcn4. Our combined results show gene-specific coactivator requirements, a surprising redundancy in activator-target interactions, and an activator-coactivator interaction mediated by multiple low-affinity protein-protein interactions.

One-step assembly of coordination complexes for versatile film and particle engineering.

PubMed

Ejima, Hirotaka; Richardson, Joseph J; Liang, Kang; Best, James P; van Koeverden, Martin P; Such, Georgina K; Cui, Jiwei; Caruso, Frank

2013-07-12

The development of facile and versatile strategies for thin-film and particle engineering is of immense scientific interest. However, few methods can conformally coat substrates of different composition, size, shape, and structure. We report the one-step coating of various interfaces using coordination complexes of natural polyphenols and Fe(III) ions. Film formation is initiated by the adsorption of the polyphenol and directed by pH-dependent, multivalent coordination bonding. Aqueous deposition is performed on a range of planar as well as inorganic, organic, and biological particle templates, demonstrating an extremely rapid technique for producing structurally diverse, thin films and capsules that can disassemble. The ease, low cost, and scalability of the assembly process, combined with pH responsiveness and negligible cytotoxicity, makes these films potential candidates for biomedical and environmental applications.

HyperForest: A high performance multi-processor architecture for real-time intelligent systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Garcia, P. Jr.; Rebeil, J.P.; Pollard, H.

1997-04-01

Intelligent Systems are characterized by the intensive use of computer power. The computer revolution of the last few years is what has made possible the development of the first generation of Intelligent Systems. Software for second generation Intelligent Systems will be more complex and will require more powerful computing engines in order to meet real-time constraints imposed by new robots, sensors, and applications. A multiprocessor architecture was developed that merges the advantages of message-passing and shared-memory structures: expendability and real-time compliance. The HyperForest architecture will provide an expandable real-time computing platform for computationally intensive Intelligent Systems and open the doorsmore » for the application of these systems to more complex tasks in environmental restoration and cleanup projects, flexible manufacturing systems, and DOE`s own production and disassembly activities.« less

A unique enhancer boundary complex on the mouse ribosomal RNA genes persists after loss of Rrn3 or UBF and the inactivation of RNA polymerase I transcription

PubMed Central

Stefanovsky, Victor Y.; Tremblay, Michel G.; Lindsay, Helen; Robinson, Mark D.

2017-01-01

Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA) genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance. Despite the need for a high level of rDNA transcription, typically only a fraction of the rDNA is transcriptionally active, and some NORs are permanently silenced by CpG methylation. Various chromatin-remodelling complexes have been implicated in counteracting silencing to maintain rDNA activity. However, the chromatin structure of the active rDNA fraction is still far from clear. Here we have combined a high-resolution ChIP-Seq protocol with conditional inactivation of key basal factors to better understand what determines active rDNA chromatin. The data resolve questions concerning the interdependence of the basal transcription factors, show that preinitiation complex formation is driven by the architectural factor UBF (UBTF) independently of transcription, and that RPI termination and release corresponds with the site of TTF1 binding. They further reveal the existence of an asymmetric Enhancer Boundary Complex formed by CTCF and Cohesin and flanked upstream by phased nucleosomes and downstream by an arrested RNA Polymerase I complex. We find that the Enhancer Boundary Complex is the only site of active histone modification in the 45kbp rDNA repeat. Strikingly, it not only delimits each functional rRNA gene, but also is stably maintained after gene inactivation and the re-establishment of surrounding repressive chromatin. Our data define a poised state of rDNA chromatin and place the Enhancer Boundary Complex as the likely entry point for chromatin remodelling complexes. PMID:28715449

A unique enhancer boundary complex on the mouse ribosomal RNA genes persists after loss of Rrn3 or UBF and the inactivation of RNA polymerase I transcription.

PubMed

Herdman, Chelsea; Mars, Jean-Clement; Stefanovsky, Victor Y; Tremblay, Michel G; Sabourin-Felix, Marianne; Lindsay, Helen; Robinson, Mark D; Moss, Tom

2017-07-01

Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA) genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance. Despite the need for a high level of rDNA transcription, typically only a fraction of the rDNA is transcriptionally active, and some NORs are permanently silenced by CpG methylation. Various chromatin-remodelling complexes have been implicated in counteracting silencing to maintain rDNA activity. However, the chromatin structure of the active rDNA fraction is still far from clear. Here we have combined a high-resolution ChIP-Seq protocol with conditional inactivation of key basal factors to better understand what determines active rDNA chromatin. The data resolve questions concerning the interdependence of the basal transcription factors, show that preinitiation complex formation is driven by the architectural factor UBF (UBTF) independently of transcription, and that RPI termination and release corresponds with the site of TTF1 binding. They further reveal the existence of an asymmetric Enhancer Boundary Complex formed by CTCF and Cohesin and flanked upstream by phased nucleosomes and downstream by an arrested RNA Polymerase I complex. We find that the Enhancer Boundary Complex is the only site of active histone modification in the 45kbp rDNA repeat. Strikingly, it not only delimits each functional rRNA gene, but also is stably maintained after gene inactivation and the re-establishment of surrounding repressive chromatin. Our data define a poised state of rDNA chromatin and place the Enhancer Boundary Complex as the likely entry point for chromatin remodelling complexes.

Hepatitis-C-virus-like internal ribosome entry sites displace eIF3 to gain access to the 40S subunit

NASA Astrophysics Data System (ADS)

Hashem, Yaser; Des Georges, Amedee; Dhote, Vidya; Langlois, Robert; Liao, Hstau Y.; Grassucci, Robert A.; Pestova, Tatyana V.; Hellen, Christopher U. T.; Frank, Joachim

2013-11-01

Hepatitis C virus (HCV) and classical swine fever virus (CSFV) messenger RNAs contain related (HCV-like) internal ribosome entry sites (IRESs) that promote 5'-end independent initiation of translation, requiring only a subset of the eukaryotic initiation factors (eIFs) needed for canonical initiation on cellular mRNAs. Initiation on HCV-like IRESs relies on their specific interaction with the 40S subunit, which places the initiation codon into the P site, where it directly base-pairs with eIF2-bound initiator methionyl transfer RNA to form a 48S initiation complex. However, all HCV-like IRESs also specifically interact with eIF3 (refs 2, 5, 6, 7, 9, 10, 11, 12), but the role of this interaction in IRES-mediated initiation has remained unknown. During canonical initiation, eIF3 binds to the 40S subunit as a component of the 43S pre-initiation complex, and comparison of the ribosomal positions of eIF3 and the HCV IRES revealed that they overlap, so that their rearrangement would be required for formation of ribosomal complexes containing both components. Here we present a cryo-electron microscopy reconstruction of a 40S ribosomal complex containing eIF3 and the CSFV IRES. Remarkably, although the position and interactions of the CSFV IRES with the 40S subunit in this complex are similar to those of the HCV IRES in the 40S-IRES binary complex, eIF3 is completely displaced from its ribosomal position in the 43S complex, and instead interacts through its ribosome-binding surface exclusively with the apical region of domain III of the IRES. Our results suggest a role for the specific interaction of HCV-like IRESs with eIF3 in preventing ribosomal association of eIF3, which could serve two purposes: relieving the competition between the IRES and eIF3 for a common binding site on the 40S subunit, and reducing formation of 43S complexes, thereby favouring translation of viral mRNAs.

HCV-like IRESs displace eIF3 to gain access to the 40S subunit

PubMed Central

Hashem, Yaser; des Georges, Amedee; Dhote, Vidya; Langlois, Robert; Liao, Hstau Y.; Grassucci, Robert A.; Pestova, Tatyana V.; Hellen, Christopher U.T.; Frank, Joachim

2014-01-01

Hepatitis C virus (HCV) and Classical swine fever virus (CSFV) mRNAs contain related (HCV-like) internal ribosome entry sites (IRESs) that promote 5’-end independent initiation of translation, requiring only a subset of the eukaryotic initiation factors (eIFs) needed for canonical initiation on cellular mRNAs1. Initiation on HCV-like IRESs relies on their specific interaction with the 40S subunit2–8, which places the initiation codon into the P site, where it directly base-pairs with eIF2-bound Met-tRNAiMet to form a 48S initiation complex. However, all HCV-like IRESs also specifically interact with eIF32,5–7,9–12, but the role of this interaction in IRES-mediated initiation has remained unknown. During canonical initiation, eIF3 binds to the 40S subunit as a component of the 43S pre-initiation complex, and comparison of the ribosomal positions of eIF313 and the HCV IRES8 revealed that they overlap, so that their rearrangement would be required for formation of ribosomal complexes containing both components13. Here, we present a cryo-electron microscopy reconstruction of a 40S ribosomal complex containing eIF3 and the CSFV IRES. Strikingly, although the position and interactions of the CSFV IRES with the 40S subunit in this complex are similar to those of the HCV IRES in the 40S/IRES binary complex8, eIF3 is completely displaced from its ribosomal position in the 43S complex, and instead interacts through its ribosome-binding surface exclusively with the apical region of domain III of the IRES. Our results suggest a role for the specific interaction of HCV-like IRESs with eIF3 in preventing ribosomal association of eIF3, which could serve two purposes: relieving the competition between the IRES and eIF3 for a common binding site on the 40S subunit, and reducing formation of 43S complexes, thereby favoring translation of viral mRNAs. PMID:24185006

Symbolic Execution Over Native x86

DTIC Science & Technology

2012-06-01

Disassembly to a Hello World Program Packed with the Ulti- mate Packer for eXecutables (UPX) (Taken from IDA Pro) . . . . . . . 7 Figure 2.3 A Simple Hello...Program Packed with the Ultimate Packer for eXecutables (UPX) (Taken from IDA Pro) operation details. However, the design of an IL language leads to...The Unofficial Guide to the World’s Most Popular Disas- sembler. No Starch Press, 2008. [13] Hex-Rays, “Interactive disassembler (ida) pro.” [Online

Evaluation of Hydroprocessed Renewable Diesel (HRD) Fuel in a Caterpillar Engine Using the 210 Hour TWV Cycle

DTIC Science & Technology

2014-05-01

TERMS Hydroprocessed Renewable Diesel , Reference Diesel Fuel, C7, emissions, power, performance, deposition, ambient, desert, synthetic fuel injector ...the engine run-in, the engine was disassembled to determine injector nozzle tip deposits, and the piston crowns and engine combustion chamber deposits...removed from the test cell and disassembled to determine injector nozzle tip and piston crown and engine combustion chamber deposits. Post- test

Early cysteine-dependent inactivation of 26S proteasomes does not involve particle disassembly.

PubMed

Hugo, Martín; Korovila, Ioanna; Köhler, Markus; García-García, Carlos; Cabrera-García, J Daniel; Marina, Anabel; Martínez-Ruiz, Antonio; Grune, Tilman

2018-06-01

Under oxidative stress 26S proteasomes suffer reversible disassembly into its 20S and 19S subunits, a process mediated by HSP70. This inhibits the degradation of polyubiquitinated proteins by the 26S proteasome and allows the degradation of oxidized proteins by a free 20S proteasome. Low fluxes of antimycin A-stimulated ROS production caused dimerization of mitochondrial peroxiredoxin 3 and cytosolic peroxiredoxin 2, but not peroxiredoxin overoxidation and overall oxidation of cellular protein thiols. This moderate redox imbalance was sufficient to inhibit the ATP stimulation of 26S proteasome activity. This process was dependent on reversible cysteine oxidation. Moreover, our results show that this early inhibition of ATP stimulation occurs previous to particle disassembly, indicating an intermediate step during the redox regulation of the 26S proteasome with special relevance under redox signaling rather than oxidative stress conditions. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Synthesis of butenes through 2-butanol dehydration over mesoporous materials produced from ferrierite

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeong, Soyeon; Kim, Hyeonjoo; Bae, Jung A.

2012-05-20

Mesoporous materials synthesized from commercial ferrierite (MMZ-FER) were applied to butanol dehydration. The MMZ-FER was produced by disassembling ferrierite into unit structures in the presence of an alkali solution, adding a surfactant as a templating material, followed by hydrothermal treatment. The effect of the alkali/(Si+Al) ratio in the disassembling step on the characteristics of the catalyst and butanol dehydration performance were investigated. The MMZ-FER materials, synthesized in a condition in which the NaOH/(Si + Al) mole ratio in the disassembling step was 0.67 and 1.0, demonstrated similar textural properties to those of MCM-41. Many weak acid sites developed on themore » MMZ-FER(0.67) and MMZ-FER(1.0) samples, which is attributed to the creation of ferrierite-induced acid sites. The MMZ-FER materials showed excellent catalytic activity, selectivity, and stability during the dehydration of 2-butanol.« less

NAD+ and SIRT3 control microtubule dynamics and reduce susceptibility to antimicrotubule agents

PubMed Central

Harkcom, William T.; Ghosh, Ananda K.; Sung, Matthew S.; Matov, Alexandre; Brown, Kevin D.; Giannakakou, Paraskevi; Jaffrey, Samie R.

2014-01-01

Nicotinamide adenine dinucleotide (NAD+) is an endogenous enzyme cofactor and cosubstrate that has effects on diverse cellular and physiologic processes, including reactive oxygen species generation, mitochondrial function, apoptosis, and axonal degeneration. A major goal is to identify the NAD+-regulated cellular pathways that may mediate these effects. Here we show that the dynamic assembly and disassembly of microtubules is markedly altered by NAD+. Furthermore, we show that the disassembly of microtubule polymers elicited by microtubule depolymerizing agents is blocked by increasing intracellular NAD+ levels. We find that these effects of NAD+ are mediated by the activation of the mitochondrial sirtuin sirtuin-3 (SIRT3). Overexpression of SIRT3 prevents microtubule disassembly and apoptosis elicited by antimicrotubule agents and knockdown of SIRT3 prevents the protective effects of NAD+ on microtubule polymers. Taken together, these data demonstrate that NAD+ and SIRT3 regulate microtubule polymerization and the efficacy of antimicrotubule agents. PMID:24889606

Termination of DNA replication forks: "Breaking up is hard to do".

PubMed

Bailey, Rachael; Priego Moreno, Sara; Gambus, Agnieszka

2015-01-01

To ensure duplication of the entire genome, eukaryotic DNA replication initiates from thousands of replication origins. The replication forks move through the chromatin until they encounter forks from neighboring origins. During replication fork termination forks converge, the replisomes disassemble and topoisomerase II resolves the daughter DNA molecules. If not resolved efficiently, terminating forks result in genomic instability through the formation of pathogenic structures. Our recent findings shed light onto the mechanism of replisome disassembly upon replication fork termination. We have shown that termination-specific polyubiquitylation of the replicative helicase component - Mcm7, leads to dissolution of the active helicase in a process dependent on the p97/VCP/Cdc48 segregase. The inhibition of terminating helicase disassembly resulted in a replication termination defect. In this extended view we present hypothetical models of replication fork termination and discuss remaining and emerging questions in the DNA replication termination field.

[Study on the liver-protective and choleretic effect of zhizi baipi soup and its disassembled prescription].

PubMed

Xiao, Xu; Zhu, Ji-Xiao; Luo, Guang-Ming; Li, Lei; Zhu, Yu-Ye; Zeng, Jin-Xiang; Wang, Xiao-Yun; Wu, Bo

2013-07-01

To investigate the effect of Zhizi Baipi soup and its disassembled prescription on protecting liver and improving choleresis and explore the regularity of Zhizi Baipi soup composition. The model of mouse liver injury induced by carbon tetraehlofide (CCl4) was used to observe the effects of Zhizi Baipi soup and its disassembled prescription by oral adminstration, the bile volume was determinied by common bile duct drainage. Zhizi Baipi soup and each treatment group with gardenia could significantly inhibit the increased serum ATL and AST activities, reduce liver MDA level, and significantly promote the bile flow and bilirubin in bile in normal rats. Zhizi Baipi soup has effects on protecting liver and increasing bile secretion, its monarch drug, gardenia plays an important role in the decoction, the effect of eliminating dampness and heat are mainly ascribed to the synergic effect of gardenia and phellodendron.

Hepatic translation control in the late-gestation fetal rat.

PubMed

Gruppuso, Philip A; Tsai, Shu-Whei; Boylan, Joan M; Sanders, Jennifer A

2008-08-01

We have investigated the regulation of translation during the period of rapid liver growth that occurs at the end of gestation in the rat. This work was based on our prior observation that fetal hepatocyte proliferation is resistant to the inhibitory effects of rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), a nutrient-sensing kinase that controls ribosome biogenesis and protein translation. We hypothesized that translation control in late-gestation fetal liver differs from that in adult liver. We first examined the ability of rapamycin to inhibit the translation of mRNAs encoding ribosomal proteins. Consistent with the effect of rapamycin on proliferation, the activation of adult liver 5'-terminal oligopyrimidine tracts (5'-TOP) translation that occurred during refeeding after food deprivation was sensitive to rapamycin. Fetal liver 5'-TOP translation was insensitive. We went on to examine the eukaryotic initiation factor (eIF) 4F cap-binding complex that controls global protein synthesis. The molecular weights of the multiple eIF4G1 isoforms present in fetal and adult liver eIF4F complexes differed. In addition, fetal liver expressed the eIF4A1 form of the eIF4A helicase, whereas adult liver contained eIF4A1 and eIF4A2. Rapamycin administration before refeeding in adult rats inhibited formation of the preinitiation complex to a much greater degree than rapamycin administration to fetal rats in situ. We conclude that there are major structural and functional differences in translation control between late-gestation fetal and adult liver. These differences may confer differential sensitivity to the growth inhibitory effects of rapamycin.

Quantitative studies of mRNA recruitment to the eukaryotic ribosome.

PubMed

Fraser, Christopher S

2015-07-01

The process of peptide bond synthesis by ribosomes is conserved between species, but the initiation step differs greatly between the three kingdoms of life. This is illustrated by the evolution of roughly an order of magnitude more initiation factor mass found in humans compared with bacteria. Eukaryotic initiation of translation is comprised of a number of sub-steps: (i) recruitment of an mRNA and initiator methionyl-tRNA to the 40S ribosomal subunit; (ii) migration of the 40S subunit along the 5' UTR to locate the initiation codon; and (iii) recruitment of the 60S subunit to form the 80S initiation complex. Although the mechanism and regulation of initiation has been studied for decades, many aspects of the pathway remain unclear. In this review, I will focus discussion on what is known about the mechanism of mRNA selection and its recruitment to the 40S subunit. I will summarize how the 43S preinitiation complex (PIC) is formed and stabilized by interactions between its components. I will discuss what is known about the mechanism of mRNA selection by the eukaryotic initiation factor 4F (eIF4F) complex and how the selected mRNA is recruited to the 43S PIC. The regulation of this process by secondary structure located in the 5' UTR of an mRNA will also be discussed. Finally, I present a possible kinetic model with which to explain the process of mRNA selection and recruitment to the eukaryotic ribosome. Copyright © 2015 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

Core Mediator structure at 3.4 Å extends model of transcription initiation complex.

PubMed

Nozawa, Kayo; Schneider, Thomas R; Cramer, Patrick

2017-05-11

Mediator is a multiprotein co-activator that binds the transcription pre-initiation complex (PIC) and regulates RNA polymerase (Pol) II. The Mediator head and middle modules form the essential core Mediator (cMed), whereas the tail and kinase modules play regulatory roles. The architecture of Mediator and its position on the PIC are known, but atomic details are limited to Mediator subcomplexes. Here we report the crystal structure of the 15-subunit cMed from Schizosaccharomyces pombe at 3.4 Å resolution. The structure shows an unaltered head module, and reveals the intricate middle module, which we show is globally required for transcription. Sites of known Mediator mutations cluster at the interface between the head and middle modules, and in terminal regions of the head subunits Med6 (ref. 16) and Med17 (ref. 17) that tether the middle module. The structure led to a model for Saccharomyces cerevisiae cMed that could be combined with the 3.6 Å cryo-electron microscopy structure of the core PIC (cPIC). The resulting atomic model of the cPIC-cMed complex informs on interactions of the submodules forming the middle module, called beam, knob, plank, connector, and hook. The hook is flexibly linked to Mediator by a conserved hinge and contacts the transcription initiation factor IIH (TFIIH) kinase that phosphorylates the carboxy (C)-terminal domain (CTD) of Pol II and was recently positioned on the PIC. The hook also contains residues that crosslink to the CTD and reside in a previously described cradle. These results provide a framework for understanding Mediator function, including its role in stimulating CTD phosphorylation by TFIIH.

Using three dimensional silicone ``boots`` to solve complex remedial design problems in curtain walls

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Y.J.

1998-12-31

Stick system curtain wall leak problems are frequently caused by water entry at the splice joints of the curtain wall frame and failure of the internal metal joinery seals. Remedial solutions involving occupied buildings inevitably face the multiple constraints of existing construction and business operations not present during the original curtain wall construction. In most cases, even partial disassembly of the curtain wall for internal seal repairs is not feasible. Remedial solutions which must be executed from the exterior of the curtain wall often involve wet-applied or preformed sealant tape bridge joints. However, some of the more complex joints cannotmore » be repaired effectively or economically with the conventional bridge joint. Fortunately, custom fabricated three-dimensional preformed sealant boots are becoming available to address these situations. This paper discusses the design considerations and the selective use of three-dimensional preformed boots in sealing complex joint geometry that would not be effective with the conventional two-dimensional bridge joint.« less

Active turnover regulates pattern formation and stress transmission in disordered acto-myosin networks

NASA Astrophysics Data System (ADS)

McCall, Patrick; Stam, Samantha; Kovar, David; Gardel, Margaret

The shape and mechanics of animal cells are controlled by a dynamic, thin network of semiflexible actin filaments and myosin-II motor proteins called the actomyosin cortex. Motor-generated stresses in the cortex drive changes in cell shape during cell division and morphogenesis, while dynamic turnover of actin filaments dissipates stress. The relative effects that force generation, force dissipation, and disassembly and reassembly of material have on motion in these networks are unknown. We find that cross-linked actin networks in vitro contract under myosin-generated stresses, resulting in partial filament disassembly, the formation of asters, and clustering of myosin motors. We observe a rapid restoration of uniform polymer density in the presence of the assembly factors which catalyze network turnover through elongation of severed actin filaments. When severing is accelerated further by the addition of a severing protein, network contraction and motor clustering are dramatically suppressed. We test the relative effects of material regeneration and force transmission using image analysis, and conclude that the dominant mechanism for this effect is relatively short-lived stresses that do not propagate over considerable distance or push network deformation into the nonlinear contractile regime we have previously characterized. Our results present a framework to understand cytoskeletal active matter that are influenced by a complex interplay between stress generation, network reorganization, and polymer turnover.

A Taz1- and Microtubule-Dependent Regulatory Relationship between Telomere and Centromere Positions in Bouquet Formation Secures Proper Meiotic Divisions

PubMed Central

Katsumata, Kazuhiro; Hirayasu, Ami; Miyoshi, Junpei; Nishi, Eriko; Ichikawa, Kento; Tateho, Kazuki; Wakuda, Airi; Matsuhara, Hirotada; Yamamoto, Ayumu

2016-01-01

During meiotic prophase, telomeres cluster, forming the bouquet chromosome arrangement, and facilitate homologous chromosome pairing. In fission yeast, bouquet formation requires switching of telomere and centromere positions. Centromeres are located at the spindle pole body (SPB) during mitotic interphase, and upon entering meiosis, telomeres cluster at the SPB, followed by centromere detachment from the SPB. Telomere clustering depends on the formation of the microtubule-organizing center at telomeres by the linker of nucleoskeleton and cytoskeleton complex (LINC), while centromere detachment depends on disassembly of kinetochores, which induces meiotic centromere formation. However, how the switching of telomere and centromere positions occurs during bouquet formation is not fully understood. Here, we show that, when impaired telomere interaction with the LINC or microtubule disruption inhibited telomere clustering, kinetochore disassembly-dependent centromere detachment and accompanying meiotic centromere formation were also inhibited. Efficient centromere detachment required telomere clustering-dependent SPB recruitment of a conserved telomere component, Taz1, and microtubules. Furthermore, when artificial SPB recruitment of Taz1 induced centromere detachment in telomere clustering-defective cells, spindle formation was impaired. Thus, detachment of centromeres from the SPB without telomere clustering causes spindle impairment. These findings establish novel regulatory mechanisms, which prevent concurrent detachment of telomeres and centromeres from the SPB during bouquet formation and secure proper meiotic divisions. PMID:27611693

27. INTERIOR VIEW TO THE WEST OF ROOM 126 AT ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

27. INTERIOR VIEW TO THE WEST OF ROOM 126 AT THE NORTH END OF THE ENTRANCE HALLWAY TO THE POST-MORTEM CELLS. IN THE CEILING IS A HATCHWAY TO THE UPPER LEVEL OF ROOM 123, THE DISASSEMBLY BAY, BY WHICH PARTS OF THE NUCLEAR REACTOR WERE PASSED FOR FURTHER DISASSEMBLY IN THE VARIOUS POST-MORTEM CELLS. - Nevada Test Site, Reactor Maintenance Assembly & Dissassembly Facility, Area 25, Jackass Flats, Junction of Roads F & G, Mercury, Nye County, NV

How do robots take two parts apart

NASA Technical Reports Server (NTRS)

Bajcsy, Ruzena K.; Tsikos, Constantine J.

1989-01-01

This research is a natural progression of efforts which begun with the introduction of a new research paradigm in machine perception, called Active Perception. There it was stated that Active Perception is a problem of intelligent control strategies applied to data acquisition processes which will depend on the current state of the data interpretation, including recognition. The disassembly/assembly problem is treated as an Active Perception problem, and a method for autonomous disassembly based on this framework is presented.

An Efficient Procedure for Removal and Inactivation of Alpha-Synuclein Assemblies from Laboratory Materials

PubMed Central

Bousset, Luc; Brundin, Patrik; Böckmann, Anja; Meier, Beat; Melki, Ronald

2015-01-01

Background: Preformed α-synuclein fibrils seed the aggregation of soluble α-synuclein in cultured cells and in vivo. This, and other findings, has kindled the idea that α-synuclein fibrils possess prion-like properties. Objective: As α-synuclein fibrils should not be considered as innocuous, there is a need for decontamination and inactivation procedures for laboratory benches and non-disposable laboratory material. Methods: We assessed the effectiveness of different procedures designed to disassemble α-synuclein fibrils and reduce their infectivity. We examined different commercially available detergents to remove α-synuclein assemblies adsorbed on materials that are not disposable and that are most found in laboratories (e.g. plastic, glass, aluminum or stainless steel surfaces). Results: We show that methods designed to decrease PrP prion infectivity neither effectively remove α-synuclein assemblies adsorbed to different materials commonly used in the laboratory nor disassemble the fibrillar form of the protein with efficiency. In contrast, both commercial detergents and SDS detached α-synuclein assemblies from contaminated surfaces and disassembled the fibrils. Conclusions: We describe three cleaning procedures that effectively remove and disassemble α-synuclein seeds. The methods rely on the use of detergents that are compatible with most non-disposable tools in a laboratory. The procedures are easy to implement and significantly decrease any potential risks associated to handling α-synuclein assemblies. PMID:26639448

Asbestos exposure from gaskets during disassembly of a medium duty diesel engine.

PubMed

Liukonen, Larry R; Weir, Francis W

2005-03-01

Diesel engines have historically used asbestos-containing gaskets leading to concerns of fiber release and mechanic exposure. Other published studies regarding asbestos fiber release during gasket removal have reported on short-duration events; were conducted under simulated work conditions; or had other limitations. There are no comprehensive studies relating to diesel engine gaskets under conditions similar to those reported herein, evaluating asbestos fiber release from gaskets during all facets of a complete disassembly and cleaning of a medium duty diesel engine in a busy repair and service shop by a journeyman mechanic. Asbestos content of all gaskets was identified; all disassembly tasks were described and timed; and personal and area air monitoring was conducted for each task. Twenty seven of thirty three gaskets contained chrysotile asbestos in concentrations that ranged from 5 to 70%. All but one air monitoring sample reported results below the limit of reliable detection even though plumes of visible dust were evident during various removal, cleaning, and buffing procedures. The detection limit for airborne asbestos fibers in this investigation was influenced by the presence of other shop dust in the air. Our investigation demonstrates that using shop-standard procedures in an established repair facility, a journeyman mechanic has very little potential for exposure to airborne asbestos fibers during disassembly of an engine, approximately 10% or less than that currently considered to be acceptable by OSHA.

HcRed, a Genetically Encoded Fluorescent Binary Cross-Linking Agent for Cross-Linking of Mitochondrial ATP Synthase in Saccharomyces cerevisiae

PubMed Central

Gong, Lan; Ramm, Georg; Devenish, Rodney J.; Prescott, Mark

2012-01-01

Genetically encoded fluorescent cross-linking agents represent powerful tools useful both for visualising and modulating protein interactions in living cells. The far-red fluorescent protein HcRed, which is fluorescent only in a dimer form, can be used to promote the homo-dimerisation of target proteins, and thereby yield useful information about biological processes. We have in yeast cells expressed HcRed fused to a subunit of mitochondrial ATP synthase (mtATPase). This resulted in cross-linking of the large multi-subunit mtATPase complex within the inner-membrane of the mitochondrion. Fluorescence microscopy revealed aberrant mitochondrial morphology, and mtATPase complexes isolated from mitochondria were recovered as fluorescent dimers under conditions where complexes from control mitochondria were recovered as monomers. When viewed by electron microscopy normal cristae were absent from mitochondria in cells in which mATPase complexes were cross-linked. mtATPase dimers are believed to be the building blocks that are assembled into supramolecular mtATPase ribbons that promote the formation of mitochondrial cristae. We propose that HcRed cross-links mATPase complexes in the mitochondrial membrane hindering the normal assembly/disassembly of the supramolecular forms of mtATPase. PMID:22496895

Disassembly of synthetic Agrobacterium T-DNA–protein complexes via the host SCFVBF ubiquitin–ligase complex pathway

PubMed Central

Zaltsman, Adi; Lacroix, Benoît; Gafni, Yedidya; Citovsky, Vitaly

2013-01-01

One the most intriguing, yet least studied, aspects of the bacterium–host plant interaction is the role of the host ubiquitin/proteasome system (UPS) in the infection process. Increasing evidence indicates that pathogenic bacteria subvert the host UPS to facilitate infection. Although both mammalian and plant bacterial pathogens are known to use the host UPS, the first prokaryotic F-box protein, an essential component of UPS, was identified in Agrobacterium. During its infection, which culminates in genetic modification of the host cell, Agrobacterium transfers its T-DNA—as a complex (T-complex) with the bacterial VirE2 and host VIP1 proteins—into the host cell nucleus. There the T-DNA is uncoated from its protein components before undergoing integration into the host genome. It has been suggested that the host UPS mediates this uncoating process, but there is no evidence indicating that this activity can unmask the T-DNA molecule. Here we provide support for the idea that the plant UPS uncoats synthetic T-complexes via the Skp1/Cullin/F-box protein VBF pathway and exposes the T-DNA molecule to external enzymatic activity. PMID:23248273

Co-assembly, spatiotemporal control and morphogenesis of a hybrid protein-peptide system.

PubMed

Inostroza-Brito, Karla E; Collin, Estelle; Siton-Mendelson, Orit; Smith, Katherine H; Monge-Marcet, Amàlia; Ferreira, Daniela S; Rodríguez, Raúl Pérez; Alonso, Matilde; Rodríguez-Cabello, José Carlos; Reis, Rui L; Sagués, Francesc; Botto, Lorenzo; Bitton, Ronit; Azevedo, Helena S; Mata, Alvaro

2015-11-01

Controlling molecular interactions between bioinspired molecules can enable the development of new materials with higher complexity and innovative properties. Here we report on a dynamic system that emerges from the conformational modification of an elastin-like protein by peptide amphiphiles and with the capacity to access, and be maintained in, non-equilibrium for substantial periods of time. The system enables the formation of a robust membrane that displays controlled assembly and disassembly capabilities, adhesion and sealing to surfaces, self-healing and the capability to undergo morphogenesis into tubular structures with high spatiotemporal control. We use advanced microscopy along with turbidity and spectroscopic measurements to investigate the mechanism of assembly and its relation to the distinctive membrane architecture and the resulting dynamic properties. Using cell-culture experiments with endothelial and adipose-derived stem cells, we demonstrate the potential of this system to generate complex bioactive scaffolds for applications such as tissue engineering.

Co-assembly, spatiotemporal control and morphogenesis of a hybrid protein-peptide system

NASA Astrophysics Data System (ADS)

Inostroza-Brito, Karla E.; Collin, Estelle; Siton-Mendelson, Orit; Smith, Katherine H.; Monge-Marcet, Amàlia; Ferreira, Daniela S.; Rodríguez, Raúl Pérez; Alonso, Matilde; Rodríguez-Cabello, José Carlos; Reis, Rui L.; Sagués, Francesc; Botto, Lorenzo; Bitton, Ronit; Azevedo, Helena S.; Mata, Alvaro

2015-11-01

Controlling molecular interactions between bioinspired molecules can enable the development of new materials with higher complexity and innovative properties. Here we report on a dynamic system that emerges from the conformational modification of an elastin-like protein by peptide amphiphiles and with the capacity to access, and be maintained in, non-equilibrium for substantial periods of time. The system enables the formation of a robust membrane that displays controlled assembly and disassembly capabilities, adhesion and sealing to surfaces, self-healing and the capability to undergo morphogenesis into tubular structures with high spatiotemporal control. We use advanced microscopy along with turbidity and spectroscopic measurements to investigate the mechanism of assembly and its relation to the distinctive membrane architecture and the resulting dynamic properties. Using cell-culture experiments with endothelial and adipose-derived stem cells, we demonstrate the potential of this system to generate complex bioactive scaffolds for applications such as tissue engineering.

Rapid Inspection of Aerospace Structures - Is It Autonomous Yet?

NASA Technical Reports Server (NTRS)

Bar-Cohen, Yoseph; Backes, Paul; Joffe, Benjamin

1996-01-01

The trend to increase the usage of aging aircraft added a great deal of urgency to the ongoing need for low-cost, rapid, simple-to-operate, reliable and efficient NDE methods for detection and characterization of flaws in aircraft structures. In many cases, the problem of inspection is complex due to the limitation of current technology and the need to disassemble aircraft structures and testing them in lab conditions. To overcome these limitations, reliable field inspection tools are being developed for rapid NDE of large and complex-shape structures, that can operate at harsh, hostal and remote conditions with minimum human interface. In recent years, to address the need for rapid inspection in field conditions, numerous portable scanners were developed using NDE methods, including ultrasonics, shearography, thermography. This paper is written with emphasis on ultrasonic NDE scanners, their evolution and the expected direction of growth.

Protoparvovirus Knocking at the Nuclear Door.

PubMed

Mäntylä, Elina; Kann, Michael; Vihinen-Ranta, Maija

2017-10-02

Protoparvoviruses target the nucleus due to their dependence on the cellular reproduction machinery during the replication and expression of their single-stranded DNA genome. In recent years, our understanding of the multistep process of the capsid nuclear import has improved, and led to the discovery of unique viral nuclear entry strategies. Preceded by endosomal transport, endosomal escape and microtubule-mediated movement to the vicinity of the nuclear envelope, the protoparvoviruses interact with the nuclear pore complexes. The capsids are transported actively across the nuclear pore complexes using nuclear import receptors. The nuclear import is sometimes accompanied by structural changes in the nuclear envelope, and is completed by intranuclear disassembly of capsids and chromatinization of the viral genome. This review discusses the nuclear import strategies of protoparvoviruses and describes its dynamics comprising active and passive movement, and directed and diffusive motion of capsids in the molecularly crowded environment of the cell.

Inter-ring rotations of AAA ATPase p97 revealed by electron cryomicroscopy

PubMed Central

Yeung, Heidi O.; Förster, Andreas; Bebeacua, Cecilia; Niwa, Hajime; Ewens, Caroline; McKeown, Ciarán; Zhang, Xiaodong; Freemont, Paul S.

2014-01-01

The type II AAA+ protein p97 is involved in numerous cellular activities, including endoplasmic reticulum-associated degradation, transcription activation, membrane fusion and cell-cycle control. These activities are at least in part regulated by the ubiquitin system, in which p97 is thought to target ubiquitylated protein substrates within macromolecular complexes and assist in their extraction or disassembly. Although ATPase activity is essential for p97 function, little is known about how ATP binding or hydrolysis is coupled with p97 conformational changes and substrate remodelling. Here, we have used single-particle electron cryomicroscopy (cryo-EM) to study the effect of nucleotides on p97 conformation. We have identified conformational heterogeneity within the cryo-EM datasets from which we have resolved two major p97 conformations. A comparison of conformations reveals inter-ring rotations upon nucleotide binding and hydrolysis that may be linked to the remodelling of target protein complexes. PMID:24598262

CALUTRON ASSEMBLING AND DISASSEMBLING MEANS

DOEpatents

Andrews, R.E.; Thornton, J.

1959-01-27

This patent relates to the assembling and disassembling of a calutron and, more specifically describes a calutron having the ion separating mechanism carried by a fuce plate removably secured to the tank. When it is desired to withdraw the ion separating mechanism from the tank, a motor is energized and a carriage attached through a bracket to the fuce plate is driven along a track. The face plate moves out from the tank in substantially a linear direction, preventing injury to the ion separating mechanism.

On the nature and shape of tubulin trails: implications on microtubule self-organization.

PubMed

Glade, Nicolas

2012-06-01

Microtubules, major elements of the cell skeleton are, most of the time, well organized in vivo, but they can also show self-organizing behaviors in time and/or space in purified solutions in vitro. Theoretical studies and models based on the concepts of collective dynamics in complex systems, reaction-diffusion processes and emergent phenomena were proposed to explain some of these behaviors. In the particular case of microtubule spatial self-organization, it has been advanced that microtubules could behave like ants, self-organizing by 'talking to each other' by way of hypothetic (because never observed) concentrated chemical trails of tubulin that are expected to be released by their disassembling ends. Deterministic models based on this idea yielded indeed like-looking spatio-temporal self-organizing behaviors. Nevertheless the question remains of whether microscopic tubulin trails produced by individual or bundles of several microtubules are intense enough to allow microtubule self-organization at a macroscopic level. In the present work, by simulating the diffusion of tubulin in microtubule solutions at the microscopic scale, we measure the shape and intensity of tubulin trails and discuss about the assumption of microtubule self-organization due to the production of chemical trails by disassembling microtubules. We show that the tubulin trails produced by individual microtubules or small microtubule arrays are very weak and not elongated even at very high reactive rates. Although the variations of concentration due to such trails are not significant compared to natural fluctuations of the concentration of tubuline in the chemical environment, the study shows that heterogeneities of biochemical composition can form due to microtubule disassembly. They could become significant when produced by numerous microtubule ends located in the same place. Their possible formation could play a role in certain conditions of reaction. In particular, it gives a mesoscopic basis to explain the collective dynamics observed in excitable microtubule solutions showing the propagation of concentration waves of microtubules at the millimeter scale, although we doubt that individual microtubules or bundles can behave like molecular ants.

Epigenetic modification of histone 3 lysine 27: mediator subunit MED25 is required for the dissociation of polycomb repressive complex 2 from the promoter of cytochrome P450 2C9.

PubMed

Englert, Neal A; Luo, George; Goldstein, Joyce A; Surapureddi, Sailesh

2015-01-23

The Mediator complex is vital for the transcriptional regulation of eukaryotic genes. Mediator binds to nuclear receptors at target response elements and recruits chromatin-modifying enzymes and RNA polymerase II. Here, we examine the involvement of Mediator subunit MED25 in the epigenetic regulation of human cytochrome P450 2C9 (CYP2C9). MED25 is recruited to the CYP2C9 promoter through association with liver-enriched HNF4α, and we show that MED25 influences the H3K27 status of the HNF4α binding region. This region was enriched for the activating marker H3K27ac and histone acetyltransferase CREBBP after MED25 overexpression but was trimethylated when MED25 expression was silenced. The epigenetic regulator Polycomb repressive complex (PRC2), which represses expression by methylating H3K27, plays an important role in target gene regulation. Silencing MED25 correlated with increased association of PRC2 not only with the promoter region chromatin but with HNF4α itself. We confirmed the involvement of MED25 for fully functional preinitiation complex recruitment and transcriptional output in vitro. Formaldehyde-assisted isolation of regulatory elements (FAIRE) revealed chromatin conformation changes that were reliant on MED25, indicating that MED25 induced a permissive chromatin state that reflected increases in CYP2C9 mRNA. For the first time, we showed evidence that a functionally relevant human gene is transcriptionally regulated by HNF4α via MED25 and PRC2. CYP2C9 is important for the metabolism of many exogenous chemicals including pharmaceutical drugs as well as endogenous substrates. Thus, MED25 is important for regulating the epigenetic landscape resulting in transcriptional activation of a highly inducible gene, CYP2C9. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Electron transfer protein complexes in the thylakoid membranes of heterocysts from the cyanobacterium Nostoc punctiforme.

PubMed

Cardona, Tanai; Battchikova, Natalia; Zhang, Pengpeng; Stensjö, Karin; Aro, Eva-Mari; Lindblad, Peter; Magnuson, Ann

2009-04-01

Filamentous, heterocystous cyanobacteria are capable of nitrogen fixation and photoautotrophic growth. Nitrogen fixation takes place in heterocysts that differentiate as a result of nitrogen starvation. Heterocysts uphold a microoxic environment to avoid inactivation of nitrogenase, e.g. by downregulation of oxygenic photosynthesis. The ATP and reductant requirement for the nitrogenase reaction is considered to depend on Photosystem I, but little is known about the organization of energy converting membrane proteins in heterocysts. We have investigated the membrane proteome of heterocysts from nitrogen fixing filaments of Nostoc punctiforme sp. PCC 73102, by 2D gel electrophoresis and mass spectrometry. The membrane proteome was found to be dominated by the Photosystem I and ATP-synthase complexes. We could identify a significant amount of assembled Photosystem II complexes containing the D1, D2, CP43, CP47 and PsbO proteins from these complexes. We could also measure light-driven in vitro electron transfer from Photosystem II in heterocyst thylakoid membranes. We did not find any partially disassembled Photosystem II complexes lacking the CP43 protein. Several subunits of the NDH-1 complex were also identified. The relative amount of NDH-1M complexes was found to be higher than NDH-1L complexes, which might suggest a role for this complex in cyclic electron transfer in the heterocysts of Nostoc punctiforme.

Single-Molecule Studies of Actin Assembly and Disassembly Factors

PubMed Central

Smith, Benjamin A.; Gelles, Jeff; Goode, Bruce L.

2014-01-01

The actin cytoskeleton is very dynamic and highly regulated by multiple associated proteins in vivo. Understanding how this system of proteins functions in the processes of actin network assembly and disassembly requires methods to dissect the mechanisms of activity of individual factors and of multiple factors acting in concert. The advent of single-filament and single-molecule fluorescence imaging methods has provided a powerful new approach to discovering actin-regulatory activities and obtaining direct, quantitative insights into the pathways of molecular interactions that regulate actin network architecture and dynamics. Here we describe techniques for acquisition and analysis of single-molecule data, applied to the novel challenges of studying the filament assembly and disassembly activities of actin-associated proteins in vitro. We discuss the advantages of single-molecule analysis in directly visualizing the order of molecular events, measuring the kinetic rates of filament binding and dissociation, and studying the coordination among multiple factors. The methods described here complement traditional biochemical approaches in elucidating actin-regulatory mechanisms in reconstituted filamentous networks. PMID:24630103

Characterization of the disassembly and reassembly of the HBV glycoprotein surface antigen, a pliable nanoparticle vaccine platform

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gallagher, John R.; Torian, Udana; McCraw, Dustin

While nanoparticle vaccine technology is gaining interest due to the success of vaccines like those for the human papillomavirus that is based on viral capsid nanoparticles, little information is available on the disassembly and reassembly of viral surface glycoprotein-based nanoparticles. One such particle is the hepatitis B virus surface antigen (sAg) that exists as nanoparticles. Here we show, using biochemical analysis coupled with electron microscopy, that sAg nanoparticle disassembly requires both reducing agent to disrupt intermolecular disulfide bonds, and detergent to disrupt hydrophobic interactions that stabilize the nanoparticle. Particles were otherwise resistant to salt and urea, suggesting the driving mechanismmore » of particle formation involves hydrophobic interactions. We reassembled isolated sAg protein into nanoparticles by detergent removal and reassembly resulted in a wider distribution of particle diameters. Knowledge of these driving forces of nanoparticle assembly and stability should facilitate construction of epitope-displaying nanoparticles that can be used as immunogens in vaccines.« less

Cofilin-2 controls actin filament length in muscle sarcomeres

PubMed Central

Kremneva, Elena; Makkonen, Maarit H.; Skwarek-Maruszewska, Aneta; Gateva, Gergana; Michelot, Alphee; Dominguez, Roberto; Lappalainen, Pekka

2014-01-01

SUMMARY ADF/cofilins drive cytoskeletal dynamics by promoting the disassembly of ‘aged’ ADP-actin filaments. Mammals express several ADF/cofilin isoforms, but their specific biochemical activities and cellular functions have not been studied in detail. Here we demonstrate that the muscle-specific isoform cofilin-2 promotes actin filament disassembly in sarcomeres to control the precise length of thin filaments in the contractile apparatus. In contrast to other isoforms, cofilin-2 efficiently binds and disassembles both ADP- and ATP/ADP-Pi-actin filaments. We mapped surface-exposed cofilin-2-specific residues required for ATP-actin binding and propose that these residues function as an ‘actin nucleotide-state sensor’ among ADF/cofilins. The results suggest that cofilin-2 evolved specific biochemical and cellular properties allowing it to control actin dynamics in sarcomeres, where filament pointed ends may contain a mixture of ADP- and ATP/ADP-Pi-actin subunits. Our findings also offer a rationale for why cofilin-2 mutations in humans lead to myopathies. PMID:25373779

A disassembly-driven mechanism explains F-actin-mediated chromosome transport in starfish oocytes

PubMed Central

Bun, Philippe; Dmitrieff, Serge; Belmonte, Julio M

2018-01-01

While contraction of sarcomeric actomyosin assemblies is well understood, this is not the case for disordered networks of actin filaments (F-actin) driving diverse essential processes in animal cells. For example, at the onset of meiosis in starfish oocytes a contractile F-actin network forms in the nuclear region transporting embedded chromosomes to the assembling microtubule spindle. Here, we addressed the mechanism driving contraction of this 3D disordered F-actin network by comparing quantitative observations to computational models. We analyzed 3D chromosome trajectories and imaged filament dynamics to monitor network behavior under various physical and chemical perturbations. We found no evidence of myosin activity driving network contractility. Instead, our observations are well explained by models based on a disassembly-driven contractile mechanism. We reconstitute this disassembly-based contractile system in silico revealing a simple architecture that robustly drives chromosome transport to prevent aneuploidy in the large oocyte, a prerequisite for normal embryonic development. PMID:29350616

Characterization of the disassembly and reassembly of the HBV glycoprotein surface antigen, a pliable nanoparticle vaccine platform.

PubMed

Gallagher, John R; Torian, Udana; McCraw, Dustin M; Harris, Audray K

2017-02-01

While nanoparticle vaccine technology is gaining interest due to the success of vaccines like those for the human papillomavirus that is based on viral capsid nanoparticles, little information is available on the disassembly and reassembly of viral surface glycoprotein-based nanoparticles. One such particle is the hepatitis B virus surface antigen (sAg) that exists as nanoparticles. Here we show, using biochemical analysis coupled with electron microscopy, that sAg nanoparticle disassembly requires both reducing agent to disrupt intermolecular disulfide bonds, and detergent to disrupt hydrophobic interactions that stabilize the nanoparticle. Particles were otherwise resistant to salt and urea, suggesting the driving mechanism of particle formation involves hydrophobic interactions. We reassembled isolated sAg protein into nanoparticles by detergent removal and reassembly resulted in a wider distribution of particle diameters. Knowledge of these driving forces of nanoparticle assembly and stability should facilitate construction of epitope-displaying nanoparticles that can be used as immunogens in vaccines. Published by Elsevier Inc.

PslG, a self-produced glycosyl hydrolase, triggers biofilm disassembly by disrupting exopolysaccharide matrix.

PubMed

Yu, Shan; Su, Tiantian; Wu, Huijun; Liu, Shiheng; Wang, Di; Zhao, Tianhu; Jin, Zengjun; Du, Wenbin; Zhu, Mei-Jun; Chua, Song Lin; Yang, Liang; Zhu, Deyu; Gu, Lichuan; Ma, Luyan Z

2015-12-01

Biofilms are surface-associated communities of microorganism embedded in extracellular matrix. Exopolysaccharide is a critical component in the extracellular matrix that maintains biofilm architecture and protects resident biofilm bacteria from antimicrobials and host immune attack. However, self-produced factors that target the matrix exopolysaccharides, are still poorly understood. Here, we show that PslG, a protein involved in the synthesis of a key biofilm matrix exopolysaccharide Psl in Pseudomonas aeruginosa, prevents biofilm formation and disassembles existing biofilms within minutes at nanomolar concentrations when supplied exogenously. The crystal structure of PslG indicates the typical features of an endoglycosidase. PslG mainly disrupts the Psl matrix to disperse bacteria from biofilms. PslG treatment markedly enhances biofilm sensitivity to antibiotics and macrophage cells, resulting in improved biofilm clearance in a mouse implant infection model. Furthermore, PslG shows biofilm inhibition and disassembly activity against a wide range of Pseudomonas species, indicating its great potential in combating biofilm-related complications.

Engineering multifunctional protein nanoparticles by in vitro disassembling and reassembling of heterologous building blocks

NASA Astrophysics Data System (ADS)

Unzueta, Ugutz; Serna, Naroa; Sánchez-García, Laura; Roldán, Mónica; Sánchez-Chardi, Alejandro; Mangues, Ramón; Villaverde, Antonio; Vázquez, Esther

2017-12-01

The engineering of protein self-assembling at the nanoscale allows the generation of functional and biocompatible materials, which can be produced by easy biological fabrication. The combination of cationic and histidine-rich stretches in fusion proteins promotes oligomerization as stable protein-only regular nanoparticles that are composed by a moderate number of building blocks. Among other applications, these materials are highly appealing as tools in targeted drug delivery once empowered with peptidic ligands of cell surface receptors. In this context, we have dissected here this simple technological platform regarding the controlled disassembling and reassembling of the composing building blocks. By applying high salt and imidazole in combination, nanoparticles are disassembled in a process that is fully reversible upon removal of the disrupting agents. By taking this approach, we accomplish here the in vitro generation of hybrid nanoparticles formed by heterologous building blocks. This fact demonstrates the capability to generate multifunctional and/or multiparatopic or multispecific materials usable in nanomedical applications.

Generation of contractile actomyosin bundles depends on mechanosensitive actin filament assembly and disassembly

PubMed Central

Tojkander, Sari; Gateva, Gergana; Husain, Amjad; Krishnan, Ramaswamy; Lappalainen, Pekka

2015-01-01

Adhesion and morphogenesis of many non-muscle cells are guided by contractile actomyosin bundles called ventral stress fibers. While it is well established that stress fibers are mechanosensitive structures, physical mechanisms by which they assemble, align, and mature have remained elusive. Here we show that arcs, which serve as precursors for ventral stress fibers, undergo lateral fusion during their centripetal flow to form thick actomyosin bundles that apply tension to focal adhesions at their ends. Importantly, this myosin II-derived force inhibits vectorial actin polymerization at focal adhesions through AMPK-mediated phosphorylation of VASP, and thereby halts stress fiber elongation and ensures their proper contractility. Stress fiber maturation additionally requires ADF/cofilin-mediated disassembly of non-contractile stress fibers, whereas contractile fibers are protected from severing. Taken together, these data reveal that myosin-derived tension precisely controls both actin filament assembly and disassembly to ensure generation and proper alignment of contractile stress fibers in migrating cells. DOI: http://dx.doi.org/10.7554/eLife.06126.001 PMID:26652273

Generation of contractile actomyosin bundles depends on mechanosensitive actin filament assembly and disassembly.

PubMed

Tojkander, Sari; Gateva, Gergana; Husain, Amjad; Krishnan, Ramaswamy; Lappalainen, Pekka

2015-12-10

Adhesion and morphogenesis of many non-muscle cells are guided by contractile actomyosin bundles called ventral stress fibers. While it is well established that stress fibers are mechanosensitive structures, physical mechanisms by which they assemble, align, and mature have remained elusive. Here we show that arcs, which serve as precursors for ventral stress fibers, undergo lateral fusion during their centripetal flow to form thick actomyosin bundles that apply tension to focal adhesions at their ends. Importantly, this myosin II-derived force inhibits vectorial actin polymerization at focal adhesions through AMPK-mediated phosphorylation of VASP, and thereby halts stress fiber elongation and ensures their proper contractility. Stress fiber maturation additionally requires ADF/cofilin-mediated disassembly of non-contractile stress fibers, whereas contractile fibers are protected from severing. Taken together, these data reveal that myosin-derived tension precisely controls both actin filament assembly and disassembly to ensure generation and proper alignment of contractile stress fibers in migrating cells.

NIa-Pro of Papaya ringspot virus interacts with Carica papaya eukaryotic translation initiation factor 3 subunit G (CpeIF3G).

PubMed

Gao, Le; Tuo, Decai; Shen, Wentao; Yan, Pu; Li, Xiaoying; Zhou, Peng

2015-02-01

The interaction of papaya eukaryotic translation initiation factor 3 subunit G (CpeIF3G) with Papaya ringspot virus (PRSV) NIa-Pro was validated using a bimolecular fluorescence complementation assay in papaya protoplasts based on the previous yeast two-hybrid assay results. The C-terminal (residues 133-239) fragment of PRSV NIa-Pro and the central domain (residues 59-167) of CpeIF3G were required for effective interaction between NIa-Pro and CpeIF3G as shown by a Sos recruitment yeast two-hybrid system with several deletion mutants of NIa-Pro and CpeIF3G. The central domain of CpeIF3G, which contains a C2HC-type zinc finger motif, is required to bind to other eIFs of the translational machinery. In addition, quantitative real-time reverse transcription PCR assay confirmed that PRSV infection leads to a 2- to 4.5-fold up-regulation of CpeIF3G mRNA in papaya. Plant eIF3G is involved in various stress response by enhancing the translation of resistance-related proteins. It is proposed that the NIa-Pro-CpeIF3G interaction may impair translation preinitiation complex assembly of defense proteins and interfere with host defense.

Distinct Annular Oligomers Captured along the Assembly and Disassembly Pathways of Transthyretin Amyloid Protofibrils

PubMed Central

Pires, Ricardo H.; Karsai, Árpád; Saraiva, Maria J.; Damas, Ana M.; Kellermayer, Miklós S. Z.

2012-01-01

Background Defects in protein folding may lead to severe degenerative diseases characterized by the appearance of amyloid fibril deposits. Cytotoxicity in amyloidoses has been linked to poration of the cell membrane that may involve interactions with amyloid intermediates of annular shape. Although annular oligomers have been detected in many amyloidogenic systems, their universality, function and molecular mechanisms of appearance are debated. Methodology/Principal Findings We investigated with high-resolution in situ atomic force microscopy the assembly and disassembly of transthyretin (TTR) amyloid protofibrils formed of the native protein by pH shift. Annular oligomers were the first morphologically distinct intermediates observed in the TTR aggregation pathway. Morphological analysis suggests that they can assemble into a double-stack of octameric rings with a 16±2 nm diameter, and displaying the tendency to form linear structures. According to light scattering data coupled to AFM imaging, annular oligomers appeared to undergo a collapse type of structural transition into spheroid oligomers containing 8–16 monomers. Disassembly of TTR amyloid protofibrils also resulted in the rapid appearance of annular oligomers but with a morphology quite distinct from that observed in the assembly pathway. Conclusions/Significance Our observations indicate that annular oligomers are key dynamic intermediates not only in the assembly but also in the disassembly of TTR protofibrils. The balance between annular and more compact forms of aggregation could be relevant for cytotoxicity in amyloidogenic disorders. PMID:22984597

Chromatin Challenges during DNA Replication: A Systems Representation

PubMed Central

Aladjem, Mirit I.; Weinstein, John N.; Pommier, Yves

2008-01-01

In a recent review, A. Groth and coworkers presented a comprehensive account of nucleosome disassembly in front of a DNA replication fork, assembly behind the replication fork, and the copying of epigenetic information onto the replicated chromatin. Understanding those processes however would be enhanced by a comprehensive graphical depiction analogous to a circuit diagram. Accordingly, we have constructed a molecular interaction map (MIM) that preserves in essentially complete detail the processes described by Groth et al. The MIM organizes and elucidates the information presented by Groth et al. on the complexities of chromatin replication, thereby providing a tool for system-level comprehension of the effects of genetic mutations, altered gene expression, and pharmacologic intervention. PMID:17959828

Self-Assembling Molecular Logic Gates Based on DNA Crossover Tiles.

PubMed

Campbell, Eleanor A; Peterson, Evan; Kolpashchikov, Dmitry M

2017-07-05

DNA-based computational hardware has attracted ever-growing attention due to its potential to be useful in the analysis of complex mixtures of biological markers. Here we report the design of self-assembling logic gates that recognize DNA inputs and assemble into crossover tiles when the output signal is high; the crossover structures disassemble to form separate DNA stands when the output is low. The output signal can be conveniently detected by fluorescence using a molecular beacon probe as a reporter. AND, NOT, and OR logic gates were designed. We demonstrate that the gates can connect to each other to produce other logic functions. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

One-step fabrication of multifunctional micromotors.

PubMed

Gao, Wenlong; Liu, Mei; Liu, Limei; Zhang, Hui; Dong, Bin; Li, Christopher Y

2015-09-07

Although artificial micromotors have undergone tremendous progress in recent years, their fabrication normally requires complex steps or expensive equipment. In this paper, we report a facile one-step method based on an emulsion solvent evaporation process to fabricate multifunctional micromotors. By simultaneously incorporating various components into an oil-in-water droplet, upon emulsification and solidification, a sphere-shaped, asymmetric, and multifunctional micromotor is formed. Some of the attractive functions of this model micromotor include autonomous movement in high ionic strength solution, remote control, enzymatic disassembly and sustained release. This one-step, versatile fabrication method can be easily scaled up and therefore may have great potential in mass production of multifunctional micromotors for a wide range of practical applications.

Safety Discrete Event Models for Holonic Cyclic Manufacturing Systems

NASA Astrophysics Data System (ADS)

Ciufudean, Calin; Filote, Constantin

In this paper the expression “holonic cyclic manufacturing systems” refers to complex assembly/disassembly systems or fork/join systems, kanban systems, and in general, to any discrete event system that transforms raw material and/or components into products. Such a system is said to be cyclic if it provides the same sequence of products indefinitely. This paper considers the scheduling of holonic cyclic manufacturing systems and describes a new approach using Petri nets formalism. We propose an approach to frame the optimum schedule of holonic cyclic manufacturing systems in order to maximize the throughput while minimize the work in process. We also propose an algorithm to verify the optimum schedule.

Regulation of platelet granule exocytosis by S-nitrosylation

PubMed Central

Morrell, Craig N.; Matsushita, Kenji; Chiles, Kelly; Scharpf, Robert B.; Yamakuchi, Munekazu; Mason, Rebecca J. A.; Bergmeier, Wolfgang; Mankowski, Joseph L.; Baldwin, William M.; Faraday, Nauder; Lowenstein, Charles J.

2005-01-01

Nitric oxide (NO) regulates platelet activation by cGMP-dependent mechanisms and by mechanisms that are not completely defined. Platelet activation includes exocytosis of platelet granules, releasing mediators that regulate interactions between platelets, leukocytes, and endothelial cells. Exocytosis is mediated in part by N-ethylmaleimide-sensitive factor (NSF), an ATPase that disassembles complexes of soluble NSF attachment protein receptors. We now demonstrate that NO inhibits exocytosis of dense granules, lysosomal granules, and α-granules from human platelets by S-nitrosylation of NSF. Platelets lacking endothelial NO synthase show increased rolling on venules, increased thrombosis in arterioles, and increased exocytosis in vivo. Regulation of exocytosis is thus a mechanism by which NO regulates thrombosis. PMID:15738422

A Stimulation Function of Synaptotagmin-1 in Ternary SNARE Complex Formation Dependent on Munc18 and Munc13

PubMed Central

Li, Yun; Wang, Shen; Li, Tianzhi; Zhu, Le; Xu, Yuanyuan; Ma, Cong

2017-01-01

The Ca2+ sensor synaptotagmin-1 (Syt1) plays an essential function in synaptic exocytosis. Recently, Syt1 has been implicated in synaptic vesicle priming, a maturation step prior to Ca2+-triggered membrane fusion that is believed to involve formation of the ternary SNARE complex and require priming proteins Munc18-1 and Munc13-1. However, the mechanisms of Syt1 in synaptic vesicle priming are still unclear. In this study, we found that Syt1 stimulates the transition from the Munc18-1/syntaxin-1 complex to the ternary SNARE complex catalyzed by Munc13-1. This stimulation can be further enhanced in a membrane-containing environment. Further, we showed that Syt1, together with Munc18-1 and Munc13-1, stimulates trans ternary SNARE complex formation on membranes in a manner resistant to disassembly factors NSF and α-SNAP. Disruption of a proposed Syt1/SNARE binding interface strongly abrogated the stimulation function of Syt1. Our results suggest that binding of Syt1 to an intermediate SNARE assembly with Munc18-1 and Munc13-1 is critical for the stimulation function of Syt1 in ternary SNARE complex formation, and this stimulation may underlie the priming function of Syt1 in synaptic exocytosis. PMID:28860966

The mRNA cap-binding protein Cbc1 is required for high and timely expression of genes by promoting the accumulation of gene-specific activators at promoters.

PubMed

Li, Tianlu; De Clercq, Nikki; Medina, Daniel A; Garre, Elena; Sunnerhagen, Per; Pérez-Ortín, José E; Alepuz, Paula

2016-02-01

The highly conserved Saccharomyces cerevisiae cap-binding protein Cbc1/Sto1 binds mRNA co-transcriptionally and acts as a key coordinator of mRNA fate. Recently, Cbc1 has also been implicated in transcription elongation and pre-initiation complex (PIC) formation. Previously, we described Cbc1 to be required for cell growth under osmotic stress and to mediate osmostress-induced translation reprogramming. Here, we observe delayed global transcription kinetics in cbc1Δ during osmotic stress that correlates with delayed recruitment of TBP and RNA polymerase II to osmo-induced promoters. Interestingly, we detect an interaction between Cbc1 and the MAPK Hog1, which controls most gene expression changes during osmostress, and observe that deletion of CBC1 delays the accumulation of the activator complex Hot1-Hog1 at osmostress promoters. Additionally, CBC1 deletion specifically reduces transcription rates of highly transcribed genes under non-stress conditions, such as ribosomal protein (RP) genes, while having low impact on transcription of weakly expressed genes. For RP genes, we show that recruitment of the specific activator Rap1, and subsequently TBP, to promoters is Cbc1-dependent. Altogether, our results indicate that binding of Cbc1 to the capped mRNAs is necessary for the accumulation of specific activators as well as PIC components at the promoters of genes whose expression requires high and rapid transcription. Copyright © 2016 Elsevier B.V. All rights reserved.

Elongation factor Ts directly facilitates the formation and disassembly of the Escherichia coli elongation factor Tu·GTP·aminoacyl-tRNA ternary complex.

PubMed

Burnett, Benjamin J; Altman, Roger B; Ferrao, Ryan; Alejo, Jose L; Kaur, Navdep; Kanji, Joshua; Blanchard, Scott C

2013-05-10

Aminoacyl-tRNA (aa-tRNA) enters the ribosome in a ternary complex with the G-protein elongation factor Tu (EF-Tu) and GTP. EF-Tu·GTP·aa-tRNA ternary complex formation and decay rates are accelerated in the presence of the nucleotide exchange factor elongation factor Ts (EF-Ts). EF-Ts directly facilitates the formation and disassociation of ternary complex. This system demonstrates a novel function of EF-Ts. Aminoacyl-tRNA enters the translating ribosome in a ternary complex with elongation factor Tu (EF-Tu) and GTP. Here, we describe bulk steady state and pre-steady state fluorescence methods that enabled us to quantitatively explore the kinetic features of Escherichia coli ternary complex formation and decay. The data obtained suggest that both processes are controlled by a nucleotide-dependent, rate-determining conformational change in EF-Tu. Unexpectedly, we found that this conformational change is accelerated by elongation factor Ts (EF-Ts), the guanosine nucleotide exchange factor for EF-Tu. Notably, EF-Ts attenuates the affinity of EF-Tu for GTP and destabilizes ternary complex in the presence of non-hydrolyzable GTP analogs. These results suggest that EF-Ts serves an unanticipated role in the cell of actively regulating the abundance and stability of ternary complex in a manner that contributes to rapid and faithful protein synthesis.

Actin Cross-link Assembly and Disassembly Mechanics for α-Actinin and Fascin*

PubMed Central

Courson, David S.; Rock, Ronald S.

2010-01-01

Self-assembly of complex structures is commonplace in biology but often poorly understood. In the case of the actin cytoskeleton, a great deal is known about the components that include higher order structures, such as lamellar meshes, filopodial bundles, and stress fibers. Each of these cytoskeletal structures contains actin filaments and cross-linking proteins, but the role of cross-linking proteins in the initial steps of structure formation has not been clearly elucidated. We employ an optical trapping assay to investigate the behaviors of two actin cross-linking proteins, fascin and α-actinin, during the first steps of structure assembly. Here, we show that these proteins have distinct binding characteristics that cause them to recognize and cross-link filaments that are arranged with specific geometries. α-Actinin is a promiscuous cross-linker, linking filaments over all angles. It retains this flexibility after cross-links are formed, maintaining a connection even when the link is rotated. Conversely, fascin is extremely selective, only cross-linking filaments in a parallel orientation. Surprisingly, bundles formed by either protein are extremely stable, persisting for over 0.5 h in a continuous wash. However, using fluorescence recovery after photobleaching and fluorescence decay experiments, we find that the stable fascin population can be rapidly competed away by free fascin. We present a simple avidity model for this cross-link dissociation behavior. Together, these results place constraints on how cytoskeletal structures assemble, organize, and disassemble in vivo. PMID:20551315

Id-1 promotes TGF-{beta}1-induced cell motility through HSP27 activation and disassembly of adherens junction in prostate epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Di Kaijun; Wong, Y.C.; Wang Xianghong

Id-1 (inhibitor of differentiation or DNA binding-1) has been positively associated with cell proliferation, cell cycle progression, and invasiveness during tumorigenesis. In addition, Id-1 has been shown to modulate cellular sensitivity to TGF-{beta}1 (transforming growth factor {beta}1). Here we demonstrate a novel role of Id-1 in promoting TGF-{beta}1-induced cell motility in a non-malignant prostate epithelial cell line, NPTX. We found that Id-1 promoted F-actin stress fiber formation in response to TGF-{beta}1, which was associated with increased cell-substrate adhesion and cell migration in NPTX cells. In addition, this positive effect of Id-1 on TGF-{beta}1-induced cell motility was mediated through activation ofmore » MEK-ERK signaling pathway and subsequent phosphorylation of HSP27 (heat shock protein 27). Furthermore, Id-1 disrupted the adherens junction complex in TGF-{beta}1-treated cells through down-regulation of E-cadherin, redistribution of {beta}-catenin, along with up-regulation of N-cadherin. These lines of evidence reveal a novel tumorigenic role of Id-1 through reorganization of actin cytoskeleton and disassembly of cell-cell adhesion in response to TGF-{beta}1 in human prostate epithelial cells, and suggest that intracellular Id-1 levels might be a determining factor for switching TGF-{beta}1 from a growth inhibitor to a tumor promoter during prostate carcinogenesis.« less

Presynaptic Filament Dynamics in Homologous Recombination and DNA Repair

PubMed Central

Liu, Jie; Ehmsen, Kirk T.; Heyer, Wolf-Dietrich; Morrical, Scott W.

2014-01-01

Homologous Recombination (HR) is an essential genome stability mechanism used for high-fidelity repair of DNA double-strand breaks and for the recovery of stalled or collapsed DNA replication forks. The crucial homology search and DNA strand exchange steps of HR are catalyzed by presynaptic filaments—helical filaments of a recombinase enzyme bound to single-stranded DNA. Presynaptic filaments are fundamentally dynamic structures, the assembly, catalytic turnover, and disassembly of which must be closely coordinated with other elements of the DNA recombination, repair, and replication machinery in order for genome maintenance functions to be effective. Here, we review the major dynamic elements controlling the assembly, activity, and disassembly of presynaptic filaments: some intrinsic such as recombinase ATP binding and hydrolytic activities, others extrinsic such as ssDNA-binding proteins, mediator proteins, and DNA motor proteins. We examine dynamic behavior on multiple levels, including atomic- and filament-level structural changes associated with ATP binding and hydrolysis as evidenced in crystal structures, as well as subunit binding and dissociation events driven by intrinsic and extrinsic factors. We examine the biochemical properties of recombination proteins from four model systems (T4 phage, E. coli, S. cerevisiae, and H. sapiens), demonstrating how their properties are tailored for the context-specific requirements in these diverse species. We propose that the presynaptic filament has evolved to rely on multiple external factors for increased multi-level regulation of HR processes in genomes with greater structural and sequence complexity. PMID:21599536

Unraveling the Fate and Transport of SrEDTA-2 and Sr+2 in Hanford Sediments

NASA Astrophysics Data System (ADS)

Pace, M. N.; Mayes, M. A.; Jardine, P. M.; Mehlhorn, T. L.; Liu, Q. G.; Yin, X. L.

2004-12-01

Accelerated migration of strontium-90 has been observed in the vadose zone beneath the Hanford tank farm. The goal of this paper is to provide an improved understanding of the hydrogeochemical processes that contribute to strontium transport in the far-field Hanford vadose zone. Laboratory scale batch, saturated packed column experiments, and an unsaturated transport experiment in an undisturbed core were conducted to quantify geochemical and hydrological processes controlling Sr+2 and SrEDTA-2 sorption to Hanford flood deposits. After experimentation, the undisturbed core was disassembled and samples were collected from different bedding units as a function of depth. Sequential extractions were then performed on the samples. It has been suggested that organic chelates such as EDTA may be responsible for the accelerated transport of strontium due to the formation of stable anionic complexes. Duplicate batch and column experiments performed with Sr+2 and SrEDTA-2 suggested that the SrEDTA-2 complex was not stable in the presence of soil and rapid dissociation allowed strontium to be transported as a divalent cation. Batch experiments indicated a decrease in sorption with increasing rock:water ratios, whereas saturated packed column experiments indicated equal retardation in columns of different lengths. This difference between the batch and column experiments is primarily due to the difference between equilibrium conditions where dissolution of cations may compete for sorption sites versus flowing conditions where any dissolved cations are flushed through the system minimizing competition for sorption sites. Unsaturated transport in the undisturbed core resulted in significant Sr+2 retardation despite the presence of physical nonequilibrium. Core disassembly and sequential extractions revealed the mass wetness distribution and reactive mineral phases associated with strontium in the core. Overall, results indicated that strontium will most likely be transported through the Hanford far-field vadose zone as a divalent cation.

Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure

PubMed Central

North, Justin A.; Šimon, Marek; Ferdinand, Michelle B.; Shoffner, Matthew A.; Picking, Jonathan W.; Howard, Cecil J.; Mooney, Alex M.; van Noort, John; Poirier, Michael G.; Ottesen, Jennifer J.

2014-01-01

Nucleosomes contain ∼146 bp of DNA wrapped around a histone protein octamer that controls DNA accessibility to transcription and repair complexes. Posttranslational modification (PTM) of histone proteins regulates nucleosome function. To date, only modest changes in nucleosome structure have been directly attributed to histone PTMs. Histone residue H3(T118) is located near the nucleosome dyad and can be phosphorylated. This PTM destabilizes nucleosomes and is implicated in the regulation of transcription and repair. Here, we report gel electrophoretic mobility, sucrose gradient sedimentation, thermal disassembly, micrococcal nuclease digestion and atomic force microscopy measurements of two DNA–histone complexes that are structurally distinct from nucleosomes. We find that H3(T118ph) facilitates the formation of a nucleosome duplex with two DNA molecules wrapped around two histone octamers, and an altosome complex that contains one DNA molecule wrapped around two histone octamers. The nucleosome duplex complex forms within short ∼150 bp DNA molecules, whereas altosomes require at least ∼250 bp of DNA and form repeatedly along 3000 bp DNA molecules. These results are the first report of a histone PTM significantly altering the nucleosome structure. PMID:24561803

Transcription-Coupled Repair and Complex Biology.

PubMed

Portman, James R; Strick, Terence R

2018-05-04

All active living organisms mitigate DNA damage via DNA repair, and the so-called nucleotide excision repair pathway (NER) represents a functionally major part of the cell's DNA repair repertoire [1]. In this pathway, the damaged strand of DNA is incised and removed before being resynthesized. This form of DNA repair requires a multitude of proteins working in a complex choreography. Repair thus typically involves detection of a DNA lesion; validation of that detection event; search for an appropriate incision site and subsequent DNA incision; DNA unwinding/removal; and DNA resynthesis and religation. These activities are ultimately the result of molecules randomly diffusing and bumping into each other and acting in succession. It is also true however that repair components are often assembled into functional complexes which may be more efficient or regular in their mode of action. Studying DNA repair complexes for their mechanisms of assembly, action, and disassembly can help address fundamental questions such as whether DNA repair pathways are branched or linear; whether for instance they tolerate fluctuations in numbers of components; and more broadly how search processes between macromolecules take place or can be enhanced. Copyright © 2018. Published by Elsevier Ltd.

QIL1 is a novel mitochondrial protein required for MICOS complex stability and cristae morphology.

PubMed

Guarani, Virginia; McNeill, Elizabeth M; Paulo, Joao A; Huttlin, Edward L; Fröhlich, Florian; Gygi, Steven P; Van Vactor, David; Harper, J Wade

2015-05-21

The mitochondrial contact site and cristae junction (CJ) organizing system (MICOS) dynamically regulate mitochondrial membrane architecture. Through systematic proteomic analysis of human MICOS, we identified QIL1 (C19orf70) as a novel conserved MICOS subunit. QIL1 depletion disrupted CJ structure in cultured human cells and in Drosophila muscle and neuronal cells in vivo. In human cells, mitochondrial disruption correlated with impaired respiration. Moreover, increased mitochondrial fragmentation was observed upon QIL1 depletion in flies. Using quantitative proteomics, we show that loss of QIL1 resulted in MICOS disassembly with the accumulation of a MIC60-MIC19-MIC25 sub-complex and degradation of MIC10, MIC26, and MIC27. Additionally, we demonstrated that in QIL1-depleted cells, overexpressed MIC10 fails to significantly restore its interaction with other MICOS subunits and SAMM50. Collectively, our work uncovers a previously unrecognized subunit of the MICOS complex, necessary for CJ integrity, cristae morphology, and mitochondrial function and provides a resource for further analysis of MICOS architecture.

QIL1 is a novel mitochondrial protein required for MICOS complex stability and cristae morphology

PubMed Central

Guarani, Virginia; McNeill, Elizabeth M; Paulo, Joao A; Huttlin, Edward L; Fröhlich, Florian; Gygi, Steven P; Van Vactor, David; Harper, J Wade

2015-01-01

The mitochondrial contact site and cristae junction (CJ) organizing system (MICOS) dynamically regulate mitochondrial membrane architecture. Through systematic proteomic analysis of human MICOS, we identified QIL1 (C19orf70) as a novel conserved MICOS subunit. QIL1 depletion disrupted CJ structure in cultured human cells and in Drosophila muscle and neuronal cells in vivo. In human cells, mitochondrial disruption correlated with impaired respiration. Moreover, increased mitochondrial fragmentation was observed upon QIL1 depletion in flies. Using quantitative proteomics, we show that loss of QIL1 resulted in MICOS disassembly with the accumulation of a MIC60-MIC19-MIC25 sub-complex and degradation of MIC10, MIC26, and MIC27. Additionally, we demonstrated that in QIL1-depleted cells, overexpressed MIC10 fails to significantly restore its interaction with other MICOS subunits and SAMM50. Collectively, our work uncovers a previously unrecognized subunit of the MICOS complex, necessary for CJ integrity, cristae morphology, and mitochondrial function and provides a resource for further analysis of MICOS architecture. DOI: http://dx.doi.org/10.7554/eLife.06265.001 PMID:25997101

Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis

PubMed Central

Singh, Prafull Kumar; Roukounakis, Aristomenis; Frank, Daniel O.; Kirschnek, Susanne; Das, Kushal Kumar; Neumann, Simon; Madl, Josef; Römer, Winfried; Zorzin, Carina; Borner, Christoph; Haimovici, Aladin; Garcia-Saez, Ana; Weber, Arnim; Häcker, Georg

2017-01-01

The Bcl-2 family protein Bim triggers mitochondrial apoptosis. Bim is expressed in nonapoptotic cells at the mitochondrial outer membrane, where it is activated by largely unknown mechanisms. We found that Bim is regulated by formation of large protein complexes containing dynein light chain 1 (DLC1). Bim rapidly inserted into cardiolipin-containing membranes in vitro and recruited DLC1 to the membrane. Bim binding to DLC1 induced the formation of large Bim complexes on lipid vesicles, on isolated mitochondria, and in intact cells. Native gel electrophoresis and gel filtration showed Bim-containing mitochondrial complexes of several hundred kilodaltons in all cells tested. Bim unable to form complexes was consistently more active than complexed Bim, which correlated with its substantially reduced binding to anti-apoptotic Bcl-2 proteins. At endogenous levels, Bim surprisingly bound only anti-apoptotic Mcl-1 but not Bcl-2 or Bcl-XL, recruiting only Mcl-1 into large complexes. Targeting of DLC1 by RNAi in human cell lines induced disassembly of Bim–Mcl-1 complexes and the proteasomal degradation of Mcl-1 and sensitized the cells to the Bcl-2/Bcl-XL inhibitor ABT-737. Regulation of apoptosis at mitochondria thus extends beyond the interaction of monomers of proapoptotic and anti-apoptotic Bcl-2 family members but involves more complex structures of proteins at the mitochondrial outer membrane, and targeting complexes may be a novel therapeutic strategy. PMID:28982759

Mucin-mediated nanocarrier disassembly for triggered uptake of oligonucleotides as a delivery strategy for the potential treatment of mucosal tumours

NASA Astrophysics Data System (ADS)

Martirosyan, A.; Olesen, M. J.; Fenton, R. A.; Kjems, J.; Howard, K. A.

2016-06-01

This work demonstrates gastric mucin-triggered nanocarrier disassembly for release of antisense oligonucleotides and consequent unassisted cellular entry as a novel oral delivery strategy. A fluorescence activation-based reporter system was used to investigate the interaction and mucin-mediated disassembly of chitosan-based nanocarriers containing a 13-mer DNA oligonucleotide with a flanked locked RNA nucleic acid gapmer design. Gastric mucins were shown to trigger gapmer release from nanocarriers that was dependent on the interaction time, mucin concentration and N : P ratio with a maximal release at N : P 10. In contrast to siRNA, naked gapmers exhibited uptake into mucus producing HT-MTX mono-cultures and HT-MTX co-cultured with the carcinoma epithelial cell line Caco-2. Importantly, in vivo gapmer uptake was observed in epithelial tissue 30 min post-injection in murine intestinal loops. The findings present a mucosal design-based system tailored for local delivery of oligonucleotides that may maximize the effectiveness of gene silencing therapeutics within tumours at mucosal sites.This work demonstrates gastric mucin-triggered nanocarrier disassembly for release of antisense oligonucleotides and consequent unassisted cellular entry as a novel oral delivery strategy. A fluorescence activation-based reporter system was used to investigate the interaction and mucin-mediated disassembly of chitosan-based nanocarriers containing a 13-mer DNA oligonucleotide with a flanked locked RNA nucleic acid gapmer design. Gastric mucins were shown to trigger gapmer release from nanocarriers that was dependent on the interaction time, mucin concentration and N : P ratio with a maximal release at N : P 10. In contrast to siRNA, naked gapmers exhibited uptake into mucus producing HT-MTX mono-cultures and HT-MTX co-cultured with the carcinoma epithelial cell line Caco-2. Importantly, in vivo gapmer uptake was observed in epithelial tissue 30 min post-injection in murine intestinal loops. The findings present a mucosal design-based system tailored for local delivery of oligonucleotides that may maximize the effectiveness of gene silencing therapeutics within tumours at mucosal sites. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr07206a

Salt Stress–Induced Disassembly of Arabidopsis Cortical Microtubule Arrays Involves 26S Proteasome–Dependent Degradation of SPIRAL1[C][W

PubMed Central

Wang, Songhu; Kurepa, Jasmina; Hashimoto, Takashi; Smalle, Jan A.

2011-01-01

The dynamic instability of cortical microtubules (MTs) (i.e., their ability to rapidly alternate between phases of growth and shrinkage) plays an essential role in plant growth and development. In addition, recent studies have revealed a pivotal role for dynamic instability in the response to salt stress conditions. The salt stress response includes a rapid depolymerization of MTs followed by the formation of a new MT network that is believed to be better suited for surviving high salinity. Although this initial depolymerization response is essential for the adaptation to salt stress, the underlying molecular mechanism has remained largely unknown. Here, we show that the MT-associated protein SPIRAL1 (SPR1) plays a key role in salt stress–induced MT disassembly. SPR1, a microtubule stabilizing protein, is degraded by the 26S proteasome, and its degradation rate is accelerated in response to high salinity. We show that accelerated SPR1 degradation is required for a fast MT disassembly response to salt stress and for salt stress tolerance. PMID:21954463

Synthesis of Zeolites Using the ADOR (Assembly-Disassembly-Organization-Reassembly) Route

PubMed Central

Wheatley, Paul S.; Čejka, Jiří; Morris, Russell E.

2016-01-01

Zeolites are an important class of materials that have wide ranging applications such as heterogeneous catalysts and adsorbents which are dependent on their framework topology. For new applications or improvements to existing ones, new zeolites with novel pore systems are desirable. We demonstrate a method for the synthesis of novel zeolites using the ADOR route. ADOR is an acronym for Assembly, Disassembly, Organization and Reassembly. This synthetic route takes advantage of the assembly of a relatively poorly stable that which can be selectively disassembled into a layered material. The resulting layered intermediate can then be organized in different manners by careful chemical manipulation and then reassembled into zeolites with new topologies. By carefully controlling the organization step of the synthetic pathway, new zeolites with never before seen topologies are capable of being synthesized. The structures of these new zeolites are confirmed using powder X-ray diffraction and further characterized by nitrogen adsorption and scanning electron microscopy. This new synthetic pathway for zeolites demonstrates its capability to produce novel frameworks that have never been prepared by traditional zeolite synthesis techniques. PMID:27078165

Synthesis of Zeolites Using the ADOR (Assembly-Disassembly-Organization-Reassembly) Route.

PubMed

Wheatley, Paul S; Čejka, Jiří; Morris, Russell E

2016-04-03

Zeolites are an important class of materials that have wide ranging applications such as heterogeneous catalysts and adsorbents which are dependent on their framework topology. For new applications or improvements to existing ones, new zeolites with novel pore systems are desirable. We demonstrate a method for the synthesis of novel zeolites using the ADOR route. ADOR is an acronym for Assembly, Disassembly, Organization and Reassembly. This synthetic route takes advantage of the assembly of a relatively poorly stable that which can be selectively disassembled into a layered material. The resulting layered intermediate can then be organized in different manners by careful chemical manipulation and then reassembled into zeolites with new topologies. By carefully controlling the organization step of the synthetic pathway, new zeolites with never before seen topologies are capable of being synthesized. The structures of these new zeolites are confirmed using powder X-ray diffraction and further characterized by nitrogen adsorption and scanning electron microscopy. This new synthetic pathway for zeolites demonstrates its capability to produce novel frameworks that have never been prepared by traditional zeolite synthesis techniques.

Integrating high-throughput genetic interaction mapping and high-content screening to explore yeast spindle morphogenesis

PubMed Central

Vizeacoumar, Franco J.; van Dyk, Nydia; S.Vizeacoumar, Frederick; Cheung, Vincent; Li, Jingjing; Sydorskyy, Yaroslav; Case, Nicolle; Li, Zhijian; Datti, Alessandro; Nislow, Corey; Raught, Brian; Zhang, Zhaolei; Frey, Brendan; Bloom, Kerry

2010-01-01

We describe the application of a novel screening approach that combines automated yeast genetics, synthetic genetic array (SGA) analysis, and a high-content screening (HCS) system to examine mitotic spindle morphogenesis. We measured numerous spindle and cellular morphological parameters in thousands of single mutants and corresponding sensitized double mutants lacking genes known to be involved in spindle function. We focused on a subset of genes that appear to define a highly conserved mitotic spindle disassembly pathway, which is known to involve Ipl1p, the yeast aurora B kinase, as well as the cell cycle regulatory networks mitotic exit network (MEN) and fourteen early anaphase release (FEAR). We also dissected the function of the kinetochore protein Mcm21p, showing that sumoylation of Mcm21p regulates the enrichment of Ipl1p and other chromosomal passenger proteins to the spindle midzone to mediate spindle disassembly. Although we focused on spindle disassembly in a proof-of-principle study, our integrated HCS-SGA method can be applied to virtually any pathway, making it a powerful means for identifying specific cellular functions. PMID:20065090

Self-assembly behavior of a linear-star supramolecular amphiphile based on host-guest complexation.

PubMed

Wang, Juan; Wang, Xing; Yang, Fei; Shen, Hong; You, Yezi; Wu, Decheng

2014-11-04

A star polymer, β-cyclodextrin-poly(l-lactide) (β-CD-PLLA), and a linear polymer, azobenzene-poly(ethylene glycol) (Azo-PEG), could self-assemble into a supramolecular amphiphilic copolymer (β-CD-PLLA@Azo-PEG) based on the host-guest interaction between β-CD and azobenzene moieties. This linear-star supramolecular amphiphilic copolymer further self-assembled into a variety of morphologies, including sphere-like micelle, carambola-like micelle, naan-like micelle, shuttle-like lamellae, tube-like fiber, and random curled-up lamellae, by tuning the length of hydrophilic or hydrophobic chains. The variation of morphology was closely related to the topological structure and block ratio of the supramolecular amphiphiles. These self-assembly structures could disassemble upon an ultraviolet (UV) light irradiation.

KSC-2011-4531

NASA Image and Video Library

2011-06-16

CAPE CANAVERAL, Fla. -- At the Shuttle Landing Facility at NASA's Kennedy Space Center in Florida, workers practice assembly and disassembling a mobile sling and crane, part of a portable mate-demate device. The portable sling and crane will be used to lift shuttles off their 747 carrier aircraft for delivery to museums next year. Shuttle Discovery will go to the Smithsonian's National Air and Space Museum, Steven F. Udvar-Hazy Center in Chantilly, Va., Endeavour will go to the California Science Center in Los Angeles and Enterprise will be moved from the Smithsonian to the Intrepid Sea, Air and Space Museum in New York. Shuttle Atlantis will go to the Kennedy Space Center Visitor Complex and won't require use of the portable sling and crane. Photo credit: NASA/Jim Grossmann

KSC-2011-4530

NASA Image and Video Library

2011-06-16

CAPE CANAVERAL, Fla. -- At the Shuttle Landing Facility at NASA's Kennedy Space Center in Florida, workers practice assembly and disassembling a mobile sling and crane, part of a portable mate-demate device. The portable sling and crane will be used to lift shuttles off their 747 carrier aircraft for delivery to museums next year. Shuttle Discovery will go to the Smithsonian's National Air and Space Museum, Steven F. Udvar-Hazy Center in Chantilly, Va., Endeavour will go to the California Science Center in Los Angeles and Enterprise will be moved from the Smithsonian to the Intrepid Sea, Air and Space Museum in New York. Shuttle Atlantis will go to the Kennedy Space Center Visitor Complex and won't require use of the portable sling and crane. Photo credit: NASA/Jim Grossmann

KSC-2011-4529

NASA Image and Video Library

2011-06-16

CAPE CANAVERAL, Fla. -- At the Shuttle Landing Facility at NASA's Kennedy Space Center in Florida, workers practice assembly and disassembling a mobile sling and crane, part of a portable mate-demate device. The portable sling and crane will be used to lift shuttles off their 747 carrier aircraft for delivery to museums next year. Shuttle Discovery will go to the Smithsonian's National Air and Space Museum, Steven F. Udvar-Hazy Center in Chantilly, Va., Endeavour will go to the California Science Center in Los Angeles and Enterprise will be moved from the Smithsonian to the Intrepid Sea, Air and Space Museum in New York. Shuttle Atlantis will go to the Kennedy Space Center Visitor Complex and won't require use of the portable sling and crane. Photo credit: NASA/Jim Grossmann

KSC-2011-4533

NASA Image and Video Library

2011-06-16

CAPE CANAVERAL, Fla. -- At the Shuttle Landing Facility at NASA's Kennedy Space Center in Florida, workers practice assembly and disassembling a mobile sling and crane, part of a portable mate-demate device. The portable sling and crane will be used to lift shuttles off their 747 carrier aircraft for delivery to museums next year. Shuttle Discovery will go to the Smithsonian's National Air and Space Museum, Steven F. Udvar-Hazy Center in Chantilly, Va., Endeavour will go to the California Science Center in Los Angeles and Enterprise will be moved from the Smithsonian to the Intrepid Sea, Air and Space Museum in New York. Shuttle Atlantis will go to the Kennedy Space Center Visitor Complex and won't require use of the portable sling and crane. Photo credit: NASA/Jim Grossmann

KSC-2011-4534

NASA Image and Video Library

2011-06-16

CAPE CANAVERAL, Fla. -- At the Shuttle Landing Facility at NASA's Kennedy Space Center in Florida, workers practice assembly and disassembling a mobile sling and crane, part of a portable mate-demate device. The portable sling and crane will be used to lift shuttles off their 747 carrier aircraft for delivery to museums next year. Shuttle Discovery will go to the Smithsonian's National Air and Space Museum, Steven F. Udvar-Hazy Center in Chantilly, Va., Endeavour will go to the California Science Center in Los Angeles and Enterprise will be moved from the Smithsonian to the Intrepid Sea, Air and Space Museum in New York. Shuttle Atlantis will go to the Kennedy Space Center Visitor Complex and won't require use of the portable sling and crane. Photo credit: NASA/Jim Grossmann

Spatial organization of xylem cell walls by ROP GTPases and microtubule-associated proteins.

PubMed

Oda, Yoshihisa; Fukuda, Hiroo

2013-12-01

Proper patterning of cellulosic cell walls is critical for cell shaping and differentiation of plant cells. Cortical microtubule arrays regulate the deposition patterns of cellulose microfibrils by controlling the targeting and trajectory of cellulose synthase complexes. Although some microtubule-associated proteins (MAPs) regulate the arrangement of cortical microtubules, knowledge about the overall mechanism governing the spacing of cortical microtubules is still limited. Recent studies reveal that ROP GTPases and MAPs spatially regulate the assembly and disassembly of cortical microtubules in developing xylem cells, in which localized secondary cell walls are deposited. Here, we review recent insights into the regulation of xylem cell wall patterning by cortical microtubules, ROP GTPases, and MAPs. Copyright © 2013 Elsevier Ltd. All rights reserved.

Metarrestin, a perinucleolar compartment inhibitor, effectively suppresses metastasis.

PubMed

Frankowski, Kevin J; Wang, Chen; Patnaik, Samarjit; Schoenen, Frank J; Southall, Noel; Li, Dandan; Teper, Yaroslav; Sun, Wei; Kandela, Irawati; Hu, Deqing; Dextras, Christopher; Knotts, Zachary; Bian, Yansong; Norton, John; Titus, Steve; Lewandowska, Marzena A; Wen, Yiping; Farley, Katherine I; Griner, Lesley Mathews; Sultan, Jamey; Meng, Zhaojing; Zhou, Ming; Vilimas, Tomas; Powers, Astin S; Kozlov, Serguei; Nagashima, Kunio; Quadri, Humair S; Fang, Min; Long, Charles; Khanolkar, Ojus; Chen, Warren; Kang, Jinsol; Huang, Helen; Chow, Eric; Goldberg, Esthermanya; Feldman, Coral; Xi, Romi; Kim, Hye Rim; Sahagian, Gary; Baserga, Susan J; Mazar, Andrew; Ferrer, Marc; Zheng, Wei; Shilatifard, Ali; Aubé, Jeffrey; Rudloff, Udo; Marugan, Juan Jose; Huang, Sui

2018-05-16

Metastasis remains a leading cause of cancer mortality due to the lack of specific inhibitors against this complex process. To identify compounds selectively targeting the metastatic state, we used the perinucleolar compartment (PNC), a complex nuclear structure associated with metastatic behaviors of cancer cells, as a phenotypic marker for a high-content screen of over 140,000 structurally diverse compounds. Metarrestin, obtained through optimization of a screening hit, disassembles PNCs in multiple cancer cell lines, inhibits invasion in vitro, suppresses metastatic development in three mouse models of human cancer, and extends survival of mice in a metastatic pancreatic cancer xenograft model with no organ toxicity or discernable adverse effects. Metarrestin disrupts the nucleolar structure and inhibits RNA polymerase (Pol) I transcription, at least in part by interacting with the translation elongation factor eEF1A2. Thus, metarrestin represents a potential therapeutic approach for the treatment of metastatic cancer. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

One-Step Generation of Multifunctional Polyelectrolyte Microcapsules via Nanoscale Interfacial Complexation in Emulsion (NICE)

DOE PAGES

Kim, Miju; Yeo, Seon Ju; Highley, Christopher B.; ...

2015-07-14

Polyelectrolyte microcapsules represent versatile stimuli-responsive structures that enable the encapsulation, protection, and release of active agents. Their conventional preparation methods, however, tend to be time-consuming, yield low encapsulation efficiency, and seldom allow for the dual incorporation of hydrophilic and hydrophobic materials, limiting their widespread utilization. In this work, we present a method to fabricate stimuli-responsive polyelectrolyte microcapsules in one step based on nanoscale interfacial complexation in emulsions (NICE) followed by spontaneous droplet hatching. NICE microcapsules can incorporate both hydrophilic and hydrophobic materials and also can be induced to trigger the release of encapsulated materials by changes in the solution pHmore » or ionic strength. We also show that NICE microcapsules can be functionalized with nanomaterials to exhibit useful functionality, such as response to a magnetic field and disassembly in response to light. NICE represents a potentially transformative method to prepare multifunctional nanoengineered polyelectrolyte microcapsules for various applications such as drug delivery and cell mimicry.« less

One-Step Generation of Multifunctional Polyelectrolyte Microcapsules via Nanoscale Interfacial Complexation in Emulsion (NICE).

PubMed

Kim, Miju; Yeo, Seon Ju; Highley, Christopher B; Burdick, Jason A; Yoo, Pil J; Doh, Junsang; Lee, Daeyeon

2015-08-25

Polyelectrolyte microcapsules represent versatile stimuli-responsive structures that enable the encapsulation, protection, and release of active agents. Their conventional preparation methods, however, tend to be time-consuming, yield low encapsulation efficiency, and seldom allow for the dual incorporation of hydrophilic and hydrophobic materials, limiting their widespread utilization. In this work, we present a method to fabricate stimuli-responsive polyelectrolyte microcapsules in one step based on nanoscale interfacial complexation in emulsions (NICE) followed by spontaneous droplet hatching. NICE microcapsules can incorporate both hydrophilic and hydrophobic materials and also can be induced to trigger the release of encapsulated materials by changes in the solution pH or ionic strength. We also show that NICE microcapsules can be functionalized with nanomaterials to exhibit useful functionality, such as response to a magnetic field and disassembly in response to light. NICE represents a potentially transformative method to prepare multifunctional nanoengineered polyelectrolyte microcapsules for various applications such as drug delivery and cell mimicry.

One-Step Generation of Multifunctional Polyelectrolyte Microcapsules via Nanoscale Interfacial Complexation in Emulsion (NICE)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Miju; Yeo, Seon Ju; Highley, Christopher B.

Polyelectrolyte microcapsules represent versatile stimuli-responsive structures that enable the encapsulation, protection, and release of active agents. Their conventional preparation methods, however, tend to be time-consuming, yield low encapsulation efficiency, and seldom allow for the dual incorporation of hydrophilic and hydrophobic materials, limiting their widespread utilization. In this work, we present a method to fabricate stimuli-responsive polyelectrolyte microcapsules in one step based on nanoscale interfacial complexation in emulsions (NICE) followed by spontaneous droplet hatching. NICE microcapsules can incorporate both hydrophilic and hydrophobic materials and also can be induced to trigger the release of encapsulated materials by changes in the solution pHmore » or ionic strength. We also show that NICE microcapsules can be functionalized with nanomaterials to exhibit useful functionality, such as response to a magnetic field and disassembly in response to light. NICE represents a potentially transformative method to prepare multifunctional nanoengineered polyelectrolyte microcapsules for various applications such as drug delivery and cell mimicry.« less

Self-assembly of an upconverting nanocomplex and its application to turn-on detection of metalloproteinase-9 in living cells

NASA Astrophysics Data System (ADS)

Nguyen, Phuong-Diem; Thanh Cong, Vu; Baek, Changyoon; Min, Junhong

2016-10-01

Upcoversion nanoparticles are an emerging luminescent nanomaterial with excellent photophysical properties that have great benefits in biological sensing. In this study, a luminescent turn-on biosensor for cell-secreted protease activity assay is established based on resonance energy transfer in an upconversion nanoparticle-graphene oxide nano-assembly. The proposed biosensor consists of a blue-emitting upconversion nanoparticle covered with a quenching complex, comprising gelatin as the proteinase substrate and graphene oxide nanosheets as luminescence acceptors. After enzymatic digestion, the upconversion nanoparticles lose the gelatin cover due to the disassembly of the quenching complex, thus the upconverting luminescence in the blue region is restored (a turn-on response). The recovered upconverting luminescence is proportional to the protease concentration; the limit of detection was 12 ng ml-1. Finally, the upconversion-graphene oxide nanocomplex was successfully applied in the detection of cell-secreted protease-metalloproteinase in MCF-7 cancer cells with high sensitivity and specificity.

CHCHD10 mutations promote loss of mitochondrial cristae junctions with impaired mitochondrial genome maintenance and inhibition of apoptosis.

PubMed

Genin, Emmanuelle C; Plutino, Morgane; Bannwarth, Sylvie; Villa, Elodie; Cisneros-Barroso, Eugenia; Roy, Madhuparna; Ortega-Vila, Bernardo; Fragaki, Konstantina; Lespinasse, Françoise; Pinero-Martos, Estefania; Augé, Gaëlle; Moore, David; Burté, Florence; Lacas-Gervais, Sandra; Kageyama, Yusuke; Itoh, Kie; Yu-Wai-Man, Patrick; Sesaki, Hiromi; Ricci, Jean-Ehrland; Vives-Bauza, Cristofol; Paquis-Flucklinger, Véronique

2016-01-01

CHCHD10-related diseases include mitochondrial DNA instability disorder, frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS) clinical spectrum, late-onset spinal motor neuropathy (SMAJ), and Charcot-Marie-Tooth disease type 2 (CMT2). Here, we show that CHCHD10 resides with mitofilin, CHCHD3 and CHCHD6 within the "mitochondrial contact site and cristae organizing system" (MICOS) complex. CHCHD10 mutations lead to MICOS complex disassembly and loss of mitochondrial cristae with a decrease in nucleoid number and nucleoid disorganization. Repair of the mitochondrial genome after oxidative stress is impaired in CHCHD10 mutant fibroblasts and this likely explains the accumulation of deleted mtDNA molecules in patient muscle. CHCHD10 mutant fibroblasts are not defective in the delivery of mitochondria to lysosomes suggesting that impaired mitophagy does not contribute to mtDNA instability. Interestingly, the expression of CHCHD10 mutant alleles inhibits apoptosis by preventing cytochrome c release. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

ERK-MAPK drives lamellipodia protrusion by activating the WAVE2 regulatory complex.

PubMed

Mendoza, Michelle C; Er, E Emrah; Zhang, Wenjuan; Ballif, Bryan A; Elliott, Hunter L; Danuser, Gaudenz; Blenis, John

2011-03-18

Cell movement begins with a leading edge protrusion, which is stabilized by nascent adhesions and retracted by mature adhesions. The ERK-MAPK (extracellular signal-regulated kinase-mitogen-activated protein kinase) localizes to protrusions and adhesions, but how it regulates motility is not understood. We demonstrate that ERK controls protrusion initiation and protrusion speed. Lamellipodial protrusions are generated via the WRC (WAVE2 regulatory complex), which activates the Arp2/3 actin nucleator for actin assembly. The WRC must be phosphorylated to be activated, but the sites and kinases that regulate its intermolecular changes and membrane recruitment are unknown. We show that ERK colocalizes with the WRC at lamellipodial leading edges and directly phosphorylates two WRC components: WAVE2 and Abi1. The phosphorylations are required for functional WRC interaction with Arp2/3 and actin during cell protrusion. Thus, ERK coordinates adhesion disassembly with WRC activation and actin polymerization to promote productive leading edge advancement during cell migration. Copyright © 2011 Elsevier Inc. All rights reserved.

ERK-MAPK Drives Lamellipodia Protrusion by Activating the WAVE2 Regulatory Complex

PubMed Central

Mendoza, Michelle C.; Emrah, E.; Zhang, Wenjuan; Ballif, Bryan A.; Elliott, Hunter L.; Danuser, Gaudenz; Blenis, John

2011-01-01

Summary Cell movement begins with a leading edge protrusion, which is stabilized by nascent adhesions and retracted by mature adhesions. The ERK-MAPK (extracellular signal regulated kinasemitogen-activated protein kinase) localizes to protrusions and adhesions, but how it regulates motility is not understood. We demonstrate ERK controls protrusion initiation and protrusion speed. Lamellipodial protrusions are generated via the WRC (WAVE2 Regulatory Complex), which activates the Arp2/3 actin nucleator for actin assembly. The WRC must be phosphorylated to be activated, but the sites and kinases that regulate its intermolecular changes and membrane recruitment are unknown. We show ERK co-localizes with the WRC at lamellipodial leading edges and directly phosphorylates two WRC components: WAVE2 and Abi1. The phosphorylations are required for functional WRC interaction with Arp2/3 and actin during cell protrusion. Thus, ERK coordinates adhesion disassembly with WRC activation and actin polymerization to promote productive leading edge advancement during cell migration. PMID:21419341

Thermally Induced Encapsulation of Food Nutrients into Phytoferritin through the Flexible Channels without Additives.

PubMed

Yang, Rui; Tian, Jing; Liu, Yuqian; Yang, Zhiying; Wu, Dandan; Zhou, Zhongkai

2017-11-22

The cavity of phytoferritin provides a nanospace to encapsulate and deliver food nutrient molecules. However, tranditional methods to prepare the ferritin-nutrient complexes must undergo acid/alkaline conditions or apply additives. In this work, we provide a novel guideline that thermal treatment at 60 °C can expand ferritin channels by uncoiling the surrounding α-helix. Upon reduction of the temperature to 20 °C, food nutrient rutin can be encapsulated in apo-soybean seed ferritin (apoSSF) at pH 7.0 through channels without disassembly of the protein cage and with no addition of additives. Results indicated that one apoSSF could encapsulate about 10.5 molecules of rutin, with an encapsulation ratio of 8.08% (w/w). In addition, the resulting rutin-loaded SSF complexes were monodispersed in a size of 12 nm in aqueous solution. This work provides a novel pathway for the encapsulation of food nutrient molecules into the nanocavity of ferritin under a neutral pH condition induced by thermal treatment.

Lysosomal multienzyme complex: pros and cons of working together.

PubMed

Bonten, Erik J; Annunziata, Ida; d'Azzo, Alessandra

2014-06-01

The ubiquitous distribution of lysosomes and their heterogeneous protein composition reflects the versatility of these organelles in maintaining cell homeostasis and their importance in tissue differentiation and remodeling. In lysosomes, the degradation of complex, macromolecular substrates requires the synergistic action of multiple hydrolases that usually work in a stepwise fashion. This catalytic machinery explains the existence of lysosomal enzyme complexes that can be dynamically assembled and disassembled to efficiently and quickly adapt to the pool of substrates to be processed or degraded, adding extra tiers to the regulation of the individual protein components. An example of such a complex is the one composed of three hydrolases that are ubiquitously but differentially expressed: the serine carboxypeptidase, protective protein/cathepsin A (PPCA), the sialidase, neuraminidase-1 (NEU1), and the glycosidase β-galactosidase (β-GAL). Next to this 'core' complex, the existence of sub-complexes, which may contain additional components, and function at the cell surface or extracellularly, suggests as yet unexplored functions of these enzymes. Here we review how studies of basic biological processes in the mouse models of three lysosomal storage disorders, galactosialidosis, sialidosis, and GM1-gangliosidosis, revealed new and unexpected roles for the three respective affected enzymes, Ppca, Neu1, and β-Gal, that go beyond their canonical degradative activities. These findings have broadened our perspective on their functions and may pave the way for the development of new therapies for these lysosomal storage disorders.

9-Triptycenecarboxylate-Bridged Diiron(II) Complexes

PubMed Central

Friedle, Simone; Kodanko, Jeremy J.; Fornace, Kyrstin L.; Lippard, Stephen J.

2008-01-01

The synthesis and characterization of diiron(II) complexes supported by 9-triptycenecarboxylate ligands (-O2CTrp) is described. The interlocking nature of the triptycenecarboxylates facilitates formation of quadruply bridged diiron(II) complexes of the type [Fe2(μ-O2CTrp)4(L)2] (L = THF, pyridine or imidazole derivative) with a paddlewheel geometry. A systematic lengthening of the Fe-Fe distance occurs with the increase in steric bulk of the neutral donor L, resulting in values of up to 3 Å without disassembly of the paddlewheel structure. Reactions with an excess of water do not lead to decomposition of the diiron(II) core, indicating that these quadruply bridged complexes are of exceptional stability. The red-colored complexes [Fe2(μ-O2CTrp)4(4-AcPy)2] (10) and [Fe2(μ-O2CTrp)4(4-CNPy)2] (11) exhibit solvent-dependent thermochromism in coordinating solvents that was studied by variable temperature UV-vis spectroscopy. Reaction of [Fe2(μ-O2CTrp)4(THF)2] with N,N,N’,N’-tetramethylethylenediamine (TMEDA), tetra-n-butyl ammonium thiocyanate, or excess 2-methylimidazole resulted in the formation of mononuclear complexes [Fe(O2CTrp)2(TMEDA)] (13), (n-Bu4N)2[Fe(O2CTrp)2(SCN)2] (14), and [Fe(O2CTrp)2(2-MeIm)2] (15) having an O4/N2 coordination sphere composition. PMID:19915653

Retention of Habitat Complexity Minimizes Disassembly of Reef Fish Communities following Disturbance: A Large-Scale Natural Experiment

PubMed Central

Emslie, Michael J.; Cheal, Alistair J.; Johns, Kerryn A.

2014-01-01

High biodiversity ecosystems are commonly associated with complex habitats. Coral reefs are highly diverse ecosystems, but are under increasing pressure from numerous stressors, many of which reduce live coral cover and habitat complexity with concomitant effects on other organisms such as reef fishes. While previous studies have highlighted the importance of habitat complexity in structuring reef fish communities, they employed gradient or meta-analyses which lacked a controlled experimental design over broad spatial scales to explicitly separate the influence of live coral cover from overall habitat complexity. Here a natural experiment using a long term (20 year), spatially extensive (∼115,000 kms2) dataset from the Great Barrier Reef revealed the fundamental importance of overall habitat complexity for reef fishes. Reductions of both live coral cover and habitat complexity had substantial impacts on fish communities compared to relatively minor impacts after major reductions in coral cover but not habitat complexity. Where habitat complexity was substantially reduced, species abundances broadly declined and a far greater number of fish species were locally extirpated, including economically important fishes. This resulted in decreased species richness and a loss of diversity within functional groups. Our results suggest that the retention of habitat complexity following disturbances can ameliorate the impacts of coral declines on reef fishes, so preserving their capacity to perform important functional roles essential to reef resilience. These results add to a growing body of evidence about the importance of habitat complexity for reef fishes, and represent the first large-scale examination of this question on the Great Barrier Reef. PMID:25140801

Retention of habitat complexity minimizes disassembly of reef fish communities following disturbance: a large-scale natural experiment.

PubMed

Emslie, Michael J; Cheal, Alistair J; Johns, Kerryn A

2014-01-01

High biodiversity ecosystems are commonly associated with complex habitats. Coral reefs are highly diverse ecosystems, but are under increasing pressure from numerous stressors, many of which reduce live coral cover and habitat complexity with concomitant effects on other organisms such as reef fishes. While previous studies have highlighted the importance of habitat complexity in structuring reef fish communities, they employed gradient or meta-analyses which lacked a controlled experimental design over broad spatial scales to explicitly separate the influence of live coral cover from overall habitat complexity. Here a natural experiment using a long term (20 year), spatially extensive (∼ 115,000 kms(2)) dataset from the Great Barrier Reef revealed the fundamental importance of overall habitat complexity for reef fishes. Reductions of both live coral cover and habitat complexity had substantial impacts on fish communities compared to relatively minor impacts after major reductions in coral cover but not habitat complexity. Where habitat complexity was substantially reduced, species abundances broadly declined and a far greater number of fish species were locally extirpated, including economically important fishes. This resulted in decreased species richness and a loss of diversity within functional groups. Our results suggest that the retention of habitat complexity following disturbances can ameliorate the impacts of coral declines on reef fishes, so preserving their capacity to perform important functional roles essential to reef resilience. These results add to a growing body of evidence about the importance of habitat complexity for reef fishes, and represent the first large-scale examination of this question on the Great Barrier Reef.

[PHAHs levels in soil samples from the E-waste disassembly sites and their sources allocation].

PubMed

Zhao, Gao-Feng; Wang, Zi-Jian

2009-06-15

Soil samples (each with 3 replicates of - 1 kg, at the top 0-5 cm layer) were collected from each of the e-waste disassembly sites and the control site. Also obtained from each disassembly site were samples (each weighing - 0.2 kg) of cable coating,stuffing powder, and circuit boards chipping. The contents of 23 PBB congeners, 12 PBDE congeners, and 27 PCB congeners in soil and in their potential sources, including e-waste residues, were measured using the GC-MS5975B technique. The highest level of PBBs was found in the cable coating among the three e-waste residues, with a concentration of 35.25 ng x g(-1). The contents of low-brominated PBBs (including monobromobiphenyls and dibromobiphenyls) accounted for 38% of the total PBBs concentration observed in cable coating sample. The highest levels of PBDEs and PBDE209 were found in the stuffing powder for electronic component among the collected e-waste residues, with a concentration of 29.71 and 4.19 x 10(3) ng x g(-1). PBDE153 and PBDE183 were the most predominant PBDE congeners, with their concentration accounting for 43% and 24% of the total PBDEs concentration observed in the stuffing powder sample, respectively. Levels of PCBs in cable coating were the highest in these e-waste residues, with a concentration of 680.02 ngx g(-1). The observed values of the three PHAHs in soils from the disassembly site were considerably higher than their corresponding values observed in the control site (p < 0.05), which indicates that these PHAHs from e-waste is the pollution source of local environment.

Depth of manual dismantling analysis: A cost–benefit approach

DOE Office of Scientific and Technical Information (OSTI.GOV)

Achillas, Ch., E-mail: c.achillas@ihu.edu.gr; Aidonis, D.; Vlachokostas, Ch.

Highlights: ► A mathematical modeling tool for OEMs. ► The tool can be used by OEMs, recyclers of electr(on)ic equipment or WEEE management systems’ regulators. ► The tool makes use of cost–benefit analysis in order to determine the optimal depth of product disassembly. ► The reusable materials and the quantity of metals and plastics recycled can be quantified in an easy-to-comprehend manner. - Abstract: This paper presents a decision support tool for manufacturers and recyclers towards end-of-life strategies for waste electrical and electronic equipment. A mathematical formulation based on the cost benefit analysis concept is herein analytically described in ordermore » to determine the parts and/or components of an obsolete product that should be either non-destructively recovered for reuse or be recycled. The framework optimally determines the depth of disassembly for a given product, taking into account economic considerations. On this basis, it embeds all relevant cost elements to be included in the decision-making process, such as recovered materials and (depreciated) parts/components, labor costs, energy consumption, equipment depreciation, quality control and warehousing. This tool can be part of the strategic decision-making process in order to maximize profitability or minimize end-of-life management costs. A case study to demonstrate the models’ applicability is presented for a typical electronic product in terms of structure and material composition. Taking into account the market values of the pilot product’s components, the manual disassembly is proven profitable with the marginal revenues from recovered reusable materials to be estimated at 2.93–23.06 €, depending on the level of disassembly.« less

Cyclic stretch-induced stress fiber dynamics - Dependence on strain rate, Rho-kinase and MLCK

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Chin-Fu; Haase, Candice; Deguchi, Shinji

2010-10-22

Research highlights: {yields} Cyclic stretch induces stress fiber disassembly, reassembly and fusion perpendicular to the direction of stretch. {yields} Stress fiber disassembly and reorientation were not induced at low stretch frequency. {yields} Stretch caused actin fiber formation parallel to stretch in distinct locations in cells treated with Rho-kinase and MLCK inhibitors. -- Abstract: Stress fiber realignment is an important adaptive response to cyclic stretch for nonmuscle cells, but the mechanism by which such reorganization occurs is not known. By analyzing stress fiber dynamics using live cell microscopy, we revealed that stress fiber reorientation perpendicular to the direction of cyclic uniaxialmore » stretching at 1 Hz did not involve disassembly of the stress fiber distal ends located at focal adhesion sites. Instead, these distal ends were often used to assemble new stress fibers oriented progressively further away from the direction of stretch. Stress fiber disassembly and reorientation were not induced when the frequency of stretch was decreased to 0.01 Hz, however. Treatment with the Rho-kinase inhibitor (Y27632) reduced stress fibers to thin fibers located in the cell periphery which bundled together to form thick fibers oriented parallel to the direction of stretching at 1 Hz. In contrast, these thin fibers remained diffuse in cells subjected to stretch at 0.01 Hz. Cyclic stretch at 1 Hz also induced actin fiber formation parallel to the direction of stretch in cells treated with the myosin light chain kinase (MLCK) inhibitor ML-7, but these fibers were located centrally rather than peripherally. These results shed new light on the mechanism by which stress fibers reorient in response to cyclic stretch in different regions of the actin cytoskeleton.« less

Tides Versus Collisions in the Primordial Main Belt

NASA Astrophysics Data System (ADS)

Asphaug, E.; Bottke, W. F., Jr.; Morbidelli, A.; Petit, J.-M.

2000-10-01

Recent numerical and theoretical developments (e.g. Wetherill 1992; Chambers and Wetherill 1998) suggest that hundreds or thousands of Moon- to Mars-sized planetary embryos may have resided between 0.5 and 4 AU during early solar system accretion, to be scattered by mutual encounters and resonant perturbations with Jupiter and Saturn. At the same time, we lack compelling scenarios leading to the origin of iron meteorites, believed to represent the cores from approximately 85 different primordial planetesimals (Kail et al. 1994). Are M-type asteroids such as Kleopatra the exposed cores of these parent bodies? Early solar system collisions have been called upon to excavate this iron (Haack et al. 1996), although numerical impact models (Asphaug 1997) have found this task difficult to achieve, particularly when it is required to occur many dozens of times, yet not a single time for asteroid Vesta. One possibility, consistent with the unusual shape of Kleopatra, is tidal disassembly of collisionally weakened differentiated planetesimals by close encounters with primordial planetary embryos. Differentiation enhances the efficacy of tidal disassembly, which is probably already comparable (Asphaug and Benz 1996) to the efficacy of collisional disassembly, but only for bodies of very low strength. Tidal disassembly has the further advantage of stripping all material from a given isosurface, whereas collisions partition energy into both fast and slow debris, leaving behind a rock mantle. To further explore this idea, in comparison with the efficacy of collisional breakup of differentiated planetesimals, we determine the minimal encounter distances between evolving asteroids and the embryos as modeled by Petit et al. (2000). We then directly simulate these tidal encounters using a smooth particle hydrocode (SPH; Benz and Asphaug 1995), and compare tidal encounters to collisional encounters using the same code.

eIF2β is critical for eIF5-mediated GDP-dissociation inhibitor activity and translational control

PubMed Central

Jennings, Martin D.; Kershaw, Christopher J.; White, Christopher; Hoyle, Danielle; Richardson, Jonathan P.; Costello, Joseph L.; Donaldson, Ian J.; Zhou, Yu; Pavitt, Graham D.

2016-01-01

In protein synthesis translation factor eIF2 binds initiator tRNA to ribosomes and facilitates start codon selection. eIF2 GDP/GTP status is regulated by eIF5 (GAP and GDI functions) and eIF2B (GEF and GDF activities), while eIF2α phosphorylation in response to diverse signals is a major point of translational control. Here we characterize a growth suppressor mutation in eIF2β that prevents eIF5 GDI and alters cellular responses to reduced eIF2B activity, including control of GCN4 translation. By monitoring the binding of fluorescent nucleotides and initiator tRNA to purified eIF2 we show that the eIF2β mutation does not affect intrinsic eIF2 affinities for these ligands, neither does it interfere with eIF2 binding to 43S pre-initiation complex components. Instead we show that the eIF2β mutation prevents eIF5 GDI stabilizing nucleotide binding to eIF2, thereby altering the off-rate of GDP from eIF2•GDP/eIF5 complexes. This enables cells to grow with reduced eIF2B GEF activity but impairs activation of GCN4 targets in response to amino acid starvation. These findings provide support for the importance of eIF5 GDI activity in vivo and demonstrate that eIF2β acts in concert with eIF5 to prevent premature release of GDP from eIF2γ and thereby ensure tight control of protein synthesis initiation. PMID:27458202

eIF2β is critical for eIF5-mediated GDP-dissociation inhibitor activity and translational control.

PubMed

Jennings, Martin D; Kershaw, Christopher J; White, Christopher; Hoyle, Danielle; Richardson, Jonathan P; Costello, Joseph L; Donaldson, Ian J; Zhou, Yu; Pavitt, Graham D

2016-11-16

In protein synthesis translation factor eIF2 binds initiator tRNA to ribosomes and facilitates start codon selection. eIF2 GDP/GTP status is regulated by eIF5 (GAP and GDI functions) and eIF2B (GEF and GDF activities), while eIF2α phosphorylation in response to diverse signals is a major point of translational control. Here we characterize a growth suppressor mutation in eIF2β that prevents eIF5 GDI and alters cellular responses to reduced eIF2B activity, including control of GCN4 translation. By monitoring the binding of fluorescent nucleotides and initiator tRNA to purified eIF2 we show that the eIF2β mutation does not affect intrinsic eIF2 affinities for these ligands, neither does it interfere with eIF2 binding to 43S pre-initiation complex components. Instead we show that the eIF2β mutation prevents eIF5 GDI stabilizing nucleotide binding to eIF2, thereby altering the off-rate of GDP from eIF2•GDP/eIF5 complexes. This enables cells to grow with reduced eIF2B GEF activity but impairs activation of GCN4 targets in response to amino acid starvation. These findings provide support for the importance of eIF5 GDI activity in vivo and demonstrate that eIF2β acts in concert with eIF5 to prevent premature release of GDP from eIF2γ and thereby ensure tight control of protein synthesis initiation. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Yeast eIF4B binds to the head of the 40S ribosomal subunit and promotes mRNA recruitment through its N-terminal and internal repeat domains.

PubMed

Walker, Sarah E; Zhou, Fujun; Mitchell, Sarah F; Larson, Victoria S; Valasek, Leos; Hinnebusch, Alan G; Lorsch, Jon R

2013-02-01

Eukaryotic translation initiation factor (eIF)4B stimulates recruitment of mRNA to the 43S ribosomal pre-initiation complex (PIC). Yeast eIF4B (yeIF4B), shown previously to bind single-stranded (ss) RNA, consists of an N-terminal domain (NTD), predicted to be unstructured in solution; an RNA-recognition motif (RRM); an unusual domain comprised of seven imperfect repeats of 26 amino acids; and a C-terminal domain. Although the mechanism of yeIF4B action has remained obscure, most models have suggested central roles for its RRM and ssRNA-binding activity. We have dissected the functions of yeIF4B's domains and show that the RRM and its ssRNA-binding activity are dispensable in vitro and in vivo. Instead, our data indicate that the 7-repeats and NTD are the most critical domains, which mediate binding of yeIF4B to the head of the 40S ribosomal subunit via interaction with Rps20. This interaction induces structural changes in the ribosome's mRNA entry channel that could facilitate mRNA loading. We also show that yeIF4B strongly promotes productive interaction of eIF4A with the 43S•mRNA PIC in a manner required for efficient mRNA recruitment.

Cellular microRNAs up-regulate transcription via interaction with promoter TATA-box motifs.

PubMed

Zhang, Yijun; Fan, Miaomiao; Zhang, Xue; Huang, Feng; Wu, Kang; Zhang, Junsong; Liu, Jun; Huang, Zhuoqiong; Luo, Haihua; Tao, Liang; Zhang, Hui

2014-12-01

The TATA box represents one of the most prevalent core promoters where the pre-initiation complexes (PICs) for gene transcription are assembled. This assembly is crucial for transcription initiation and well regulated. Here we show that some cellular microRNAs (miRNAs) are associated with RNA polymerase II (Pol II) and TATA box-binding protein (TBP) in human peripheral blood mononuclear cells (PBMCs). Among them, let-7i sequence specifically binds to the TATA-box motif of interleukin-2 (IL-2) gene and elevates IL-2 mRNA and protein production in CD4(+) T-lymphocytes in vitro and in vivo. Through direct interaction with the TATA-box motif, let-7i facilitates the PIC assembly and transcription initiation of IL-2 promoter. Several other cellular miRNAs, such as mir-138, mir-92a or mir-181d, also enhance the promoter activities via binding to the TATA-box motifs of insulin, calcitonin or c-myc, respectively. In agreement with the finding that an HIV-1-encoded miRNA could enhance viral replication through targeting the viral promoter TATA-box motif, our data demonstrate that the interaction with core transcription machinery is a novel mechanism for miRNAs to regulate gene expression. © 2014 Zhang et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Dynein light chain 1 induces assembly of large Bim complexes on mitochondria that stabilize Mcl-1 and regulate apoptosis.

PubMed

Singh, Prafull Kumar; Roukounakis, Aristomenis; Frank, Daniel O; Kirschnek, Susanne; Das, Kushal Kumar; Neumann, Simon; Madl, Josef; Römer, Winfried; Zorzin, Carina; Borner, Christoph; Haimovici, Aladin; Garcia-Saez, Ana; Weber, Arnim; Häcker, Georg

2017-09-01

The Bcl-2 family protein Bim triggers mitochondrial apoptosis. Bim is expressed in nonapoptotic cells at the mitochondrial outer membrane, where it is activated by largely unknown mechanisms. We found that Bim is regulated by formation of large protein complexes containing dynein light chain 1 (DLC1). Bim rapidly inserted into cardiolipin-containing membranes in vitro and recruited DLC1 to the membrane. Bim binding to DLC1 induced the formation of large Bim complexes on lipid vesicles, on isolated mitochondria, and in intact cells. Native gel electrophoresis and gel filtration showed Bim-containing mitochondrial complexes of several hundred kilodaltons in all cells tested. Bim unable to form complexes was consistently more active than complexed Bim, which correlated with its substantially reduced binding to anti-apoptotic Bcl-2 proteins. At endogenous levels, Bim surprisingly bound only anti-apoptotic Mcl-1 but not Bcl-2 or Bcl-X L , recruiting only Mcl-1 into large complexes. Targeting of DLC1 by RNAi in human cell lines induced disassembly of Bim-Mcl-1 complexes and the proteasomal degradation of Mcl-1 and sensitized the cells to the Bcl-2/Bcl-X L inhibitor ABT-737. Regulation of apoptosis at mitochondria thus extends beyond the interaction of monomers of proapoptotic and anti-apoptotic Bcl-2 family members but involves more complex structures of proteins at the mitochondrial outer membrane, and targeting complexes may be a novel therapeutic strategy. © 2017 Singh et al.; Published by Cold Spring Harbor Laboratory Press.

Binding of Substrates to the Central Pore of the Vps4 ATPase Is Autoinhibited by the Microtubule Interacting and Trafficking (MIT) Domain and Activated by MIT Interacting Motifs (MIMs).

PubMed

Han, Han; Monroe, Nicole; Votteler, Jörg; Shakya, Binita; Sundquist, Wesley I; Hill, Christopher P

2015-05-22

The endosomal sorting complexes required for transport (ESCRT) pathway drives reverse topology membrane fission events within multiple cellular pathways, including cytokinesis, multivesicular body biogenesis, repair of the plasma membrane, nuclear membrane vesicle formation, and HIV budding. The AAA ATPase Vps4 is recruited to membrane necks shortly before fission, where it catalyzes disassembly of the ESCRT-III lattice. The N-terminal Vps4 microtubule-interacting and trafficking (MIT) domains initially bind the C-terminal MIT-interacting motifs (MIMs) of ESCRT-III subunits, but it is unclear how the enzyme then remodels these substrates in response to ATP hydrolysis. Here, we report quantitative binding studies that demonstrate that residues from helix 5 of the Vps2p subunit of ESCRT-III bind to the central pore of an asymmetric Vps4p hexamer in a manner that is dependent upon the presence of flexible nucleotide analogs that can mimic multiple states in the ATP hydrolysis cycle. We also find that substrate engagement is autoinhibited by the Vps4p MIT domain and that this inhibition is relieved by binding of either Type 1 or Type 2 MIM elements, which bind the Vps4p MIT domain through different interfaces. These observations support the model that Vps4 substrates are initially recruited by an MIM-MIT interaction that activates the Vps4 central pore to engage substrates and generate force, thereby triggering ESCRT-III disassembly. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The adherens junction is lost during normal pregnancy but not during ovarian hyperstimulated pregnancy.

PubMed

Dowland, Samson N; Madawala, Romanthi J; Lindsay, Laura A; Murphy, Christopher R

2016-03-01

During early pregnancy in the rat, the luminal uterine epithelial cells (UECs) must transform to a receptive state to permit blastocyst attachment and implantation. The implantation process involves penetration of the epithelial barrier, so it is expected that the transformation of UECs includes alterations in the lateral junctional complex. Previous studies have demonstrated a deepening of the tight junction (zonula occludens) and a reduction in the number of desmosomes (macula adherens) in UECs at the time of implantation. However, the adherens junction (zonula adherens), which is primarily responsible for cell-cell adhesion, has been little studied during early pregnancy. This study investigated the adherens junction in rat UECs during the early stages of normal pregnancy and ovarian hyperstimulated (OH) pregnancy using transmission electron microscopy. The adherens junction is present in UECs at the time of fertilisation, but is lost at the time of blastocyst implantation during normal pregnancy. Interestingly, at the time of implantation after OH, adherens junctions are retained and may impede blastocyst penetration of the epithelium. The adherens junction anchors the actin-based terminal web, which is known to be disrupted in UECs during early pregnancy. However, artificial disruption of the terminal web, using cytochalasin D, did not cause removal of the adherens junction in UECs. This study revealed that adherens junction disassembly occurs during early pregnancy, but that this process does not occur during OH pregnancy. Such disassembly does not appear to depend on the disruption of the terminal web. Copyright © 2015 Elsevier GmbH. All rights reserved.

Binding of Substrates to the Central Pore of the Vps4 ATPase Is Autoinhibited by the Microtubule Interacting and Trafficking (MIT) Domain and Activated by MIT Interacting Motifs (MIMs)*

PubMed Central

Han, Han; Monroe, Nicole; Votteler, Jörg; Shakya, Binita; Sundquist, Wesley I.; Hill, Christopher P.

2015-01-01

The endosomal sorting complexes required for transport (ESCRT) pathway drives reverse topology membrane fission events within multiple cellular pathways, including cytokinesis, multivesicular body biogenesis, repair of the plasma membrane, nuclear membrane vesicle formation, and HIV budding. The AAA ATPase Vps4 is recruited to membrane necks shortly before fission, where it catalyzes disassembly of the ESCRT-III lattice. The N-terminal Vps4 microtubule-interacting and trafficking (MIT) domains initially bind the C-terminal MIT-interacting motifs (MIMs) of ESCRT-III subunits, but it is unclear how the enzyme then remodels these substrates in response to ATP hydrolysis. Here, we report quantitative binding studies that demonstrate that residues from helix 5 of the Vps2p subunit of ESCRT-III bind to the central pore of an asymmetric Vps4p hexamer in a manner that is dependent upon the presence of flexible nucleotide analogs that can mimic multiple states in the ATP hydrolysis cycle. We also find that substrate engagement is autoinhibited by the Vps4p MIT domain and that this inhibition is relieved by binding of either Type 1 or Type 2 MIM elements, which bind the Vps4p MIT domain through different interfaces. These observations support the model that Vps4 substrates are initially recruited by an MIM-MIT interaction that activates the Vps4 central pore to engage substrates and generate force, thereby triggering ESCRT-III disassembly. PMID:25833946

Optimizing decision making at the end of life of a product

NASA Astrophysics Data System (ADS)

Gonzalez-Torre, Beatriz; Adenso-Diaz, Belarmino

2004-02-01

European environmental legislation has significantly evolved over the last few years, forcing manufacturers to be more environmentally aware and to introduce ecological criteria in their traditional practices. One of the most important goals of this set of regulations is to reduce the amount of solid waste generated per unit of time by promoting recycling, repair, reuse and other recovery strategies at the product end of life (EOL). However, one of the most difficult steps for manufacturers is that of deciding which of these options or which combination of them should be implemented to get the maximum recovery value taking into account the specific characteristics of each product. In this paper, a recurrent algorithm is proposed to determine the optimal end-of-life strategy. On the basis of the product bill of materials and its graphical CAD/CAM representation, the model will determine to what extent the product should be disassembled and what the final end of each disassembled part should be (reuse, recycling or disposal). The paper starts by presenting an overview of the model, to then focus on the CAD-integrated algorithm for determining the optimum disassembly sequence, a necessary step in EOL decision-making.

Induced Förster resonance energy transfer by encapsulation of DNA-scaffold based probes inside a plant virus based protein cage

NASA Astrophysics Data System (ADS)

de Ruiter, Mark V.; Overeem, Nico J.; Singhai, Gaurav; Cornelissen, Jeroen J. L. M.

2018-05-01

Insight into the assembly and disassembly of viruses can play a crucial role in developing cures for viral diseases. Specialized fluorescent probes can benefit the study of interactions within viruses, especially during cell studies. In this work, we developed a strategy based on Förster resonance energy transfer (FRET) to study the assembly of viruses without labeling the exterior of viruses. Instead, we exploit their encapsulation of nucleic cargo, using three different fluorescent ATTO dyes linked to single-stranded DNA oligomers, which are hybridised to a longer DNA strand. FRET is induced upon assembly of the cowpea chlorotic mottle virus, which forms monodisperse icosahedral particles of about 22 nm, thereby increasing the FRET efficiency by a factor of 8. Additionally, encapsulation of the dyes in virus-like particles induces a two-step FRET. When the formed constructs are disassembled, this FRET signal is fully reduced to the value before encapsulation. This reversible behavior makes the system a good probe for studying viral assembly and disassembly. It, furthermore, shows that multi-component supramolecular materials are stabilized in the confinement of a protein cage.

Structure of clathrin coat with bound Hsc70 and auxilin: mechanism of Hsc70-facilitated disassembly

PubMed Central

Xing, Yi; Böcking, Till; Wolf, Matthias; Grigorieff, Nikolaus; Kirchhausen, Tomas; Harrison, Stephen C

2010-01-01

The chaperone Hsc70 drives the clathrin assembly–disassembly cycle forward by stimulating dissociation of a clathrin lattice. A J-domain containing co-chaperone, auxilin, associates with a freshly budded clathrin-coated vesicle, or with an in vitro assembled clathrin coat, and recruits Hsc70 to its specific heavy-chain-binding site. We have determined by electron cryomicroscopy (cryoEM), at about 11 Å resolution, the structure of a clathrin coat (in the D6-barrel form) with specifically bound Hsc70 and auxilin. The Hsc70 binds a previously analysed site near the C-terminus of the heavy chain, with a stoichiometry of about one per three-fold vertex. Its binding is accompanied by a distortion of the clathrin lattice, detected by a change in the axial ratio of the D6 barrel. We propose that when Hsc70, recruited to a position close to its target by the auxilin J-domain, splits ATP, it clamps firmly onto its heavy-chain site and locks in place a transient fluctuation. Accumulation of the local strain thus imposed at multiple vertices can then lead to disassembly. PMID:20033059

Spiropyran-Decorated SiO₂-Pt Janus Micromotor: Preparation and Light-Induced Dynamic Self-Assembly and Disassembly.

PubMed

Zhang, Qilu; Dong, Renfeng; Chang, Xueyi; Ren, Biye; Tong, Zhen

2015-11-11

The controlled self-assembly of self-propelled Janus micromotors may give the micromotors some potential applications in many fields. In this work, we design a kind of SiO2-Pt Janus catalytic micromotor functionalized by spiropyran (SP) moieties on the surface of the SiO2 hemisphere. The spiropyran-modified SiO2-Pt Janus micromotor exhibits autonomous self-propulsion in the presence of hydrogen peroxide fuel in N,N-dimethylformamide (DMF)/H2O (1:1 in volume) mixture. We demonstrate that the self-propelled Janus micromotors can dynamically assemble into multiple motors because of the electrostatic attractions and π-π stacking between MC molecules induced by UV light irradiation (λ = 365 nm) and also quickly disassemble into mono motors when the light is switched to green light (λ = 520 nm) for the first time. Furthermore, the assembled Janus motors can move together automatically with different motion patterns propelled by the hydrogen peroxide fuels upon UV irradiation. The work provides a new approach not only to the development of the potential application of Janus motors but also to the fundamental science of reversible self-assembly and disassembly of Janus micromotors.

Prey and mound disassembly, manipulation and transport by fire ant collectives

NASA Astrophysics Data System (ADS)

Dutta, Bahnisikha; Monaenkova, Daria; Goodisman, Michael A.; Goldman, Daniel

Fire ants inhabit subterranean nests covered by a hemispherical mound of soil permeated by narrow ( 1 body length diameter) tunnels. Fire ants can use their mound for long-term food storage [Gayahan &Tschinkel, J. Insect Sci.,2008]. Since mound tunnels are narrow, we expect that in addition to prey manipulation, mound reconfiguration could also be an important aspect of the food storage strategy. Ant colonies collected from wild were allowed to build nests in containers filled with clay soil in the laboratory. These colonies were offered diverse prey embedded with lead markers, including mealworms, crickets and shrimp. Ant-prey-soil interactions on the nest surface were recorded using overhead video and subsurface using x-ray imaging. Individual ants involved in prey storage exhibited three distinct behaviors: prey maneuvering, prey dissection and mound reconfiguration. Small prey (e.g. mealworms) were collectively carried intact into the mound through a tunnel, and then disassembled within the mound. Larger prey (e.g. shrimp) were dismantled into small pieces above the surface and carried to mound tunnels. The bodies of hard medium-sized prey (e.g. crickets) were buried after limb removal and then disassembled and moved into tunnels. Soil reconfiguration occurred in all cases.

In-situ coupling between kinase activities and protein dynamics within single focal adhesions

NASA Astrophysics Data System (ADS)

Wu, Yiqian; Zhang, Kaiwen; Seong, Jihye; Fan, Jason; Chien, Shu; Wang, Yingxiao; Lu, Shaoying

2016-07-01

The dynamic activation of oncogenic kinases and regulation of focal adhesions (FAs) are crucial molecular events modulating cell adhesion in cancer metastasis. However, it remains unclear how these events are temporally coordinated at single FA sites. Therefore, we targeted fluorescence resonance energy transfer (FRET)-based biosensors toward subcellular FAs to report local molecular events during cancer cell adhesion. Employing single FA tracking and cross-correlation analysis, we quantified the dynamic coupling characteristics between biochemical kinase activities and structural FA within single FAs. We show that kinase activations and FA assembly are strongly and sequentially correlated, with the concurrent FA assembly and Src activation leading focal adhesion kinase (FAK) activation by 42.6 ± 12.6 sec. Strikingly, the temporal coupling between kinase activation and individual FA assembly reflects the fate of FAs at later stages. The FAs with a tight coupling tend to grow and mature, while the less coupled FAs likely disassemble. During FA disassembly, however, kinase activations lead the disassembly, with FAK being activated earlier than Src. Therefore, by integrating subcellularly targeted FRET biosensors and computational analysis, our study reveals intricate interplays between Src and FAK in regulating the dynamic life of single FAs in cancer cells.

Depth of manual dismantling analysis: a cost-benefit approach.

PubMed

Achillas, Ch; Aidonis, D; Vlachokostas, Ch; Karagiannidis, A; Moussiopoulos, N; Loulos, V

2013-04-01

This paper presents a decision support tool for manufacturers and recyclers towards end-of-life strategies for waste electrical and electronic equipment. A mathematical formulation based on the cost benefit analysis concept is herein analytically described in order to determine the parts and/or components of an obsolete product that should be either non-destructively recovered for reuse or be recycled. The framework optimally determines the depth of disassembly for a given product, taking into account economic considerations. On this basis, it embeds all relevant cost elements to be included in the decision-making process, such as recovered materials and (depreciated) parts/components, labor costs, energy consumption, equipment depreciation, quality control and warehousing. This tool can be part of the strategic decision-making process in order to maximize profitability or minimize end-of-life management costs. A case study to demonstrate the models' applicability is presented for a typical electronic product in terms of structure and material composition. Taking into account the market values of the pilot product's components, the manual disassembly is proven profitable with the marginal revenues from recovered reusable materials to be estimated at 2.93-23.06 €, depending on the level of disassembly. Copyright © 2013 Elsevier Ltd. All rights reserved.

Microscale Mechanics of Actin Networks During Dynamic Assembly and Dissociation

NASA Astrophysics Data System (ADS)

Gurmessa, Bekele; Robertson-Anderson, Rae; Ross, Jennifer; Nguyen, Dan; Saleh, Omar

Actin is one of the key components of the cytoskeleton, enabling cells to move and divide while maintaining shape by dynamic polymerization, dissociation and crosslinking. Actin polymerization and network formation is driven by ATP hydrolysis and varies depending on the concentrations of actin monomers and crosslinking proteins. The viscoelastic properties of steady-state actin networks have been well-characterized, yet the mechanical properties of these non-equilibrium systems during dynamic assembly and disassembly remain to be understood. We use semipermeable microfluidic devices to induce in situ dissolution and re-polymerization of entangled and crosslinked actin networks, by varying ATP concentrations in real-time, while measuring the mechanical properties during disassembly and re-assembly. We use optical tweezers to sinusoidally oscillate embedded microspheres and measure the resulting force at set time-intervals and in different regions of the network during cyclic assembly/disassembly. We determine the time-dependent viscoelastic properties of non-equilibrium network intermediates and the reproducibility and homogeneity of network formation and dissolution. Results inform the role that cytoskeleton reorganization plays in the dynamic multifunctional mechanics of cells. NSF CAREER Award (DMR-1255446) and a Scialog Collaborative Innovation Award funded by Research Corporation for Scientific Advancement (Grant No. 24192).

Xyloglucan mobilisation in cotyledons of developing plantlets of Hymenaea courbaril L. (Leguminosae-Caesalpinoideae).

PubMed

Tiné; Cortelazzo; Buckeridge

2000-05-29

Many seeds contain storage compounds that are used by the embryo/plantlet as a source of nutrients after germination. In seeds of Hymenaea courbaril, a leguminous tree, the main reserve consists of a structurally unusual xyloglucan stored in thickened walls of the cotyledon cells. The present work aimed to study H. courbaril xyloglucan metabolism during and after germination in order to compare its degrading system with the other known xyloglucan containing seeds. Polysaccharide degradation occurred after germination between 35 and 55 days after planting. The activities of alpha-xylosidase, beta-glucosidase, beta-galactosidase and XET rose during the period of xyloglucan disassembling but a low level of endo-beta-glucanase activity was detected, suggesting that this XET has high affinity for the oligosaccharides. The pH optimum of beta-galactosidase was different from the alpha-xylosidase, beta-glucosidase and XET optima suggesting that the former may be important in the control of the mobilisation process. A tentative model for xyloglucan disassembling in vivo is proposed, where beta-galactosidase allows the free oligosaccharides to bypass a transglycosylation cycle and be disassembled by the other exo-enzymes. Some ecophysiological comparisons among H. courbaril and other xyloglucan storing seeds are discussed.

LEAP into the Pfizer Global Virtual Library (PGVL) space: creation of readily synthesizable design ideas automatically.

PubMed

Hu, Qiyue; Peng, Zhengwei; Kostrowicki, Jaroslav; Kuki, Atsuo

2011-01-01

Pfizer Global Virtual Library (PGVL) of 10(13) readily synthesizable molecules offers a tremendous opportunity for lead optimization and scaffold hopping in drug discovery projects. However, mining into a chemical space of this size presents a challenge for the concomitant design informatics due to the fact that standard molecular similarity searches against a collection of explicit molecules cannot be utilized, since no chemical information system could create and manage more than 10(8) explicit molecules. Nevertheless, by accepting a tolerable level of false negatives in search results, we were able to bypass the need for full 10(13) enumeration and enabled the efficient similarity search and retrieval into this huge chemical space for practical usage by medicinal chemists. In this report, two search methods (LEAP1 and LEAP2) are presented. The first method uses PGVL reaction knowledge to disassemble the incoming search query molecule into a set of reactants and then uses reactant-level similarities into actual available starting materials to focus on a much smaller sub-region of the full virtual library compound space. This sub-region is then explicitly enumerated and searched via a standard similarity method using the original query molecule. The second method uses a fuzzy mapping onto candidate reactions and does not require exact disassembly of the incoming query molecule. Instead Basis Products (or capped reactants) are mapped into the query molecule and the resultant asymmetric similarity scores are used to prioritize the corresponding reactions and reactant sets. All sets of Basis Products are inherently indexed to specific reactions and specific starting materials. This again allows focusing on a much smaller sub-region for explicit enumeration and subsequent standard product-level similarity search. A set of validation studies were conducted. The results have shown that the level of false negatives for the disassembly-based method is acceptable when the query molecule can be recognized for exact disassembly, and the fuzzy reaction mapping method based on Basis Products has an even better performance in terms of lower false-negative rate because it is not limited by the requirement that the query molecule needs to be recognized by any disassembly algorithm. Both search methods have been implemented and accessed through a powerful desktop molecular design tool (see ref. (33) for details). The chapter will end with a comparison of published search methods against large virtual chemical space.

Myogenin Recruits the Histone Chaperone Facilitates Chromatin Transcription (FACT) to Promote Nucleosome Disassembly at Muscle-specific Genes*

PubMed Central

Lolis, Alexandra A.; Londhe, Priya; Beggs, Benjamin C.; Byrum, Stephanie D.; Tackett, Alan J.; Davie, Judith K.

2013-01-01

Facilitates chromatin transcription (FACT) functions to reorganize nucleosomes by acting as a histone chaperone that destabilizes and restores nucleosomal structure. The FACT complex is composed of two subunits: SSRP1 and SPT16. We have discovered that myogenin interacts with the FACT complex. Transfection of FACT subunits with myogenin is highly stimulatory for endogenous muscle gene expression in 10T1/2 cells. We have also found that FACT subunits do not associate with differentiation-specific genes while C2C12 cells are proliferating but are recruited to muscle-specific genes as differentiation initiates and then dissociate as differentiation proceeds. The recruitment is dependent on myogenin, as knockdowns of myogenin show no recruitment of the FACT complex. These data suggest that FACT is involved in the early steps of gene activation through its histone chaperone activities that serve to open the chromatin structure and facilitate transcription. Consistent with this hypothesis, we find that nucleosomes are depleted at muscle-specific promoters upon differentiation and that this activity is dependent on the presence of FACT. Our results show that the FACT complex promotes myogenin-dependent transcription and suggest that FACT plays an important role in the establishment of the appropriate transcription profile in a differentiated muscle cell. PMID:23364797

Centromere pairing – tethering partner chromosomes in meiosis I

PubMed Central

Kurdzo, Emily L; Dawson, Dean S

2015-01-01

In meiosis, homologous chromosomes face the obstacle of finding, holding onto and segregating away from their partner chromosome. There is increasing evidence, in a diverse range of organisms, that centromere–centromere interactions that occur in late prophase are an important mechanism in ensuring segregation fidelity. Centromere pairing appears to initiate when homologous chromosomes synapse in meiotic prophase. Structural proteins of the synaptonemal complex have been shown to help mediate centromere pairing, but how the structure that maintains centromere pairing differs from the structure of the synaptonemal complex along the chromosomal arms remains unknown. When the synaptonemal complex proteins disassemble from the chromosome arms in late prophase, some of these synaptonemal complex components persist at the centromeres. In yeast and Drosophila these centromere-pairing behaviors promote the proper segregation of chromosome partners that have failed to become linked by chiasmata. Recent studies of mouse spermatocytes have described centromere pairing behaviors that are similar in several respects to what has been described in the fly and yeast systems. In humans, chromosomes that fail to experience crossovers in meiosis are error-prone and are a major source of aneuploidy. The finding that centromere pairing is a conserved phenomenon raises the possibility that it may play a role in promoting the segregation fidelity of non-exchange chromosome pairs in humans. PMID:25817724

Assembly and control of large microtubule complexes

NASA Astrophysics Data System (ADS)

Korolev, Kirill; Ishihara, Keisuke; Mitchison, Timothy

Motility, division, and other cellular processes require rapid assembly and disassembly of microtubule structures. We report a new mechanism for the formation of asters, radial microtubule complexes found in very large cells. The standard model of aster growth assumes elongation of a fixed number of microtubules originating from the centrosomes. However, aster morphology in this model does not scale with cell size, and we found evidence for microtubule nucleation away from centrosomes. By combining polymerization dynamics and auto-catalytic nucleation of microtubules, we developed a new biophysical model of aster growth. The model predicts an explosive transition from an aster with a steady-state radius to one that expands as a travelling wave. At the transition, microtubule density increases continuously, but aster growth rate discontinuously jumps to a nonzero value. We tested our model with biochemical perturbations in egg extract and confirmed main theoretical predictions including the jump in the growth rate. Our results show that asters can grow even though individual microtubules are short and unstable. The dynamic balance between microtubule collapse and nucleation could be a general framework for the assembly and control of large microtubule complexes. NIH GM39565; Simons Foundation 409704; Honjo International 486 Scholarship Foundation.

Metal-Organic Polyhedron Capped with Cucurbit[8]uril Delivers Doxorubicin to Cancer Cells.

PubMed

Samanta, Soumen K; Moncelet, Damien; Briken, Volker; Isaacs, Lyle

2016-11-02

Self-assembly of ligand 1 and Pd(NO 3 ) 2 delivers Fujita-type metal-organic polyhedron (MOP) 3 which bears 24 covalently attached methyl viologen units on its external surface, as evidenced by 1 H NMR, diffusion-ordered spectroscopy NMR, electrospray mass spectrometry, transmission electron microscopy, and atomic force microscopy measurements. MOP 3 undergoes noncovalent complexation with cucurbit[n]urils to yield MOPs 4-6 with diameter ≈5-6 nm. MOP 5 can be fully loaded with doxorubicin (DOX) prodrug 2 via hetero-ternary complex formation to yield 7. The MOPs exhibit excellent stability toward neutral to slightly acidic pH in 10 mM sodium phosphate buffer, mitigating the concern of disassembly during circulation. The results of MTS assays show that MOP 7 is 10-fold more cytotoxic toward HeLa cells than equimolar quantities of DOX prodrug 2. The enhanced cytotoxicity can be traced to a combination of enhanced cellular uptake of 7 and DOX release as demonstrated by flow cytometry and confocal fluorescence microscopy. The confluence of properties imparted by the polycationic MOP architecture and plug-and-play CB[n] complexation provides a potent new platform for drug delivery application.

Both cyclin A and cyclin E have S-phase promoting (SPF) activity in Xenopus egg extracts.

PubMed

Strausfeld, U P; Howell, M; Descombes, P; Chevalier, S; Rempel, R E; Adamczewski, J; Maller, J L; Hunt, T; Blow, J J

1996-06-01

Extracts of activated Xenopus eggs in which protein synthesis has been inhibited support a single round of chromosomal DNA replication. Affinity-depletion of cyclin dependent kinases (Cdks) from these extracts blocks the initiation of DNA replication. We define 'S-phase promoting factor' (SPF) as the Cdk activity required for DNA replication in these Cdk-depleted extracts. Recombinant cyclins A and E, but not cyclin B, showed significant SPF activity. High concentrations of cyclin A promoted entry into mitosis, which inhibited DNA replication. In contrast, high concentrations of cyclin E1 promoted neither nuclear envelope disassembly nor full chromosome condensation. In the early embryo cyclin E1 complexes exclusively with Cdk2 and cyclin A is complexed predominantly with Cdc2; only later in development does cyclin A associate with Cdk2. We show that baculovirus-produced complexes of cyclin A-Cd2, cyclin A-Cdk2 and cyclin E-Cdk2 could each provide SPF activity. These results suggest that although in the early Xenopus embryo cyclin E1-Cdk2 is sufficient to support entry into S-phase, cyclin A-Cdc2 provides a significant additional quantity of SPF as its levels rise during S phase.

Stability of spermine oxidase to thermal and chemical denaturation: comparison with bovine serum amine oxidase.

PubMed

Cervelli, Manuela; Leonetti, Alessia; Cervoni, Laura; Ohkubo, Shinji; Xhani, Marla; Stano, Pasquale; Federico, Rodolfo; Polticelli, Fabio; Mariottini, Paolo; Agostinelli, Enzo

2016-10-01

Spermine oxidase (SMOX) is a flavin-containing enzyme that specifically oxidizes spermine to produce spermidine, 3-aminopropanaldehyde and hydrogen peroxide. While no crystal structure is available for any mammalian SMOX, X-ray crystallography showed that the yeast Fms1 polyamine oxidase has a dimeric structure. Based on this scenario, we have investigated the quaternary structure of the SMOX protein by native gel electrophoresis, which revealed a composite gel band pattern, suggesting the formation of protein complexes. All high-order protein complexes are sensitive to reducing conditions, showing that disulfide bonds were responsible for protein complexes formation. The major gel band other than the SMOX monomer is the covalent SMOX homodimer, which was disassembled by increasing the reducing conditions, while being resistant to other denaturing conditions. Homodimeric and monomeric SMOXs are catalytically active, as revealed after gel staining for enzymatic activity. An engineered SMOX mutant deprived of all but two cysteine residues was prepared and characterized experimentally, resulting in a monomeric species. High-sensitivity differential scanning calorimetry of SMOX was compared with that of bovine serum amine oxidase, to analyse their thermal stability. Furthermore, enzymatic activity assays and fluorescence spectroscopy were used to gain insight into the unfolding process.

GDP-tubulin incorporation into growing microtubules modulates polymer stability.

PubMed

Valiron, Odile; Arnal, Isabelle; Caudron, Nicolas; Job, Didier

2010-06-04

Microtubule growth proceeds through the endwise addition of nucleotide-bound tubulin dimers. The microtubule wall is composed of GDP-tubulin subunits, which are thought to come exclusively from the incorporation of GTP-tubulin complexes at microtubule ends followed by GTP hydrolysis within the polymer. The possibility of a direct GDP-tubulin incorporation into growing polymers is regarded as hardly compatible with recent structural data. Here, we have examined GTP-tubulin and GDP-tubulin incorporation into polymerizing microtubules using a minimal assembly system comprised of nucleotide-bound tubulin dimers, in the absence of free nucleotide. We find that GDP-tubulin complexes can efficiently co-polymerize with GTP-tubulin complexes during microtubule assembly. GDP-tubulin incorporation into microtubules occurs with similar efficiency during bulk microtubule assembly as during microtubule growth from seeds or centrosomes. Microtubules formed from GTP-tubulin/GDP-tubulin mixtures display altered microtubule dynamics, in particular a decreased shrinkage rate, apparently due to intrinsic modifications of the polymer disassembly properties. Thus, although microtubules polymerized from GTP-tubulin/GDP-tubulin mixtures or from homogeneous GTP-tubulin solutions are both composed of GDP-tubulin subunits, they have different dynamic properties, and this may reveal a novel form of microtubule "structural plasticity."

Mechanisms of cooperation in cancer nanomedicine: towards systems nanotechnology.

PubMed

Hauert, Sabine; Bhatia, Sangeeta N

2014-09-01

Nanoparticles are designed to deliver therapeutics and diagnostics selectively to tumors. Their size, shape, charge, material, coating, and cargo determine their individual functionalities. A systems approach could help predict the behavior of trillions of nanoparticles interacting in complex tumor environments. Engineering these nanosystems may lead to biomimetic strategies where interactions between nanoparticles and their environment give rise to cooperative behaviors typically seen in natural self-organized systems. Examples include nanoparticles that communicate the location of a tumor to amplify tumor homing or self-assemble and disassemble to optimize nanoparticle transport. The challenge is to discover which nanoparticle designs lead to a desired system behavior. To this end, novel nanomaterials, deep understanding of biology, and computational tools are emerging as the next frontier. Copyright © 2014 Elsevier Ltd. All rights reserved.

Nano-encrypted Morse code: a versatile approach to programmable and reversible nanoscale assembly and disassembly.

PubMed

Wong, Ngo Yin; Xing, Hang; Tan, Li Huey; Lu, Yi

2013-02-27

While much work has been devoted to nanoscale assembly of functional materials, selective reversible assembly of components in the nanoscale pattern at selective sites has received much less attention. Exerting such a reversible control of the assembly process will make it possible to fine-tune the functional properties of the assembly and to realize more complex designs. Herein, by taking advantage of different binding affinities of biotin and desthiobiotin toward streptavidin, we demonstrate selective and reversible decoration of DNA origami tiles with streptavidin, including revealing an encrypted Morse code "NANO" and reversible exchange of uppercase letter "I" with lowercase "i". The yields of the conjugations are high (>90%), and the process is reversible. We expect this versatile conjugation technique to be widely applicable with different nanomaterials and templates.

NM-Scale Anatomy of an Entire Stardust Carrot Track

NASA Technical Reports Server (NTRS)

Nakamura-Messenger, K.; Keller, L. P.; Clemett, S. J.; Messenger, S.

2009-01-01

Comet Wild-2 samples collected by NASA s Stardust mission are extremely complex, heterogeneous, and have experienced wide ranges of alteration during the capture process. There are two major types of track morphologies: "carrot" and "bulbous," that reflect different structural/compositional properties of the impactors. Carrot type tracks are typically produced by compact or single mineral grains which survive essentially intact as a single large terminal particle. Bulbous tracks are likely produced by fine-grained or organic-rich impactors [1]. Owing to their challenging nature and especially high value of Stardust samples, we have invested considerable effort in developing both sample preparation and analytical techniques tailored for Stardust sample analyses. Our report focuses on our systematic disassembly and coordinated analysis of Stardust carrot track #112 from the mm to nm-scale.

KSC-2011-4532

NASA Image and Video Library

2011-06-16

CAPE CANAVERAL, Fla. -- At the Shuttle Landing Facility at NASA's Kennedy Space Center in Florida, workers practice assembly and disassembling a mobile sling and crane, part of a portable mate-demate device (image left). The portable sling and crane will be used to lift shuttles off their 747 carrier aircraft for delivery to museums next year. Shuttle Discovery will go to the Smithsonian's National Air and Space Museum, Steven F. Udvar-Hazy Center in Chantilly, Va., Endeavour will go to the California Science Center in Los Angeles and Enterprise will be moved from the Smithsonian to the Intrepid Sea, Air and Space Museum in New York. Shuttle Atlantis will go to the Kennedy Space Center Visitor Complex and won't require use of the portable sling and crane. Photo credit: NASA/Jim Grossmann

E3 ligase Hei10: a multifaceted structure-based signaling molecule with roles within and beyond meiosis

PubMed Central

De Muyt, Arnaud; Zhang, Liangran; Piolot, Tristan; Kleckner, Nancy; Espagne, Eric; Zickler, Denise

2014-01-01

Human enhancer of invasion-10 (Hei10) mediates meiotic recombination and also plays roles in cell proliferation. Here we explore Hei10’s roles throughout the sexual cycle of the fungus Sordaria with respect to localization and effects of null, RING-binding, and putative cyclin-binding (RXL) domain mutations. Hei10 makes three successive types of foci. Early foci form along synaptonemal complex (SC) central regions. At some of these positions, depending on its RING and RXL domains, Hei10 mediates development and turnover of two sequential types of recombination complexes, each demarked by characteristic amplified Hei10 foci. Integration with ultrastructural data for recombination nodules further reveals that recombination complexes differentiate into three types, one of which corresponds to crossover recombination events during or prior to SC formation. Finally, Hei10 positively and negatively modulates SUMO localization along SCs by its RING and RXL domains, respectively. The presented findings suggest that Hei10 integrates signals from the SC, associated recombination complexes, and the cell cycle to mediate both the development and programmed turnover/evolution of recombination complexes via SUMOylation/ubiquitination. Analogous cell cycle-linked assembly/disassembly switching could underlie localization and roles for Hei10 in centrosome/spindle pole body dynamics and associated nuclear trafficking. We suggest that Hei10 is a unique type of structure-based signal transduction protein. PMID:24831702

Study to define an approach for developing a computer-based system capable of automatic, unattended assembly/disassembly of spacecraft, phase 1

NASA Technical Reports Server (NTRS)

Nevins, J. L.; Defazio, T. L.; Seltzer, D. S.; Whitney, D. E.

1981-01-01

The initial set of requirements for additional studies necessary to implement a space-borne, computer-based work system capable of achieving assembly, disassembly, repair, or maintenance in space were developed. The specific functions required of a work system to perform repair and maintenance were discussed. Tasks and relevant technologies were identified and delineated. The interaction of spacecraft design and technology options, including a consideration of the strategic issues of repair versus retrieval-replacement or destruction by removal were considered along with the design tradeoffs for accomplishing each of the options. A concept system design and its accompanying experiment or test plan were discussed.

Proteins from disassembled microtubules characterized by oligospecific antisera.

PubMed

Meier, E; Jorgensen, O S

1977-10-26

The immunochemical properties of in vitro reassembled microtubules were investigated by immunoelectrophoretic techniques. The tubulin dimer gave no measurable immunochemical response, but the tubulin oligomer, the tau-factor and an antigen of about 135 000 daltons all gave precipitating antibodies. Those four proteins were investigated in reassembled microtubules, in DEAE-cellulose purified tubulin, and after molecular sieve chromatography of disassembled and NaCl-dissociated microtubules. Reconstitution of tubulin oligomer from tubulin dimer and tau-factor was also performed. The presence of a unique antigenic structure on tubulin oligomer which was not found in the dissociated components and the role of this aggregate as a nucleation center or intermediate in the assembly of microtubules is discussed.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Norris, R.S.; Arkin, W.M.

The US. nuclear stockpile is at its lowest level since late 1958 or early 1959. In the past year, many weapons were returned to central military storage depots in the United States and funneled to the Energy Department's Pantex facility for final disassembly and disposal. This article presents a table showing the author's current estimate of the composition of the current operational stockpile, which contains some 10,500 warheads. Also categorized are warheads in [open quotes]inactive reserve[close quotes] and warheads awaiting eventual disassembly. The warheads are generally grouped as bombs, submarine-launched ballistic missiles, intercontinental ballistic missiles, air-launched cruise missiles, and sea-launchedmore » cruise missiles. Initial production dates and yield are listed for the warheads.« less

Colossal Tooling Design: 3D Simulation for Ergonomic Analysis

NASA Technical Reports Server (NTRS)

Hunter, Steve L.; Dischinger, Charles; Thomas, Robert E.; Babai, Majid

2003-01-01

The application of high-level 3D simulation software to the design phase of colossal mandrel tooling for composite aerospace fuel tanks was accomplished to discover and resolve safety and human engineering problems. The analyses were conducted to determine safety, ergonomic and human engineering aspects of the disassembly process of the fuel tank composite shell mandrel. Three-dimensional graphics high-level software, incorporating various ergonomic analysis algorithms, was utilized to determine if the process was within safety and health boundaries for the workers carrying out these tasks. In addition, the graphical software was extremely helpful in the identification of material handling equipment and devices for the mandrel tooling assembly/disassembly process.

Examination of SR101 shipping packages

DOE Office of Scientific and Technical Information (OSTI.GOV)

Daugherty, W. L.

Four SR101 shipping packages were removed from service and provided for disassembly and examination of the internal fiberboard assemblies. These packages were 20 years old, and had experienced varying levels of degradation. Two of the packages were successfully disassembled and fiberboard samples were removed from these packages and tested. Mechanical and thermal property values are generally comparable to or higher than baseline values measured on fiberboard from 9975 packages, which differs primarily in the specified density range. While baseline data for the SR101 material is not available, this comparison with 9975 material suggests that the material properties of the SR101more » fiberboard have not significantly degraded.« less

Observation of the noncovalent assembly and disassembly pathways of the chaperone complex MtGimC by mass spectrometry

PubMed Central

Fändrich, Marcus; Tito, Mark A.; Leroux, Michel R.; Rostom, Adam A.; Hartl, F. Ulrich; Dobson, Christopher M.; Robinson, Carol V.

2000-01-01

We have analyzed a newly described archaeal GimC/prefoldin homologue, termed MtGimC, by using nanoflow electrospray coupled with time-of-flight MS. The molecular weight of the complex from Methanobacterium thermoautotrophicum corresponds to a well-defined hexamer of two α subunits and four β subunits. Dissociation of the complex within the gas phase reveals a quaternary arrangement of two central subunits, both α, and four peripheral β subunits. By constructing a thermally controlled nanoflow device, we have monitored the thermal stability of the complex by MS. The results of these experiments demonstrate that a significant proportion of the MtGimC hexamer remains intact under low-salt conditions at elevated temperatures. This finding is supported by data from CD spectroscopy, which show that at physiological salt concentrations, the complex remains stable at temperatures above 65°C. Mass spectrometric methods were developed to monitor in real time the assembly of the MtGimC hexamer from its component subunits. By using this methodology, the mass spectra recorded throughout the time course of the experiment showed the absence of any significantly populated intermediates, demonstrating that the assembly process is highly cooperative. Taken together, these data show that the complex is stable under the elevated temperatures that are appropriate for its hyperthermophile host and demonstrate that the assembly pathway leads exclusively to the hexamer, which is likely to be a structural unit in vivo. PMID:11087821

Lysosomal Multienzyme Complex: Pros and Cons of Working Together

PubMed Central

Bonten, Erik J.; Annunziata, Ida; d’Azzo, Alessandra

2014-01-01

The ubiquitous distribution of lysosomes and their heterogeneous protein composition reflects the versatility of these organelles in maintaining cell homeostasis and their importance in tissue differentiation and remodeling. In lysosomes, the degradation of complex, macromolecular substrates requires the synergistic action of multiple hydrolases that usually work in a stepwise fashion. This catalytic machinery explains the existence of lysosomal enzyme complexes that can be dynamically assembled and disassembled to efficiently and quickly adapt to the pool of substrates to be processed or degraded, adding extra tiers to the regulation of the individual protein components. An example of such a complex is the one composed of three hydrolases that are ubiquitously but differentially expressed: the serine carboxypeptidase, Protective Protein/Cathepsin A (PPCA), the sialidase, Neuraminidase-1 (NEU1), and the glycosidase β-Galactosidase (β-GAL). Next to this ‘core’ complex, the existence of sub-complexes, that may contain additional components, and function at the cell surface or extracellularly, suggests as yet unexplored functions of these enzymes. Here we review how studies of basic biological processes in the mouse models of three lysosomal storage disorders, galactosialidosis, sialidosis, and GM1-gangliosidosis, revealed new and unexpected roles for the three respective affected enzymes, Ppca, Neu1 and β-Gal, that go beyond their canonical degradative activities. These findings have broadened our perspective on their functions and may pave the way for the development of new therapies for these lysosomal storage disorders. PMID:24337808

Structural insights into the p97-Ufd1-Npl4 complex

PubMed Central

Pye, Valerie E.; Beuron, Fabienne; Keetch, Catherine A.; McKeown, Ciaran; Robinson, Carol V.; Meyer, Hemmo H.; Zhang, Xiaodong; Freemont, Paul S.

2007-01-01

p97/VCP (Cdc48 in yeast) is an essential and abundant member of the AAA+ family of ATPases and is involved in a number of diverse cellular pathways through interactions with different adaptor proteins. The two most characterized adaptors for p97 are p47 and the Ufd1 (ubiquitin fusion degradation 1)-Npl4 (nuclear protein localization 4) complex. p47 directs p97 to membrane fusion events and has been shown to be involved in protein degradation. The Ufd1-Npl4 complex directs p97 to an essential role in endoplasmic reticulum-associated degradation and an important role in mitotic spindle disassembly postmitosis. Here we describe the structural features of the Ufd1-Npl4 complex and its interaction with p97 with the aid of EM and other biophysical techniques. The Ufd1-Npl4 heterodimer has an elongated bilobed structure that is ≈80 × 30 Å in dimension. One Ufd1-Npl4 heterodimer is shown to interact with one p97 hexamer to form the p97-Ufd1-Npl4 complex. The Ufd1-Npl4 heterodimer emanates from one region on the periphery of the N-D1 plane of the p97 hexamer. Intriguingly, the p97-p47 and the p97-Ufd1-Npl4 complexes are significantly different in stoichiometry, symmetry, and quaternary arrangement, reflecting their specific actions and their ability to interact with additional cofactors that cooperate with p97 in diverse cellular pathways. PMID:17202270

Structural mechanisms of DREAM complex assembly and regulation

PubMed Central

Guiley, Keelan Z.; Liban, Tyler J.; Felthousen, Jessica G.; Ramanan, Parameshwaran

2015-01-01

The DREAM complex represses cell cycle genes during quiescence through scaffolding MuvB proteins with E2F4/5 and the Rb tumor suppressor paralog p107 or p130. Upon cell cycle entry, MuvB dissociates from p107/p130 and recruits B-Myb and FoxM1 for up-regulating mitotic gene expression. To understand the biochemical mechanisms underpinning DREAM function and regulation, we investigated the structural basis for DREAM assembly. We identified a sequence in the MuvB component LIN52 that binds directly to the pocket domains of p107 and p130 when phosphorylated on the DYRK1A kinase site S28. A crystal structure of the LIN52–p107 complex reveals that LIN52 uses a suboptimal LxSxExL sequence together with the phosphate at nearby S28 to bind the LxCxE cleft of the pocket domain with high affinity. The structure explains the specificity for p107/p130 over Rb in the DREAM complex and how the complex is disrupted by viral oncoproteins. Based on insights from the structure, we addressed how DREAM is disassembled upon cell cycle entry. We found that p130 and B-Myb can both bind the core MuvB complex simultaneously but that cyclin-dependent kinase phosphorylation of p130 weakens its association. Together, our data inform a novel target interface for studying MuvB and p130 function and the design of inhibitors that prevent tumor escape in quiescence. PMID:25917549

Flight set 360T004 (STS-30)

NASA Technical Reports Server (NTRS)

Nelsen, Lowell V.

1990-01-01

The performance of 360T004, Forth Flight, Redesigned Solid Rocket Motors (RSRM) is assessed in respect to joint sealing issues as seen from post-test inspection of the seals and sealing surfaces. The factory joint disassembly inspections for this flight set were omitted. The decision was based on the rational that there is sufficient information in the present data base, and this would give H-7 refurbishment operations faster turn around time for this set of hardware. The factory joint disassembly inspections will resume for 360H005, Fifth Flight, through 360L007, Seventh Flight, due to a new grease application being in effect during the assembly process. The left hand nozzle was forced into the snubbed position upon splash down. This required unique tooling to be manufactured to perform the disassembly of the internal nozzle joints. This was completed on February 5 and 6, 1990 at the H-5 Clearfield, Utah facility. The RSRM consisting of capture feature, field joints with the J-joint insulation configuration is illustrated. The nozzle-to-case joint design, which includes 100, 7/8-inch radial bolts in conjunction with a wiper O-ring and modified insulation design is also illustrated, as is the ignition system seals and a cross section of the igniter. The configuration of all internal nozzle joints is shown.

Postflight hardware evaluation 360T026 (RSRM-26, STS-47)

NASA Technical Reports Server (NTRS)

Nielson, Greg

1993-01-01

The final report for the Clearfield disassembly evaluation and a continuation of the KSC postflight assessment for the 360T026 (STS-47) Redesigned Solid Rocket Motor (RSRM) flight set is provided. All observed hardware conditions were documented on PFOR's and are included in Appendices A, B, and C. Appendices D and E contain the measurements and safety factor data for the nozzle and insulation components. This report, along with the KSC Ten-Day Postflight Hardware Evaluation Report (TWR-64203), represents a summary of the 360T026 hardware evaluation. The as-flown hardware configuration is documented in TWR-60472. Disassembly evaluation photograph numbers are logged in TWA-1987. The 360T026 flight set disassembly evaluations described were performed at the RSRM Refurbishment Facility in Clearfield, Utah. The final factory joint demate occurred on 12 April 1993. Detailed evaluations were performed in accordance with the Clearfield Postflight Engineering Evaluation Plan (PEEP), TWR-50051, Revision A. All observations were compared against limits that are also defined in the PEEP. These limits outline the criteria for categorizing the observations as acceptable, reportable, or critical. Hardware conditions that were unexpected and/or determined to be reportable or critical were evaluated by the applicable CPT and tracked through the PFAR system.

Problems Encountered During the Recertification of the GLORY Solar Array Dual Axis Gimbal Drive Actuators

NASA Technical Reports Server (NTRS)

Saltzman, Marc; Schepis, Jospeh P.; Bruckner, Michael J.

2009-01-01

The Glory observatory is the current incarnation of the Vegetation Canopy Lidar (VCL) mission spacecraft bus. The VCL spacecraft bus, having been cancelled for programmatic reasons in 2000, was nearly integrated when it was put into storage for possible future use. The Glory mission was a suitable candidate for using this spacecraft and in 2006 an effort to recertify the two axis solar array gimbal drive after its extended storage was begun. What was expected to be a simple performance validation of the two dual axis gimbal stepper motors became a serious test, diagnosis and repair task once questions arose on the flight worthiness of the hardware. A significant test program logic flow was developed which identified decisions that could be made based on the results of individual recertification tests. Without disassembling the bi-axial gimbals, beginning with stepper motor threshold voltage measurements and relating these to powered drive torque measurements, both performed at the spacecraft integrator s facility, a confusing picture of the health of the actuators came to light. Tests at the gimbal assembly level and tests of the disassembled actuators were performed by the manufacturer to validate our results and torque discrepancies were noted. Further disassembly to the component level of the actuator revealed the source of the torque loss.

Stimuli-disassembling gold nanoclusters for diagnosis of early stage oral cancer by optical coherence tomography

NASA Astrophysics Data System (ADS)

Kim, Chang Soo; Ingato, Dominique; Wilder-Smith, Petra; Chen, Zhongping; Kwon, Young Jik

2018-01-01

A key design consideration in developing contrast agents is obtaining distinct, multiple signal changes in diseased tissue. Plasmonic gold nanoparticles (Au NPs) have been developed as contrast agents due to their strong surface plasmon resonance (SPR). This study aims to demonstrate that stimuli-responsive plasmonic Au nanoclusters (Au NCs) can be used as a contrast agent for optical coherence tomography (OCT) in detecting early-stage cancer. Au NPs were clustered via acid-cleavable linkers to synthesize Au NCs that disassemble under mildly acidic conditions into individual Au NPs, simultaneously diminishing SPR effect (quantified by scattering intensity) and increasing Brownian motion (quantified by Doppler variance). The acid-triggered morphological and accompanying optico-physical property changes of the acid-disassembling Au NCs were confirmed by TEM, DLS, UV/Vis, and OCT. Stimuli-responsive Au NCs were applied in a hamster check pouch model carrying early-stage squamous carcinoma tissue. The tissue was visualized by OCT imaging, which showed reduced scattering intensity and increased Doppler variance in the dysplastic tissue. This study demonstrates the promise of diagnosing early-stage cancer using molecularly programmable, inorganic nanomaterial-based contrast agents that are capable of generating multiple, stimuli-triggered diagnostic signals in early-stage cancer.[Figure not available: see fulltext.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tao, Jinhui; Buchko, Garry W.; Shaw, Wendy J.

The interactions between proteins and surfaces are critical to a number of important processes including biomineralization, the biocompatibility of biomaterials, and the function of biosensors. Although many proteins exist as monomers or small oligomers, amelogenin is a unique protein that self-assembles into supramolecular structures called “nanospheres,” aggregates of 100’s of monomers that are 20-60 nm in diameter. The nanosphere quaternary structure is observed in solution, however, the quaternary structure of amelogenin adsorbed onto hydroxyapatite (HAP) surfaces is not known even though it may be important to amelogenin’s function in forming highly elongated and intricately assembled HAP crystallites during enamel formation.more » We report studies of the interactions of the enamel protein, amelogenin (rpM179), with a well-defined (100) face prepared by synthesis of large crystals of HAP. High resolution, in-situ atomic force microscopy (AFM) was used to directly observe protein adsorption onto HAP at the molecular level within an aqueous solution environment. Our study shows that the amelogenin nanospheres disassemble onto the HAP surface, breaking down into oligomeric (25-mer) subunits of the larger nanosphere. In some cases, the disassembly event is directly observed by in situ imaging for the first time. Quantification of the adsorbate amounts by size analysis led to the determination of a protein binding energy (17.1 kbT) to a specific face of HAP (100). The kinetics of disassembly are greatly slowed in aged solutions, indicating there are time-dependent increases in oligomer-oligomer binding interactions within the nanosphere. A small change in the sequence of amelogenin by the attachment of a histidine tag to the N-terminus of rpM179 to form rp(H)M180 results in the adsorption of a complete second layer on top of the underlying first layer. Our research elucidates how supramolecular protein structures interact and break down at surfaces and how small changes in the primary sequence of amelogenin can affect the disassembly process.« less

MgATP hydrolysis destabilizes the interaction between subunit H and yeast V1-ATPase, highlighting H's role in V-ATPase regulation by reversible disassembly.

PubMed

Sharma, Stuti; Oot, Rebecca A; Wilkens, Stephan

2018-05-12

Vacuolar H+-ATPases (V-ATPases; V1Vo-ATPases) are rotary motor proton pumps that acidify intracellular compartments and in some tissues, the extracellular space. V-ATPase is regulated by reversible disassembly into autoinhibited V1-ATPase and Vo proton channel sectors. An important player in V-ATPase regulation is subunit H, which binds at the interface of V1 and Vo. H is required for MgATPase activity in holo V-ATPase, but also for stabilizing the MgADP inhibited state in membrane detached V1. However, how H fulfills these two functions is poorly understood. To characterize the H-V1 interaction and its role in reversible disassembly, we determined binding affinities of full length H and its N-terminal domain (HNT) for an isolated heterodimer of subunits E and G (EG), the N-terminal domain of subunit a (aNT), and V1 lacking subunit H (V1ΔH). Using isothermal titration calorimetry (ITC) and biolayer interferometry (BLI), we show that HNT binds EG with moderate affinity, that full length H binds aNT weakly, and that both H and HNT bind V1ΔH with high affinity. We also found that only one molecule of HNT binds V1ΔH with high affinity, suggesting conformational asymmetry of the three EG heterodimers in V1ΔH. Moreover, MgATP hydrolysis-driven conformational changes in V1 destabilized the interaction of H, or HNT, with V1ΔH, suggesting an interplay between MgADP inhibition and subunit H. Our observation that H binding is affected by MgATP hydrolysis in V1 points to H's role in the mechanism of reversible disassembly. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Fruit softening and pectin disassembly: an overview of nanostructural pectin modifications assessed by atomic force microscopy

PubMed Central

Paniagua, Candelas; Posé, Sara; Morris, Victor J.; Kirby, Andrew R.; Quesada, Miguel A.; Mercado, José A.

2014-01-01

Background One of the main factors that reduce fruit quality and lead to economically important losses is oversoftening. Textural changes during fruit ripening are mainly due to the dissolution of the middle lamella, the reduction of cell-to-cell adhesion and the weakening of parenchyma cell walls as a result of the action of cell wall modifying enzymes. Pectins, major components of fruit cell walls, are extensively modified during ripening. These changes include solubilization, depolymerization and the loss of neutral side chains. Recent evidence in strawberry and apple, fruits with a soft or crisp texture at ripening, suggests that pectin disassembly is a key factor in textural changes. In both these fruits, softening was reduced as result of antisense downregulation of polygalacturonase genes. Changes in pectic polymer size, composition and structure have traditionally been studied by conventional techniques, most of them relying on bulk analysis of a population of polysaccharides, and studies focusing on modifications at the nanostructural level are scarce. Atomic force microscopy (AFM) allows the study of individual polymers at high magnification and with minimal sample preparation; however, AFM has rarely been employed to analyse pectin disassembly during fruit ripening. Scope In this review, the main features of the pectin disassembly process during fruit ripening are first discussed, and then the nanostructural characterization of fruit pectins by AFM and its relationship with texture and postharvest fruit shelf life is reviewed. In general, fruit pectins are visualized under AFM as linear chains, a few of which show long branches, and aggregates. Number- and weight-average values obtained from these images are in good agreement with chromatographic analyses. Most AFM studies indicate reductions in the length of individual pectin chains and the frequency of aggregates as the fruits ripen. Pectins extracted with sodium carbonate, supposedly located within the primary cell wall, are the most affected. PMID:25063934

The neuroregenerative mechanism mediated by the Hsp90-binding immunophilin FKBP52 resembles the early steps of neuronal differentiation

PubMed Central

Quintá, HR; Galigniana, MD

2012-01-01

BACKGROUND AND PURPOSE The immunosuppressive macrolide FK506 (tacrolimus) shows neuroregenerative action by a mechanism that appears to involve the Hsp90-binding immunophilin FKBP52. This study analyses some aspects of the early steps of neuronal differentiation and neuroregeneration. EXPERIMENTAL APPROACH Undifferentiated murine neuroblastoma cells and hippocampal neurones isolated from embryonic day-17 rat embryos were induced to differentiate with FK506. Subcellular relocalization of FKBP52, Hsp90 and its co-chaperone p23 was analysed by indirect immunofluorescence confocal microscopy and by Western blots of axonal fractions isolated from cells grown on a porous transwell cell culture chamber. Neuroregeneration was evaluated using a scratch-wound assay. KEY RESULTS In undifferentiated cells, FKBP52, Hsp90 and p23 are located in the cell nucleus, forming an annular structure that disassembles when the differentiation process is triggered by FK506. This was observed in the N2a cell line and in hippocampal neurones. More importantly, the annular structure of chaperones is reassembled after damaging the neurones, whereas FK506 prompts their rapid regeneration, a process linked to the subcellular redistribution of the heterocomplex. CONCLUSIONS AND IMPLICATIONS There is a direct relationship between the disassembly of the chaperone complex and the progression of neuronal differentiation upon stimulation with the immunophilin ligand FK506. Both neuronal differentiation and neuroregeneration appear to be mechanistically linked, so the elucidation of one mechanism may lead to unravel the properties of the other. This study also implies that the discovery of FK506 derivatives, devoid of immunosuppressive action, would be therapeutically significant for neurotrophic use. PMID:22091865

The characean internodal cell as a model system for studying wound healing

PubMed Central

Foissner, I.; Wasteneys, G.O.

2012-01-01

Summary This work describes the characean internodal cell as a model system for the study of wound healing and compares wounds induced by certain chemicals and UV irradiation with wounds occurring in the natural environment. We review the existing literature and define three types of wound response: 1) cortical window formation characterized by disassembly of microtubules, transient inhibition of actin-dependent cytoplasmic streaming and chloroplast detachment, 2) fibrillar wound walls characterized by exocytosis of vesicles carrying wall polysaccharides and membrane-bound cellulose synthase complexes coupled with endocytosis of surplus membrane and 3) amorphous, callose- and membrane-containing wound walls characterized by exocytosis of vesicles and endoplasmic reticulum (ER) cisternae in the absence of membrane recycling. We hypothesize that these three wound responses reflect the extent of damage, probably Ca2+ influx, and that the secretion of Ca2+ - loaded ER cisternae is an emergency reaction in case of severe Ca2+ load. Microtubules are not required for wound healing but their disassembly could have a signalling function. Transient reorganization of the actin cytoskeleton into a meshwork of randomly oriented filaments is required for the migration of wound wall forming organelles, just as occurs in tip-growing plant cells. New data presented in this study show that during the deposition of an amorphous wound wall numerous actin rings are present, which may indicate specific ion fluxes and/or a storage form for actin. In addition, we present new evidence for the exocytosis of FM1-43-stained organelles, putative endosomes, required for plasma membrane repair during wound healing. Finally we show that quickly growing fibrillar wound walls, even when deposited in the absence of microtubules, have a highly ordered helical structure of consistent handedness comprised of cellulose microfibrils. PMID:22118365

Effects of Plectin Depletion on Keratin Network Dynamics and Organization

PubMed Central

Moch, Marcin; Windoffer, Reinhard; Schwarz, Nicole; Pohl, Raphaela; Omenzetter, Andreas; Schnakenberg, Uwe; Herb, Fabian; Chaisaowong, Kraisorn; Merhof, Dorit; Ramms, Lena; Fabris, Gloria; Hoffmann, Bernd; Merkel, Rudolf; Leube, Rudolf E.

2016-01-01

The keratin intermediate filament cytoskeleton protects epithelial cells against various types of stress and is involved in fundamental cellular processes such as signaling, differentiation and organelle trafficking. These functions rely on the cell type-specific arrangement and plasticity of the keratin system. It has been suggested that these properties are regulated by a complex cycle of assembly and disassembly. The exact mechanisms responsible for the underlying molecular processes, however, have not been clarified. Accumulating evidence implicates the cytolinker plectin in various aspects of the keratin cycle, i.e., by acting as a stabilizing anchor at hemidesmosomal adhesion sites and the nucleus, by affecting keratin bundling and branching and by linkage of keratins to actin filament and microtubule dynamics. In the present study we tested these hypotheses. To this end, plectin was downregulated by shRNA in vulvar carcinoma-derived A431 cells. As expected, integrin β4- and BPAG-1-positive hemidesmosomal structures were strongly reduced and cytosolic actin stress fibers were increased. In addition, integrins α3 and β1 were reduced. The experiments furthermore showed that loss of plectin led to a reduction in keratin filament branch length but did not alter overall mechanical properties as assessed by indentation analyses using atomic force microscopy and by displacement analyses of cytoplasmic superparamagnetic beads using magnetic tweezers. An increase in keratin movement was observed in plectin-depleted cells as was the case in control cells lacking hemidesmosome-like structures. Yet, keratin turnover was not significantly affected. We conclude that plectin alone is not needed for keratin assembly and disassembly and that other mechanisms exist to guarantee proper keratin cycling under steady state conditions in cultured single cells. PMID:27007410

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marcianò, G.; Huang, D. T., E-mail: d.huang@beatson.gla.ac.uk

The Spt16–SSRP1 heterodimer is a histone chaperone that plays an important role in regulating chromatin assembly. Here, a crystal structure of the N-terminal domain of human Spt16 is presented and it is shown that this domain may contribute to histone binding. The histone chaperone FACT plays an important role in facilitating nucleosome assembly and disassembly during transcription. FACT is a heterodimeric complex consisting of Spt16 and SSRP1. The N-terminal domain of Spt16 resembles an inactive aminopeptidase. How this domain contributes to the histone chaperone activity of FACT remains elusive. Here, the crystal structure of the N-terminal domain (NTD) of humanmore » Spt16 is reported at a resolution of 1.84 Å. The structure adopts an aminopeptidase-like fold similar to those of the Saccharomyces cerevisiae and Schizosaccharomyces pombe Spt16 NTDs. Isothermal titration calorimetry analyses show that human Spt16 NTD binds histones H3/H4 with low-micromolar affinity, suggesting that Spt16 NTD may contribute to histone binding in the FACT complex. Surface-residue conservation and electrostatic analysis reveal a conserved acidic patch that may be involved in histone binding.« less

The selfish yeast plasmid uses the nuclear motor Kip1p but not Cin8p for its localization and equal segregation.

PubMed

Cui, Hong; Ghosh, Santanu K; Jayaram, Makkuni

2009-04-20

The 2 micron plasmid of Saccharomyces cerevisiae uses the Kip1 motor, but not the functionally redundant Cin8 motor, for its precise nuclear localization and equal segregation. The timing and lifetime of Kip1p association with the plasmid partitioning locus STB are consistent with Kip1p being an authentic component of the plasmid partitioning complex. Kip1-STB association is not blocked by disassembling the mitotic spindle. Lack of Kip1p disrupts recruitment of the cohesin complex at STB and cohesion of replicated plasmid molecules. Colocalization of a 2 micron reporter plasmid with Kip1p in close proximity to the spindle pole body is reminiscent of that of a CEN reporter plasmid. Absence of Kip1p displaces the plasmid from this nuclear address, where it has the potential to tether to a chromosome or poach chromosome segregation factors. Exploiting Kip1p, which is subsidiary to Cin8p for chromosome segregation, to direct itself to a "partitioning center" represents yet another facet of the benign parasitism of the yeast plasmid.

Testis-specific ATP synthase peripheral stalk subunits required for tissue-specific mitochondrial morphogenesis in Drosophila.

PubMed

Sawyer, Eric M; Brunner, Elizabeth C; Hwang, Yihharn; Ivey, Lauren E; Brown, Olivia; Bannon, Megan; Akrobetu, Dennis; Sheaffer, Kelsey E; Morgan, Oshauna; Field, Conroy O; Suresh, Nishita; Gordon, M Grace; Gunnell, E Taylor; Regruto, Lindsay A; Wood, Cricket G; Fuller, Margaret T; Hales, Karen G

2017-03-23

In Drosophila early post-meiotic spermatids, mitochondria undergo dramatic shaping into the Nebenkern, a spherical body with complex internal structure that contains two interwrapped giant mitochondrial derivatives. The purpose of this study was to elucidate genetic and molecular mechanisms underlying the shaping of this structure. The knotted onions (knon) gene encodes an unconventionally large testis-specific paralog of ATP synthase subunit d and is required for internal structure of the Nebenkern as well as its subsequent disassembly and elongation. Knon localizes to spermatid mitochondria and, when exogenously expressed in flight muscle, alters the ratio of ATP synthase complex dimers to monomers. By RNAi knockdown we uncovered mitochondrial shaping roles for other testis-expressed ATP synthase subunits. We demonstrate the first known instance of a tissue-specific ATP synthase subunit affecting tissue-specific mitochondrial morphogenesis. Since ATP synthase dimerization is known to affect the degree of inner mitochondrial membrane curvature in other systems, the effect of Knon and other testis-specific paralogs of ATP synthase subunits may be to mediate differential membrane curvature within the Nebenkern.

An essential and NSF independent role for α-SNAP in store-operated calcium entry.

PubMed

Miao, Yong; Miner, Cathrine; Zhang, Lei; Hanson, Phyllis I; Dani, Adish; Vig, Monika

2013-07-16

Store-operated calcium entry (SOCE) by calcium release activated calcium (CRAC) channels constitutes a primary route of calcium entry in most cells. Orai1 forms the pore subunit of CRAC channels and Stim1 is the endoplasmic reticulum (ER) resident Ca(2+) sensor. Upon store-depletion, Stim1 translocates to domains of ER adjacent to the plasma membrane where it interacts with and clusters Orai1 hexamers to form the CRAC channel complex. Molecular steps enabling activation of SOCE via CRAC channel clusters remain incompletely defined. Here we identify an essential role of α-SNAP in mediating functional coupling of Stim1 and Orai1 molecules to activate SOCE. This role for α-SNAP is direct and independent of its known activity in NSF dependent SNARE complex disassembly. Importantly, Stim1-Orai1 clustering still occurs in the absence of α-SNAP but its inability to support SOCE reveals that a previously unsuspected molecular re-arrangement within CRAC channel clusters is necessary for SOCE. DOI:http://dx.doi.org/10.7554/eLife.00802.001.

Effects of Temperature on Microstructure and Wear of Salt Bath Nitrided 17-4PH Stainless Steel

NASA Astrophysics Data System (ADS)

Wang, Jun; Lin, Yuanhua; Fan, Hongyuan; Zeng, Dezhi; Peng, Qian; Shen, Baoluo

2012-08-01

Salt bath nitriding of 17-4 PH martensitic precipitation hardening stainless steels was conducted at 610, 630, and 650 °C for 2 h using a complex salt bath heat-treatment, and the properties of the nitrided surface were systematically evaluated. Experimental results revealed that the microstructure and phase constituents of the nitrided surface alloy are highly process condition dependent. When 17-4PH stainless steel was subjected to complex salt bathing nitriding, the main phase of the nitrided layer was expanded martensite (α'), expanded austenite (γN), CrN, Fe4N, and (Fe,Cr) x O y . In the sample nitrided above 610 °C, the expanded martensite transformed into expanded austenite. But in the sample nitrided at 650 °C, the expanded austenite decomposed into αN and CrN. The decomposed αN then disassembled into CrN and alpha again. The nitrided layer depth thickened intensively with the increasing nitriding temperature. The activation energy of nitriding in this salt bath was 125 ± 5 kJ/mol.

Identification of a major polypeptide of the nuclear pore complex

PubMed Central

1982-01-01

The nuclear pore complex is a prominent structural component of the nuclear envelope that appears to regulate nucleoplasmic molecular movement. Up to now, none of its polypeptides have been defined. To identify possible pore complex proteins, we fractionated rat liver nuclear envelopes and microsomal membranes with strong protein perturbants into peripheral and intrinsic membrane proteins, and compared these fractions on SDS gels. From this analysis, we identified a prominent 190-kilodalton intrinsic membrane polypeptide that occurs specifically in nuclear envelopes. Lectin binding studies indicate that this polypeptide (gp 190) is the major nuclear envelope glycoprotein. Upon treatment of nuclear envelopes with Triton X-100, gp 190 remains associated with a protein substructure of the nuclear envelope consisting of pore complexes and nuclear lamina. We prepared monospecific antibodies to gp 190 for immunocytochemical localization. Immunofluorescence staining of tissue culture cells suggests that gp 190 occurs exclusively in the nucleus during interphase. This polypeptide becomes dispersed throughout the cell in mitotic prophase when the nuclear envelope is disassembled, and subsequently returns to the nuclear surfaces during telophase when the nuclear envelope is reconstructed. Immunoferritin labeling of Triton-treated rat liver nuclei demonstrates that gp 190 occurs exclusively in the nuclear pore complex, in the regions of the cytoplasmic (and possibly nucleoplasmic) pore complex annuli. A polypeptide that cross-reacts with gp 190 is present in diverse vertebrate species, as shown by antibody labeling of nitrocellulose SDS gel transfers. On the basis of its biochemical characteristics, we suggest that gp 190 may be involved in anchoring the pore complex to nuclear envelope membranes. PMID:7153248

Accelerated Disassembly of IgE:Receptor Complexes by a Disruptive Macromolecular Inhibitor

PubMed Central

Kim, Beomkyu; Eggel, Alexander; Tarchevskaya, Svetlana S.; Vogel, Monique; Prinz, Heino; Jardetzky, Theodore S.

2012-01-01

IgE antibodies bind the high affinity IgE Fc receptor (FcεRI), found primarily on mast cells and basophils, and trigger inflammatory cascades of the allergic response1,2. Inhibitors of IgE:FcεRI binding have been identified and an anti-IgE therapeutic antibody (omalizumab) is used to treat severe allergic asthma3,4. However, preformed IgE:FcεRI complexes that prime cells prior to allergen exposure dissociate extremely slowly5 and cannot be disrupted by strictly competitive inhibitors. IgE-Fc conformational flexibility indicated that inhibition could be mediated by allosteric or other non-classical mechanisms6–8. Here we demonstrate that an engineered protein inhibitor, DARPin E2_799–11, acts through a non-classical inhibition mechanism, not only blocking IgE:FcεRI interactions, but actively stimulating the dissociation of preformed ligand-receptor complexes. The structure of the E2_79:IgE-Fc3-4 complex predicts the presence of two non-equivalent E2_79 sites in the asymmetric IgE:FcεRI complex, with Site 1 distant from the receptor and Site 2 exhibiting partial steric overlap. While the structure is suggestive of an allosteric inhibition mechanism, mutational studies and quantitative kinetic modeling indicate that E2_79 acts through a facilitated dissociation mechanism at Site 2 alone. These results demonstrate that high affinity IgE:FcεRI complexes can be actively dissociated to block the allergic response and suggest that protein:protein complexes may be more generally amenable to active disruption by macromolecular inhibitors. PMID:23103871

Disassembly line balancing.

DOT National Transportation Integrated Search

2007-01-01

Manufacturers are increasingly recycling and remanufacturing their postconsumed : products due to new, more rigid environmental legislation, : increased public awareness, and extended manufacturer responsibility. In : addition, the economic attractiv...

Theoretical study of cut area of reduction of large surfaces of rotation parts on machines with rotary cutters “Extra”

NASA Astrophysics Data System (ADS)

Bondarenko, J. A.; Fedorenko, M. A.; Pogonin, A. A.

2018-03-01

Large parts can be treated without disassembling machines using “Extra”, having technological and design challenges, which differ from the challenges in the processing of these components on the stationary machine. Extension machines are used to restore large parts up to the condition allowing one to use them in a production environment. To achieve the desired accuracy and surface roughness parameters, the surface after rotary grinding becomes recoverable, which greatly increases complexity. In order to improve production efficiency and productivity of the process, the qualitative rotary processing of the machined surface is applied. The rotary cutting process includes a continuous change of the cutting edge surfaces. The kinematic parameters of a rotary cutting define its main features and patterns, the cutting operation of the rotary cutting instrument.

Nano-Encrypted Morse Code: A Versatile Approach to Programmable and Reversible Nanoscale Assembly and Disassembly

PubMed Central

Wong, Ngo Yin; Xing, Hang; Tan, Li Huey; Lu, Yi

2013-01-01

While much work has been devoted to nanoscale assembly of functional materials, selective reversible assembly of components in the nanoscale pattern at selective sites has received much less attention. Exerting such a reversible control of the assembly process will make it possible to fine-tune the functional properties of the assembly and to realize more complex designs. Herein, by taking advantage of different binding affinities of biotin and desthiobiotin toward streptavidin, we demonstrate selective and reversible decoration of DNA origami tiles with streptavidin, including revealing an encrypted Morse code “NANO” and reversible exchange of uppercase letter “I” with lowercase “i”. The yields of the conjugations are high (> 90%) and the process is reversible. We expect this versatile conjugation technique to be widely applicable with different nanomaterials and templates. PMID:23373425

Disassembly Control of Saccharide-Based Amphiphiles Driven by Electrostatic Repulsion.

PubMed

Yamada, Taihei; Kokado, Kenta; Sada, Kazuki

2017-03-14

According to the design of disassembly using electrostatic repulsion, novel amphiphiles consisting of a lipophilic ion part and a hydrophilic saccharide part were synthesized via the facile copper-catalyzed click reaction, and their molecular assemblies in water and chloroform were studied. The amphiphiles exhibited a molecular orientation opposite to that of the conventional amphiphiles in each case. ζ Potential measurements indicated that the lipophilic ion part is exposed outside in chloroform. The size of a solvophobic part in the amphiphiles dominates the size of an assembling structure; that is, in water, these amphiphiles tethering different lengths of the saccharide part exhibited almost identical assembling size, whereas in chloroform, the size depends on the length of the saccharide part in the amphiphiles.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Washiya, Tadahiro; Komaki, Jun; Funasaka, Hideyuki

Japan Atomic Energy Agency (JAEA) has been developing the new aqueous reprocessing system named 'NEXT' (New Extraction system for TRU recovery)1-2, which provides many advantages as waste volume reduction, cost savings by advanced components and simplification of process operation. Advanced head-end systems in the 'NEXT' process consist of fuel disassembly system, fuel shearing system and continuous dissolver system. We developed reliable fuel disassembly system with innovative procedure, and short-length shearing system and continuous dissolver system can be provided highly concentrated dissolution to adapt to the uranium crystallization process. We have carried out experimental studies, and fabrication of engineering-scale test devicesmore » to confirm the systems performance. In this paper, research and development of advanced head-end systems are described. (authors)« less

Yeast eIF4B binds to the head of the 40S ribosomal subunit and promotes mRNA recruitment through its N-terminal and internal repeat domains

PubMed Central

Walker, Sarah E.; Zhou, Fujun; Mitchell, Sarah F.; Larson, Victoria S.; Valasek, Leos; Hinnebusch, Alan G.; Lorsch, Jon R.

2013-01-01

Eukaryotic translation initiation factor (eIF)4B stimulates recruitment of mRNA to the 43S ribosomal pre-initiation complex (PIC). Yeast eIF4B (yeIF4B), shown previously to bind single-stranded (ss) RNA, consists of an N-terminal domain (NTD), predicted to be unstructured in solution; an RNA-recognition motif (RRM); an unusual domain comprised of seven imperfect repeats of 26 amino acids; and a C-terminal domain. Although the mechanism of yeIF4B action has remained obscure, most models have suggested central roles for its RRM and ssRNA-binding activity. We have dissected the functions of yeIF4B’s domains and show that the RRM and its ssRNA-binding activity are dispensable in vitro and in vivo. Instead, our data indicate that the 7-repeats and NTD are the most critical domains, which mediate binding of yeIF4B to the head of the 40S ribosomal subunit via interaction with Rps20. This interaction induces structural changes in the ribosome’s mRNA entry channel that could facilitate mRNA loading. We also show that yeIF4B strongly promotes productive interaction of eIF4A with the 43S•mRNA PIC in a manner required for efficient mRNA recruitment. PMID:23236192

A TATA binding protein mutant with increased affinity for DNA directs transcription from a reversed TATA sequence in vivo.

PubMed

Spencer, J Vaughn; Arndt, Karen M

2002-12-01

The TATA-binding protein (TBP) nucleates the assembly and determines the position of the preinitiation complex at RNA polymerase II-transcribed genes. We investigated the importance of two conserved residues on the DNA binding surface of Saccharomyces cerevisiae TBP to DNA binding and sequence discrimination. Because they define a significant break in the twofold symmetry of the TBP-TATA interface, Ala100 and Pro191 have been proposed to be key determinants of TBP binding orientation and transcription directionality. In contrast to previous predictions, we found that substitution of an alanine for Pro191 did not allow recognition of a reversed TATA box in vivo; however, the reciprocal change, Ala100 to proline, resulted in efficient utilization of this and other variant TATA sequences. In vitro assays demonstrated that TBP mutants with the A100P and P191A substitutions have increased and decreased affinity for DNA, respectively. The TATA binding defect of TBP with the P191A mutation could be intragenically suppressed by the A100P substitution. Our results suggest that Ala100 and Pro191 are important for DNA binding and sequence recognition by TBP, that the naturally occurring asymmetry of Ala100 and Pro191 is not essential for function, and that a single amino acid change in TBP can lead to elevated DNA binding affinity and recognition of a reversed TATA sequence.

The beta -globin locus control region (LCR) functions primarily by enhancing the transition from transcription initiation to elongation.

PubMed

Sawado, Tomoyuki; Halow, Jessica; Bender, M A; Groudine, Mark

2003-04-15

To investigate the molecular basis of beta-globin gene activation, we analyzed factor recruitment and histone modification at the adult beta-globin gene in wild-type (WT)/locus control region knockout (DeltaLCR) heterozygous mice and in murine erythroleukemia (MEL) cells. Although histone acetylation and methylation (Lys 4) are high before and after MEL differentiation, recruitment of the erythroid-specific activator NF-E2 to the promoter and preinitiation complex (PIC) assembly occur only after differentiation. We reported previously that targeted deletion of the LCR reduces beta-globin gene expression to 1%-4% of WT without affecting promoter histone acetylation. Here, we report that NF-E2 is recruited equally efficiently to the adult beta-globin promoters of the DeltaLCR and WT alleles. Moreover, the LCR deletion reduces PIC assembly only twofold, but has a dramatic effect on Ser 5 phosphorylation of RNA polymerase II and transcriptional elongation. Our results suggest at least three distinct stages in beta-globin gene activation: (1) an LCR-independent chromatin opening stage prior to NF-E2 recruitment to the promoter and PIC assembly; (2) an intermediate stage in which NF-E2 binding (LCR-independent) and PIC assembly (partially LCR-dependent) occur; and (3) an LCR-dependent fully active stage characterized by efficient pol II elongation. Thus, in its native location the LCR functions primarily downstream of activator recruitment and PIC assembly.

Modulation of chromatin structure by the FACT histone chaperone complex regulates HIV-1 integration.

PubMed

Matysiak, Julien; Lesbats, Paul; Mauro, Eric; Lapaillerie, Delphine; Dupuy, Jean-William; Lopez, Angelica P; Benleulmi, Mohamed Salah; Calmels, Christina; Andreola, Marie-Line; Ruff, Marc; Llano, Manuel; Delelis, Olivier; Lavigne, Marc; Parissi, Vincent

2017-07-28

Insertion of retroviral genome DNA occurs in the chromatin of the host cell. This step is modulated by chromatin structure as nucleosomes compaction was shown to prevent HIV-1 integration and chromatin remodeling has been reported to affect integration efficiency. LEDGF/p75-mediated targeting of the integration complex toward RNA polymerase II (polII) transcribed regions ensures optimal access to dynamic regions that are suitable for integration. Consequently, we have investigated the involvement of polII-associated factors in the regulation of HIV-1 integration. Using a pull down approach coupled with mass spectrometry, we have selected the FACT (FAcilitates Chromatin Transcription) complex as a new potential cofactor of HIV-1 integration. FACT is a histone chaperone complex associated with the polII transcription machinery and recently shown to bind LEDGF/p75. We report here that a tripartite complex can be formed between HIV-1 integrase, LEDGF/p75 and FACT in vitro and in cells. Biochemical analyzes show that FACT-dependent nucleosome disassembly promotes HIV-1 integration into chromatinized templates, and generates highly favored nucleosomal structures in vitro. This effect was found to be amplified by LEDGF/p75. Promotion of this FACT-mediated chromatin remodeling in cells both increases chromatin accessibility and stimulates HIV-1 infectivity and integration. Altogether, our data indicate that FACT regulates HIV-1 integration by inducing local nucleosomes dissociation that modulates the functional association between the incoming intasome and the targeted nucleosome.

Yeast cohesin complex embraces 2 micron plasmid sisters in a tri-linked catenane complex

PubMed Central

Ghosh, Santanu K.; Huang, Chu-Chun; Hajra, Sujata; Jayaram, Makkuni

2010-01-01

Sister chromatid cohesion, crucial for faithful segregation of replicated chromosomes in eukaryotes, is mediated by the multi-subunit protein complex cohesin. The Saccharomyces cerevisiae plasmid 2 micron circle mimics chromosomes in assembling cohesin at its partitioning locus. The plasmid is a multi-copy selfish DNA element that resides in the nucleus and propagates itself stably, presumably with assistance from cohesin. In metaphase cell lysates, or fractions enriched for their cohesed state by sedimentation, plasmid molecules are trapped topologically by the protein ring formed by cohesin. They can be released from cohesin’s embrace either by linearizing the DNA or by cleaving a cohesin subunit. Assays using two distinctly tagged cohesin molecules argue against the hand-cuff (an associated pair of monomeric cohesin rings) or the bracelet (a dimeric cohesin ring) model as responsible for establishing plasmid cohesion. Our cumulative results most easily fit a model in which a single monomeric cohesin ring, rather than a series of such rings, conjoins a pair of sister plasmids. These features of plasmid cohesion account for its sister-to-sister mode of segregation by cohesin disassembly during anaphase. The mechanistic similarities of cohesion between mini-chromosome sisters and 2 micron plasmid sisters suggest a potential kinship between the plasmid partitioning locus and centromeres. PMID:19920123

Structural dissection of a complex Bacteroides ovatus gene locus conferring xyloglucan metabolism in the human gut.

PubMed

Hemsworth, Glyn R; Thompson, Andrew J; Stepper, Judith; Sobala, Łukasz F; Coyle, Travis; Larsbrink, Johan; Spadiut, Oliver; Goddard-Borger, Ethan D; Stubbs, Keith A; Brumer, Harry; Davies, Gideon J

2016-07-01

The human gastrointestinal tract harbours myriad bacterial species, collectively termed the microbiota, that strongly influence human health. Symbiotic members of our microbiota play a pivotal role in the digestion of complex carbohydrates that are otherwise recalcitrant to assimilation. Indeed, the intrinsic human polysaccharide-degrading enzyme repertoire is limited to various starch-based substrates; more complex polysaccharides demand microbial degradation. Select Bacteroidetes are responsible for the degradation of the ubiquitous vegetable xyloglucans (XyGs), through the concerted action of cohorts of enzymes and glycan-binding proteins encoded by specific xyloglucan utilization loci (XyGULs). Extending recent (meta)genomic, transcriptomic and biochemical analyses, significant questions remain regarding the structural biology of the molecular machinery required for XyG saccharification. Here, we reveal the three-dimensional structures of an α-xylosidase, a β-glucosidase, and two α-l-arabinofuranosidases from the Bacteroides ovatus XyGUL. Aided by bespoke ligand synthesis, our analyses highlight key adaptations in these enzymes that confer individual specificity for xyloglucan side chains and dictate concerted, stepwise disassembly of xyloglucan oligosaccharides. In harness with our recent structural characterization of the vanguard endo-xyloglucanse and cell-surface glycan-binding proteins, the present analysis provides a near-complete structural view of xyloglucan recognition and catalysis by XyGUL proteins. © 2016 The Authors.

Supramolecular assembly of the beta-catenin destruction complex and the effect of Wnt signaling on its localization, molecular size, and activity in vivo

PubMed Central

Schaefer, Kristina N.; Williams, Clara E.; Roberts, David M.; McKay, Daniel J.

2018-01-01

Wnt signaling provides a paradigm for cell-cell signals that regulate embryonic development and stem cell homeostasis and are inappropriately activated in cancers. The tumor suppressors APC and Axin form the core of the multiprotein destruction complex, which targets the Wnt-effector beta-catenin for phosphorylation, ubiquitination and destruction. Based on earlier work, we hypothesize that the destruction complex is a supramolecular entity that self-assembles by Axin and APC polymerization, and that regulating assembly and stability of the destruction complex underlie its function. We tested this hypothesis in Drosophila embryos, a premier model of Wnt signaling. Combining biochemistry, genetic tools to manipulate Axin and APC2 levels, advanced imaging and molecule counting, we defined destruction complex assembly, stoichiometry, and localization in vivo, and its downregulation in response to Wnt signaling. Our findings challenge and revise current models of destruction complex function. Endogenous Axin and APC2 proteins and their antagonist Dishevelled accumulate at roughly similar levels, suggesting competition for binding may be critical. By expressing Axin:GFP at near endogenous levels we found that in the absence of Wnt signals, Axin and APC2 co-assemble into large cytoplasmic complexes containing tens to hundreds of Axin proteins. Wnt signals trigger recruitment of these to the membrane, while cytoplasmic Axin levels increase, suggesting altered assembly/disassembly. Glycogen synthase kinase3 regulates destruction complex recruitment to the membrane and release of Armadillo/beta-catenin from the destruction complex. Manipulating Axin or APC2 levels had no effect on destruction complex activity when Wnt signals were absent, but, surprisingly, had opposite effects on the destruction complex when Wnt signals were present. Elevating Axin made the complex more resistant to inactivation, while elevating APC2 levels enhanced inactivation. Our data suggest both absolute levels and the ratio of these two core components affect destruction complex function, supporting models in which competition among Axin partners determines destruction complex activity. PMID:29641560

Structural rearrangements in chloroplast thylakoid membranes revealed by differential scanning calorimetry and circular dichroism spectroscopy. Thermo-optic effect.

PubMed

Dobrikova, Anelia G; Várkonyi, Zsuzsanna; Krumova, Sashka B; Kovács, László; Kostov, Georgi K; Todinova, Svetla J; Busheva, Mira C; Taneva, Stefka G; Garab, Gyozo

2003-09-30

The thermo-optic mechanism in thylakoid membranes was earlier identified by measuring the thermal and light stabilities of pigment arrays with different levels of structural complexity [Cseh, Z., et al. (2000) Biochemistry 39, 15250-15257]. (According to the thermo-optic mechanism, fast local thermal transients, arising from the dissipation of excess, photosynthetically not used, excitation energy, induce elementary structural changes due to the "built-in" thermal instabilities of the given structural units.) The same mechanism was found to be responsible for the light-induced trimer-to-monomer transition in LHCII, the main chlorophyll a/b light-harvesting antenna of photosystem II (PSII) [Garab, G., et al. (2002) Biochemistry 41, 15121-15129]. In this paper, differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy on thylakoid membranes of barley and pea are used to correlate the thermo-optically inducible structural changes with well-discernible calorimetric transitions. The thylakoid membranes exhibited six major DSC bands, with maxima between about 43 and 87 degrees C. The heat sorption curves were analyzed both by mathematical deconvolution of the overall endotherm and by a successive annealing procedure; these yielded similar thermodynamic parameters, transition temperature and calorimetric enthalpy. A systematic comparison of the DSC and CD data on samples with different levels of complexity revealed that the heat-induced disassembly of chirally organized macrodomains contributes profoundly to the first endothermic event, a weak and broad DSC band between 43 and 48 degrees C. Similarly to the main macrodomain-associated CD signals, this low enthalpy band could be diminished by prolonged photoinhibitory preillumination, the extent of which depended on the temperature of preillumination. By means of nondenaturing, "green" gel electrophoresis and CD fingerprinting, it is shown that the second main endotherm, around 60 degrees C, originates to a large extent from the monomerization of LHCII trimers. The main DSC band, around 70 degrees C, which exhibits the highest enthalpy change, and another band around 75-77 degrees C relate to the dismantling of LHCII and other pigment-protein complexes, which under physiologically relevant conditions cannot be induced by light. The currently available data suggest the following sequence of events of thermo-optically inducible changes: (i) unstacking of membranes, followed by (ii) lateral disassembly of the chiral macrodomains and (iii) monomerization of LHCII trimers. We propose that thermo-optical structural reorganizations provide a structural flexibility, which is proportional to the intensity of the excess excitation, while for their localized nature, the structural stability of the system can be retained.

Progress in joining, reuse, and customization of WR284 waveguide in the laboratory

NASA Astrophysics Data System (ADS)

Clark, Mike; Flanagan, Ken; Milhone, Jason; Nonn, Paul; Forest, Cary

2017-10-01

A system of five 20 kW magnetrons is being installed for the Big Red Ball (BRB) to produce and heat the plasma with 2.45GHz RF energy. An existing system of two 6 kW magentrons of the same frequency is actively used for the same purpose on Plasma Couette Experiment Upgrade (PCX-U). In each experiment, the RF is transmitted to the vessel via WR284 waveguide. Waveguide occasionally needs to be disassembled, modified and rebuilt for different reasons such as physics interests, ongoing problems (arcing), or efficient utilization of laboratory space. Reuse of disassembled waveguide parts is desirable for cost savings. Methods of assembly, disassembly, and modification of waveguide will be discussed. Also, frequently used designs of chokes, windows, and limiters will be shown. Materials used include copper, brass, and even aluminum. The vacuum vessel of PCX-U is a 1 meter diameter, 1 meter tall cylinder comprised of ¼'' thick stainless steel. PCX-U has one removable end. The vacuum vessel of the BRB is a 3 meter diameter, sphere comprised of two hemispheres of 1-¼'' thick cast A356 aluminum. Rings comprised of hundreds of SmCo magnets in each vessel create a cusp field to contain the plasma and provide a resonance surface for the RF. Supported by NSF and DoE.

Timely Endocytosis of Cytokinetic Enzymes Prevents Premature Spindle Breakage during Mitotic Exit

PubMed Central

Onishi, Masayuki; Yeong, Foong May

2016-01-01

Cytokinesis requires the spatio-temporal coordination of membrane deposition and primary septum (PS) formation at the division site to drive acto-myosin ring (AMR) constriction. It has been demonstrated that AMR constriction invariably occurs only after the mitotic spindle disassembly. It has also been established that Chitin Synthase II (Chs2p) neck localization precedes mitotic spindle disassembly during mitotic exit. As AMR constriction depends upon PS formation, the question arises as to how chitin deposition is regulated so as to prevent premature AMR constriction and mitotic spindle breakage. In this study, we propose that cells regulate the coordination between spindle disassembly and AMR constriction via timely endocytosis of cytokinetic enzymes, Chs2p, Chs3p, and Fks1p. Inhibition of endocytosis leads to over accumulation of cytokinetic enzymes during mitotic exit, which accelerates the constriction of the AMR, and causes spindle breakage that eventually could contribute to monopolar spindle formation in the subsequent round of cell division. Intriguingly, the mitotic spindle breakage observed in endocytosis mutants can be rescued either by deleting or inhibiting the activities of, CHS2, CHS3 and FKS1, which are involved in septum formation. The findings from our study highlight the importance of timely endocytosis of cytokinetic enzymes at the division site in safeguarding mitotic spindle integrity during mitotic exit. PMID:27447488

Deubiquitylases USP5 and USP13 are recruited to and regulate heat-induced stress granules through their deubiquitylating activities.

PubMed

Xie, Xuan; Matsumoto, Shunsuke; Endo, Akinori; Fukushima, Toshiaki; Kawahara, Hiroyuki; Saeki, Yasushi; Komada, Masayuki

2018-04-12

Stress granules are transient cytoplasmic foci induced by various stresses that contain translation-stalled mRNAs and RNA-binding proteins. They are proposed to modulate mRNA translation and stress responses. Here, we show that the deubiquitylases USP5 and USP13 are recruited to heat-induced stress granules. Heat-induced stress granules also contained K48- and K63-linked ubiquitin chains. Depletion of USP5 or USP13 resulted in elevated ubiquitin chain levels and accelerated assembly of heat-induced stress granules, suggesting that these enzymes regulate the stability of the stress granules through their ubiquitin isopeptidase activity. Moreover, disassembly of heat-induced stress granules after returning the cells to normal temperatures was markedly repressed by individual depletion of USP5 or USP13. Finally, overexpression of a ubiquitin mutant lacking the C-terminal diglycine motif caused the accumulation of unanchored ubiquitin chains and the repression of the disassembly of heat-induced stress granules. As unanchored ubiquitin chains are preferred substrates for USP5, we suggest that USP5 regulates the assembly and disassembly of heat-induced stress granules by mediating the hydrolysis of unanchored ubiquitin chains while USP13 regulates stress granules through deubiquitylating protein-conjugated ubiquitin chains.This article has an associated First Person interview with the first author of the paper. © 2018. Published by The Company of Biologists Ltd.

In-service inspection of steam turbine blades without disassembly

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reinhart, E.R.

1987-01-01

Loss of utility plant availability as a result of failure-causing cracks in steam turbine blades makes early detection of this problem critical. An Electric Power Research Institute survey, conducted as part of project RP 1266-24, indicated that 72% of turbine blade failures in fossil power plants occur in low-pressure (LP) turbines with half of all blade failures occurring in the last two blade stages (L-0 and L-1 rows). Failures are generally associated with blade tailing edges and root areas. Project RP 1266-24 also found that 79% of the blade problems in LP turbines were cracks. A turbine design of particularmore » concern has been the Westinghouse Building Block (B.B.) 73. Reinhart and Associates has successfully inspected seven in-place B.B. 73 units for six utilities during the past 3 yr, as well as several disassembled turbines of other manufacturers and designs. These examinations consisted of visual and eddy-current examinations of the blade roots and trailing edges. The in-place inspections were performed using prototype manipulation devices to gain access to the blades through the hand holes. The only disassembly required to gain access for the examinations was the removal of the man-way covers on the main shell and the hand-hole covers on the outer cylinder covering the L-0 and L-1 blade rows.« less

Final postflight hardware evaluation report RSRM-28 (STS-53)

NASA Technical Reports Server (NTRS)

Starrett, William David, Jr.

1993-01-01

The final report for the Clearfield disassembly evaluation and a continuation of the KSC postflight assessment for the RSRM-28 (STS-53) RSRM flight set is presented. All observed hardware conditions were documented on PFOR's and are included in Appendices A through C. Appendices D and E contain the measurements and safety factor data for the nozzle and insulation components. This report, along with the KSC Ten-Day Postflight Hardware Evaluation Report (TWR-64215), represents a summary of the RSRM-28 hardware evaluation. The as-flown hardware configuration is documented in TWR-63638. Disassembly evaluation photograph numbers are logged in TWA-1989. The RSRM-28 flight set disassembly evaluations described were performed at the RSRM Refurbishment Facility in Clearfield, Utah. The final factory joint demate occurred on July 15, 1993. Additional time was required to perform the evaluation of the stiffener rings per special issue 4.1.5.2 because of the washout schedule. The release of this report was after completion of all special issues per program management direction. Detailed evaluations were performed in accordance with the Clearfield PEEP, TWR-50051, Revision A. All observations were compared against limits that are also defined in the PEEP. These limits outline the criteria for categorizing the observations as acceptable, reportable, or critical. Hardware conditions that were unexpected and/or determined to be reportable or critical were evaluated by the applicable team and tracked through the PFAR system.

Plastics disassembly versus bulk recycling: engineering design for end-of-life electronics resource recovery.

PubMed

Rios, Pedro; Stuart, Julie Ann; Grant, Ed

2003-12-01

Annual plastic flows through the business and consumer electronics manufacturing supply chain include nearly 3 billion lb of high-value engineering plastics derived from petroleum. The recovery of resource value from this stream presents critical challenges in areas of materials identification and recycling process design that demand new green engineering technologies applied together with life cycle assessment and ecological supply chain analysis to create viable plastics-to-plastics supply cycles. The sustainable recovery of potentially high-value engineering plastics streams requires that recyclers either avoid mixing plastic parts or purify later by separating smaller plastic pieces created in volume reduction (shredding) steps. Identification and separation constitute significant barriers in the plastics-to-plastics recycling value proposition. In the present work, we develop a model that accepts randomly arriving electronic products to study scenarios by which a recycler might identify and separate high-value engineering plastics as well as metals. Using discrete eventsimulation,we compare current mixed plastics recovery with spectrochemical plastic resin identification and subsequent sorting. Our results show that limited disassembly with whole-part identification can produce substantial yields in separated streams of recovered engineering thermoplastics. We find that disassembly with identification does not constitute a bottleneck, but rather, with relatively few workers, can be configured to pull the process and thus decrease maximum staging space requirements.

The Aminoacyl-tRNA Synthetase Complex.

PubMed

Mirande, Marc

2017-01-01

Aminoacyl-tRNA synthetases (AARSs) are essential enzymes that specifically aminoacylate one tRNA molecule by the cognate amino acid. They are a family of twenty enzymes, one for each amino acid. By coupling an amino acid to a specific RNA triplet, the anticodon, they are responsible for interpretation of the genetic code. In addition to this translational, canonical role, several aminoacyl-tRNA synthetases also fulfill nontranslational, moonlighting functions. In mammals, nine synthetases, those specific for amino acids Arg, Asp, Gln, Glu, Ile, Leu, Lys, Met and Pro, associate into a multi-aminoacyl-tRNA synthetase complex, an association which is believed to play a key role in the cellular organization of translation, but also in the regulation of the translational and nontranslational functions of these enzymes. Because the balance between their alternative functions rests on the assembly and disassembly of this supramolecular entity, it is essential to get precise insight into the structural organization of this complex. The high-resolution 3D-structure of the native particle, with a molecular weight of about 1.5 MDa, is not yet known. Low-resolution structures of the multi-aminoacyl-tRNA synthetase complex, as determined by cryo-EM or SAXS, have been reported. High-resolution data have been reported for individual enzymes of the complex, or for small subcomplexes. This review aims to present a critical view of our present knowledge of the aminoacyl-tRNA synthetase complex in 3D. These preliminary data shed some light on the mechanisms responsible for the balance between the translational and nontranslational functions of some of its components.

AGR-3/4 Irradiation Test Train Disassembly and Component Metrology First Look Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stempien, John Dennis; Rice, Francine Joyce; Harp, Jason Michael

2016-03-01

The AGR-3/4 experiment was designed to study fission product transport within graphitic matrix material and nuclear-grade graphite. To this end, this experiment consisted of 12 capsules, each fueled with 4 compacts containing UCO TRISO particles as driver fuel and 20 UCO designed-to-fail (DTF) fuel particles in each compact. The DTF fuel was fabricated with a thin pyrocarbon layer which was intended to fail during irradiation and provide a source of fission products. These fission products could then migrate through the compact and into the surrounding concentric rings of graphitic matrix material and/or nuclear graphite. Through post-irradiation examination (PIE) of themore » rings (including physical sampling and gamma scanning) fission product concentration profiles within the rings can be determined. These data can be used to elucidate fission product transport parameters (e.g. diffusion coefficients within the test materials) which will be used to inform and refine models of fission product transport. After irradiation in the Advanced Test Reactor (ATR) had been completed in April 2014, the AGR-3/4 experiment was shipped to the Hot Fuel Examination Facility (HFEF) at the Materials and Fuels Complex (MFC) for inspection, disassembly, and metrology. The AGR-3/4 test train was received at MFC in two separate shipments between February and April 2015. Visual examinations of the test train exterior did not indicate dimensional distortion, and only two small discolored areas were observed at the bottom of Capsules 8 and 9. No corresponding discoloration was found on the inside of these capsules, however. Prior to disassembly, the two test train sections were subject to analysis via the Precision Gamma Scanner (PGS), which did not indicate that any gross fuel relocation had occurred. A series of specialized tools (including clamps, cutters, and drills) had been designed and fabricated in order to carry out test train disassembly and recovery of capsule components (graphite rings and fuel compacts). This equipment performed well for separating each capsule in the test train and extracting the capsule components. Only a few problems were encountered. In one case, the outermost ring (the sink ring) was cracked during removal of the capsule through tubes. Although the sink ring will be analyzed in order to obtain a mass balance of fission products in the experiment, these cracks do not pose a major concern because the sink ring will not be analyzed in detail to obtain the spatial distribution of fission products. In Capsules 4 and 5, the compacts could not be removed from the inner rings. Strategies for removing the compacts are being evaluated. Sampling the inner rings with the compacts in-place is also an option. Dimensional measurements were made on the compacts, inner rings, outer rings, and sink rings. The diameters of all compacts decreased by 0.5 to 2.0 %. Generally, the extent of diametric shrinkage increased linearly with increasing neutron fluence. Most compact lengths also decreased. Compact lengths decreased with increasing fluence, reaching maximum shrinkage of about 0.9 % at a fast fluence of 4.0x10 25 n/m 2 E > 0.18 MeV. Above this fluence, the extent of length shrinkage appeared to decrease with fluence, and two compacts from Capsule 7 were found to have slightly increased in length (< 0.1 %) after a fluence of 5.2x10 25 n/m 2.« less

AGR-3/4 Irradiation Test Train Disassembly and Component Metrology First Look Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stempien, John Dennis; Rice, Francine Joyce; Harp, Jason Michael

The AGR-3/4 experiment was designed to study fission product transport within graphitic matrix material and nuclear-grade graphite. To this end, this experiment consisted of 12 capsules, each fueled with 4 compacts containing UCO TRISO particles as driver fuel and 20 UCO designed-to-fail (DTF) fuel particles in each compact. The DTF fuel was fabricated with a thin pyrocarbon layer which was intended to fail during irradiation and provide a source of fission products. These fission products could then migrate through the compact and into the surrounding concentric rings of graphitic matrix material and/or nuclear graphite. Through post-irradiation examination (PIE) of themore » rings (including physical sampling and gamma scanning) fission product concentration profiles within the rings can be determined. These data can be used to elucidate fission product transport parameters (e.g. diffusion coefficients within the test materials) which will be used to inform and refine models of fission product transport. After irradiation in the Advanced Test Reactor (ATR) had been completed in April 2014, the AGR-3/4 experiment was shipped to the Hot Fuel Examination Facility (HFEF) at the Materials and Fuels Complex (MFC) for inspection, disassembly, and metrology. The AGR-3/4 test train was received at MFC in two separate shipments between February and April 2015. Visual examinations of the test train exterior did not indicate dimensional distortion, and only two small discolored areas were observed at the bottom of Capsules 8 and 9. No corresponding discoloration was found on the inside of these capsules, however. Prior to disassembly, the two test train sections were subject to analysis via the Precision Gamma Scanner (PGS), which did not indicate that any gross fuel relocation had occurred. A series of specialized tools (including clamps, cutters, and drills) had been designed and fabricated in order to carry out test train disassembly and recovery of capsule components (graphite rings and fuel compacts). This equipment performed well for separating each capsule in the test train and extracting the capsule components. Only a few problems were encountered. In one case, the outermost ring (the sink ring) was cracked during removal of the capsule through tubes. Although the sink ring will be analyzed in order to obtain a mass balance of fission products in the experiment, these cracks do not pose a major concern because the sink ring will not be analyzed in detail to obtain the spatial distribution of fission products. In Capsules 4 and 5, the compacts could not be removed from the inner rings. Strategies for removing the compacts are being evaluated. Sampling the inner rings with the compacts in-place is also an option. Dimensional measurements were made on the compacts, inner rings, outer rings, and sink rings. The diameters of all compacts decreased by 0.5 to 2.0 %. Generally, the extent of diametric shrinkage increased linearly with increasing neutron fluence. Most compact lengths also decreased. Compact lengths decreased with increasing fluence, reaching maximum shrinkage of about 0.9 % at a fast fluence of 4.0x1025 n/m2 E > 0.18 MeV. Above this fluence, the extent of length shrinkage appeared to decrease with fluence, and two compacts from Capsule 7 were found to have slightly increased in length (< 0.1 %) after a fluence of 5.2x1025 n/m2.« less

48 CFR 252.228-7001 - Ground and flight risk.

Code of Federal Regulations, 2014 CFR

2014-10-01

... aircraft in the process of being manufactured, disassembled, or reassembled; provided that an engine... airships, unmanned aerial vehicles, or other nonconventional aircraft specified in this contract. (2...

48 CFR 252.228-7001 - Ground and flight risk.

Code of Federal Regulations, 2013 CFR

2013-10-01

... aircraft in the process of being manufactured, disassembled, or reassembled; provided that an engine... airships, unmanned aerial vehicles, or other nonconventional aircraft specified in this contract. (2...

48 CFR 252.228-7001 - Ground and flight risk.

Code of Federal Regulations, 2012 CFR

2012-10-01

... aircraft in the process of being manufactured, disassembled, or reassembled; provided that an engine... airships, unmanned aerial vehicles, or other nonconventional aircraft specified in this contract. (2...

48 CFR 252.228-7001 - Ground and flight risk.

Code of Federal Regulations, 2011 CFR

2011-10-01

... aircraft in the process of being manufactured, disassembled, or reassembled; provided that an engine... airships, unmanned aerial vehicles, or other nonconventional aircraft specified in this contract. (2...

A micromotor based on polymer single crystals and nanoparticles: toward functional versatility

NASA Astrophysics Data System (ADS)

Liu, Mei; Liu, Limei; Gao, Wenlong; Su, Miaoda; Ge, Ya; Shi, Lili; Zhang, Hui; Dong, Bin; Li, Christopher Y.

2014-07-01

We report a multifunctional micromotor fabricated by the self-assembly technique using multifunctional materials, i.e. polymer single crystals and nanoparticles, as basic building blocks. Not only can this micromotor achieve autonomous and directed movement, it also possesses unprecedented functions, including enzymatic degradation-induced micromotor disassembly, sustained release and molecular detection.We report a multifunctional micromotor fabricated by the self-assembly technique using multifunctional materials, i.e. polymer single crystals and nanoparticles, as basic building blocks. Not only can this micromotor achieve autonomous and directed movement, it also possesses unprecedented functions, including enzymatic degradation-induced micromotor disassembly, sustained release and molecular detection. Electronic supplementary information (ESI) available: Experimental section, Fig. S1-S8 and Video S1-S4. See DOI: 10.1039/c4nr02593h

Knowledge of damage identification about tensegrities via flexibility disassembly

NASA Astrophysics Data System (ADS)

Jiang, Ge; Feng, Xiaodong; Du, Shigui

2017-12-01

Tensegrity structures composing of continuous cables and discrete struts are under tension and compression, respectively. In order to determine the damage extents of tensegrity structures, a new method for tensegrity structural damage identification is presented based on flexibility disassembly. To decompose a tensegrity structural flexibility matrix into the matrix represention of the connectivity between degress-of-freedoms and the diagonal matrix comprising of magnitude informations. Step 1: Calculate perturbation flexibility; Step 2: Compute the flexibility connectivity matrix and perturbation flexibility parameters; Step 3: Calculate the perturbation stiffness parameters. The efficiency of the proposed method is demonstrated by a numeical example comprising of 12 cables and 4 struts with pretensioned. Accurate identification of local damage depends on the availability of good measured data, an accurate and reasonable algorithm.

Design of an interventional magnetic resonance imaging coil for cerebral surgery

NASA Astrophysics Data System (ADS)

Xu, Yue; Wang, Wen-Tao; Wang, Wei-Min

2012-11-01

In clinical magnetic resonance imaging (MRI), the design of the radiofrequency (RF) coil is very important. For certain applications, the appropriate coil can produce an improved image quality. However, it is difficult to achieve a uniform B1 field and a high signal-to-noise ratio (SNR) simultaneously. In this article, we design an interventional transmitter-and-receiver RF coil for cerebral surgery. This coil adopts a disassembly structure that can be assembled and disassembled repeatedly on the cerebral surgery gantry to reduce the amount of interference from the MRI during surgery. The simulation results and the imaging experiments demonstrate that this coil can produce a uniform RF field, a high SNR, and a large imaging range to meet the requirements of the cerebral surgery.

NEDDylation promotes stress granule assembly.

PubMed

Jayabalan, Aravinth Kumar; Sanchez, Anthony; Park, Ra Young; Yoon, Sang Pil; Kang, Gum-Yong; Baek, Je-Hyun; Anderson, Paul; Kee, Younghoon; Ohn, Takbum

2016-07-06

Stress granules (SGs) harbour translationally stalled messenger ribonucleoproteins and play important roles in regulating gene expression and cell fate. Here we show that neddylation promotes SG assembly in response to arsenite-induced oxidative stress. Inhibition or depletion of key components of the neddylation machinery concomitantly inhibits stress-induced polysome disassembly and SG assembly. Affinity purification and subsequent mass-spectrometric analysis of Nedd8-conjugated proteins from translationally stalled ribosomal fractions identified ribosomal proteins, translation factors and RNA-binding proteins (RBPs), including SRSF3, a previously known SG regulator. We show that SRSF3 is selectively neddylated at Lys85 in response to arsenite. A non-neddylatable SRSF3 (K85R) mutant do not prevent arsenite-induced polysome disassembly, but fails to support the SG assembly, suggesting that the neddylation pathway plays an important role in SG assembly.

Conditional Inactivation of Upstream Binding Factor Reveals Its Epigenetic Functions and the Existence of a Somatic Nucleolar Precursor Body

PubMed Central

Hamdane, Nourdine; Stefanovsky, Victor Y.; Tremblay, Michel G.; Németh, Attila; Paquet, Eric; Lessard, Frédéric; Sanij, Elaine; Hannan, Ross; Moss, Tom

2014-01-01

Upstream Binding Factor (UBF) is a unique multi-HMGB-box protein first identified as a co-factor in RNA polymerase I (RPI/PolI) transcription. However, its poor DNA sequence selectivity and its ability to generate nucleosome-like nucleoprotein complexes suggest a more generalized role in chromatin structure. We previously showed that extensive depletion of UBF reduced the number of actively transcribed ribosomal RNA (rRNA) genes, but had little effect on rRNA synthesis rates or cell proliferation, leaving open the question of its requirement for RPI transcription. Using gene deletion in mouse, we now show that UBF is essential for embryo development beyond morula. Conditional deletion in cell cultures reveals that UBF is also essential for transcription of the rRNA genes and that it defines the active chromatin conformation of both gene and enhancer sequences. Loss of UBF prevents formation of the SL1/TIF1B pre-initiation complex and recruitment of the RPI-Rrn3/TIF1A complex. It is also accompanied by recruitment of H3K9me3, canonical histone H1 and HP1α, but not by de novo DNA methylation. Further, genes retain penta-acetyl H4 and H2A.Z, suggesting that even in the absence of UBF the rRNA genes can maintain a potentially active state. In contrast to canonical histone H1, binding of H1.4 is dependent on UBF, strongly suggesting that it plays a positive role in gene activity. Unexpectedly, arrest of rRNA synthesis does not suppress transcription of the 5S, tRNA or snRNA genes, nor expression of the several hundred mRNA genes implicated in ribosome biogenesis. Thus, rRNA gene activity does not coordinate global gene expression for ribosome biogenesis. Loss of UBF also unexpectedly induced the formation in cells of a large sub-nuclear structure resembling the nucleolar precursor body (NPB) of oocytes and early embryos. These somatic NPBs contain rRNA synthesis and processing factors but do not associate with the rRNA gene loci (NORs). PMID:25121932

49 CFR 572.190 - Incorporated materials.

Code of Federal Regulations, 2012 CFR

2012-10-01

... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) ANTHROPOMORPHIC TEST DEVICES IIsD Side Impact Crash Test... procedures manual entitled, “Procedures for Assembly, Disassembly, and Inspection (PADI) of the SID-IIsD Side...

49 CFR 572.190 - Incorporated materials.

Code of Federal Regulations, 2014 CFR

2014-10-01

... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) ANTHROPOMORPHIC TEST DEVICES SID-IIsD Side Impact Crash... procedures manual entitled, “Procedures for Assembly, Disassembly, and Inspection (PADI) of the SID-IIsD Side...

49 CFR 572.190 - Incorporated materials.

Code of Federal Regulations, 2013 CFR

2013-10-01

... ADMINISTRATION, DEPARTMENT OF TRANSPORTATION (CONTINUED) ANTHROPOMORPHIC TEST DEVICES SID-IIsD Side Impact Crash... procedures manual entitled, “Procedures for Assembly, Disassembly, and Inspection (PADI) of the SID-IIsD Side...

Mitochondrial hepato-encephalopathy due to deficiency of QIL1/MIC13 (C19orf70), a MICOS complex subunit.

PubMed

Zeharia, Avraham; Friedman, Jonathan R; Tobar, Ana; Saada, Ann; Konen, Osnat; Fellig, Yacov; Shaag, Avraham; Nunnari, Jodi; Elpeleg, Orly

2016-12-01

The mitochondrial inner membrane possesses distinct subdomains including cristae, which are lamellar structures invaginated into the mitochondrial matrix and contain the respiratory complexes. Generation of inner membrane domains requires the complex interplay between the respiratory complexes, mitochondrial lipids and the recently identified mitochondrial contact site and cristae organizing system (MICOS) complex. Proper organization of the mitochondrial inner membrane has recently been shown to be important for respiratory function in yeast. Here we aimed at a molecular diagnosis in a brother and sister from a consanguineous family who presented with a neurodegenerative disorder accompanied by hyperlactatemia, 3-methylglutaconic aciduria, disturbed hepatocellular function with abnormal cristae morphology in liver and cerebellar and vermis atrophy, which suggest mitochondrial dysfunction. Using homozygosity mapping and exome sequencing the patients were found to be homozygous for the p.(Gly15Glufs*75) variant in the QIL1/MIC13 (C19orf70) gene. QIL1/MIC13 is a constituent of MICOS, a six subunit complex that helps to form and/or stabilize cristae junctions and determine the placement, distribution and number of cristae within mitochondria. In patient fibroblasts both MICOS subunits QIL1/MIC13 and MIC10 were absent whereas MIC60 was present in a comparable abundance to that of the control. We conclude that QIL1/MIC13 deficiency in human, is associated with disassembly of the MICOS complex, with the associated aberration of cristae morphology and mitochondrial respiratory dysfunction. 3-Methylglutaconic aciduria is associated with variants in genes encoding mitochondrial inner membrane organizing determinants, including TAZ, DNAJC19, SERAC1 and QIL1/MIC13.

Crystal structure of the Xpo1p nuclear export complex bound to the SxFG/PxFG repeats of the nucleoporin Nup42p.

PubMed

Koyama, Masako; Hirano, Hidemi; Shirai, Natsuki; Matsuura, Yoshiyuki

2017-10-01

Xpo1p (yeast CRM1) is the major nuclear export receptor that carries a plethora of proteins and ribonucleoproteins from the nucleus to cytoplasm. The passage of the Xpo1p nuclear export complex through nuclear pore complexes (NPCs) is facilitated by interactions with nucleoporins (Nups) containing extensive repeats of phenylalanine-glycine (so-called FG repeats), although the precise role of each Nup in the nuclear export reaction remains incompletely understood. Here we report structural and biochemical characterization of the interactions between the Xpo1p nuclear export complex and the FG repeats of Nup42p, a nucleoporin localized at the cytoplasmic face of yeast NPCs and has characteristic SxFG/PxFG sequence repeat motif. The crystal structure of Xpo1p-PKI-Nup42p-Gsp1p-GTP complex identified three binding sites for the SxFG/PxFG repeats on HEAT repeats 14-20 of Xpo1p. Mutational analyses of Nup42p showed that the conserved serines and prolines in the SxFG/PxFG repeats contribute to Xpo1p-Nup42p binding. Our structural and biochemical data suggest that SxFG/PxFG-Nups such as Nup42p and Nup159p at the cytoplasmic face of NPCs provide high-affinity docking sites for the Xpo1p nuclear export complex in the terminal stage of NPC passage and that subsequent disassembly of the nuclear export complex facilitates recycling of free Xpo1p back to the nucleus. © 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Initiation of lambda DNA replication. The Escherichia coli small heat shock proteins, DnaJ and GrpE, increase DnaK's affinity for the lambda P protein.

PubMed

Osipiuk, J; Georgopoulos, C; Zylicz, M

1993-03-05

It is known that the initiation of bacteriophage lambda replication requires the orderly assembly of the lambda O.lambda P.DnaB helicase protein preprimosomal complex at the ori lambda DNA site. The DnaK, DnaJ, and GrpE heat shock proteins act together to destabilize the lambda P.DnaB complex, thus freeing DnaB and allowing it to unwind lambda DNA near the ori lambda site. The first step of this disassembly reaction is the binding of DnaK to the lambda P protein. In this report, we examined the influence of the DnaJ and GrpE proteins on the stability of the lambda P.DnaK complex. We present evidence for the existence of the following protein-protein complexes: lambda P.DnaK, lambda P.DnaJ, DnaJ.DnaK, DnaK.GrpE, and lambda P.DnaK.GrpE. Our results suggest that the presence of GrpE alone destabilizes the lambda P.DnaK complex, whereas the presence of DnaJ alone stabilizes the lambda P.DnaK complex. Using immunoprecipitation, we show that in the presence of GrpE, DnaK exhibits a higher affinity for the lambda P.DnaJ complex than it does alone. Using cross-linking with glutaraldehyde, we show that oligomeric forms of DnaK exhibit a higher affinity for lambda P than monomeric DnaK. However, in the presence of GrpE, monomeric DnaK can efficiently bind lambda P protein. These findings help explain our previous results, namely that in the GrpE-dependent lambda DNA replication system, the DnaK protein requirement can be reduced up to 10-fold.

Old foes, new understandings: nuclear entry of small non-enveloped DNA viruses.

PubMed

Fay, Nikta; Panté, Nelly

2015-06-01

The nuclear import of viral genomes is an important step of the infectious cycle for viruses that replicate in the nucleus of their host cells. Although most viruses use the cellular nuclear import machinery or some components of this machinery, others have developed sophisticated ways to reach the nucleus. Some of these have been known for some time; however, recent studies have changed our understanding of how some non-enveloped DNA viruses access the nucleus. For example, parvoviruses enter the nucleus through small disruptions of the nuclear membranes and nuclear lamina, and adenovirus tugs at the nuclear pore complex, using kinesin-1, to disassemble their capsids and deliver viral proteins and genomes into the nucleus. Here we review recent findings of the nuclear import strategies of three small non-enveloped DNA viruses, including adenovirus, parvovirus, and the polyomavirus simian virus 40. Copyright © 2015 Elsevier B.V. All rights reserved.

Plant signalling: the opportunities and dangers of chemical communication.

PubMed

Adler, Frederick R

2011-04-23

The notion of chemical communication between plants and other organisms has gone from being viewed as a fringe idea to an accepted ecological phenomenon only recently. An Organized Oral Session at the August 2010 Ecological Society of America meeting in Pittsburgh examined the role of plant signalling both within and between plants, with speakers addressing the remarkably wide array of effects that plant signals have on plant physiology, species interactions and entire communities. In addition to the familiar way that plants communicate with mutualists like pollinators and fruit dispersers through both chemical and visual cues, speakers at this session described how plants communicate with themselves, with each other, with herbivores and with predators of those herbivores. These plant signals create a complex odour web superimposed upon the more classical food web itself, with its own dynamics in the face of exotic species and rapid community assembly and disassembly.

Guide of good practices for occupational radiological protection in plutonium facilities

DOE Office of Scientific and Technical Information (OSTI.GOV)

NONE

1998-06-01

This Technical Standard (TS) does not contain any new requirements. Its purpose is to provide guides to good practice, update existing reference material, and discuss practical lessons learned relevant to the safe handling of plutonium. the technical rationale is given to allow US Department of Energy (DOE) health physicists to adapt the recommendations to similar situations throughout the DOE complex. Generally, DOE contractor health physicists will be responsible to implement radiation protection activities at DOE facilities and DOE health physicists will be responsible for oversight of those activities. This guidance is meant to be useful for both efforts. This TSmore » replaces PNL-6534, Health Physics Manual of Good Practices for Plutonium Facilities, by providing more complete and current information and by emphasizing the situations that are typical of DOE`s current plutonium operations; safe storage, decontamination, and decommissioning (environmental restoration); and weapons disassembly.« less

Phospho-control of TGF-β superfamily signaling

PubMed Central

Wrighton, Katharine H; Lin, Xia; Feng, Xin-Hua

2010-01-01

Members of the transforming growth factor-β (TGF-β) family control a broad range of cellular responses in metazoan organisms via autocrine, paracrine, and endocrine modes. Thus, aberrant TGF-β signaling can play a key role in the pathogenesis of several diseases, including cancer. TGF-β signaling pathways are activated by a short phospho-cascade, from receptor phosphorylation to the subsequent phosphorylation and activation of downstream signal transducers called R-Smads. R-Smad phosphorylation state determines Smad complex assembly/disassembly, nuclear import/export, transcriptional activity and stability, and is thus the most critical event in TGF-β signaling. Dephosphorylation of R-Smads by specific phosphatases prevents or terminates TGF-β signaling, highlighting the need to consider Smad (de)phosphorylation as a tightly controlled and dynamic event. This article illustrates the essential roles of reversible phosphorylation in controlling the strength and duration of TGF-β signaling and the ensuing physiological responses. PMID:19114991