Hsp90 is required for the activity of a hepatitis B virus reverse transcriptase.

PubMed Central

Hu, J; Seeger, C

1996-01-01

The heat shock protein Hsp90 is known as an essential component of several signal transduction pathways and has now been identified as an essential host factor for hepatitis B virus replication. Hsp90 interacts with the viral reverse transcriptase to facilitate the formation of a ribonucleoprotein (RNP) complex between the polymerase and an RNA ligand. This RNP complex is required early in replication for viral assembly and initiation of DNA synthesis through a protein-priming mechanism. These results thus invoke a role for the Hsp90 pathway in the formation of an RNP. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8577714

Mechanisms of Translation Control Underlying Long-lasting Synaptic Plasticity and the Consolidation of Long-term Memory

PubMed Central

Santini, Emanuela; Huynh, Thu N.; Klann, Eric

2018-01-01

The complexity of memory formation and its persistence is a phenomenon that has been studied intensely for centuries. Memory exists in many forms and is stored in various brain regions. Generally speaking, memories are reorganized into broadly distributed cortical networks over time through systems level consolidation. At the cellular level, storage of information is believed to initially occur via altered synaptic strength by processes such as long-term potentiation (LTP). New protein synthesis is required for long-lasting synaptic plasticity as well as for the formation of long-term memory. The mammalian target of rapamycin complex 1 (mTORC1) is a critical regulator of cap-dependent protein synthesis and is required for numerous forms of long-lasting synaptic plasticity and long-term memory. As such, the study of mTORC1 and protein factors that control translation initiation and elongation have enhanced our understanding of how the process of protein synthesis is regulated during memory formation. Herein we will discuss the molecular mechanisms that regulate protein synthesis as well as pharmacological and genetic manipulations that demonstrate the requirement for proper translational control in long-lasting synaptic plasticity and long-term memory formation. PMID:24484700

In vivo characterization of the Drosophila mRNA 3′ end processing core cleavage complex

PubMed Central

Michalski, Daniel; Steiniger, Mindy

2015-01-01

A core cleavage complex (CCC) consisting of CPSF73, CPSF100, and Symplekin is required for cotranscriptional 3′ end processing of all metazoan pre-mRNAs, yet little is known about the in vivo molecular interactions within this complex. The CCC is a component of two distinct complexes, the cleavage/polyadenylation complex and the complex that processes nonpolyadenylated histone pre-mRNAs. RNAi-depletion of CCC factors in Drosophila culture cells causes reduction of CCC processing activity on histone mRNAs, resulting in read through transcription. In contrast, RNAi-depletion of factors only required for histone mRNA processing allows use of downstream cryptic polyadenylation signals to produce polyadenylated histone mRNAs. We used Dmel-2 tissue culture cells stably expressing tagged CCC components to determine that amino acids 272–1080 of Symplekin and the C-terminal approximately 200 amino acids of both CPSF73 and CPSF100 are required for efficient CCC formation in vivo. Additional experiments reveal that the C-terminal 241 amino acids of CPSF100 are sufficient for histone mRNA processing indicating that the first 524 amino acids of CPSF100 are dispensable for both CCC formation and histone mRNA 3′ end processing. CCCs containing deletions of Symplekin lacking the first 271 amino acids resulted in dramatic increased use of downstream polyadenylation sites for histone mRNA 3′ end processing similar to RNAi-depletion of histone-specific 3′ end processing factors FLASH, SLBP, and U7 snRNA. We propose a model in which CCC formation is mediated by CPSF73, CPSF100, and Symplekin C-termini, and the N-terminal region of Symplekin facilitates cotranscriptional 3′ end processing of histone mRNAs. PMID:26081560

A Meier-Gorlin syndrome mutation in a conserved C-terminal helix of Orc6 impedes origin recognition complex formation.

PubMed

Bleichert, Franziska; Balasov, Maxim; Chesnokov, Igor; Nogales, Eva; Botchan, Michael R; Berger, James M

2013-10-08

In eukaryotes, DNA replication requires the origin recognition complex (ORC), a six-subunit assembly that promotes replisome formation on chromosomal origins. Despite extant homology between certain subunits, the degree of structural and organizational overlap between budding yeast and metazoan ORC has been unclear. Using 3D electron microscopy, we determined the subunit organization of metazoan ORC, revealing that it adopts a global architecture very similar to the budding yeast complex. Bioinformatic analysis extends this conservation to Orc6, a subunit of somewhat enigmatic function. Unexpectedly, a mutation in the Orc6 C-terminus linked to Meier-Gorlin syndrome, a dwarfism disorder, impedes proper recruitment of Orc6 into ORC; biochemical studies reveal that this region of Orc6 associates with a previously uncharacterized domain of Orc3 and is required for ORC function and MCM2-7 loading in vivo. Together, our results suggest that Meier-Gorlin syndrome mutations in Orc6 impair the formation of ORC hexamers, interfering with appropriate ORC functions. DOI:http://dx.doi.org/10.7554/eLife.00882.001.

Interference-mediated synaptonemal complex formation with embedded crossover designation

PubMed Central

Zhang, Liangran; Espagne, Eric; de Muyt, Arnaud; Zickler, Denise; Kleckner, Nancy E.

2014-01-01

Biological systems exhibit complex patterns at length scales ranging from the molecular to the organismic. Along chromosomes, events often occur stochastically at different positions in different nuclei but nonetheless tend to be relatively evenly spaced. Examples include replication origin firings, formation of chromatin loops along chromosome axes and, during meiosis, localization of crossover recombination sites (“crossover interference”). We present evidence in the fungus Sordaria macrospora that crossover interference is part of a broader pattern that includes synaptonemal complex (SC) nucleation. This pattern comprises relatively evenly spaced SC nucleation sites, among which a subset are crossover sites that show a classical interference distribution. This pattern ensures that SC forms regularly along the entire length of the chromosome as required for the maintenance of homolog pairing while concomitantly having crossover interactions locally embedded within the SC structure as required for both DNA recombination and structural events of chiasma formation. This pattern can be explained by a threshold-based designation and spreading interference process. This model can be generalized to give diverse types of related and/or partially overlapping patterns, in two or more dimensions, for any type of object. PMID:25380597

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

Role of IκB kinase β in regulating the remodeling of the CARMA1-Bcl10-MALT1 complex.

PubMed

Karim, Zubair A; Hensch, Nicole R; Qasim, Hanan; Alshbool, Fatima Z; Khasawneh, Fadi T

2018-06-02

The current work investigates the notion that inducible clustering of signaling mediators of the IKK pathway is important for platelet activation. Thus, while the CARMA1, Bcl10, and MALT1 (CBM) complex is essential for triggering IKK/NF-κB activation upon platelet stimulation, the signals that elicit its formation and downstream effector activation remain elusive. We demonstrate herein that IKKβ is involved in membrane fusion, and serves as a critical protein kinase required for initial formation and the regulation of the CARMA1/MALT1/Bcl10/CBM complex in platelets. We also show that IKKβ regulates these processes via modulation of phosphorylation of Bcl10 and IKKγ polyubiquitination. Collectively, our data demonstrate that IKKβ regulates membrane fusion and the remodeling of the CBM complex formation. Copyright © 2018 Elsevier Inc. All rights reserved.

The prokaryotic enhancer binding protein NTRC has an ATPase activity which is phosphorylation and DNA dependent.

PubMed Central

Austin, S; Dixon, R

1992-01-01

The prokaryotic activator protein NTRC binds to enhancer-like elements and activates transcription in response to nitrogen limitation by catalysing open complex formation by sigma 54 RNA polymerase holoenzyme. Formation of open complexes requires the phosphorylated form of NTRC and the reaction is ATP dependent. We find that NTRC has an ATPase activity which is activated by phosphorylation and is strongly stimulated by the presence of DNA containing specific NTRC binding sites. Images PMID:1534752

Conformation-Directed Formation of Self-Healing Diblock Copolypeptide Hydrogels via Polyion Complexation.

PubMed

Sun, Yintao; Wollenberg, Alexander L; O'Shea, Timothy Mark; Cui, Yanxiang; Zhou, Z Hong; Sofroniew, Michael V; Deming, Timothy J

2017-10-25

Synthetic diblock copolypeptides were designed to incorporate oppositely charged ionic segments that form β-sheet-structured hydrogel assemblies via polyion complexation when mixed in aqueous media. The observed chain conformation directed assembly was found to be required for efficient hydrogel formation and provided distinct and useful properties to these hydrogels, including self-healing after deformation, microporous architecture, and stability against dilution in aqueous media. While many promising self-assembled materials have been prepared using disordered or liquid coacervate polyion complex (PIC) assemblies, the use of ordered chain conformations in PIC assemblies to direct formation of new supramolecular morphologies is unprecedented. The promising attributes and unique features of the β-sheet-structured PIC hydrogels described here highlight the potential of harnessing conformational order derived from PIC assembly to create new supramolecular materials.

Inhibition of amyloid peptide fibril formation by gold-sulfur complexes.

PubMed

Wang, Wenji; Zhao, Cong; Zhu, Dengsen; Gong, Gehui; Du, Weihong

2017-06-01

Amyloid-related diseases are characterized by protein conformational change and amyloid fibril deposition. Metal complexes are potential inhibitors of amyloidosis. Nitrogen-coordinated gold complexes have been used to disaggregate prion neuropeptide (PrP106-126) and human islet amyloid polypeptide (hIAPP). However, the roles of metal complexes in peptide fibril formation and related bioactivity require further exploration. In this work, we investigated the interactions of amyloid peptides PrP106-126 and hIAPP with two tetracoordinated gold-sulfur complexes, namely, dichloro diethyl dithiocarbamate gold complex and dichloro pyrrolidine dithiocarbamate gold complex. We also determined the effects of these complexes on peptide-induced cytotoxicity. Thioflavin T assay, morphological characterization, and particle size analysis indicated that the two gold-sulfur complexes effectively inhibited the fibrillation of the amyloid peptides, which led to the formation of nanoscale particles. The complexes reduced the cytotoxicity induced by the amyloid peptides. Intrinsic fluorescence, nuclear magnetic resonance, and mass spectrometry revealed that the complexes interacted with PrP106-126 and hIAPP via metal coordination and hydrophobic interaction, which improved the inhibition and binding of the two gold-sulfur compounds. Our study provided new insights into the use of tetracoordinated gold-sulfur complexes as drug candidates against protein conformational disorders. Copyright © 2017 Elsevier Inc. All rights reserved.

HDAC3 and the Molecular Brake Pad Hypothesis

PubMed Central

McQuown, Susan C.; Wood, Marcelo A.

2011-01-01

Successful transcription of specific genes required for long-term memory processes involves the orchestrated effort of not only transcription factors, but also very specific enzymatic protein complexes that modify chromatin structure. Chromatin modification has been identified as a pivotal molecular mechanism underlying certain forms of synaptic plasticity and memory. The best-studied form of chromatin modification in the learning and memory field is histone acetylation, which is regulated by histone acetyltransferases and histone deacetylases (HDACs). HDAC inhibitors have been shown to strongly enhance long-term memory processes, and recent work has aimed to identify contributions of individual HDACs. In this review, we focus on HDAC3 and discuss its recently defined role as a negative regulator of long-term memory formation. HDAC3 is part of a corepressor complex and has direct interactions with class II HDACs that may be important for its molecular and behavioral consequences. And last, we propose the “molecular brake pad” hypothesis of HDAC function. The HDACs and associated corepressor complexes may function in neurons, in part, as “molecular brake pads.” HDACs are localized to promoters of active genes and act as a persistent clamp that requires strong activity-dependent signaling to temporarily release these complexes (or brake pads) to activate gene expression required for long-term memory formation. Thus, HDAC inhibition removes the “molecular brake pads” constraining the processes necessary for long-term memory and results in strong, persistent memory formation. PMID:21521655

Assembly and stoichiometry of the core structure of the bacterial flagellar type III export gate complex.

PubMed

Fukumura, Takuma; Makino, Fumiaki; Dietsche, Tobias; Kinoshita, Miki; Kato, Takayuki; Wagner, Samuel; Namba, Keiichi; Imada, Katsumi; Minamino, Tohru

2017-08-01

The bacterial flagellar type III export apparatus, which is required for flagellar assembly beyond the cell membranes, consists of a transmembrane export gate complex and a cytoplasmic ATPase complex. FlhA, FlhB, FliP, FliQ, and FliR form the gate complex inside the basal body MS ring, although FliO is required for efficient export gate formation in Salmonella enterica. However, it remains unknown how they form the gate complex. Here we report that FliP forms a homohexameric ring with a diameter of 10 nm. Alanine substitutions of conserved Phe-137, Phe-150, and Glu-178 residues in the periplasmic domain of FliP (FliPP) inhibited FliP6 ring formation, suppressing flagellar protein export. FliO formed a 5-nm ring structure with 3 clamp-like structures that bind to the FliP6 ring. The crystal structure of FliPP derived from Thermotoga maritia, and structure-based photo-crosslinking experiments revealed that Phe-150 and Ser-156 of FliPP are involved in the FliP-FliP interactions and that Phe-150, Arg-152, Ser-156, and Pro-158 are responsible for the FliP-FliO interactions. Overexpression of FliP restored motility of a ∆fliO mutant to the wild-type level, suggesting that the FliP6 ring is a functional unit in the export gate complex and that FliO is not part of the final gate structure. Copurification assays revealed that FlhA, FlhB, FliQ, and FliR are associated with the FliO/FliP complex. We propose that the assembly of the export gate complex begins with FliP6 ring formation with the help of the FliO scaffold, followed by FliQ, FliR, and FlhB and finally FlhA during MS ring formation.

Assembly and stoichiometry of the core structure of the bacterial flagellar type III export gate complex

PubMed Central

Fukumura, Takuma; Makino, Fumiaki; Dietsche, Tobias; Kinoshita, Miki; Kato, Takayuki; Wagner, Samuel; Namba, Keiichi; Imada, Katsumi

2017-01-01

The bacterial flagellar type III export apparatus, which is required for flagellar assembly beyond the cell membranes, consists of a transmembrane export gate complex and a cytoplasmic ATPase complex. FlhA, FlhB, FliP, FliQ, and FliR form the gate complex inside the basal body MS ring, although FliO is required for efficient export gate formation in Salmonella enterica. However, it remains unknown how they form the gate complex. Here we report that FliP forms a homohexameric ring with a diameter of 10 nm. Alanine substitutions of conserved Phe-137, Phe-150, and Glu-178 residues in the periplasmic domain of FliP (FliPP) inhibited FliP6 ring formation, suppressing flagellar protein export. FliO formed a 5-nm ring structure with 3 clamp-like structures that bind to the FliP6 ring. The crystal structure of FliPP derived from Thermotoga maritia, and structure-based photo-crosslinking experiments revealed that Phe-150 and Ser-156 of FliPP are involved in the FliP–FliP interactions and that Phe-150, Arg-152, Ser-156, and Pro-158 are responsible for the FliP–FliO interactions. Overexpression of FliP restored motility of a ∆fliO mutant to the wild-type level, suggesting that the FliP6 ring is a functional unit in the export gate complex and that FliO is not part of the final gate structure. Copurification assays revealed that FlhA, FlhB, FliQ, and FliR are associated with the FliO/FliP complex. We propose that the assembly of the export gate complex begins with FliP6 ring formation with the help of the FliO scaffold, followed by FliQ, FliR, and FlhB and finally FlhA during MS ring formation. PMID:28771466

The acetate/ACSS2 switch regulates HIF-2 stress signaling in the tumor cell microenvironment.

PubMed

Chen, Rui; Xu, Min; Nagati, Jason S; Hogg, Richard T; Das, Alok; Gerard, Robert D; Garcia, Joseph A

2015-01-01

Optimal stress signaling by Hypoxia Inducible Factor 2 (HIF-2) during low oxygen states or hypoxia requires coupled actions of a specific coactivator/lysine acetyltransferase, Creb binding protein (CBP), and a specific deacetylase, Sirtuin 1 (SIRT1). We recently reported that acetylation of HIF-2 by CBP also requires a specific acetyl CoA generator, acetate-dependent acetyl CoA synthetase 2 (ACSS2). In this study, we demonstrate that ACSS2/HIF-2 signaling is active not only during hypoxia, but also during glucose deprivation. Acetate levels increase during stress and coincide with maximal HIF-2α acetylation and CBP/HIF-2α complex formation. Exogenous acetate induces HIF-2α acetylation, CBP/HIF-2α complex formation, and HIF-2 signaling. ACSS2 and HIF-2 are required for maximal colony formation, proliferation, migration, and invasion during stress. Acetate also stimulates flank tumor growth and metastasis in mice in an ACSS2 and HIF-2 dependent manner. Thus, ACSS2/CBP/SIRT1/HIF-2 signaling links nutrient sensing and stress signaling with cancer growth and progression in mammals.

The Acetate/ACSS2 Switch Regulates HIF-2 Stress Signaling in the Tumor Cell Microenvironment

PubMed Central

Chen, Rui; Xu, Min; Nagati, Jason S.; Hogg, Richard T.; Das, Alok; Gerard, Robert D.; Garcia, Joseph A.

2015-01-01

Optimal stress signaling by Hypoxia Inducible Factor 2 (HIF-2) during low oxygen states or hypoxia requires coupled actions of a specific coactivator/lysine acetyltransferase, Creb binding protein (CBP), and a specific deacetylase, Sirtuin 1 (SIRT1). We recently reported that acetylation of HIF-2 by CBP also requires a specific acetyl CoA generator, acetate-dependent acetyl CoA synthetase 2 (ACSS2). In this study, we demonstrate that ACSS2/HIF-2 signaling is active not only during hypoxia, but also during glucose deprivation. Acetate levels increase during stress and coincide with maximal HIF-2α acetylation and CBP/HIF-2α complex formation. Exogenous acetate induces HIF-2α acetylation, CBP/HIF-2α complex formation, and HIF-2 signaling. ACSS2 and HIF-2 are required for maximal colony formation, proliferation, migration, and invasion during stress. Acetate also stimulates flank tumor growth and metastasis in mice in an ACSS2 and HIF-2 dependent manner. Thus, ACSS2/CBP/SIRT1/HIF-2 signaling links nutrient sensing and stress signaling with cancer growth and progression in mammals. PMID:25689462

Nup155 regulates nuclear envelope and nuclear pore complex formation in nematodes and vertebrates

PubMed Central

Franz, Cerstin; Askjaer, Peter; Antonin, Wolfram; Iglesias, Carmen López; Haselmann, Uta; Schelder, Malgorzata; de Marco, Ario; Wilm, Matthias; Antony, Claude; Mattaj, Iain W

2005-01-01

Nuclear envelope (NE) formation during cell division in multicellular organisms is a central yet poorly understood biological process. We report that the conserved nucleoporin Nup155 has an essential function in NE formation in Caenorhabditis elegans embryos and in Xenopus laevis egg extracts. In vivo depletion of Nup155 led to failure of nuclear lamina formation and defects in chromosome segregation at anaphase. Nup155 depletion inhibited accumulation of nucleoporins at the nuclear periphery, including those recruited to chromatin early in NE formation. Electron microscopy analysis revealed that Nup155 is also required for the formation of a continuous nuclear membrane in vivo and in vitro. Time-course experiments indicated that Nup155 is recruited to chromatin at the time of NE sealing, suggesting that nuclear pore complex assembly has to progress to a relatively late stage before NE membrane assembly occurs. PMID:16193066

Structural requirements of endopolygalacturonase for the interaction with PGIP (polygalacturonase-inhibiting protein)

PubMed Central

Federici, L.; Caprari, C.; Mattei, B.; Savino, C.; Di Matteo, A.; De Lorenzo, G.; Cervone, F.; Tsernoglou, D.

2001-01-01

To invade a plant tissue, phytopathogenic fungi produce several cell wall-degrading enzymes; among them, endopolygalacturonase (PG) catalyzes the fragmentation and solubilization of homogalacturonan. Polygalacturonase-inhibiting proteins (PGIPs), found in the cell wall of many plants, counteract fungal PGs by forming specific complexes with them. We report the crystal structure at 1.73 Å resolution of PG from the phytopathogenic fungus Fusarium moniliforme (FmPG). The structure of FmPG was useful to study the mode of interaction of the enzyme with PGIP-2 from Phaseolus vulgaris. Several amino acids of FmPG were mutated, and their contribution to the formation of the complex with PGIP-2 was investigated by surface plasmon resonance. The residues Lys-269 and Arg-267, located inside the active site cleft, and His-188, at the edge of the active site cleft, are critical for the formation of the complex, which is consistent with the observed competitive inhibition of the enzyme played by PGIP-2. The replacement of His-188 with a proline or the insertion of a tryptophan after position 270, variations that both occur in plant PGs, interferes with the formation of the complex. We suggest that these variations are important structural requirements of plant PGs to prevent PGIP binding. PMID:11687632

Monkeypox Virus Host Factor Screen Using Haploid Cells Identifies Essential Role of GARP Complex in Extracellular Virus Formation.

PubMed

Realegeno, Susan; Puschnik, Andreas S; Kumar, Amrita; Goldsmith, Cynthia; Burgado, Jillybeth; Sambhara, Suryaprakash; Olson, Victoria A; Carroll, Darin; Damon, Inger; Hirata, Tetsuya; Kinoshita, Taroh; Carette, Jan E; Satheshkumar, Panayampalli Subbian

2017-06-01

Monkeypox virus (MPXV) is a human pathogen that is a member of the Orthopoxvirus genus, which includes Vaccinia virus and Variola virus (the causative agent of smallpox). Human monkeypox is considered an emerging zoonotic infectious disease. To identify host factors required for MPXV infection, we performed a genome-wide insertional mutagenesis screen in human haploid cells. The screen revealed several candidate genes, including those involved in Golgi trafficking, glycosaminoglycan biosynthesis, and glycosylphosphatidylinositol (GPI)-anchor biosynthesis. We validated the role of a set of vacuolar protein sorting (VPS) genes during infection, VPS51 to VPS54 (VPS51-54), which comprise the Golgi-associated retrograde protein (GARP) complex. The GARP complex is a tethering complex involved in retrograde transport of endosomes to the trans -Golgi apparatus. Our data demonstrate that VPS52 and VPS54 were dispensable for mature virion (MV) production but were required for extracellular virus (EV) formation. For comparison, a known antiviral compound, ST-246, was used in our experiments, demonstrating that EV titers in VPS52 and VPS54 knockout (KO) cells were comparable to levels exhibited by ST-246-treated wild-type cells. Confocal microscopy was used to examine actin tail formation, one of the viral egress mechanisms for cell-to-cell dissemination, and revealed an absence of actin tails in VPS52KO- or VPS54KO-infected cells. Further evaluation of these cells by electron microscopy demonstrated a decrease in levels of wrapped viruses (WVs) compared to those seen with the wild-type control. Collectively, our data demonstrate the role of GARP complex genes in double-membrane wrapping of MVs necessary for EV formation, implicating the host endosomal trafficking pathway in orthopoxvirus infection. IMPORTANCE Human monkeypox is an emerging zoonotic infectious disease caused by Monkeypox virus (MPXV). Of the two MPXV clades, the Congo Basin strain is associated with severe disease, increased mortality, and increased human-to-human transmission relative to the West African strain. Monkeypox is endemic in regions of western and central Africa but was introduced into the United States in 2003 from the importation of infected animals. The threat of MPXV and other orthopoxviruses is increasing due to the absence of routine smallpox vaccination leading to a higher proportion of naive populations. In this study, we have identified and validated candidate genes that are required for MPXV infection, specifically, those associated with the Golgi-associated retrograde protein (GARP) complex. Identifying host targets required for infection that prevents extracellular virus formation such as the GARP complex or the retrograde pathway can provide a potential target for antiviral therapy. Copyright © 2017 American Society for Microbiology.

Simulator validation results and proposed reporting format from flight testing a software model of a complex, high-performance airplane.

DOT National Transportation Integrated Search

2008-01-01

Computer simulations are often used in aviation studies. These simulation tools may require complex, high-fidelity aircraft models. Since many of the flight models used are third-party developed products, independent validation is desired prior to im...

High-frequency promoter firing links THO complex function to heavy chromatin formation.

PubMed

Mouaikel, John; Causse, Sébastien Z; Rougemaille, Mathieu; Daubenton-Carafa, Yves; Blugeon, Corinne; Lemoine, Sophie; Devaux, Frédéric; Darzacq, Xavier; Libri, Domenico

2013-11-27

The THO complex is involved in transcription, genome stability, and messenger ribonucleoprotein (mRNP) formation, but its precise molecular function remains enigmatic. Under heat shock conditions, THO mutants accumulate large protein-DNA complexes that alter the chromatin density of target genes (heavy chromatin), defining a specific biochemical facet of THO function and a powerful tool of analysis. Here, we show that heavy chromatin distribution is dictated by gene boundaries and that the gene promoter is necessary and sufficient to convey THO sensitivity in these conditions. Single-molecule fluorescence in situ hybridization measurements show that heavy chromatin formation correlates with an unusually high firing pace of the promoter with more than 20 transcription events per minute. Heavy chromatin formation closely follows the modulation of promoter firing and strongly correlates with polymerase occupancy genome wide. We propose that the THO complex is required for tuning the dynamic of gene-nuclear pore association and mRNP release to the same high pace of transcription initiation. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Single-stranded nucleic acids promote SAMHD1 complex formation.

PubMed

Tüngler, Victoria; Staroske, Wolfgang; Kind, Barbara; Dobrick, Manuela; Kretschmer, Stefanie; Schmidt, Franziska; Krug, Claudia; Lorenz, Mike; Chara, Osvaldo; Schwille, Petra; Lee-Kirsch, Min Ae

2013-06-01

SAM domain and HD domain-containing protein 1 (SAMHD1) is a dGTP-dependent triphosphohydrolase that degrades deoxyribonucleoside triphosphates (dNTPs) thereby limiting the intracellular dNTP pool. Mutations in SAMHD1 cause Aicardi-Goutières syndrome (AGS), an inflammatory encephalopathy that mimics congenital viral infection and that phenotypically overlaps with the autoimmune disease systemic lupus erythematosus. Both disorders are characterized by activation of the antiviral cytokine interferon-α initiated by immune recognition of self nucleic acids. Here we provide first direct evidence that SAMHD1 associates with endogenous nucleic acids in situ. Using fluorescence cross-correlation spectroscopy, we demonstrate that SAMHD1 specifically interacts with ssRNA and ssDNA and establish that nucleic acid-binding and formation of SAMHD1 complexes are mutually dependent. Interaction with nucleic acids and complex formation do not require the SAM domain, but are dependent on the HD domain and the C-terminal region of SAMHD1. We finally demonstrate that mutations associated with AGS exhibit both impaired nucleic acid-binding and complex formation implicating that interaction with nucleic acids is an integral aspect of SAMHD1 function.

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

SAD-3, a Putative Helicase Required for Meiotic Silencing by Unpaired DNA, Interacts with Other Components of the Silencing Machinery

PubMed Central

Hammond, Thomas M.; Xiao, Hua; Boone, Erin C.; Perdue, Tony D.; Pukkila, Patricia J.; Shiu, Patrick K. T.

2011-01-01

In Neurospora crassa, genes lacking a pairing partner during meiosis are suppressed by a process known as meiotic silencing by unpaired DNA (MSUD). To identify novel MSUD components, we have developed a high-throughput reverse-genetic screen for use with the N. crassa knockout library. Here we describe the screening method and the characterization of a gene (sad-3) subsequently discovered. SAD-3 is a putative helicase required for MSUD and sexual spore production. It exists in a complex with other known MSUD proteins in the perinuclear region, a center for meiotic silencing activity. Orthologs of SAD-3 include Schizosaccharomyces pombe Hrr1, a helicase required for RNAi-induced heterochromatin formation. Both SAD-3 and Hrr1 interact with an RNA-directed RNA polymerase and an Argonaute, suggesting that certain aspects of silencing complex formation may be conserved between the two fungal species. PMID:22384347

Status of the Planet Formation Imager (PFI) concept

NASA Astrophysics Data System (ADS)

Ireland, Michael J.; Monnier, John D.; Kraus, Stefan; Isella, Andrea; Minardi, Stefano; Petrov, Romain; ten Brummelaar, Theo; Young, John; Vasisht, Gautam; Mozurkewich, David; Rinehart, Stephen; Michael, Ernest A.; van Belle, Gerard; Woillez, Julien

2016-08-01

The Planet Formation Imager (PFI) project aims to image the period of planet assembly directly, resolving structures as small as a giant planet's Hill sphere. These images will be required in order to determine the key mechanisms for planet formation at the time when processes of grain growth, protoplanet assembly, magnetic fields, disk/planet dynamical interactions and complex radiative transfer all interact - making some planetary systems habitable and others inhospitable. We will present the overall vision for the PFI concept, focusing on the key technologies and requirements that are needed to achieve the science goals. Based on these key requirements, we will define a cost envelope range for the design and highlight where the largest uncertainties lie at this conceptual stage.

SMC5/6 is required for the formation of segregation-competent bivalent chromosomes during meiosis I in mouse oocytes

PubMed Central

Hwang, Grace; Sun, Fengyun; Eppig, John J.; Handel, Mary Ann

2017-01-01

SMC complexes include three major classes: cohesin, condensin and SMC5/6. However, the localization pattern and genetic requirements for the SMC5/6 complex during mammalian oogenesis have not previously been examined. In mouse oocytes, the SMC5/6 complex is enriched at the pericentromeric heterochromatin, and also localizes along chromosome arms during meiosis. The infertility phenotypes of females with a Zp3-Cre-driven conditional knockout (cKO) of Smc5 demonstrated that maternally expressed SMC5 protein is essential for early embryogenesis. Interestingly, protein levels of SMC5/6 complex components in oocytes decline as wild-type females age. When SMC5/6 complexes were completely absent in oocytes during meiotic resumption, homologous chromosomes failed to segregate accurately during meiosis I. Despite what appears to be an inability to resolve concatenation between chromosomes during meiosis, localization of topoisomerase IIα to bivalents was not affected; however, localization of condensin along the chromosome axes was perturbed. Taken together, these data demonstrate that the SMC5/6 complex is essential for the formation of segregation-competent bivalents during meiosis I, and findings suggest that age-dependent depletion of the SMC5/6 complex in oocytes could contribute to increased incidence of oocyte aneuploidy and spontaneous abortion in aging females. PMID:28302748

The Allende multicompound chondrule (ACC)—Chondrule formation in a local super-dense region of the early solar system

NASA Astrophysics Data System (ADS)

Bischoff, Addi; Wurm, Gerhard; Chaussidon, Marc; Horstmann, Marian; Metzler, Knut; Weyrauch, Mona; Weinauer, Julia

2017-05-01

In Allende, a very complex compound chondrule (Allende compound chondrule; ACC) was found consisting of at least 16 subchondrules (14 siblings and 2 independents). Its overall texture can roughly be described as a barred olivine object (BO). The BO texture is similar in all siblings, but does not exist in the two independents, which appear as relatively compact olivine-rich units. Because of secondary alteration of pristine Allende components and the ACC in particular, only limited predictions can be made concerning the original compositions of the colliding melt droplets. Based on textural and mineralogical characteristics, the siblings must have been formed on a very short time scale in a dense, local environment. This is also supported by oxygen isotope systematics showing similar compositions for all 16 subchondrules. Furthermore, the ACC subchondrules are isotopically distinct from typical Allende chondrules, indicating formation in or reaction with a more 16O-poor reservoir. We modeled constraints on the particle density required at the ACC formation location, using textural, mineral-chemical, and isotopic observations on this multicompound chondrule to define melt droplet collision conditions. In this context, we discuss the possible relationship between the formation of complex chondrules and the formation of macrochondrules and cluster chondrites. While macrochondrules may have formed under similar or related conditions as complex chondrules, cluster chondrites certainly require different formation conditions. Cluster chondrites represent a mixture of viscously deformed, seemingly young chondrules of different chemical and textural types and a population of older chondrules. Concerning the formation of ACC calculations suggest the existence of very local, kilometer-sized, and super-dense chondrule-forming regions with extremely high solid-to-gas mass ratios of 1000 or more.

Complex formation between the protein components of methane monooxygenase from Methylosinus trichosporium OB3b. Identification of sites of component interaction.

PubMed

Fox, B G; Liu, Y; Dege, J E; Lipscomb, J D

1991-01-05

Kinetic, spectroscopic, and chemical evidence for the formation of specific catalytically essential complexes between the three protein components of the soluble form of methane monooxygenase from Methylosinus trichosporium OB3b is reported. The effects of the concentrations of the reductase and component B on the hydroxylation activity of the reconstituted enzyme system has been numerically simulated based on a kinetic model which assumes formation of multiple high affinity complexes with the hydroxylase component during catalysis. The formation of several of these complexes has been directly demonstrated. By using EPR spectroscopy, the binding of approximately 2 mol of component B/mol of hydroxylase (subunit structure (alpha beta gamma)2) is shown to significantly change the electronic environment of the mu-(H/R)-oxo-bridged binuclear iron cluster of the hydroxylase in both the mixed valent (Fe(II).Fe(III)) and fully reduced (Fe(II).Fe(II)) states. Protein-protein complexes between the reductase and component B as well as between the reductase and hydroxylase have been shown to form by monitoring quenching of the tryptophan fluorescence spectrum of either the component B (KD approximately 0.4 microM) or hydroxylase (two binding sites, KDa approximately 10 nM, KDb approximately 8 microM). The observed KD values are in agreement with the best fit values from the kinetic simulation. Through the use of the covalent zero length cross-linking reagent 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), the binding sites of the component B and reductase were shown to be on the hydroxylase alpha and beta subunits, respectively. The alpha and beta subunits of the hydroxylase are cross-linked by EDC suggesting that they are juxtaposed. EDC also caused the rapid loss of the ability of the monomeric component B to stimulate the hydroxylation reaction suggesting that cross-linking of reactive groups on the protein surface had occurred. This effect was inhibited by the presence of hydroxylase and was accompanied by a loss of the ability of the component B to bind to the hydroxylase. Thus, formation of a component B-hydroxylase complex is apparently required for effective catalysis linked to NADH oxidation. When present in concentrations greater than required to saturate the initial hydroxylase complex, component B inhibited both the rate of the enzymic reaction and the cross-linking of the reductase to the hydroxylase. This suggests that a second complex involving component B can form that negatively regulates catalysis by preventing formation of the reductase-hydroxylase complex.

Porcine endothelium induces DNA-histone complex formation in human whole blood: a harmful effect of histone on coagulation and endothelial activation.

PubMed

Yoo, Hyun Ju; Kim, Ji-Eun; Gu, Ja Yoon; Lee, Sae Bom; Lee, Hyun Joo; Hwang, Ho Young; Hwang, Yoohwa; Kim, Young Tae; Kim, Hyun Kyung

2016-11-01

Neutrophils play a role in xenograft rejection. When neutrophils are stimulated, they eject the DNA-histone complex into the extracellular space, called neutrophil extracellular traps (NET). We investigated whether NET formation actively occurs in the xenograft and contributes to coagulation and endothelial activation. Human whole blood was incubated with porcine aortic endothelial cells (pEC) from wild-type or α1,3-galactosyltransferase gene-knockout (GTKO) pigs. In the supernatant plasma from human blood, the level of the DNA-histone complex was measured by ELISA, and thrombin generation was measured using a calibrated automated thrombogram. Histone-induced tissue factor and adhesion molecule expression were measured by flow cytometry. pEC from both wild-type and GTKO pigs significantly induced DNA-histone complex formation in human whole blood. The DNA-histone complex produced shortened the thrombin generation time and clotting time. Histone alone dose-dependently induced tissue factor and adhesion molecule expression in pEC. Aurintricarboxylic acid pretreatment partially inhibited pEC-induced DNA-histone complex formation. DNA-histone complex actively generated upon xenotransplantation is a novel target to inhibit coagulation and endothelial activation. To prevent tissue factor and adhesion molecule expression, a strategy to block soluble histone may be required in xenotransplantation. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

ATP can be dispensable for prespliceosome formation in yeast

PubMed Central

Perriman, Rhonda; Ares, Manuel

2000-01-01

The first ATP-dependent step in pre-mRNA splicing involves the stable binding of U2 snRNP to form the prespliceosome. We show that a prespliceosome-like complex forms in the absence of ATP in yeast extracts lacking the U2 suppressor protein CUS2. These complexes display the same pre-mRNA and U snRNA requirements as authentic prespliceosomes and can be chased through the splicing pathway, indicating that they are a functional intermediate in the spliceosome assembly pathway. ATP-independent prespliceosome-like complexes are also observed in extracts containing a mutant U2 snRNA. Loss of CUS2 does not bypass the role of PRP5, an RNA helicase family member required for ATP-dependent prespliceosome formation. Genetic interactions between CUS2 and a heat-sensitive prp5 allele parallel those observed between CUS2 and U2, and suggest that CUS2 mediates functional interactions between U2 RNA and PRP5. We propose that CUS2 enforces ATP dependence during formation of the prespliceosome by brokering an interaction between PRP5 and the U2 snRNP that depends on correct U2 RNA structure. PMID:10640279

Quantitative assessment of Hox complex expression in the indirect development of the polychaete annelid Chaetopterus sp

NASA Technical Reports Server (NTRS)

Peterson, K. J.; Irvine, S. Q.; Cameron, R. A.; Davidson, E. H.

2000-01-01

A prediction from the set-aside theory of bilaterian origins is that pattern formation processes such as those controlled by the Hox cluster genes are required specifically for adult body plan formation. This prediction can be tested in animals that use maximal indirect development, in which the embryonic formation of the larva and the postembryonic formation of the adult body plan are temporally and spatially distinct. To this end, we quantitatively measured the amount of transcripts for five Hox genes in embryos of a lophotrochozoan, the polychaete annelid Chaetopterus sp. The polychaete Hox complex is shown not to be expressed during embryogenesis, but transcripts of all measured Hox complex genes are detected at significant levels during the initial stages of adult body plan formation. Temporal colinearity in the sequence of their activation is observed, so that activation follows the 3'-5' arrangement of the genes. Moreover, Hox gene expression is spatially localized to the region of teloblastic set-aside cells of the later-stage embryos. This study shows that an indirectly developing lophotrochozoan shares with an indirectly developing deuterostome, the sea urchin, a common mode of Hox complex utilization: construction of the larva, whether a trochophore or dipleurula, does not involve Hox cluster expression, but in both forms the complex is expressed in the set-aside cells from which the adult body plan derives.

Complex structure of the fission yeast SREBP-SCAP binding domains reveals an oligomeric organization.

PubMed

Gong, Xin; Qian, Hongwu; Shao, Wei; Li, Jingxian; Wu, Jianping; Liu, Jun-Jie; Li, Wenqi; Wang, Hong-Wei; Espenshade, Peter; Yan, Nieng

2016-11-01

Sterol regulatory element-binding protein (SREBP) transcription factors are master regulators of cellular lipid homeostasis in mammals and oxygen-responsive regulators of hypoxic adaptation in fungi. SREBP C-terminus binds to the WD40 domain of SREBP cleavage-activating protein (SCAP), which confers sterol regulation by controlling the ER-to-Golgi transport of the SREBP-SCAP complex and access to the activating proteases in the Golgi. Here, we biochemically and structurally show that the carboxyl terminal domains (CTD) of Sre1 and Scp1, the fission yeast SREBP and SCAP, form a functional 4:4 oligomer and Sre1-CTD forms a dimer of dimers. The crystal structure of Sre1-CTD at 3.5 Å and cryo-EM structure of the complex at 5.4 Å together with in vitro biochemical evidence elucidate three distinct regions in Sre1-CTD required for Scp1 binding, Sre1-CTD dimerization and tetrameric formation. Finally, these structurally identified domains are validated in a cellular context, demonstrating that the proper 4:4 oligomeric complex formation is required for Sre1 activation.

PriC-mediated DNA replication restart requires PriC complex formation with the single-stranded DNA-binding protein.

PubMed

Wessel, Sarah R; Marceau, Aimee H; Massoni, Shawn C; Zhou, Ruobo; Ha, Taekjip; Sandler, Steven J; Keck, James L

2013-06-14

Frequent collisions between cellular DNA replication complexes (replisomes) and obstacles such as damaged DNA or frozen protein complexes make DNA replication fork progression surprisingly sporadic. These collisions can lead to the ejection of replisomes prior to completion of replication, which, if left unrepaired, results in bacterial cell death. As such, bacteria have evolved DNA replication restart mechanisms that function to reload replisomes onto abandoned DNA replication forks. Here, we define a direct interaction between PriC, a key Escherichia coli DNA replication restart protein, and the single-stranded DNA-binding protein (SSB), a protein that is ubiquitously associated with DNA replication forks. PriC/SSB complex formation requires evolutionarily conserved residues from both proteins, including a pair of Arg residues from PriC and the C terminus of SSB. In vitro, disruption of the PriC/SSB interface by sequence changes in either protein blocks the first step of DNA replication restart, reloading of the replicative DnaB helicase onto an abandoned replication fork. Consistent with the critical role of PriC/SSB complex formation in DNA replication restart, PriC variants that cannot bind SSB are non-functional in vivo. Single-molecule experiments demonstrate that PriC binding to SSB alters SSB/DNA complexes, exposing single-stranded DNA and creating a platform for other proteins to bind. These data lead to a model in which PriC interaction with SSB remodels SSB/DNA structures at abandoned DNA replication forks to create a DNA structure that is competent for DnaB loading.

Amine-free reversible hydrogen storage in formate salts catalyzed by ruthenium pincer complex without pH control or solvent change.

PubMed

Kothandaraman, Jotheeswari; Czaun, Miklos; Goeppert, Alain; Haiges, Ralf; Jones, John-Paul; May, Robert B; Prakash, G K Surya; Olah, George A

2015-04-24

Due to the intermittent nature of most renewable energy sources, such as solar and wind, energy storage is increasingly required. Since electricity is difficult to store, hydrogen obtained by electrochemical water splitting has been proposed as an energy carrier. However, the handling and transportation of hydrogen in large quantities is in itself a challenge. We therefore present here a method for hydrogen storage based on a CO2 (HCO3 (-) )/H2 and formate equilibrium. This amine-free and efficient reversible system (>90 % yield in both directions) is catalyzed by well-defined and commercially available Ru pincer complexes. The formate dehydrogenation was triggered by simple pressure swing without requiring external pH control or the change of either the solvent or the catalyst. Up to six hydrogenation-dehydrogenation cycles were performed and the catalyst performance remained steady with high selectivity (CO free H2 /CO2 mixture was produced). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Studies of the Escherichia coli Rsd-sigma70 complex.

PubMed

Westblade, Lars F; Ilag, Leopold L; Powell, Andrew K; Kolb, Annie; Robinson, Carol V; Busby, Stephen J W

2004-01-16

Escherichia coli Rsd protein was previously identified on the basis of its binding to the RNA polymerase sigma(70) subunit. The Rsd-sigma(70) complex has been studied using different methods. Our data show that Rsd associates with sigma(70) to form a complex with a stoichiometry of 1:1. Alanine scanning and deletion mutagenesis were used to locate regions of sigma(70) that are required for the formation of the Rsd-sigma(70) complex.

An endosomal syntaxin and the AP-3 complex are required for formation and maturation of candidate lysosome-related secretory organelles (mucocysts) in Tetrahymena thermophila

PubMed Central

Kaur, Harsimran; Sparvoli, Daniela; Osakada, Hiroko; Iwamoto, Masaaki; Haraguchi, Tokuko; Turkewitz, Aaron P.

2017-01-01

The ciliate Tetrahymena thermophila synthesizes large secretory vesicles called mucocysts. Mucocyst biosynthesis shares features with dense core granules (DCGs) in animal cells, including proteolytic processing of cargo proteins during maturation. However, other molecular features have suggested relatedness to lysosome-related organelles (LROs). LROs, which include diverse organelles in animals, are formed via convergence of secretory and endocytic trafficking. Here we analyzed Tetrahymena syntaxin 7-like 1 (Stx7l1p), a Qa-SNARE whose homologues in other lineages are linked with vacuoles/LROs. Stx7l1p is targeted to both immature and mature mucocysts and is essential in mucocyst formation. In STX7L1-knockout cells, the two major classes of mucocyst cargo proteins localize independently, accumulating in largely nonoverlapping vesicles. Thus initial formation of immature mucocysts involves heterotypic fusion, in which a subset of mucocyst proteins is delivered via an endolysosomal compartment. Further, we show that subsequent maturation requires AP-3, a complex widely implicated in LRO formation. Knockout of the µ-subunit gene does not impede delivery of any known mucocyst cargo but nonetheless arrests mucocyst maturation. Our data argue that secretory organelles in ciliates may represent a new class of LROs and reveal key roles of an endosomal syntaxin and AP-3 in the assembly of this complex compartment. PMID:28381425

Mycobacterial biofilms: a greasy way to hold it together.

PubMed

Zambrano, María Mercedes; Kolter, Roberto

2005-12-02

Microorganisms growing on surfaces can form biofilms under certain conditions. In this issue of Cell, Ojha et al. (2005) investigate biofilm formation in mycobacteria. They identify new cell-wall components that are required for the formation of architecturally complex mature biofilms in these bacteria and the surprising involvement of a chaperone protein in this process.

Assembly of the epithelial Na+ channel evaluated using sucrose gradient sedimentation analysis.

PubMed

Cheng, C; Prince, L S; Snyder, P M; Welsh, M J

1998-08-28

Three subunits, alpha, beta, and gamma, contribute to the formation of the epithelial Na+ channel. To investigate the oligomeric assembly of the channel complex, we used sucrose gradient sedimentation analysis to determine the sedimentation properties of individual subunits and heteromultimers comprised of multiple subunits. When the alpha subunit was expressed alone, it first formed an oligomeric complex with a sedimentation coefficient of 11 S, and then generated a higher order multimer of 25 S. In contrast, individual beta and gamma subunits predominately assembled into 11 S complexes. We obtained similar results with expression in cells and in vitro. When we co-expressed beta with alpha or with alpha plus gamma, the beta subunit assembled into a 25 S complex. Glycosylation of the alpha subunit was not required for assembly into a 25 S complex. We found that the alpha subunit formed intra-chain disulfide bonds. Although such bonds were not required to generate an oligomeric complex, under nonreducing conditions the alpha subunit formed a complex that migrated more homogeneously at 25 S. This suggests that intra-chain disulfide bonds may stabilize the complex. These data suggest that the epithelial Na+ channel subunits form high order oligomeric complexes and that the alpha subunit contains the information that facilitates such formation. Interestingly, the ability of the alpha, but not the beta or gamma, subunit to assemble into a 25 S homomeric complex correlates with the ability of these subunits to generate functional channels when expressed alone.

Effect of phospholipase A treatment of low density lipoproteins on the dextran sulfate--lipoprotein interaction.

PubMed

Nishida, T

1968-09-01

The effect of phospholipase A on the interaction of low density lipoproteins of the S(f) 0-10 class with dextran sulfate was studied in phosphate buffer of pH 7.4, ionic strength 0.1, by chemical, spectrophotometric, and centrifugal methods. When low density lipoproteins that had been treated with phospholipase A were substituted for untreated lipoproteins, the amount of insoluble dextran sulfate-lipoprotein complex formed was greatly reduced. Hydrolysis of over 20% of the lecithin and phosphatidyl ethanolamine constituents of the lipoproteins prevented the formation of insoluble complex. However, even the lipoproteins in which almost all the phosphoglycerides were hydrolyzed produced soluble complex, which was converted to insoluble complex upon addition of magnesium sulfate. It is apparent that the lipoproteins altered extensively by treatment with phospholipase A retain many characteristic properties of native low density lipoproteins. Fatty acids, but not lysolecithin, released by the action of phospholipase A interfered with the formation of insoluble complex; this interference was due to association of the fatty acids with the lipoproteins. With increases in the concentration of the associated fatty acids, the amounts of magnesium ion required for the conversion of soluble complex to insoluble complex increased progressively. Charge interaction is evidently of paramount importance in the formation of sulfated polysaccharide-lipoprotein complexes.

The Evolutionarily Conserved Protein LAS1 Is Required for Pre-rRNA Processing at Both Ends of ITS2

PubMed Central

Schillewaert, Stéphanie; Wacheul, Ludivine; Lhomme, Frédéric

2012-01-01

Ribosome synthesis entails the formation of mature rRNAs from long precursor molecules, following a complex pre-rRNA processing pathway. Why the generation of mature rRNA ends is so complicated is unclear. Nor is it understood how pre-rRNA processing is coordinated at distant sites on pre-rRNA molecules. Here we characterized, in budding yeast and human cells, the evolutionarily conserved protein Las1. We found that, in both species, Las1 is required to process ITS2, which separates the 5.8S and 25S/28S rRNAs. In yeast, Las1 is required for pre-rRNA processing at both ends of ITS2. It is required for Rrp6-dependent formation of the 5.8S rRNA 3′ end and for Rat1-dependent formation of the 25S rRNA 5′ end. We further show that the Rat1-Rai1 5′-3′ exoribonuclease (exoRNase) complex functionally connects processing at both ends of the 5.8S rRNA. We suggest that pre-rRNA processing is coordinated at both ends of 5.8S rRNA and both ends of ITS2, which are brought together by pre-rRNA folding, by an RNA processing complex. Consistently, we note the conspicuous presence of ∼7- or 8-nucleotide extensions on both ends of 5.8S rRNA precursors and at the 5′ end of pre-25S RNAs suggestive of a protected spacer fragment of similar length. PMID:22083961

Hrs regulates early endosome fusion by inhibiting formation of an endosomal SNARE complex

PubMed Central

Sun, Wei; Yan, Qing; Vida, Thomas A.; Bean, Andrew J.

2003-01-01

Movement through the endocytic pathway occurs principally via a series of membrane fusion and fission reactions that allow sorting of molecules to be recycled from those to be degraded. Endosome fusion is dependent on SNARE proteins, although the nature of the proteins involved and their regulation has not been fully elucidated. We found that the endosome-associated hepatocyte responsive serum phosphoprotein (Hrs) inhibited the homotypic fusion of early endosomes. A region of Hrs predicted to form a coiled coil required for binding the Q-SNARE, SNAP-25, mimicked the inhibition of endosome fusion produced by full-length Hrs, and was sufficient for endosome binding. SNAP-25, syntaxin 13, and VAMP2 were bound from rat brain membranes to the Hrs coiled-coil domain. Syntaxin 13 inhibited early endosomal fusion and botulinum toxin/E inhibition of early endosomal fusion was reversed by addition of SNAP-25(150–206), confirming a role for syntaxin 13, and establishing a role for SNAP-25 in endosomal fusion. Hrs inhibited formation of the syntaxin 13–SNAP-25–VAMP2 complex by displacing VAMP2 from the complex. These data suggest that SNAP-25 is a receptor for Hrs on early endosomal membranes and that the binding of Hrs to SNAP-25 on endosomal membranes inhibits formation of a SNARE complex required for homotypic endosome fusion. PMID:12847087

14-3-3 Regulates Actin Filament Formation in the Deep-Branching Eukaryote Giardia lamblia

PubMed Central

Xu, Jennifer; Steele-Ogus, Melissa; Alas, Germain C. M.

2017-01-01

ABSTRACT The phosphoserine/phosphothreonine-binding protein 14-3-3 is known to regulate actin; this function has been previously attributed to sequestration of phosphorylated cofilin. 14-3-3 was identified as an actin-associated protein in the deep-branching eukaryote Giardia lamblia; however, Giardia lacks cofilin and all other canonical actin-binding proteins (ABPs). Thus, the role of G. lamblia 14-3-3 (Gl-14-3-3) in actin regulation was unknown. Gl-14-3-3 depletion resulted in an overall disruption of actin organization characterized by ectopically distributed short actin filaments. Using phosphatase and kinase inhibitors, we demonstrated that actin phosphorylation correlated with destabilization of the actin network and increased complex formation with 14-3-3, while blocking actin phosphorylation stabilized actin filaments and attenuated complex formation. Giardia’s sole Rho family GTPase, Gl-Rac, modulates Gl-14-3-3’s association with actin, providing the first connection between Gl-Rac and the actin cytoskeleton in Giardia. Giardia actin (Gl-actin) contains two putative 14-3-3 binding motifs, one of which (S330) is conserved in mammalian actin. Mutation of these sites reduced, but did not completely disrupt, the association with 14-3-3. Native gels and overlay assays indicate that intermediate proteins are required to support complex formation between 14-3-3 and actin. Overall, our results support a role for 14-3-3 as a regulator of actin; however, the presence of multiple 14-3-3–actin complexes suggests a more complex regulatory relationship than might be expected for a minimalistic parasite. IMPORTANCE Giardia lacks canonical actin-binding proteins. Gl-14-3-3 was identified as an actin interactor, but the significance of this interaction was unknown. Loss of Gl-14-3-3 results in ectopic short actin filaments, indicating that Gl-14-3-3 is an important regulator of the actin cytoskeleton in Giardia. Drug studies indicate that Gl-14-3-3 complex formation is in part phospho-regulated. We demonstrate that complex formation is downstream of Giardia’s sole Rho family GTPase, Gl-Rac. This result provides the first mechanistic connection between Gl-Rac and Gl-actin in Giardia. Native gels and overlay assays indicate intermediate proteins are required to support the interaction between Gl-14-3-3 and Gl-actin, suggesting that Gl-14-3-3 is regulating multiple Gl-actin complexes. PMID:28932813

Hemolymph Melanization in the Silkmoth Bombyx mori Involves Formation of a High Molecular Mass Complex That Metabolizes Tyrosine*

PubMed Central

Clark, Kevin D.; Strand, Michael R.

2013-01-01

The phenoloxidase (PO) cascade regulates the melanization of blood (hemolymph) in insects and other arthropods. Most studies indicate that microbial elicitors activate the PO cascade, which results in processing of the zymogen PPO to PO. PO is then thought to oxidize tyrosine and o-diphenols to quinones, which leads to melanin. However, different lines of investigation raise questions as to whether these views are fully correct. Here we report that hemolymph from the silkmoth, Bombyx mori, rapidly melanizes after collection from a wound site. Prior studies indicated that in vitro activated PPO hydroxylates Tyr inefficiently. Measurement of in vivo substrate titers, however, suggested that Tyr was the only PO substrate initially present in B. mori plasma and that it is rapidly metabolized by PO. Fractionation of plasma by gel filtration chromatography followed by bioassays indicated that melanization activity was primarily associated with a high mass complex (∼670 kDa) that contained PO. The prophenoloxidase-activating protease inhibitor Egf1.0 blocked formation of this complex and Tyr metabolism, but the addition of phenylthiourea to plasma before fractionation enhanced complex formation and Tyr metabolism. Mass spectrometry analysis indicated that the complex contained PO plus other proteins. Taken together, our results indicate that wounding alone activates the PO cascade in B. mori. They also suggest that complex formation is required for efficient use of Tyr as a substrate. PMID:23553628

Nonadiabatic one-electron transfer mechanism for the O-O bond formation in the oxygen-evolving complex of photosystem II

NASA Astrophysics Data System (ADS)

Shoji, Mitsuo; Isobe, Hiroshi; Shigeta, Yasuteru; Nakajima, Takahito; Yamaguchi, Kizashi

2018-04-01

The reaction mechanism of the O2 formation in the S4 state of the oxygen-evolving complex of photosystem II was clarified at the quantum mechanics/molecular mechanics (QM/MM) level. After the Yz (Y161) oxidation and the following proton transfer in the S3 state, five reaction steps are required to produce the molecular dioxygen. The highest barrier step is the first proton transfer reaction (0 → 1). The following reactions involving electron transfers were precisely analyzed in terms of their energies, structures and spin densities. We found that the one-electron transfer from the Mn4Ca cluster to Y161 triggers the O-O sigma bond formation.

SMC5/6 is required for the formation of segregation-competent bivalent chromosomes during meiosis I in mouse oocytes.

PubMed

Hwang, Grace; Sun, Fengyun; O'Brien, Marilyn; Eppig, John J; Handel, Mary Ann; Jordan, Philip W

2017-05-01

SMC complexes include three major classes: cohesin, condensin and SMC5/6. However, the localization pattern and genetic requirements for the SMC5/6 complex during mammalian oogenesis have not previously been examined. In mouse oocytes, the SMC5/6 complex is enriched at the pericentromeric heterochromatin, and also localizes along chromosome arms during meiosis. The infertility phenotypes of females with a Zp3-Cre -driven conditional knockout (cKO) of Smc5 demonstrated that maternally expressed SMC5 protein is essential for early embryogenesis. Interestingly, protein levels of SMC5/6 complex components in oocytes decline as wild-type females age. When SMC5/6 complexes were completely absent in oocytes during meiotic resumption, homologous chromosomes failed to segregate accurately during meiosis I. Despite what appears to be an inability to resolve concatenation between chromosomes during meiosis, localization of topoisomerase IIα to bivalents was not affected; however, localization of condensin along the chromosome axes was perturbed. Taken together, these data demonstrate that the SMC5/6 complex is essential for the formation of segregation-competent bivalents during meiosis I, and findings suggest that age-dependent depletion of the SMC5/6 complex in oocytes could contribute to increased incidence of oocyte aneuploidy and spontaneous abortion in aging females. © 2017. Published by The Company of Biologists Ltd.

PAS Kinase Promotes Cell Survival and Growth Through Activation of Rho1

PubMed Central

Cardon, Caleb M.; Beck, Thomas; Hall, Michael N.; Rutter, Jared

2014-01-01

In Saccharomyces cerevisiae, phosphorylation of Ugp1 by either of the yeast PASK family protein kinases (yPASK), Psk1 or Psk2, directs this metabolic enzyme to deliver glucose to the periphery for synthesis of the cell wall. However, we isolated PSK1 and PSK2 in a high-copy suppressor screen of a temperature-sensitive mutant of target of rapamycin 2 (TOR2). Posttranslational activation of yPASK, either by cell integrity stress or by growth on nonfermentative carbon sources, also suppressed the growth defect resulting from tor2 mutation. Although suppression of the tor2 mutant growth phenotype by activation of the kinase activity of yPASK required phosphorylation of the metabolic enzyme Ugp1 on serine 11, this resulted in the formation of a complex that induced Rho1 activation, rather than required the glucose partitioning function of Ugp1. In addition to phosphorylated Ugp1, this complex contained Rom2, a Rho1 guanine nucleotide exchange factor, and Ssd1, an mRNA-binding protein. Activation of yPASK-dependent Ugp1 phosphorylation, therefore, enables two processes that are required for cell growth and stress resistance: synthesis of the cell wall through partitioning glucose to the periphery and the formation of the signaling complex with Rom2 and Ssd1 to promote Rho1-dependent polarized cell growth. This complex may integrate metabolic and signaling responses required for cell growth and survival in suboptimal conditions. PMID:22296835

The interaction of DiaA and DnaA regulates the replication cycle in E. coli by directly promoting ATP–DnaA-specific initiation complexes

PubMed Central

Keyamura, Kenji; Fujikawa, Norie; Ishida, Takuma; Ozaki, Shogo; Su’etsugu, Masayuki; Fujimitsu, Kazuyuki; Kagawa, Wataru; Yokoyama, Shigeyuki; Kurumizaka, Hitoshi; Katayama, Tsutomu

2007-01-01

Escherichia coli DiaA is a DnaA-binding protein that is required for the timely initiation of chromosomal replication during the cell cycle. In this study, we determined the crystal structure of DiaA at 1.8 Å resolution. DiaA forms a homotetramer consisting of a symmetrical pair of homodimers. Mutational analysis revealed that the DnaA-binding activity and formation of homotetramers are required for the stimulation of initiation by DiaA. DiaA tetramers can bind multiple DnaA molecules simultaneously. DiaA stimulated the assembly of multiple DnaA molecules on oriC, conformational changes in ATP–DnaA-specific initiation complexes, and unwinding of oriC duplex DNA. The mutant DiaA proteins are defective in these stimulations. DiaA associated also with ADP–DnaA, and stimulated the assembly of inactive ADP–DnaA–oriC complexes. Specific residues in the putative phosphosugar-binding motif of DiaA were required for the stimulation of initiation and formation of ATP–DnaA-specific–oriC complexes. Our data indicate that DiaA regulates initiation by a novel mechanism, in which DiaA tetramers most likely bind to multiple DnaA molecules and stimulate the assembly of specific ATP–DnaA–oriC complexes. These results suggest an essential role for DiaA in the promotion of replication initiation in a cell cycle coordinated manner. PMID:17699754

Formation of virions is strictly required for turnip yellows virus long-distance movement in plants.

PubMed

Hipper, Clémence; Monsion, Baptiste; Bortolamiol-Bécet, Diane; Ziegler-Graff, Véronique; Brault, Véronique

2014-02-01

Viral genomic RNA of the Turnip yellows virus (TuYV; genus Polerovirus; family Luteoviridae) is protected in virions formed by the major capsid protein (CP) and the minor component, the readthrough (RT*) protein. Long-distance transport, used commonly by viruses to systemically infect host plants, occurs in phloem sieve elements and two viral forms of transport have been described: virions and ribonucleoprotein (RNP) complexes. With regard to poleroviruses, virions have always been presumed to be the long-distance transport form, but the potential role of RNP complexes has not been investigated. Here, we examined the requirement of virions for polerovirus systemic movement by analysing CP-targeted mutants that were unable to form viral particles. We confirmed that TuYV mutants that cannot encapsidate into virions are not able to reach systemic leaves. To completely discard the possibility that the introduced mutations in CP simply blocked the formation or the movement of RNP complexes, we tested in trans complementation of TuYV CP mutants by providing WT CP expressed in transgenic plants. WT CP was able to facilitate systemic movement of TuYV CP mutants and this observation was always correlated with the formation of virions. This demonstrated clearly that virus particles are essential for polerovirus systemic movement.

Multiple domains of fission yeast Cdc19p (MCM2) are required for its association with the core MCM complex.

PubMed

Sherman, D A; Pasion, S G; Forsburg, S L

1998-07-01

The members of the MCM protein family are essential eukaryotic DNA replication factors that form a six-member protein complex. In this study, we use antibodies to four MCM proteins to investigate the structure of and requirements for the formation of fission yeast MCM complexes in vivo, with particular regard to Cdc19p (MCM2). Gel filtration analysis shows that the MCM protein complexes are unstable and can be broken down to subcomplexes. Using coimmunoprecipitation, we find that Mis5p (MCM6) and Cdc21p (MCM4) are tightly associated with one another in a core complex with which Cdc19p loosely associates. Assembly of Cdc19p with the core depends upon Cdc21p. Interestingly, there is no obvious change in Cdc19p-containing MCM complexes through the cell cycle. Using a panel of Cdc19p mutants, we find that multiple domains of Cdc19p are required for MCM binding. These studies indicate that MCM complexes in fission yeast have distinct substructures, which may be relevant for function.

Multiple Domains of Fission Yeast Cdc19p (MCM2) Are Required for Its Association with the Core MCM Complex

PubMed Central

Sherman, Daniel A.; Pasion, Sally G.; Forsburg, Susan L.

1998-01-01

The members of the MCM protein family are essential eukaryotic DNA replication factors that form a six-member protein complex. In this study, we use antibodies to four MCM proteins to investigate the structure of and requirements for the formation of fission yeast MCM complexes in vivo, with particular regard to Cdc19p (MCM2). Gel filtration analysis shows that the MCM protein complexes are unstable and can be broken down to subcomplexes. Using coimmunoprecipitation, we find that Mis5p (MCM6) and Cdc21p (MCM4) are tightly associated with one another in a core complex with which Cdc19p loosely associates. Assembly of Cdc19p with the core depends upon Cdc21p. Interestingly, there is no obvious change in Cdc19p-containing MCM complexes through the cell cycle. Using a panel of Cdc19p mutants, we find that multiple domains of Cdc19p are required for MCM binding. These studies indicate that MCM complexes in fission yeast have distinct substructures, which may be relevant for function. PMID:9658174

Elements of a next generation time-series ASCII data file format for Earth Sciences

NASA Astrophysics Data System (ADS)

Webster, C. J.

2015-12-01

Data in ASCII comma separated value (CSV) format are recognized as the most simple, straightforward and readable type of data present in the geosciences. Many scientific workflows developed over the years rely on data using this simple format. However, there is a need for a lightweight ASCII header format standard that is easy to create and easy to work with. Current OGC grade XML standards are complex and difficult to implement for researchers with few resources. Ideally, such a format should provide the data in CSV for easy consumption by generic applications such as spreadsheets. The format should use an existing time standard. The header should be easily human readable as well as machine parsable. The metadata format should be extendable to allow vocabularies to be adopted as they are created by external standards bodies. The creation of such a format will increase the productivity of software engineers and scientists because fewer translators and checkers would be required. Data in ASCII comma separated value (CSV) format are recognized as the most simple, straightforward and readable type of data present in the geosciences. Many scientific workflows developed over the years rely on data using this simple format. However, there is a need for a lightweight ASCII header format standard that is easy to create and easy to work with. Current OGC grade XML standards are complex and difficult to implement for researchers with few resources. Ideally, such a format would provide the data in CSV for easy consumption by generic applications such as spreadsheets. The format would use existing time standard. The header would be easily human readable as well as machine parsable. The metadata format would be extendable to allow vocabularies to be adopted as they are created by external standards bodies. The creation of such a format would increase the productivity of software engineers and scientists because fewer translators would be required.

Neurobeachin is required postsynaptically for electrical and chemical synapse formation

PubMed Central

Miller, Adam C.; Voelker, Lisa H.; Shah, Arish N.; Moens, Cecilia B.

2014-01-01

Summary Background Neural networks and their function are defined by synapses, which are adhesions specialized for intercellular communication that can be either chemical or electrical. At chemical synapses transmission between neurons is mediated by neurotransmitters, while at electrical synapses direct ionic and metabolic coupling occurs via gap junctions between neurons. The molecular pathways required for electrical synaptogenesis are not well understood and whether they share mechanisms of formation with chemical synapses is not clear. Results Here, using a forward genetic screen in zebrafish we find that the autism-associated gene neurobeachin (nbea), which encodes a BEACH-domain containing protein implicated in endomembrane trafficking, is required for both electrical and chemical synapse formation. Additionally, we find that nbea is dispensable for axonal formation and early dendritic outgrowth, but is required to maintain dendritic complexity. These synaptic and morphological defects correlate with deficiencies in behavioral performance. Using chimeric animals in which individually identifiable neurons are either mutant or wildtype we find that Nbea is necessary and sufficient autonomously in the postsynaptic neuron for both synapse formation and dendritic arborization. Conclusions Our data identify a surprising link between electrical and chemical synapse formation and show that Nbea acts as a critical regulator in the postsynaptic neuron for the coordination of dendritic morphology with synaptogenesis. PMID:25484298

Photosynthesis. Electronic structure of the oxygen-evolving complex in photosystem II prior to O-O bond formation.

PubMed

Cox, Nicholas; Retegan, Marius; Neese, Frank; Pantazis, Dimitrios A; Boussac, Alain; Lubitz, Wolfgang

2014-08-15

The photosynthetic protein complex photosystem II oxidizes water to molecular oxygen at an embedded tetramanganese-calcium cluster. Resolving the geometric and electronic structure of this cluster in its highest metastable catalytic state (designated S3) is a prerequisite for understanding the mechanism of O-O bond formation. Here, multifrequency, multidimensional magnetic resonance spectroscopy reveals that all four manganese ions of the catalyst are structurally and electronically similar immediately before the final oxygen evolution step; they all exhibit a 4+ formal oxidation state and octahedral local geometry. Only one structural model derived from quantum chemical modeling is consistent with all magnetic resonance data; its formation requires the binding of an additional water molecule. O-O bond formation would then proceed by the coupling of two proximal manganese-bound oxygens in the transition state of the cofactor. Copyright © 2014, American Association for the Advancement of Science.

BAG3 regulates formation of the SNARE complex and insulin secretion

PubMed Central

Iorio, V; Festa, M; Rosati, A; Hahne, M; Tiberti, C; Capunzo, M; De Laurenzi, V; Turco, M C

2015-01-01

Insulin release in response to glucose stimulation requires exocytosis of insulin-containing granules. Glucose stimulation of beta cells leads to focal adhesion kinase (FAK) phosphorylation, which acts on the Rho family proteins (Rho, Rac and Cdc42) that direct F-actin remodeling. This process requires docking and fusion of secretory vesicles to the release sites at the plasma membrane and is a complex mechanism that is mediated by SNAREs. This transiently disrupts the F-actin barrier and allows the redistribution of the insulin-containing granules to more peripheral regions of the β cell, hence facilitating insulin secretion. In this manuscript, we show for the first time that BAG3 plays an important role in this process. We show that BAG3 downregulation results in increased insulin secretion in response to glucose stimulation and in disruption of the F-actin network. Moreover, we show that BAG3 binds to SNAP-25 and syntaxin-1, two components of the t-SNARE complex preventing the interaction between SNAP-25 and syntaxin-1. Upon glucose stimulation BAG3 is phosphorylated by FAK and dissociates from SNAP-25 allowing the formation of the SNARE complex, destabilization of the F-actin network and insulin release. PMID:25766323

CAPS drives trans-SNARE complex formation and membrane fusion through syntaxin interactions.

PubMed

James, Declan J; Kowalchyk, Judith; Daily, Neil; Petrie, Matt; Martin, Thomas F J

2009-10-13

Ca(2+)-dependent activator protein for secretion (CAPS) is an essential factor for regulated vesicle exocytosis that functions in priming reactions before Ca(2+)-triggered fusion of vesicles with the plasma membrane. However, the precise events that CAPS regulates to promote vesicle fusion are unclear. In the current work, we reconstituted CAPS function in a SNARE-dependent liposome fusion assay using VAMP2-containing donor and syntaxin-1/SNAP-25-containing acceptor liposomes. The CAPS stimulation of fusion required PI(4,5)P(2) in acceptor liposomes and was independent of Ca(2+), but Ca(2+) dependence was restored by inclusion of synaptotagmin. CAPS stimulated trans-SNARE complex formation concomitant with the stimulation of full membrane fusion at physiological SNARE densities. CAPS bound syntaxin-1, and CAPS truncations that competitively inhibited syntaxin-1 binding also inhibited CAPS-dependent fusion. The results revealed an unexpected activity of a priming protein to accelerate fusion by efficiently promoting trans-SNARE complex formation. CAPS may function in priming by organizing SNARE complexes on the plasma membrane.

A positive-strand RNA virus uses alternative protein-protein interactions within a viral protease/cofactor complex to switch between RNA replication and virion morphogenesis.

PubMed

Dubrau, Danilo; Tortorici, M Alejandra; Rey, Félix A; Tautz, Norbert

2017-02-01

The viruses of the family Flaviviridae possess a positive-strand RNA genome and express a single polyprotein which is processed into functional proteins. Initially, the nonstructural (NS) proteins, which are not part of the virions, form complexes capable of genome replication. Later on, the NS proteins also play a critical role in virion formation. The molecular basis to understand how the same proteins form different complexes required in both processes is so far unknown. For pestiviruses, uncleaved NS2-3 is essential for virion morphogenesis while NS3 is required for RNA replication but is not functional in viral assembly. Recently, we identified two gain of function mutations, located in the C-terminal region of NS2 and in the serine protease domain of NS3 (NS3 residue 132), which allow NS2 and NS3 to substitute for uncleaved NS2-3 in particle assembly. We report here the crystal structure of pestivirus NS3-4A showing that the NS3 residue 132 maps to a surface patch interacting with the C-terminal region of NS4A (NS4A-kink region) suggesting a critical role of this contact in virion morphogenesis. We show that destabilization of this interaction, either by alanine exchanges at this NS3/4A-kink interface, led to a gain of function of the NS3/4A complex in particle formation. In contrast, RNA replication and thus replicase assembly requires a stable association between NS3 and the NS4A-kink region. Thus, we propose that two variants of NS3/4A complexes exist in pestivirus infected cells each representing a basic building block required for either RNA replication or virion morphogenesis. This could be further corroborated by trans-complementation studies with a replication-defective NS3/4A double mutant that was still functional in viral assembly. Our observations illustrate the presence of alternative overlapping surfaces providing different contacts between the same proteins, allowing the switch from RNA replication to virion formation.

Novel interactions of CAPS (Ca2+-dependent activator protein for secretion) with the three neuronal SNARE proteins required for vesicle fusion.

PubMed

Daily, Neil J; Boswell, Kristin L; James, Declan J; Martin, Thomas F J

2010-11-12

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca(2+)-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis.

Novel Interactions of CAPS (Ca2+-dependent Activator Protein for Secretion) with the Three Neuronal SNARE Proteins Required for Vesicle Fusion*

PubMed Central

Daily, Neil J.; Boswell, Kristin L.; James, Declan J.; Martin, Thomas F. J.

2010-01-01

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca2+-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis. PMID:20826818

An Epichloë festucae homologue of MOB3, a component of the STRIPAK complex, is required for the establishment of a mutualistic symbiotic interaction with Lolium perenne

PubMed Central

Green, Kimberly A.; Becker, Yvonne; Fitzsimons, Helen L.

2016-01-01

Summary In both Sordaria macrospora and Neurospora crassa, components of the conserved STRIPAK (striatin‐interacting phosphatase and kinase) complex regulate cell–cell fusion, hyphal network development and fruiting body formation. Interestingly, a number of Epichloë festucae genes that are required for hyphal cell–cell fusion, such as noxA, noxR, proA, mpkA and mkkA, are also required for the establishment of a mutualistic symbiotic interaction with Lolium perenne. To determine whether MobC, a homologue of the STRIPAK complex component MOB3 in S. macrospora and N. crassa, is required for E. festucae hyphal fusion and symbiosis, a mobC deletion strain was generated. The ΔmobC mutant showed reduced rates of hyphal cell–cell fusion, formed intrahyphal hyphae and exhibited enhanced conidiation. Plants infected with ΔmobC were severely stunted. Hyphae of ΔmobC showed a proliferative pattern of growth within the leaves of Lolium perenne with increased colonization of the intercellular spaces and vascular bundles. Although hyphae were still able to form expressoria, structures allowing the colonization of the leaf surface, the frequency of formation was significantly reduced. Collectively, these results show that the STRIPAK component MobC is required for the establishment of a mutualistic symbiotic association between E. festucae and L. perenne, and plays an accessory role in the regulation of hyphal cell–cell fusion and expressorium development in E. festucae. PMID:27277141

Confluence and convergence: team effectiveness in complex systems.

PubMed

Porter-OʼGrady, Tim

2015-01-01

Complex adaptive systems require nursing leadership to rethink organizational work and the viability and effectiveness of teams. Much of emergent thinking about complexity and systems and organizations alter the understanding of the nature and function of teamwork and the configuration and leadership of team effort. Reflecting on basic concepts of complexity and their application to team formation, dynamics, and outcomes lays an important foundation for effectively guiding the strategic activity of systems through the focused tactical action of teams. Basic principles of complexity, their impact on teams, and the fundamental elements of team effectiveness are explored.

Functional requirements of AID's higher order structures and their interaction with RNA-binding proteins.

PubMed

Mondal, Samiran; Begum, Nasim A; Hu, Wenjun; Honjo, Tasuku

2016-03-15

Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. Although both the N and C termini of AID have unique functions in DNA cleavage and recombination, respectively, during SHM and CSR, their molecular mechanisms are poorly understood. Using a bimolecular fluorescence complementation (BiFC) assay combined with glycerol gradient fractionation, we revealed that the AID C terminus is required for a stable dimer formation. Furthermore, AID monomers and dimers form complexes with distinct heterogeneous nuclear ribonucleoproteins (hnRNPs). AID monomers associate with DNA cleavage cofactor hnRNP K whereas AID dimers associate with recombination cofactors hnRNP L, hnRNP U, and Serpine mRNA-binding protein 1. All of these AID/ribonucleoprotein associations are RNA-dependent. We propose that AID's structure-specific cofactor complex formations differentially contribute to its DNA-cleavage and recombination functions.

Functional requirements of AID’s higher order structures and their interaction with RNA-binding proteins

PubMed Central

Mondal, Samiran; Begum, Nasim A.; Hu, Wenjun; Honjo, Tasuku

2016-01-01

Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. Although both the N and C termini of AID have unique functions in DNA cleavage and recombination, respectively, during SHM and CSR, their molecular mechanisms are poorly understood. Using a bimolecular fluorescence complementation (BiFC) assay combined with glycerol gradient fractionation, we revealed that the AID C terminus is required for a stable dimer formation. Furthermore, AID monomers and dimers form complexes with distinct heterogeneous nuclear ribonucleoproteins (hnRNPs). AID monomers associate with DNA cleavage cofactor hnRNP K whereas AID dimers associate with recombination cofactors hnRNP L, hnRNP U, and Serpine mRNA-binding protein 1. All of these AID/ribonucleoprotein associations are RNA-dependent. We propose that AID’s structure-specific cofactor complex formations differentially contribute to its DNA-cleavage and recombination functions. PMID:26929374

Vascular lumen formation.

PubMed

Lammert, Eckhard; Axnick, Jennifer

2012-04-01

The vascular system developed early in evolution. It is required in large multicellular organisms for the transport of nutrients, oxygen, and waste products to and from tissues. The vascular system is composed of hollow tubes, which have a high level of complexity in vertebrates. Vasculogenesis describes the de novo formation of blood vessels, e.g., aorta formation in vertebrate embryogenesis. In contrast, angiogenesis is the formation of blood vessels from preexisting ones, e.g., sprouting of intersomitic blood vessels from the aorta. Importantly, the lumen of all blood vessels in vertebrates is lined and formed by endothelial cells. In both vasculogenesis and angiogenesis, lumen formation takes place in a cord of endothelial cells. It involves a complex molecular mechanism composed of endothelial cell repulsion at the cell-cell contacts within the endothelial cell cords, junctional rearrangement, and endothelial cell shape change. As the vascular system also participates in the course of many diseases, such as cancer, stroke, and myocardial infarction, it is important to understand and make use of the molecular mechanisms of blood vessel formation to better understand and manipulate the pathomechanisms involved.

Replication Protein A2c Coupled with Replication Protein A1c Regulates Crossover Formation during Meiosis in Rice[C][W][OPEN

PubMed Central

Li, Xingwang; Chang, Yuxiao; Xin, Xiaodong; Zhu, Chunmei; Li, Xianghua; Higgins, James D.; Wu, Changyin

2013-01-01

Replication protein A (RPA) is a conserved heterotrimeric protein complex comprising RPA1, RPA2, and RPA3 subunits involved in multiple DNA metabolism pathways attributable to its single-stranded DNA binding property. Unlike other species possessing a single RPA2 gene, rice (Oryza sativa) possesses three RPA2 paralogs, but their functions remain unclear. In this study, we identified RPA2c, a rice gene preferentially expressed during meiosis. A T-DNA insertional mutant (rpa2c) exhibited reduced bivalent formation, leading to chromosome nondisjunction. In rpa2c, chiasma frequency is reduced by ∼78% compared with the wild type and is accompanied by loss of the obligate chiasma. The residual ∼22% chiasmata fit a Poisson distribution, suggesting loss of crossover control. RPA2c colocalized with the meiotic cohesion subunit REC8 and the axis-associated protein PAIR2. Localization of REC8 was necessary for loading of RPA2c to the chromosomes. In addition, RPA2c partially colocalized with MER3 during late leptotene, thus indicating that RPA2c is required for class I crossover formation at a late stage of homologous recombination. Furthermore, we identified RPA1c, an RPA1 subunit with nearly overlapping distribution to RPA2c, required for ∼79% of chiasmata formation. Our results demonstrate that an RPA complex comprising RPA2c and RPA1c is required to promote meiotic crossovers in rice. PMID:24122830

Designing Infographics to support teaching complex science subject: A comparison between static and animated Infographics

NASA Astrophysics Data System (ADS)

Hassan, Hesham Galal

This thesis explores the proper principles and rules for creating excellent infographics that communicate information successfully and effectively. Not only does this thesis examine the creation of Infographics, it also tries to answer which format, Static or Animated Infographics, is the most effective when used as a teaching-aid framework for complex science subjects, and if compelling Infographics in the preferred format facilitate the learning experience. The methodology includes the creation of infographic using two formats (Static and Animated) of a fairly complex science subject (Phases Of The Moon), which were then tested for their efficacy as a whole, and the two formats were compared in terms of information comprehension and retention. My hypothesis predicts that the creation of an infographic using the animated format would be more effective in communicating a complex science subject (Phases Of The Moon), specifically when using 3D computer animation to visualize the topic. This would also help different types of learners to easily comprehend science subjects. Most of the animated infographics produced nowadays are created for marketing and business purposes and do not implement the analytical design principles required for creating excellent information design. I believe that science learners are still in need of more variety in their methods of learning information, and that infographics can be of great assistance. The results of this thesis study suggests that using properly designed infographics would be of great help in teaching complex science subjects that involve spatial and temporal data. This could facilitate learning science subjects and consequently impact the interest of young learners in STEM.

Trimeric association of Hox and TALE homeodomain proteins mediates Hoxb2 hindbrain enhancer activity.

PubMed

Jacobs, Y; Schnabel, C A; Cleary, M L

1999-07-01

Pbx/exd proteins modulate the DNA binding affinities and specificities of Hox proteins and contribute to the execution of Hox-dependent developmental programs in arthropods and vertebrates. Pbx proteins also stably heterodimerize and bind DNA with Meis and Pknox1-Prep1, additional members of the TALE (three-amino-acid loop extension) superclass of homeodomain proteins that function on common genetic pathways with a subset of Hox proteins. In this study, we demonstrated that Pbx and Meis bind DNA as heterotrimeric complexes with Hoxb1 on a genetically defined Hoxb2 enhancer, r4, that mediates the cross-regulatory transcriptional effects of Hoxb1 in vivo. The DNA binding specificity of the heterotrimeric complex for r4 is mediated by a Pbx-Hox site in conjunction with a distal Meis site, which we showed to be required for ternary complex formation and Meis-enhanced transcription. Formation of heterotrimeric complexes in which all three homeodomains bind their cognate DNA sites is topologically facilitated by the ability of Pbx and Meis to interact through their amino termini and bind DNA without stringent half-site orientation and spacing requirements. Furthermore, Meis site mutation in the Hoxb2 enhancer phenocopies Pbx-Hox site mutation to abrogate enhancer-directed expression of a reporter transgene in the murine embryonic hindbrain, demonstrating that DNA binding by all three proteins is required for trimer function in vivo. Our data provide in vitro and in vivo evidence for the combinatorial regulation of Hox and TALE protein functions that are mediated, in part, by their interdependent DNA binding activities as ternary complexes. As a consequence, Hoxb1 employs Pbx and Meis-related proteins, as a pair of essential cofactors in a higher-order molecular complex, to mediate its transcriptional effects on an endogenous Hox response element.

User's guide for a computer program for calculating the zero-lift wave drag of complex aircraft configurations

NASA Technical Reports Server (NTRS)

Craidon, C. B.

1983-01-01

A computer program was developed to extend the geometry input capabilities of previous versions of a supersonic zero lift wave drag computer program. The arbitrary geometry input description is flexible enough to describe almost any complex aircraft concept, so that highly accurate wave drag analysis can now be performed because complex geometries can be represented accurately and do not have to be modified to meet the requirements of a restricted input format.

MURC/Cavin-4 and cavin family members form tissue-specific caveolar complexes.

PubMed

Bastiani, Michele; Liu, Libin; Hill, Michelle M; Jedrychowski, Mark P; Nixon, Susan J; Lo, Harriet P; Abankwa, Daniel; Luetterforst, Robert; Fernandez-Rojo, Manuel; Breen, Michael R; Gygi, Steven P; Vinten, Jorgen; Walser, Piers J; North, Kathryn N; Hancock, John F; Pilch, Paul F; Parton, Robert G

2009-06-29

Polymerase I and transcript release factor (PTRF)/Cavin is a cytoplasmic protein whose expression is obligatory for caveola formation. Using biochemistry and fluorescence resonance energy transfer-based approaches, we now show that a family of related proteins, PTRF/Cavin-1, serum deprivation response (SDR)/Cavin-2, SDR-related gene product that binds to C kinase (SRBC)/Cavin-3, and muscle-restricted coiled-coil protein (MURC)/Cavin-4, forms a multiprotein complex that associates with caveolae. This complex can constitutively assemble in the cytosol and associate with caveolin at plasma membrane caveolae. Cavin-1, but not other cavins, can induce caveola formation in a heterologous system and is required for the recruitment of the cavin complex to caveolae. The tissue-restricted expression of cavins suggests that caveolae may perform tissue-specific functions regulated by the composition of the cavin complex. Cavin-4 is expressed predominantly in muscle, and its distribution is perturbed in human muscle disease associated with Caveolin-3 dysfunction, identifying Cavin-4 as a novel muscle disease candidate caveolar protein.

Global Analysis of Yeast Endosomal Transport Identifies the Vps55/68 Sorting Complex

PubMed Central

Schluter, Cayetana; Lam, Karen K.Y.; Brumm, Jochen; Wu, Bella W.; Saunders, Matthew; Stevens, Tom H.

2008-01-01

Endosomal transport is critical for cellular processes ranging from receptor down-regulation and retroviral budding to the immune response. A full understanding of endosome sorting requires a comprehensive picture of the multiprotein complexes that orchestrate vesicle formation and fusion. Here, we use unsupervised, large-scale phenotypic analysis and a novel computational approach for the global identification of endosomal transport factors. This technique effectively identifies components of known and novel protein assemblies. We report the characterization of a previously undescribed endosome sorting complex that contains two well-conserved proteins with four predicted membrane-spanning domains. Vps55p and Vps68p form a complex that acts with or downstream of ESCRT function to regulate endosomal trafficking. Loss of Vps68p disrupts recycling to the TGN as well as onward trafficking to the vacuole without preventing the formation of lumenal vesicles within the MVB. Our results suggest the Vps55/68 complex mediates a novel, conserved step in the endosomal maturation process. PMID:18216282

SCRATCHING THE SURFACE: Expectations of USAFRICOM

DTIC Science & Technology

2008-04-28

approaches. In response, the U.S. is taking a different approach with the formation of the United States Africa Command (USAFRICOM). USAFRICOM has been...undertaking that requires much more than narrow military focused approaches. In response, the U.S. is taking a different approach with the formation of...complex and intricate problems. "[Africa is a] continent characterized by a high degree of income inequality and is prone to conflict,,2 internally

From the Teachers' Eyes: An Ethnographic-Case Study on Developing Models of Informal Formative Assessments (IFA) and Understanding the Challenges to Effective Implementation in Science Classrooms

ERIC Educational Resources Information Center

Sezen, Asli

2011-01-01

The emphasis on socio-cultural theories of learning has required the understanding of multi-dimensional, dynamic and social nature of acquiring the scientific knowledge and practices. Recent policy documents suggest a focus on formative and dynamic assessment practices that will help understand and improve the complex nature of scientific learning…

Architecture and ssDNA interaction of the Timeless-Tipin-RPA complex

PubMed Central

Witosch, Justine; Wolf, Eva; Mizuno, Naoko

2014-01-01

The Timeless-Tipin (Tim-Tipin) complex, also referred to as the fork protection complex, is involved in coordination of DNA replication. Tim-Tipin is suggested to be recruited to replication forks via Replication Protein A (RPA) but details of the interaction are unknown. Here, using cryo-EM and biochemical methods, we characterized complex formation of Tim-Tipin, RPA and single-stranded DNA (ssDNA). Tim-Tipin and RPA form a 258 kDa complex with a 1:1:1 stoichiometry. The cryo-EM 3D reconstruction revealed a globular architecture of the Tim-Tipin-RPA complex with a ring-like and a U-shaped domain covered by a RPA lid. Interestingly, RPA in the complex adopts a horse shoe-like shape resembling its conformation in the presence of long ssDNA (>30 nucleotides). Furthermore, the recruitment of the Tim-Tipin-RPA complex to ssDNA is modulated by the RPA conformation and requires RPA to be in the more compact 30 nt ssDNA binding mode. The dynamic formation and disruption of the Tim-Tipin-RPA-ssDNA complex implicates the RPA-based recruitment of Tim-Tipin to the replication fork. PMID:25348395

Differential identity of Filopodia and Tunneling Nanotubes revealed by the opposite functions of actin regulatory complexes.

PubMed

Delage, Elise; Cervantes, Diégo Cordero; Pénard, Esthel; Schmitt, Christine; Syan, Sylvie; Disanza, Andrea; Scita, Giorgio; Zurzolo, Chiara

2016-12-23

Tunneling Nanotubes (TNTs) are actin enriched filopodia-like protrusions that play a pivotal role in long-range intercellular communication. Different pathogens use TNT-like structures as "freeways" to propagate across cells. TNTs are also implicated in cancer and neurodegenerative diseases, making them promising therapeutic targets. Understanding the mechanism of their formation, and their relation with filopodia is of fundamental importance to uncover their physiological function, particularly since filopodia, differently from TNTs, are not able to mediate transfer of cargo between distant cells. Here we studied different regulatory complexes of actin, which play a role in the formation of both these structures. We demonstrate that the filopodia-promoting CDC42/IRSp53/VASP network negatively regulates TNT formation and impairs TNT-mediated intercellular vesicle transfer. Conversely, elevation of Eps8, an actin regulatory protein that inhibits the extension of filopodia in neurons, increases TNT formation. Notably, Eps8-mediated TNT induction requires Eps8 bundling but not its capping activity. Thus, despite their structural similarities, filopodia and TNTs form through distinct molecular mechanisms. Our results further suggest that a switch in the molecular composition in common actin regulatory complexes is critical in driving the formation of either type of membrane protrusion.

Multiple roles for the Na,K-ATPase subunits, Atp1a1 and Fxyd1, during brain ventricle development

PubMed Central

Chang, Jessica T.; Lowery, Laura Anne; Sive, Hazel

2012-01-01

Formation of the vertebrate brain ventricles requires both production of cerebrospinal fluid (CSF), and its retention in the ventricles. The Na,K-ATPase is required for brain ventricle development, and we show here that this protein complex impacts three associated processes. The first requires both the alpha subunit (Atp1a1) and the regulatory subunit, Fxyd1, and leads to formation of a cohesive neuroepithelium, with continuous apical junctions. The second process leads to modulation of neuroepithelial permeability, and requires Atp1a1, which increases permeability with partial loss of function and decreases it with overexpression. In contrast, fxyd1 overexpression does not alter neuroepithelial permeability, suggesting that its activity is limited to neuroepithelium formation. RhoA regulates both neuroepithelium formation and permeability, downstream of the Na,K-ATPase. A third process, likely to be CSF production, is RhoA-independent, requiring Atp1a1, but not Fxyd1. Consistent with a role for Na,K-ATPase pump function, the inhibitor ouabain prevents neuroepithelium formation, while intracellular Na+ increases after Atp1a1 and Fxyd1 loss of function. These data include the first reported role for Fxyd1 in the developing brain, and indicate that the Na,K-ATPase regulates three aspects of brain ventricle development essential for normal function - formation of a cohesive neuroepithelium, restriction of neuroepithelial permeability, and production of CSF. PMID:22683378

Characterization of active reverse transcriptase and nucleoprotein complexes of the yeast retrotransposon Ty3 in vitro.

PubMed

Cristofari, G; Gabus, C; Ficheux, D; Bona, M; Le Grice, S F; Darlix, J L

1999-12-17

Human immunodeficiency virus (HIV) and the distantly related yeast Ty3 retrotransposon encode reverse transcriptase (RT) and a nucleic acid-binding protein designated nucleocapsid protein (NCp) with either one or two zinc fingers, required for HIV-1 replication and Ty3 transposition, respectively. In vitro binding of HIV-1 NCp7 to viral 5' RNA and primer tRNA(3)(Lys) catalyzes formation of nucleoprotein complexes resembling the virion nucleocapsid. Nucleocapsid complex formation functions in viral RNA dimerization and tRNA annealing to the primer binding site (PBS). RT is recruited in these nucleoprotein complexes and synthesizes minus-strand cDNA initiated at the PBS. Recent results on yeast Ty3 have shown that the homologous NCp9 promotes annealing of primer tRNA(i)(Met) to a 5'-3' bipartite PBS, allowing RNA:tRNA dimer formation and initiation of cDNA synthesis at the 5' PBS (). To compare specific cDNA synthesis in a retrotransposon and HIV-1, we have established a Ty3 model system comprising Ty3 RNA with the 5'-3' PBS, primer tRNA(i)(Met), NCp9, and for the first time, highly purified Ty3 RT. Here we report that Ty3 RT is as active as retroviral HIV-1 or murine leukemia virus RT using a synthetic template-primer system. Moreover, and in contrast to what was found with retroviral RTs, retrotransposon Ty3 RT was unable to direct cDNA synthesis by self-priming. We also show that Ty3 nucleoprotein complexes were formed in vitro and that the N terminus of NCp9, but not the zinc finger, is required for complex formation, tRNA annealing to the PBS, RNA dimerization, and primer tRNA-directed cDNA synthesis by Ty3 RT. These results indicate that NCp9 chaperones bona fide cDNA synthesis by RT in the yeast Ty3 retrotransposon, as illustrated for NCp7 in HIV-1, reinforcing the notion that Ty3 NCp9 is an ancestor of HIV-1 NCp7.

Candidate gene screen in the red flour beetle Tribolium reveals six3 as ancient regulator of anterior median head and central complex development.

PubMed

Posnien, Nico; Koniszewski, Nikolaus Dieter Bernhard; Hein, Hendrikje Jeannette; Bucher, Gregor

2011-12-01

Several highly conserved genes play a role in anterior neural plate patterning of vertebrates and in head and brain patterning of insects. However, head involution in Drosophila has impeded a systematic identification of genes required for insect head formation. Therefore, we use the red flour beetle Tribolium castaneum in order to comprehensively test the function of orthologs of vertebrate neural plate patterning genes for a function in insect head development. RNAi analysis reveals that most of these genes are indeed required for insect head capsule patterning, and we also identified several genes that had not been implicated in this process before. Furthermore, we show that Tc-six3/optix acts upstream of Tc-wingless, Tc-orthodenticle1, and Tc-eyeless to control anterior median development. Finally, we demonstrate that Tc-six3/optix is the first gene known to be required for the embryonic formation of the central complex, a midline-spanning brain part connected to the neuroendocrine pars intercerebralis. These functions are very likely conserved among bilaterians since vertebrate six3 is required for neuroendocrine and median brain development with certain mutations leading to holoprosencephaly.

Candidate Gene Screen in the Red Flour Beetle Tribolium Reveals Six3 as Ancient Regulator of Anterior Median Head and Central Complex Development

PubMed Central

Hein, Hendrikje Jeannette; Bucher, Gregor

2011-01-01

Several highly conserved genes play a role in anterior neural plate patterning of vertebrates and in head and brain patterning of insects. However, head involution in Drosophila has impeded a systematic identification of genes required for insect head formation. Therefore, we use the red flour beetle Tribolium castaneum in order to comprehensively test the function of orthologs of vertebrate neural plate patterning genes for a function in insect head development. RNAi analysis reveals that most of these genes are indeed required for insect head capsule patterning, and we also identified several genes that had not been implicated in this process before. Furthermore, we show that Tc-six3/optix acts upstream of Tc-wingless, Tc-orthodenticle1, and Tc-eyeless to control anterior median development. Finally, we demonstrate that Tc-six3/optix is the first gene known to be required for the embryonic formation of the central complex, a midline-spanning brain part connected to the neuroendocrine pars intercerebralis. These functions are very likely conserved among bilaterians since vertebrate six3 is required for neuroendocrine and median brain development with certain mutations leading to holoprosencephaly. PMID:22216011

Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly

PubMed Central

Didychuk, Allison L.; Montemayor, Eric J.; Brow, David A.; Butcher, Samuel E.

2016-01-01

Base-pairing of U4 and U6 snRNAs during di-snRNP assembly requires large-scale remodeling of RNA structure that is chaperoned by the U6 snRNP protein Prp24. We investigated the mechanism of U4/U6 annealing in vitro using an assay that enables visualization of ribonucleoprotein complexes and faithfully recapitulates known in vivo determinants for the process. We find that annealing, but not U6 RNA binding, is highly dependent on the electropositive character of a 20 Å-wide groove on the surface of Prp24. During annealing, we observe the formation of a stable ternary complex between U4 and U6 RNAs and Prp24, indicating that displacement of Prp24 in vivo requires additional factors. Mutations that stabilize the U6 ‘telestem’ helix increase annealing rates by up to 15-fold, suggesting that telestem formation is rate-limiting for U4/U6 pairing. The Lsm2–8 complex, which binds adjacent to the telestem at the 3′ end of U6, provides a comparable rate enhancement. Collectively, these data identify domains of the U6 snRNP that are critical for one of the first steps in assembly of the megaDalton U4/U6.U5 tri-snRNP complex, and lead to a dynamic model for U4/U6 pairing that involves a striking degree of evolved cooperativity between protein and RNA. PMID:26673715

SufD and SufC ATPase activity are required for iron acquisition during in vivo Fe-S cluster formation on SufB.

PubMed

Saini, Avneesh; Mapolelo, Daphne T; Chahal, Harsimranjit K; Johnson, Michael K; Outten, F Wayne

2010-11-02

In vivo biogenesis of Fe-S cluster cofactors requires complex biosynthetic machinery to limit release of iron and sulfide, to protect the Fe-S cluster from oxidation, and to target the Fe-S cluster to the correct apoenzyme. The SufABCDSE pathway for Fe-S cluster assembly in Escherichia coli accomplishes these tasks under iron starvation and oxidative stress conditions that disrupt Fe-S cluster metabolism. Although SufB, SufC, and SufD are all required for in vivo Suf function, their exact roles are unclear. Here we show that SufB, SufC, and SufD, coexpressed with the SufS-SufE sulfur transfer pair, purify as two distinct complexes (SufBC(2)D and SufB(2)C(2)) that contain Fe-S clusters and FADH(2). These studies also show that SufC and SufD are required for in vivo Fe-S cluster formation on SufB. Furthermore, while SufD is dispensable for in vivo sulfur transfer, it is absolutely required for in vivo iron acquisition. Finally, we demonstrate for the first time that the ATPase activity of SufC is necessary for in vivo iron acquisition during Fe-S cluster assembly.

Volumetric analysis of formation of the complex of G-quadruplex DNA with hemin using high pressure.

PubMed

Takahashi, Shuntaro; Bhowmik, Sudipta; Sugimoto, Naoki

2017-01-01

DNA guanine-quadruplexes (G-quadruplexes) complexed with the Fe-containing porphyrin, hemin (iron(III)-protoporphyrin IX), can catalyze oxidation reactions. This so-called DNAzyme has been widely used in the field of DNA nanotechnology. To improve DNAzyme properties, we sought to elucidate the interaction mechanism between G-quadruplex DNA and hemin. Here, we performed volumetric analyses of formation of the complex between an oligonucleotide with the sequence of human telomeric DNA (h-telo) and hemin. The G-quadruplex DNA alone and the G-quadruplex DNA-hemin complex were destabilized with increasing pressure in Na + buffer. The pressure required to destabilize the h-telo-hemin complex was less in K + -containing buffer than in buffer with Na + , which indicates that there was a smaller volumetric change upon h-telo formation in K + buffer than in Na + buffer. The calculated change in h-telo-hemin binding volume (∆V b ) in the Na + buffer was 2.5mLmol -1 , whereas it was -41.7 in mLmol -1 the K + buffer. The DNAzyme activity in the K + buffer was higher than that in the Na + buffer at atmospheric pressure. Interestingly, the pressure effect on the destabilization of the h-telo-hemin complex in the presence of poly(ethylene glycol)200 (PEG200) was repressed compared to that in the absence of PEG200. These results suggest that differences in volumetric parameters reflect different mechanisms of interaction between hemin and h-telo due to differences in both the fit of hemin into the h-telo structure and hydration. Thus, the pressure-based thermodynamic analysis provided important information about complex formation and could be a useful index to improve function of DNAzymes. Copyright © 2016 Elsevier Inc. All rights reserved.

Determinants for membrane association and permeabilization of the coxsackievirus 2B protein and the identification of the Golgi complex as the target organelle.

PubMed

de Jong, Arjan S; Wessels, Els; Dijkman, Henri B P M; Galama, Jochem M D; Melchers, Willem J G; Willems, Peter H G M; van Kuppeveld, Frank J M

2003-01-10

The 2B protein of enterovirus is responsible for the alterations in the permeability of secretory membranes and the plasma membrane in infected cells. The structural requirements for the membrane association and the subcellular localization of this essential virus protein, however, have not been defined. Here, we provide evidence that the 2B protein is an integral membrane protein in vivo that is predominantly localized at the Golgi complex upon individual expression. Addition of organelle-specific targeting signals to the 2B protein revealed that the Golgi localization is an absolute prerequisite for the ability of the protein to modify plasma membrane permeability. Expression of deletion mutants and heterologous proteins containing specific domains of the 2B protein demonstrated that each of the two hydrophobic regions could mediate membrane binding individually. However, the presence of both hydrophobic regions was required for the correct membrane association, efficient Golgi targeting, and the membrane-permeabilizing activity of the 2B protein, suggesting that the two hydrophobic regions are cooperatively involved in the formation of a membrane-integral complex. The formation of membrane-integral pores by the 2B protein in the Golgi complex and the possible mechanism by which a Golgi-localized virus protein modifies plasma membrane permeability are discussed.

File formats commonly used in mass spectrometry proteomics.

PubMed

Deutsch, Eric W

2012-12-01

The application of mass spectrometry (MS) to the analysis of proteomes has enabled the high-throughput identification and abundance measurement of hundreds to thousands of proteins per experiment. However, the formidable informatics challenge associated with analyzing MS data has required a wide variety of data file formats to encode the complex data types associated with MS workflows. These formats encompass the encoding of input instruction for instruments, output products of the instruments, and several levels of information and results used by and produced by the informatics analysis tools. A brief overview of the most common file formats in use today is presented here, along with a discussion of related topics.

The plant cell cycle: Pre-Replication complex formation and controls

PubMed Central

Brasil, Juliana Nogueira; Costa, Carinne N. Monteiro; Cabral, Luiz Mors; Ferreira, Paulo C. G.; Hemerly, Adriana S.

2017-01-01

Abstract The multiplication of cells in all living organisms requires a tight regulation of DNA replication. Several mechanisms take place to ensure that the DNA is replicated faithfully and just once per cell cycle in order to originate through mitoses two new daughter cells that contain exactly the same information from the previous one. A key control mechanism that occurs before cells enter S phase is the formation of a pre-replication complex (pre-RC) that is assembled at replication origins by the sequential association of the origin recognition complex, followed by Cdt1, Cdc6 and finally MCMs, licensing DNA to start replication. The identification of pre-RC members in all animal and plant species shows that this complex is conserved in eukaryotes and, more importantly, the differences between kingdoms might reflect their divergence in strategies on cell cycle regulation, as it must be integrated and adapted to the niche, ecosystem, and the organism peculiarities. Here, we provide an overview of the knowledge generated so far on the formation and the developmental controls of the pre-RC mechanism in plants, analyzing some particular aspects in comparison to other eukaryotes. PMID:28304073

S100A13-C2A binary complex structure-a key component in the acidic fibroblast growth factor for the non-classical pathway

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

Mohan, Sepuru K.; Rani, Sandhya G.; Kumar, Sriramoju M.

2009-03-13

Fibroblast growth factors (FGFs) are key regulators of cell proliferation, differentiation, tumor-induced angiogenesis and migration. FGFs are essential for early embryonic development, organ formation and angiogenesis. They play important roles in tumor formation, inflammation, wound healing and restenosis. The biological effects of FGFs are mediated through the activation of the four transmembrane phosphotyrosine kinase receptors (FGFRs) in the presence of heparin sulfate proteoglycans (HSPGs) and therefore require the release of FGFs into the extracellular space. However, FGF-1 lacks the signal peptide required for the releasing of these proteins through the classical endoplasmic reticulum (ER)-Golgi secretary pathway. Maciag et al. demonstratedmore » that FGF-1 is exported through a non-classical release pathway involving the formation of a specific multiprotein complex [M. Landriscina, R. Soldi, C. Bagala, I. Micucci, S. Bellum, F. Tarantini, I. Prudovsky, T. Maciag, S100A13 participates in the release of fibroblast growth factor 1 in response to heat shock in vitro, J. Biol. Chem. 276 (2001) 22544-22552; C.M. Carreira, T.M. LaVallee, F. Tarantini, A. Jackson, J.T. Lathrop, B. Hampton, W.H. Burgess, T. Maciag, S100A13 is involved in the regulation of fibroblast growth factor-1 and p40 synaptotagmin-1 release in vitro, J. Biol. Chem. 273 (1998) 22224-22231; T.M. LaValle, F. Tarantini, S. Gamble, C.M. Carreira, A. Jackson, T. Maciag, Synaptotagmin-1 is required for fibroblast growth factor-1 release, J. Biol. Chem. 273 (1998) 22217-22223; C. Bagala, V. Kolev, A. Mandinova, R. Soldi, C. Mouta, I. Graziani, I, Prudovsky, T. Maciag, The alternative translation of synaptotagmin 1 mediates the non-classical release of FGF1, Biochem. Biophys. Res. Commun. 310 (2003) 1041-1047]. The protein constituents of this complex include FGF-1, S100A13 (a Ca{sup 2+}-binding protein), and the p40 form of synaptotagmin 1 (Syt1). To understand the molecular events in the FGF-1 releasing pathway, we have studied the interactions of S100A13 with C2A by {sup 1}H-{sup 15}N HSQC titration and 3D-filtered NOESY experiments. We characterized the binary complex structure of S100A13-C2A by using a variety of multi-dimensional NMR experiments. This complex acts as a template for FGF-1 dimerization and multiprotein complex formation.« less

Complexation of Contaminants and Aqueous-Phase Ozone with Cyclodextrin for Emerging Contaminant Oxidative Degradation

NASA Astrophysics Data System (ADS)

Khan, N. A.; Carroll, K. C.

2016-12-01

Recalcitrant emerging contaminants in groundwater, such as 1,4-dioxane, require strong oxidants for complete mineralization, whereas strong oxidant efficacy for in-situ chemical oxidation (ISCO) is limited by oxidant decay, reactivity, and non-specificity. Hydroxypropyl-β-cyclodextrin (HPβCD) was examined for its ability to stabilize aqueous phase ozone (O3) and prolong oxidation potential through inclusion complex formation. Partial transformation of HPβCD by O3 was observed but HPβCD proved to be sufficiently resilient and only partially degraded in the presence of O3. The formation of a HPβCD:O3 inclusion clathrate complex was observed, and multiple methods for binding constant measurements carried out and compared for HPβCD complexes with O3 and multiple contaminants. The presence of HPβCD increased the O3 half-life linearly with increasing HPβCD:O3 molar ratio. The O3 half-life in solutions increased by as much as 40-fold relative to HPβCD-free O3 solutions, and complexation reversibility was confirmed. Decay rate coefficients increased for 1,4-dioxane, trichloroethene, and trichloroethane likely due to the formation of HPβCD-O3-contaminant ternary complexes. These results suggest that the use of clathrate stabilizers, such as HPβCD, can support the development of a facilitated-transport enabled ISCO for the O3 treatment of groundwater impacted by recalcitrant emerging contaminants.

Formation and dissociation of proteasome storage granules are regulated by cytosolic pH.

PubMed

Peters, Lee Zeev; Hazan, Rotem; Breker, Michal; Schuldiner, Maya; Ben-Aroya, Shay

2013-05-27

The 26S proteasome is the major protein degradation machinery of the cell and is regulated at many levels. One mode of regulation involves accumulation of proteasomes in proteasome storage granules (PSGs) upon glucose depletion. Using a systematic robotic screening approach in yeast, we identify trans-acting proteins that regulate the accumulation of proteasomes in PSGs. Our dataset was enriched for subunits of the vacuolar adenosine triphosphatase (V-ATPase) complex, a proton pump required for vacuole acidification. We show that the impaired ability of V-ATPase mutants to properly govern intracellular pH affects the kinetics of PSG formation. We further show that formation of other protein aggregates upon carbon depletion also is triggered in mutants with impaired activity of the plasma membrane proton pump and the V-ATPase complex. We thus identify cytosolic pH as a specific cellular signal involved both in the glucose sensing that mediates PSG formation and in a more general mechanism for signaling carbon source exhaustion.

Deformational history of part of the Acatlán Complex: Late Ordovician Early Silurian and Early Permian orogenesis in southern Mexico

NASA Astrophysics Data System (ADS)

Malone, J. R.; Nance, R. D.; Keppie, J. D.; Dostal, J.

2002-10-01

The Paleozoic Acatlán Complex of southern Mexico comprises polydeformed metasedimentary, granitoid, and mafic-ultramafic rocks variously interpreted as recording the closure of the Iapetus, Rheic, and Ouachitan Oceans. The complex is tectonically juxtaposed on its eastern margin against Grenville-age gneisses (Oaxacan Complex) that are unconformably overlain by Lower Paleozoic strata containing fossils of Gondwanan affinity. A thick siliciclastic unit (Chazumba and Cosoltepec Formations) at the base of the complex is considered part of a Lower Paleozoic accretionary prism with a provenance that isotopically resembles the Oaxacan Complex. This unit is tectonically overridden by a locally eclogitic mafic-ultramafic unit interpreted as a westward-obducted ophiolite, the emplacement of which was synchronous with mylonitic granitoid intrusion at ca. 440 Ma. Both units are unconformably overlain by a deformed volcano-sedimentary sequence (Tecomate Formation) attributed to a volcanic arc of presumed Devonian age. Deformed granitoids in contact with this sequence have been dated at ca. 371 (La Noria granite) and 287 Ma (Totoltepec pluton). Three phases of penetrative deformation (D 1-3) affect the Cosoltepec Formation; the last two correlate with two penetrative deformational phases that affect the Tecomate Formation. D 1 is of unknown kinematics but predates deposition of the Tecomate Formation and likely records obduction at ca. 440 Ma (Acatecan orogeny). A folded foliation in the Totoltepec pluton appears to record both deformational phases in the Tecomate Formation, bracketing D 2 and D 3 between 287 Ma and the deposition of the nonconformably overlying Leonardian Matzitzi Formation. D 2 records north-south dextral transpression and south-vergent thrusting and is attributed to the collision of Gondwana and southern Laurentia (Ouachitan orogeny) at ca. 290 Ma, the kinematics being consistent with the northward motion of Mexico that is required by most continental reconstructions for the final assembly of Pangea. D 3, which produced broadly north-south, upright folds, is also attributed to this collision and likely followed D 2 closely in the latest Paleozoic.

Integrating a geographic information system, a scientific visualization system and an orographic precipitation model

USGS Publications Warehouse

Hay, L.; Knapp, L.

1996-01-01

Investigating natural, potential, and man-induced impacts on hydrological systems commonly requires complex modelling with overlapping data requirements, and massive amounts of one- to four-dimensional data at multiple scales and formats. Given the complexity of most hydrological studies, the requisite software infrastructure must incorporate many components including simulation modelling, spatial analysis and flexible, intuitive displays. There is a general requirement for a set of capabilities to support scientific analysis which, at this time, can only come from an integration of several software components. Integration of geographic information systems (GISs) and scientific visualization systems (SVSs) is a powerful technique for developing and analysing complex models. This paper describes the integration of an orographic precipitation model, a GIS and a SVS. The combination of these individual components provides a robust infrastructure which allows the scientist to work with the full dimensionality of the data and to examine the data in a more intuitive manner.

A divalent interaction between HPS1 and HPS4 is required for the formation of the biogenesis of lysosome-related organelle complex-3 (BLOC-3).

PubMed

Carmona-Rivera, Carmelo; Simeonov, Dimitre R; Cardillo, Nicholas D; Gahl, William A; Cadilla, Carmen L

2013-03-01

Hermansky-Pudlak syndrome (HPS) is a group of rare autosomal recessive disorders characterized by oculocutaneous albinism, a bleeding tendency, and sporadic pulmonary fibrosis, granulomatous colitis or infections. Nine HPS-causing genes have been identified in humans. HPS-1 is the most severe subtype with a prevalence of ~1/1800 in northwest Puerto Rico due to a founder mutation in the HPS1 gene. Mutations in HPS genes affect the biogenesis of lysosome-related organelles such as melanosomes in melanocytes and platelet dense granules. Two of these genes (HPS1 and HPS4) encode the HPS1 and HPS4 proteins, which assemble to form a complex known as Biogenesis of Lysosome-related Organelle Complex 3 (BLOC-3). We report the identification of the interacting regions in HPS1 and HPS4 required for the formation of this complex. Two regions in HPS1, spanning amino acids 1-249 and 506-700 are required for binding to HPS4; the middle portion of HPS1 (residues 250-505) is not required for this interaction. Further interaction studies showed that the N-termini of HPS1 and HPS4 interact with each other and that a discrete region of HPS4 (residues 340-528) interacts with both the N- and C-termini of the HPS1 protein. Several missense mutations found in HPS-1 patients did not affect interaction with HPS4, but some mutations involving regions interacting with HPS4 caused instability of HPS1. These observations extend our understanding of BLOC-3 assembly and represent an important first step in the identification of domains responsible for the biogenesis of lysosome-related organelles. Copyright © 2012 Elsevier B.V. All rights reserved.

Stomatin-Like Protein 2 Is Required for In Vivo Mitochondrial Respiratory Chain Supercomplex Formation and Optimal Cell Function

PubMed Central

Mitsopoulos, Panagiotis; Chang, Yu-Han; Wai, Timothy; König, Tim; Dunn, Stanley D.; Langer, Thomas

2015-01-01

Stomatin-like protein 2 (SLP-2) is a mainly mitochondrial protein that is widely expressed and is highly conserved across evolution. We have previously shown that SLP-2 binds the mitochondrial lipid cardiolipin and interacts with prohibitin-1 and -2 to form specialized membrane microdomains in the mitochondrial inner membrane, which are associated with optimal mitochondrial respiration. To determine how SLP-2 functions, we performed bioenergetic analysis of primary T cells from T cell-selective Slp-2 knockout mice under conditions that forced energy production to come almost exclusively from oxidative phosphorylation. These cells had a phenotype characterized by increased uncoupled mitochondrial respiration and decreased mitochondrial membrane potential. Since formation of mitochondrial respiratory chain supercomplexes (RCS) may correlate with more efficient electron transfer during oxidative phosphorylation, we hypothesized that the defect in mitochondrial respiration in SLP-2-deficient T cells was due to deficient RCS formation. We found that in the absence of SLP-2, T cells had decreased levels and activities of complex I-III2 and I-III2-IV1-3 RCS but no defects in assembly of individual respiratory complexes. Impaired RCS formation in SLP-2-deficient T cells correlated with significantly delayed T cell proliferation in response to activation under conditions of limiting glycolysis. Altogether, our findings identify SLP-2 as a key regulator of the formation of RCS in vivo and show that these supercomplexes are required for optimal cell function. PMID:25776552

The role of hybrid ubiquitin chains in the MyD88 and other innate immune signalling pathways.

PubMed

Cohen, Philip; Strickson, Sam

2017-07-01

The adaptor protein MyD88 is required for signal transmission by toll-like receptors and receptors of the interleukin-1 family of cytokines. MyD88 signalling triggers the formation of Lys63-linked and Met1-linked ubiquitin (K63-Ub, M1-Ub) chains within minutes. The K63-Ub chains, which are formed by the E3 ubiquitin ligases TRAF6, Pellino1 and Pellino2, activate TAK1, the master kinase that switches on mitogen-activated protein (MAP) kinase cascades and initiates activation of the canonical IκB kinase (IKK) complex. The M1-Ub chains, which are formed by the linear ubiquitin chain assembly complex (LUBAC), bind to the NEMO (NF-κB essential modulator) component of the IKK complex and are required for TAK1 to activate IKKs, but not MAP kinases. An essential E3 ligase-independent role of TRAF6 is to recruit LUBAC into the MyD88 signalling complex, where it recognises preformed K63-Ub chains attached to protein components of these complexes, such as IRAK1 (IL-1 receptor-associated kinase), producing ubiquitin chains containing both types of linkage, termed K63/M1-Ub hybrids. The formation of K63/M1-Ub hybrids, which is a feature of several innate immune signalling pathways, permits the co-recruitment of proteins that interact with either K63-Ub or M1-Ub chains. Two likely roles for K63/M1-Ub hybrids are to facilitate the TAK1-dependent activation of the IKK complex and to prevent the hyperactivation of these kinases by recruiting A20 and A20-binding inhibitor of NF-κB1 (ABIN1). These proteins restrict activation of the TAK1 and IKK complexes, probably by competing with them for binding to K63/M1-Ub hybrids. The formation of K63/M1-Ub hybrids may also regulate the rate at which the ubiquitin linkages in these chains are hydrolysed. The IKK-catalysed phosphorylation of some of its substrates permits their recognition by the E3 ligase SCF βTRCP , leading to their Lys48-linked ubiquitylation and proteasomal degradation. Innate immune signalling is therefore controlled by the formation and destruction of three different types of ubiquitin linkage.

The role of hybrid ubiquitin chains in the MyD88 and other innate immune signalling pathways

PubMed Central

Cohen, Philip; Strickson, Sam

2017-01-01

The adaptor protein MyD88 is required for signal transmission by toll-like receptors and receptors of the interleukin-1 family of cytokines. MyD88 signalling triggers the formation of Lys63-linked and Met1-linked ubiquitin (K63-Ub, M1-Ub) chains within minutes. The K63-Ub chains, which are formed by the E3 ubiquitin ligases TRAF6, Pellino1 and Pellino2, activate TAK1, the master kinase that switches on mitogen-activated protein (MAP) kinase cascades and initiates activation of the canonical IκB kinase (IKK) complex. The M1-Ub chains, which are formed by the linear ubiquitin chain assembly complex (LUBAC), bind to the NEMO (NF-κB essential modulator) component of the IKK complex and are required for TAK1 to activate IKKs, but not MAP kinases. An essential E3 ligase-independent role of TRAF6 is to recruit LUBAC into the MyD88 signalling complex, where it recognises preformed K63-Ub chains attached to protein components of these complexes, such as IRAK1 (IL-1 receptor-associated kinase), producing ubiquitin chains containing both types of linkage, termed K63/M1-Ub hybrids. The formation of K63/M1-Ub hybrids, which is a feature of several innate immune signalling pathways, permits the co-recruitment of proteins that interact with either K63-Ub or M1-Ub chains. Two likely roles for K63/M1-Ub hybrids are to facilitate the TAK1-dependent activation of the IKK complex and to prevent the hyperactivation of these kinases by recruiting A20 and A20-binding inhibitor of NF-κB1 (ABIN1). These proteins restrict activation of the TAK1 and IKK complexes, probably by competing with them for binding to K63/M1-Ub hybrids. The formation of K63/M1-Ub hybrids may also regulate the rate at which the ubiquitin linkages in these chains are hydrolysed. The IKK-catalysed phosphorylation of some of its substrates permits their recognition by the E3 ligase SCFβTRCP, leading to their Lys48-linked ubiquitylation and proteasomal degradation. Innate immune signalling is therefore controlled by the formation and destruction of three different types of ubiquitin linkage. PMID:28475177

Phosphoinositide Signaling Regulates the Exocyst Complex and Polarized Integrin Trafficking in Directionally Migrating Cells

PubMed Central

Thapa, Narendra; Sun, Yue; Schramp, Mark; Choi, Suyoung; Ling, Kun; Anderson, Richard A

2011-01-01

Summary Polarized delivery of signaling and adhesion molecules to the leading edge is required for directional migration of cells. Here, we describe a role for the PIP2 synthesizing enzyme, PIPKIγi2, in regulation of exocyst complex control of cell polarity and polarized integrin trafficking during migration. Loss of PIPKIγi2 impaired directional migration, formation of cell polarity, and integrin trafficking to the leading edge. Upon initiation of directional migration PIPKIγi2 via PIP2 generation controls the integration of the exocyst complex into an integrin-containing trafficking compartment(s) that requires the talin-binding ability of PIPKIγi2, and talin for integrin recruitment to the leading edge. A PIP2 requirement is further emphasized by inhibition of PIPKIγi2-regulated directional migration by an Exo70 mutant deficient in PIP2 binding. These results reveal how phosphoinositide generation orchestrates polarized trafficking of integrin in coordination with talin that links integrins to the actin cytoskeleton, processes that are required for directional migration. PMID:22264730

Distinguishing Grenvillian basement from pre-Taconian cover rocks in the Northern Appalachians

USGS Publications Warehouse

Karabinos, P.; Aleinikoff, J.N.; Fanning, C.M.

1999-01-01

Distinguishing Grenvillian basement rocks from pre-Taconian cover sequences in the Appalachians is a first-order problem essential for accurate structural interpretations. The Cavendish Formation in southeastern Vermont presents a classic example of this problem. Doll and others (1961) showed the Cavendish Formation as younger than the Middle Proterozoic Mount Holly Complex but older than the lithologically similar Cambrian Tyson and Hoosac Formations. More recently, the name Cavendish Formation has been informally abandoned, and its metasedimentary units have been mapped as the Tyson and Hoosac Formations of Late Proterozoic to Cambrian age. In a radical departure from these interpretations, Ratcliffe and others (1997) reassigned metasedimentary rocks of the Cavendish Formation to the Mount Holly Complex based on an inferred intrusive relationship between them and a 1.42 Ga tonalite. This new age assignment, if correct, requires a completely new structural interpretation of the region. SHRIMP and Pb evaporation ages of detrital zircons extracted from a quartzite layer from Cavendish Gorge near the proposed intrusive contact with the tonalite constrain the time of deposition of the Cavendish Formation. Grain shapes of the zircons vary from euhedral to nearly spherical. Virtually all the grains have pitted surfaces and show at least some rounding of edges and terminations; grains exhibit oscillatory zoning typical of zircons that crystallized from a magma. Single-grain Pb evaporation analyses of ten zircons and SHRIMP analyses of 15 zircons all yield ages less than 1.42 Ga. Seven of the grains are consistent with derivation from the Bull Hill Gneiss that postdates the Grenville orogenic cycle and predates deposition of the Cavendish Formation. Thus, the metasedimentary units of the Cavendish Formation should not be assigned to the Mount Holly Complex.

ISO 19115 Experiences in NASA's Earth Observing System (EOS) ClearingHOuse (ECHO)

NASA Astrophysics Data System (ADS)

Cechini, M. F.; Mitchell, A.

2011-12-01

Metadata is an important entity in the process of cataloging, discovering, and describing earth science data. As science research and the gathered data increases in complexity, so does the complexity and importance of descriptive metadata. To meet these growing needs, the metadata models required utilize richer and more mature metadata attributes. Categorizing, standardizing, and promulgating these metadata models to a politically, geographically, and scientifically diverse community is a difficult process. An integral component of metadata management within NASA's Earth Observing System Data and Information System (EOSDIS) is the Earth Observing System (EOS) ClearingHOuse (ECHO). ECHO is the core metadata repository for the EOSDIS data centers providing a centralized mechanism for metadata and data discovery and retrieval. ECHO has undertaken an internal restructuring to meet the changing needs of scientists, the consistent advancement in technology, and the advent of new standards such as ISO 19115. These improvements were based on the following tenets for data discovery and retrieval: + There exists a set of 'core' metadata fields recommended for data discovery. + There exists a set of users who will require the entire metadata record for advanced analysis. + There exists a set of users who will require a 'core' set metadata fields for discovery only. + There will never be a cessation of new formats or a total retirement of all old formats. + Users should be presented metadata in a consistent format of their choosing. In order to address the previously listed items, ECHO's new metadata processing paradigm utilizes the following approach: + Identify a cross-format set of 'core' metadata fields necessary for discovery. + Implement format-specific indexers to extract the 'core' metadata fields into an optimized query capability. + Archive the original metadata in its entirety for presentation to users requiring the full record. + Provide on-demand translation of 'core' metadata to any supported result format. Lessons learned by the ECHO team while implementing its new metadata approach to support usage of the ISO 19115 standard will be presented. These lessons learned highlight some discovered strengths and weaknesses in the ISO 19115 standard as it is introduced to an existing metadata processing system.

Polycomb-Mediated Repression and Sonic Hedgehog Signaling Interact to Regulate Merkel Cell Specification during Skin Development

PubMed Central

Bar, Carmit; Tsai, Pai-Chi; Valdes, Victor J.; Cohen, Idan; Santoriello, Francis J.; Zhao, Dejian; Hsu, Ya-Chieh; Ezhkova, Elena

2016-01-01

An increasing amount of evidence indicates that developmental programs are tightly regulated by the complex interplay between signaling pathways, as well as transcriptional and epigenetic processes. Here, we have uncovered coordination between transcriptional and morphogen cues to specify Merkel cells, poorly understood skin cells that mediate light touch sensations. In murine dorsal skin, Merkel cells are part of touch domes, which are skin structures consisting of specialized keratinocytes, Merkel cells, and afferent neurons, and are located exclusively around primary hair follicles. We show that the developing primary hair follicle functions as a niche required for Merkel cell specification. We find that intraepidermal Sonic hedgehog (Shh) signaling, initiated by the production of Shh ligand in the developing hair follicles, is required for Merkel cell specification. The importance of Shh for Merkel cell formation is further reinforced by the fact that Shh overexpression in embryonic epidermal progenitors leads to ectopic Merkel cells. Interestingly, Shh signaling is common to primary, secondary, and tertiary hair follicles, raising the possibility that there are restrictive mechanisms that regulate Merkel cell specification exclusively around primary hair follicles. Indeed, we find that loss of Polycomb repressive complex 2 (PRC2) in the epidermis results in the formation of ectopic Merkel cells that are associated with all hair types. We show that PRC2 loss expands the field of epidermal cells competent to differentiate into Merkel cells through the upregulation of key Merkel-differentiation genes, which are known PRC2 targets. Importantly, PRC2-mediated repression of the Merkel cell differentiation program requires inductive Shh signaling to form mature Merkel cells. Our study exemplifies how the interplay between epigenetic and morphogen cues regulates the complex patterning and formation of the mammalian skin structures. PMID:27414999

Polycomb-Mediated Repression and Sonic Hedgehog Signaling Interact to Regulate Merkel Cell Specification during Skin Development.

PubMed

Perdigoto, Carolina N; Dauber, Katherine L; Bar, Carmit; Tsai, Pai-Chi; Valdes, Victor J; Cohen, Idan; Santoriello, Francis J; Zhao, Dejian; Zheng, Deyou; Hsu, Ya-Chieh; Ezhkova, Elena

2016-07-01

An increasing amount of evidence indicates that developmental programs are tightly regulated by the complex interplay between signaling pathways, as well as transcriptional and epigenetic processes. Here, we have uncovered coordination between transcriptional and morphogen cues to specify Merkel cells, poorly understood skin cells that mediate light touch sensations. In murine dorsal skin, Merkel cells are part of touch domes, which are skin structures consisting of specialized keratinocytes, Merkel cells, and afferent neurons, and are located exclusively around primary hair follicles. We show that the developing primary hair follicle functions as a niche required for Merkel cell specification. We find that intraepidermal Sonic hedgehog (Shh) signaling, initiated by the production of Shh ligand in the developing hair follicles, is required for Merkel cell specification. The importance of Shh for Merkel cell formation is further reinforced by the fact that Shh overexpression in embryonic epidermal progenitors leads to ectopic Merkel cells. Interestingly, Shh signaling is common to primary, secondary, and tertiary hair follicles, raising the possibility that there are restrictive mechanisms that regulate Merkel cell specification exclusively around primary hair follicles. Indeed, we find that loss of Polycomb repressive complex 2 (PRC2) in the epidermis results in the formation of ectopic Merkel cells that are associated with all hair types. We show that PRC2 loss expands the field of epidermal cells competent to differentiate into Merkel cells through the upregulation of key Merkel-differentiation genes, which are known PRC2 targets. Importantly, PRC2-mediated repression of the Merkel cell differentiation program requires inductive Shh signaling to form mature Merkel cells. Our study exemplifies how the interplay between epigenetic and morphogen cues regulates the complex patterning and formation of the mammalian skin structures.

Helical assembly in the MyD88-IRAK4-IRAK2 complex in TLR/IL-1R signalling

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

Lin, Su-Chang; Lo, Yu-Chih; Wu, Hao

2010-08-23

MyD88, IRAK4 and IRAK2 are critical signalling mediators of the TLR/IL1-R superfamily. Here we report the crystal structure of the MyD88-IRAK4-IRAK2 death domain (DD) complex, which surprisingly reveals a left-handed helical oligomer that consists of 6 MyD88, 4 IRAK4 and 4 IRAK2 DDs. Assembly of this helical signalling tower is hierarchical, in which MyD88 recruits IRAK4 and the MyD88-IRAK4 complex recruits the IRAK4 substrates IRAK2 or the related IRAK1. Formation of these Myddosome complexes brings the kinase domains of IRAKs into proximity for phosphorylation and activation. Composite binding sites are required for recruitment of the individual DDs in the complex,more » which are confirmed by mutagenesis and previously identified signalling mutations. Specificities in Myddosome formation are dictated by both molecular complementarity and correspondence of surface electrostatics. The MyD88-IRAK4-IRAK2 complex provides a template for Toll signalling in Drosophila and an elegant mechanism for versatile assembly and regulation of DD complexes in signal transduction.« less

Roles of the Nuclear Lamina in Stable Nuclear Association and Assembly of a Herpesviral Transactivator Complex on Viral Immediate-Early Genes

PubMed Central

Silva, Lindsey; Oh, Hyung Suk; Chang, Lynne; Yan, Zhipeng; Triezenberg, Steven J.; Knipe, David M.

2012-01-01

ABSTRACT Little is known about the mechanisms of gene targeting within the nucleus and its effect on gene expression, but most studies have concluded that genes located near the nuclear periphery are silenced by heterochromatin. In contrast, we found that early herpes simplex virus (HSV) genome complexes localize near the nuclear lamina and that this localization is associated with reduced heterochromatin on the viral genome and increased viral immediate-early (IE) gene transcription. In this study, we examined the mechanism of this effect and found that input virion transactivator protein, virion protein 16 (VP16), targets sites adjacent to the nuclear lamina and is required for targeting of the HSV genome to the nuclear lamina, exclusion of heterochromatin from viral replication compartments, and reduction of heterochromatin on the viral genome. Because cells infected with the VP16 mutant virus in1814 showed a phenotype similar to that of lamin A/C−/− cells infected with wild-type virus, we hypothesized that the nuclear lamina is required for VP16 activator complex formation. In lamin A/C−/− mouse embryo fibroblasts, VP16 and Oct-1 showed reduced association with the viral IE gene promoters, the levels of VP16 and HCF-1 stably associated with the nucleus were lower than in wild-type cells, and the association of VP16 with HCF-1 was also greatly reduced. These results show that the nuclear lamina is required for stable nuclear localization and formation of the VP16 activator complex and provide evidence for the nuclear lamina being the site of assembly of the VP16 activator complex. PMID:22251972

Inhibitory effect of phosphates on magnesium lactate efflorescence formation in dry-fermented sausages.

PubMed

Walz, Felix H; Gibis, Monika; Schrey, Pia; Herrmann, Kurt; Reichert, Corina L; Hinrichs, Jörg; Weiss, Jochen

2017-10-01

This study aimed to prevent the phenomena of efflorescence formation on the surface of dry fermented sausages due to the complexation of efflorescence forming cations with phosphates. Efflorescence formation is a critical issue constituting a major quality defect, especially of dry fermented sausages. Different phosphates (di- and hexametaphosphate) were added (3.0g/kg) to the sausage batter. As a hypothesis, these additives should complex with one of the main efflorescence-causing substances such as magnesium. The formation of efflorescences was determined for dry fermented sausages without phosphate addition, with diphosphate, or hexametaphosphate addition during 8weeks of storage under modified atmosphere. The visual analyses of the sausage surface revealed high amounts of efflorescences for the control (42.2%) and for the sausages with added diphosphate (40.9%), whereas the sausages containing hexametaphosphate had significantly reduced amounts of efflorescence formation, showing only 11.9% efflorescences after 8weeks of storage. This inhibition was a result of strong complexation of hexametaphosphate with magnesium ions, thus preventing the diffusion of magnesium towards the sausage surface. This can be explained by the magnesium content on the sausage surface that increased by 163.9, 127.8, and 52.8% for the sausages without phosphate, diphosphate, and hexametaphosphate addition, respectively. The mass transport of lactate and creatine was not affected by phosphate addition. Isothermal titration calorimetry confirmed that, theoretically, 4.5g/kg of diphosphate or 2.8g/kg hexametaphosphate are required to complex 0.2g/kg magnesium ions naturally occurring in dry fermented sausages and, thus, the chosen overall phosphate concentration of 3.0g/kg was enough when adding hexametaphosphate, but not for diphosphate, to inhibit the efflorescence formation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Ballbot-type motion of N-heterocyclic carbenes on gold surfaces

NASA Astrophysics Data System (ADS)

Wang, Gaoqiang; Rühling, Andreas; Amirjalayer, Saeed; Knor, Marek; Ernst, Johannes Bruno; Richter, Christian; Gao, Hong-Jun; Timmer, Alexander; Gao, Hong-Ying; Doltsinis, Nikos L.; Glorius, Frank; Fuchs, Harald

2017-02-01

Recently, N-heterocyclic carbenes (NHCs) were introduced as alternative anchors for surface modifications and so offered many attractive features, which might render them superior to thiol-based systems. However, little effort has been made to investigate the self-organization process of NHCs on surfaces, an important aspect for the formation of self-assembled monolayers (SAMs), which requires molecular mobility. Based on investigations with scanning tunnelling microscopy and first-principles calculations, we provide an understanding of the microscopic mechanism behind the high mobility observed for NHCs. These NHCs extract a gold atom from the surface, which leads to the formation of an NHC-gold adatom complex that displays a high surface mobility by a ballbot-type motion. Together with their high desorption barrier this enables the formation of ordered and strongly bound SAMs. In addition, this mechanism allows a complementary surface-assisted synthesis of dimeric and hitherto unknown trimeric NHC gold complexes on the surface.

Ballbot-type motion of N-heterocyclic carbenes on gold surfaces.

PubMed

Wang, Gaoqiang; Rühling, Andreas; Amirjalayer, Saeed; Knor, Marek; Ernst, Johannes Bruno; Richter, Christian; Gao, Hong-Jun; Timmer, Alexander; Gao, Hong-Ying; Doltsinis, Nikos L; Glorius, Frank; Fuchs, Harald

2017-02-01

Recently, N-heterocyclic carbenes (NHCs) were introduced as alternative anchors for surface modifications and so offered many attractive features, which might render them superior to thiol-based systems. However, little effort has been made to investigate the self-organization process of NHCs on surfaces, an important aspect for the formation of self-assembled monolayers (SAMs), which requires molecular mobility. Based on investigations with scanning tunnelling microscopy and first-principles calculations, we provide an understanding of the microscopic mechanism behind the high mobility observed for NHCs. These NHCs extract a gold atom from the surface, which leads to the formation of an NHC-gold adatom complex that displays a high surface mobility by a ballbot-type motion. Together with their high desorption barrier this enables the formation of ordered and strongly bound SAMs. In addition, this mechanism allows a complementary surface-assisted synthesis of dimeric and hitherto unknown trimeric NHC gold complexes on the surface.

Analysis of interstellar cloud structure based on IRAS images

NASA Technical Reports Server (NTRS)

Scalo, John M.

1992-01-01

The goal of this project was to develop new tools for the analysis of the structure of densely sampled maps of interstellar star-forming regions. A particular emphasis was on the recognition and characterization of nested hierarchical structure and fractal irregularity, and their relation to the level of star formation activity. The panoramic IRAS images provided data with the required range in spatial scale, greater than a factor of 100, and in column density, greater than a factor of 50. In order to construct densely sampled column density maps of star-forming clouds, column density images of four nearby cloud complexes were constructed from IRAS data. The regions have various degrees of star formation activity, and most of them have probably not been affected much by the disruptive effects of young massive stars. The largest region, the Scorpius-Ophiuchus cloud complex, covers about 1000 square degrees (it was subdivided into a few smaller regions for analysis). Much of the work during the early part of the project focused on an 80 square degree region in the core of the Taurus complex, a well-studied region of low-mass star formation.

Nucleoprotein of influenza B virus binds to its type A counterpart and disrupts influenza A viral polymerase complex formation

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

Jaru-ampornpan, Peera, E-mail: peera.jar@biotec.or.th; Narkpuk, Jaraspim; Wanitchang, Asawin

Highlights: •FluB nucleoprotein (BNP) can bind to FluA nucleoprotein (ANP). •BNP–ANP interaction inhibits FluA polymerase activity. •BNP binding prevents ANP from forming a functional FluA polymerase complex. •Nuclear localization of BNP is necessary for FluA polymerase inhibition. •Viral RNA is not required for the BNP–ANP interaction. -- Abstract: Upon co-infection with influenza B virus (FluB), influenza A virus (FluA) replication is substantially impaired. Previously, we have shown that the nucleoprotein of FluB (BNP) can inhibit FluA polymerase machinery, retarding the growth of FluA. However, the molecular mechanism underlying this inhibitory action awaited further investigation. Here, we provide evidence that BNPmore » hinders the proper formation of FluA polymerase complex by competitively binding to the nucleoprotein of FluA. To exert this inhibitory effect, BNP must be localized in the nucleus. The interaction does not require the presence of the viral RNA but needs an intact BNP RNA-binding motif. The results highlight the novel role of BNP as an anti-influenza A viral agent and provide insights into the mechanism of intertypic interference.« less

Architecture and ssDNA interaction of the Timeless-Tipin-RPA complex.

PubMed

Witosch, Justine; Wolf, Eva; Mizuno, Naoko

2014-11-10

The Timeless-Tipin (Tim-Tipin) complex, also referred to as the fork protection complex, is involved in coordination of DNA replication. Tim-Tipin is suggested to be recruited to replication forks via Replication Protein A (RPA) but details of the interaction are unknown. Here, using cryo-EM and biochemical methods, we characterized complex formation of Tim-Tipin, RPA and single-stranded DNA (ssDNA). Tim-Tipin and RPA form a 258 kDa complex with a 1:1:1 stoichiometry. The cryo-EM 3D reconstruction revealed a globular architecture of the Tim-Tipin-RPA complex with a ring-like and a U-shaped domain covered by a RPA lid. Interestingly, RPA in the complex adopts a horse shoe-like shape resembling its conformation in the presence of long ssDNA (>30 nucleotides). Furthermore, the recruitment of the Tim-Tipin-RPA complex to ssDNA is modulated by the RPA conformation and requires RPA to be in the more compact 30 nt ssDNA binding mode. The dynamic formation and disruption of the Tim-Tipin-RPA-ssDNA complex implicates the RPA-based recruitment of Tim-Tipin to the replication fork. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Defect states of complexes involving a vacancy on the boron site in boronitrene

NASA Astrophysics Data System (ADS)

Ngwenya, T. B.; Ukpong, A. M.; Chetty, N.

2011-12-01

First principles calculations have been performed to investigate the ground state properties of freestanding monolayer hexagonal boronitrene (h-BN). We have considered monolayers that contain native point defects and their complexes, which form when the point defects bind with the boron vacancy on the nearest-neighbor position. The changes in the electronic structure are analyzed to show the extent of localization of the defect-induced midgap states. The variations in formation energies suggest that defective h-BN monolayers that contain carbon substitutional impurities are the most stable structures, irrespective of the changes in growth conditions. The high energies of formation of the boron vacancy complexes suggest that they are less stable, and their creation by ion bombardment would require high-energy ions compared to point defects. Using the relative positions of the derived midgap levels for the double vacancy complex, it is shown that the quasi-donor-acceptor pair interpretation of optical transitions is consistent with stimulated transitions between electron and hole states in boronitrene.

Assembly dynamics and stability of the pneumococcal epsilon zeta antitoxin toxin (PezAT) system from Streptococcus pneumoniae.

PubMed

Mutschler, Hannes; Reinstein, Jochen; Meinhart, Anton

2010-07-09

The pneumococcal epsilon zeta antitoxin toxin (PezAT) system is a chromosomally encoded, class II toxin antitoxin system from the human pathogen Streptococcus pneumnoniae. Neutralization of the bacteriotoxic protein PezT is carried out by complex formation with its cognate antitoxin PezA. Here we study the stability of the inhibitory complex in vivo and in vitro. We found that toxin release is impeded in Escherichia coli and Bacillus subtilis due to the proteolytic resistance of PezA once bound to PezT. These findings are supported by in vitro experiments demonstrating a strong thermodynamic stabilization of both proteins upon binding. A detailed kinetic analysis of PezAT assembly revealed that these particular features of PezAT are based on a strong, electrostatically guided binding mechanism leading to a stable toxin antitoxin complex with femtomolar affinity. Our data show that PezAT complex formation is distinct to all other conventional toxin antitoxin modules and a controlled mode of toxin release is required for activation.

[Mechanistic modelling allows to assess pathways of DNA lesion interactions underlying chromosome aberration formation].

PubMed

Eĭdel'man, Iu A; Slanina, S V; Sal'nikov, I V; Andreev, S G

2012-12-01

The knowledge of radiation-induced chromosomal aberration (CA) mechanisms is required in many fields of radiation genetics, radiation biology, biodosimetry, etc. However, these mechanisms are yet to be quantitatively characterised. One of the reasons is that the relationships between primary lesions of DNA/chromatin/chromosomes and dose-response curves for CA are unknown because the pathways of lesion interactions in an interphase nucleus are currently inaccessible for direct experimental observation. This article aims for the comparative analysis of two principally different scenarios of formation of simple and complex interchromosomal exchange aberrations: by lesion interactions at chromosome territories' surface vs. in the whole space of the nucleus. The analysis was based on quantitative mechanistic modelling of different levels of structures and processes involved in CA formation: chromosome structure in an interphase nucleus, induction, repair and interactions of DNA lesions. It was shown that the restricted diffusion of chromosomal loci, predicted by computational modelling of chromosome organization, results in lesion interactions in the whole space of the nucleus being impossible. At the same time, predicted features of subchromosomal dynamics agrees well with in vivo observations and does not contradict the mechanism of CA formation at the surface of chromosome territories. On the other hand, the "surface mechanism" of CA formation, despite having certain qualities, proved to be insufficient to explain high frequency of complex exchange aberrations observed by mFISH technique. The alternative mechanism, CA formation on nuclear centres is expected to be sufficient to explain frequent complex exchanges.

Forming metal-intermetallic or metal-ceramic composites by self-propagating high-temperature reactions

DOEpatents

Rawers, James C.; Alman, David E.; Petty, Jr., Arthur V.

1996-01-01

Industrial applications of composites often require that the final product have a complex shape. In this invention intermetallic or ceramic phases are formed from sheets of unreacted elemental metals. The process described in this invention allows the final product shape be formed prior to the formation of the composite. This saves energy and allows formation of shaped articles of metal-intermetallic composites composed of brittle materials that cannot be deformed without breaking.

An endosomal syntaxin and the AP-3 complex are required for formation and maturation of candidate lysosome-related secretory organelles (mucocysts) in Tetrahymena thermophila.

PubMed

Kaur, Harsimran; Sparvoli, Daniela; Osakada, Hiroko; Iwamoto, Masaaki; Haraguchi, Tokuko; Turkewitz, Aaron P

2017-06-01

The ciliate Tetrahymena thermophila synthesizes large secretory vesicles called mucocysts. Mucocyst biosynthesis shares features with dense core granules (DCGs) in animal cells, including proteolytic processing of cargo proteins during maturation. However, other molecular features have suggested relatedness to lysosome-related organelles (LROs). LROs, which include diverse organelles in animals, are formed via convergence of secretory and endocytic trafficking. Here we analyzed Tetrahymena syntaxin 7-like 1 (Stx7l1p), a Qa-SNARE whose homologues in other lineages are linked with vacuoles/LROs. Stx7l1p is targeted to both immature and mature mucocysts and is essential in mucocyst formation. In STX7L1 -knockout cells, the two major classes of mucocyst cargo proteins localize independently, accumulating in largely nonoverlapping vesicles. Thus initial formation of immature mucocysts involves heterotypic fusion, in which a subset of mucocyst proteins is delivered via an endolysosomal compartment. Further, we show that subsequent maturation requires AP-3, a complex widely implicated in LRO formation. Knockout of the µ-subunit gene does not impede delivery of any known mucocyst cargo but nonetheless arrests mucocyst maturation. Our data argue that secretory organelles in ciliates may represent a new class of LROs and reveal key roles of an endosomal syntaxin and AP-3 in the assembly of this complex compartment. © 2017 Kaur et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

MURC/Cavin-4 and cavin family members form tissue-specific caveolar complexes

PubMed Central

Bastiani, Michele; Liu, Libin; Hill, Michelle M.; Jedrychowski, Mark P.; Nixon, Susan J.; Lo, Harriet P.; Abankwa, Daniel; Luetterforst, Robert; Fernandez-Rojo, Manuel; Breen, Michael R.; Gygi, Steven P.; Vinten, Jorgen; Walser, Piers J.; North, Kathryn N.; Hancock, John F.; Pilch, Paul F.

2009-01-01

Polymerase I and transcript release factor (PTRF)/Cavin is a cytoplasmic protein whose expression is obligatory for caveola formation. Using biochemistry and fluorescence resonance energy transfer–based approaches, we now show that a family of related proteins, PTRF/Cavin-1, serum deprivation response (SDR)/Cavin-2, SDR-related gene product that binds to C kinase (SRBC)/Cavin-3, and muscle-restricted coiled-coil protein (MURC)/Cavin-4, forms a multiprotein complex that associates with caveolae. This complex can constitutively assemble in the cytosol and associate with caveolin at plasma membrane caveolae. Cavin-1, but not other cavins, can induce caveola formation in a heterologous system and is required for the recruitment of the cavin complex to caveolae. The tissue-restricted expression of cavins suggests that caveolae may perform tissue-specific functions regulated by the composition of the cavin complex. Cavin-4 is expressed predominantly in muscle, and its distribution is perturbed in human muscle disease associated with Caveolin-3 dysfunction, identifying Cavin-4 as a novel muscle disease candidate caveolar protein. PMID:19546242

Crystal structures of the CPAP/STIL complex reveal its role in centriole assembly and human microcephaly

PubMed Central

Cottee, Matthew A; Muschalik, Nadine; Wong, Yao Liang; Johnson, Christopher M; Johnson, Steven; Andreeva, Antonina; Oegema, Karen; Lea, Susan M; Raff, Jordan W; van Breugel, Mark

2013-01-01

Centrioles organise centrosomes and template cilia and flagella. Several centriole and centrosome proteins have been linked to microcephaly (MCPH), a neuro-developmental disease associated with small brain size. CPAP (MCPH6) and STIL (MCPH7) are required for centriole assembly, but it is unclear how mutations in them lead to microcephaly. We show that the TCP domain of CPAP constitutes a novel proline recognition domain that forms a 1:1 complex with a short, highly conserved target motif in STIL. Crystal structures of this complex reveal an unusual, all-β structure adopted by the TCP domain and explain how a microcephaly mutation in CPAP compromises complex formation. Through point mutations, we demonstrate that complex formation is essential for centriole duplication in vivo. Our studies provide the first structural insight into how the malfunction of centriole proteins results in human disease and also reveal that the CPAP–STIL interaction constitutes a conserved key step in centriole biogenesis. DOI: http://dx.doi.org/10.7554/eLife.01071.001 PMID:24052813

Sam37 is crucial for formation of the mitochondrial TOM-SAM supercomplex, thereby promoting β-barrel biogenesis.

PubMed

Wenz, Lena-Sophie; Ellenrieder, Lars; Qiu, Jian; Bohnert, Maria; Zufall, Nicole; van der Laan, Martin; Pfanner, Nikolaus; Wiedemann, Nils; Becker, Thomas

2015-09-28

Biogenesis of mitochondrial β-barrel proteins requires two preprotein translocases, the general translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). TOM and SAM form a supercomplex that promotes transfer of β-barrel precursors. The SAM core complex contains the channel protein Sam50, which cooperates with Sam35 in precursor recognition, and the peripheral membrane protein Sam37. The molecular function of Sam37 has been unknown. We report that Sam37 is crucial for formation of the TOM-SAM supercomplex. Sam37 interacts with the receptor domain of Tom22 on the cytosolic side of the mitochondrial outer membrane and links TOM and SAM complexes. Sam37 thus promotes efficient transfer of β-barrel precursors to the SAM complex. We conclude that Sam37 functions as a coupling factor of the translocase supercomplex of the mitochondrial outer membrane. © 2015 Wenz et al.

Effects of the dielectric properties of the ceramic-solvent interface on the binding of proteins to oxide ceramics: a non-local electrostatic approach.

PubMed

Rubinstein, Alexander I; Sabirianov, Renat F; Namavar, Fereydoon

2016-10-14

The rapid development of nanoscience and nanotechnology has raised many fundamental questions that significantly impede progress in these fields. In particular, understanding the physicochemical processes at the interface in aqueous solvents requires the development and application of efficient and accurate methods. In the present work we evaluate the electrostatic contribution to the energy of model protein-ceramic complex formation in an aqueous solvent. We apply a non-local (NL) electrostatic approach that accounts for the effects of the short-range structure of the solvent on the electrostatic interactions of the interfacial systems. In this approach the aqueous solvent is considered as a non-ionic liquid, with the rigid and strongly correlated dipoles of the water molecules. We have found that an ordered interfacial aqueous solvent layer at the protein- and ceramic-solvent interfaces reduces the charging energy of both the ceramic and the protein in the solvent, and significantly increases the electrostatic contribution to their association into a complex. This contribution in the presented NL approach was found to be significantly shifted with respect to the classical model at any dielectric constant value of the ceramics. This implies a significant increase of the adsorption energy in the protein-ceramic complex formation for any ceramic material. We show that for several biocompatible ceramics (for example HfO2, ZrO2, and Ta2O5) the above effect predicts electrostatically induced protein-ceramic complex formation. However, in the framework of the classical continuum electrostatic model (the aqueous solvent as a uniform dielectric medium with a high dielectric constant ∼80) the above ceramics cannot be considered as suitable for electrostatically induced complex formation. Our results also show that the protein-ceramic electrostatic interactions can be strong enough to compensate for the unfavorable desolvation effect in the process of protein-ceramic complex formation.

Effects of the dielectric properties of the ceramic-solvent interface on the binding of proteins to oxide ceramics: a non-local electrostatic approach

NASA Astrophysics Data System (ADS)

Rubinstein, Alexander I.; Sabirianov, Renat F.; Namavar, Fereydoon

2016-10-01

The rapid development of nanoscience and nanotechnology has raised many fundamental questions that significantly impede progress in these fields. In particular, understanding the physicochemical processes at the interface in aqueous solvents requires the development and application of efficient and accurate methods. In the present work we evaluate the electrostatic contribution to the energy of model protein-ceramic complex formation in an aqueous solvent. We apply a non-local (NL) electrostatic approach that accounts for the effects of the short-range structure of the solvent on the electrostatic interactions of the interfacial systems. In this approach the aqueous solvent is considered as a non-ionic liquid, with the rigid and strongly correlated dipoles of the water molecules. We have found that an ordered interfacial aqueous solvent layer at the protein- and ceramic-solvent interfaces reduces the charging energy of both the ceramic and the protein in the solvent, and significantly increases the electrostatic contribution to their association into a complex. This contribution in the presented NL approach was found to be significantly shifted with respect to the classical model at any dielectric constant value of the ceramics. This implies a significant increase of the adsorption energy in the protein-ceramic complex formation for any ceramic material. We show that for several biocompatible ceramics (for example HfO2, ZrO2, and Ta2O5) the above effect predicts electrostatically induced protein-ceramic complex formation. However, in the framework of the classical continuum electrostatic model (the aqueous solvent as a uniform dielectric medium with a high dielectric constant ˜80) the above ceramics cannot be considered as suitable for electrostatically induced complex formation. Our results also show that the protein-ceramic electrostatic interactions can be strong enough to compensate for the unfavorable desolvation effect in the process of protein-ceramic complex formation.

Induction of cell-cell fusion by ectromelia virus is not inhibited by its fusion inhibitory complex.

PubMed

Erez, Noam; Paran, Nir; Maik-Rachline, Galia; Politi, Boaz; Israely, Tomer; Schnider, Paula; Fuchs, Pinhas; Melamed, Sharon; Lustig, Shlomo

2009-09-29

Ectromelia virus, a member of the Orthopox genus, is the causative agent of the highly infectious mousepox disease. Previous studies have shown that different poxviruses induce cell-cell fusion which is manifested by the formation of multinucleated-giant cells (polykaryocytes). This phenomenon has been widely studied with vaccinia virus in conditions which require artificial acidification of the medium. We show that Ectromelia virus induces cell-cell fusion under neutral pH conditions and requires the presence of a sufficient amount of viral particles on the plasma membrane of infected cells. This could be achieved by infection with a replicating virus and its propagation in infected cells (fusion "from within") or by infection with a high amount of virus particles per cell (fusion "from without"). Inhibition of virus maturation or inhibition of virus transport on microtubules towards the plasma membrane resulted in a complete inhibition of syncytia formation. We show that in contrast to vaccinia virus, Ectromelia virus induces cell-cell fusion irrespectively of its hemagglutination properties and cell-surface expression of the orthologs of the fusion inhibitory complex, A56 and K2. Additionally, cell-cell fusion was also detected in mice lungs following lethal respiratory infection. Ectromelia virus induces spontaneous cell-cell fusion in-vitro and in-vivo although expressing an A56/K2 fusion inhibitory complex. This syncytia formation property cannot be attributed to the 37 amino acid deletion in ECTV A56.

Cytosolic Extract Induces Tir Translocation and Pedestals in EPEC-Infected Red Blood Cells

PubMed Central

Swimm, Alyson I; Kalman, Daniel

2008-01-01

Enteropathogenic Escherichia coli (EPEC) are deadly contaminants in water and food, and induce protrusion of actin-filled membranous pedestals beneath themselves upon attachment to intestinal epithelia. Pedestal formation requires clustering of Tir and subsequent recruitment of cellular tyrosine kinases including Abl, Arg, and Etk as well as signaling molecules Nck, N-WASP, and Arp2/3 complex. We have developed a cytosolic extract-based cellular system that recapitulates actin pedestal formation in permeabilized red blood cells (RBC) infected with EPEC. RBC support attachment of EPEC and translocation of virulence factors, but not pedestal formation. We show here that extract induces a rapid Ca++-dependent release of Tir from the EPEC Type III secretion system, and that cytoplasmic factor(s) present in the extract facilitate translocation of Tir into the RBC plasma membrane. We show that Abl and related kinases in the extract phosphorylate Tir and that actin polymerization can be reconstituted in infected RBC following addition of cytosolic extract. Reconstitution requires the bacterial virulence factors Tir and intimin, and phosphorylation of Tir on tyrosine residue 474 results in the recruitment of Nck, N-WASP, and Arp2/3 complex beneath attached bacteria at sites of actin polymerization. Together these data describe a biochemical system for dissection of host components that mediate Type III secretion and the mechanisms by which complexes of proteins are recruited to discrete sites within the plasma membrane to initiate localized actin polymerization and morphological changes. PMID:18208322

Inflammasome Assembly in the Chorioamniotic Membranes during Spontaneous Labor at Term

PubMed Central

Gomez-Lopez, Nardhy; Romero, Roberto; Xu, Yi; Garcia-Flores, Valeria; Leng, Yaozhu; Panaitescu, Bogdan; Miller, Derek; Abrahams, Vikki M.; Hassan, Sonia S.

2017-01-01

Problem Inflammasome activation requires two steps: priming and assembly of the multimeric complex. The second step includes assembly of the sensor molecule and adaptor protein ASC (an apoptosis-associated speck-like protein containing a CARD), which results in ASC speck formation and the recruitment of caspase (CASP)-1. Herein, we investigated whether there is inflammasome assembly in the chorioamniotic membranes and choriodecidual leukocytes from women who underwent spontaneous labor at term. Method of Study Using in situ proximity ligation assays, ASC/CASP-1 complexes were determined in the chorioamniotic membranes from women who delivered at term without labor or underwent spontaneous labor at term with or without acute histologic chorioamnionitis (n=10–11 each). Also, ASC speck formation was determined by flow cytometry in the choriodecidual leukocytes isolated from women who delivered at term with or without spontaneous labor (n=9–12 each). Results 1) ASC/CASP-1 complexes were detected in the chorioamniotic membranes; 2) ASC/CASP-1 complexes were greater in the chorioamniotic membranes from women who underwent spontaneous labor at term than in those without labor; 3) ASC/CASP-1 complexes were even more abundant in the chorioamniotic membranes from women who underwent spontaneous labor at term with acute histologic chorioamnionitis than in those without this placental lesion; 4) ASC speck formation was detected in the choriodecidual leukocytes; and 5) ASC speck formation was greater in the choriodecidual leukocytes isolated from women who underwent spontaneous labor at term than in those without labor. Conclusion There is inflammasome assembly in the chorioamniotic membranes and choriodecidual leukocytes during spontaneous labor at term. PMID:28233423

File Formats Commonly Used in Mass Spectrometry Proteomics*

PubMed Central

Deutsch, Eric W.

2012-01-01

The application of mass spectrometry (MS) to the analysis of proteomes has enabled the high-throughput identification and abundance measurement of hundreds to thousands of proteins per experiment. However, the formidable informatics challenge associated with analyzing MS data has required a wide variety of data file formats to encode the complex data types associated with MS workflows. These formats encompass the encoding of input instruction for instruments, output products of the instruments, and several levels of information and results used by and produced by the informatics analysis tools. A brief overview of the most common file formats in use today is presented here, along with a discussion of related topics. PMID:22956731

The Nuclear Transport Factor Kap121 Is Required for Stability of the Dam1 Complex and Mitotic Kinetochore Bi-orientation.

PubMed

Cairo, Lucas V; Wozniak, Richard W

2016-03-15

The karyopherin (Kap) family of nuclear transport factors facilitates macromolecular transport through nuclear pore complexes (NPCs). The binding of Kaps to their cargos can also regulate, both temporally and spatially, the interactions of the cargo protein with interacting partners. Here, we show that the essential yeast Kap, Kap121, binds Dam1 and Duo1, components of the microtubule (MT)-associated Dam1 complex required for linking dynamic MT ends with kinetochores (KTs). Like mutations in the Dam1 complex, loss of Kap121 function compromises the formation of normal KT-MT attachments during mitosis. We show that the stability of the Dam1 complex in vivo is dependent on its association with Kap121. Furthermore, we show that the Kap121/Duo1 complex is maintained in the presence of RanGTP but Kap121 is released by the cooperative actions of RanGTP and tubulin. We propose that Kap121 stabilizes the Dam1 complex and participates in escorting it to spindle MTs. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Whirlin and PDZ domain-containing 7 (PDZD7) proteins are both required to form the quaternary protein complex associated with Usher syndrome type 2.

PubMed

Chen, Qian; Zou, Junhuang; Shen, Zuolian; Zhang, Weiping; Yang, Jun

2014-12-26

Usher syndrome (USH) is the leading genetic cause of combined hearing and vision loss. Among the three USH clinical types, type 2 (USH2) occurs most commonly. USH2A, GPR98, and WHRN are three known causative genes of USH2, whereas PDZD7 is a modifier gene found in USH2 patients. The proteins encoded by these four USH genes have been proposed to form a multiprotein complex, the USH2 complex, due to interactions found among some of these proteins in vitro, their colocalization in vivo, and mutual dependence of some of these proteins for their normal in vivo localizations. However, evidence showing the formation of the USH2 complex is missing, and details on how this complex is formed remain elusive. Here, we systematically investigated interactions among the intracellular regions of the four USH proteins using colocalization, yeast two-hybrid, and pull-down assays. We show that multiple domains of the four USH proteins interact among one another. Importantly, both WHRN and PDZD7 are required for the complex formation with USH2A and GPR98. In this USH2 quaternary complex, WHRN prefers to bind to USH2A, whereas PDZD7 prefers to bind to GPR98. Interaction between WHRN and PDZD7 is the bridge between USH2A and GPR98. Additionally, the USH2 quaternary complex has a variable stoichiometry. These findings suggest that a non-obligate, short term, and dynamic USH2 quaternary protein complex may exist in vivo. Our work provides valuable insight into the physiological role of the USH2 complex in vivo and informs possible reconstruction of the USH2 complex for future therapy. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Stellar properties of dwarf galaxies and their connections with the Milky Way halo

NASA Astrophysics Data System (ADS)

Revaz, Yves; Pascale Jablonka

2018-06-01

In this talk, relying on recent chemo-dynamical simulations, I will describe the stellar properties and in particular the abundances ratios of dwarf galaxies emerging from a LCDM framework. Faint systems quenched by the UV-background as well as luminous ones exhibiting an extended star formation history nicely reproduce observations, without necessary requiring a strong interaction with the Milky Way. However, dwarf galaxies with complex star formation histories like Carina and Fornax are much more difficult to reproduce. Those systems are often believed to result from an interaction with the Milky Way. I will show that when such interaction is taken into account in our high resolution simulations through ram pressure stripping, a much more complex reality appears.

Understanding the visual resource

Treesearch

Floyd L. Newby

1971-01-01

Understanding our visual resources involves a complex interweaving of motivation and cognitive recesses; but, more important, it requires that we understand and can identify those characteristics of a landscape that influence the image formation process. From research conducted in Florida, three major variables were identified that appear to have significant effect...

Importance of Highly Conserved Peptide Sites of Human Cytomegalovirus gO for Formation of the gH/gL/gO Complex

PubMed Central

Stegmann, Cora; Abdellatif, Mohamed E. A.; Laib Sampaio, Kerstin; Walther, Paul

2016-01-01

ABSTRACT The glycoprotein O (gO) is betaherpesvirus specific. Together with the viral glycoproteins H and L, gO forms a covalent trimeric complex that is part of the viral envelope. This trimer is crucial for cell-free infectivity of human cytomegalovirus (HCMV) but dispensable for cell-associated spread. We hypothesized that the amino acids that are conserved among gOs of different cytomegaloviruses are important for the formation of the trimeric complex and hence for efficient virus spread. In a mutational approach, nine peptide sites, containing all 13 highly conserved amino acids, were analyzed in the context of HCMV strain TB40-BAC4 with regard to infection efficiency and formation of the gH/gL/gO complex. Mutation of amino acids (aa) 181 to 186 or aa 193 to 198 resulted in the loss of the trimer and a complete small-plaque phenotype, whereas mutation of aa 108 or aa 249 to 254 caused an intermediate phenotype. While individual mutations of the five conserved cysteines had little impact, their relevance was revealed in a combined mutation, which abrogated both complex formation and cell-free infectivity. C343 was unique, as it was sufficient and necessary for covalent binding of gO to gH/gL. Remarkably, however, C218 together with C167 rescued infectivity in the absence of detectable covalent complex formation. We conclude that all highly conserved amino acids contribute to the function of gO to some extent but that aa 181 to 198 and cysteines 343, 218, and 167 are particularly relevant. Surprisingly, covalent binding of gO to gH/gL is required neither for its incorporation into virions nor for proper function in cell-free infection. IMPORTANCE Like all herpesviruses, the widespread human pathogen HCMV depends on glycoproteins gB, gH, and gL for entry into target cells. Additionally, gH and gL have to bind gO in a trimeric complex for efficient cell-free infection. Homologs of gO are shared by all cytomegaloviruses, with 13 amino acids being highly conserved. In a mutational approach we analyzed these amino acids to elucidate their role in the function of gO. All conserved amino acids contributed either to formation of the trimeric complex or to cell-free infection. Notably, these two phenotypes were not inevitably linked as the mutation of a charged cluster in the center of gO abrogated cell-free infection while trimeric complexes were still being formed. Cysteine 343 was essential for covalent binding of gO to gH/gL; however, noncovalent complex formation in the absence of cysteine 343 also allowed for cell-free infectivity. PMID:27795411

Formation and biochemical characterization of tube/pelle death domain complexes: critical regulators of postreceptor signaling by the Drosophila toll receptor.

PubMed

Schiffmann, D A; White, J H; Cooper, A; Nutley, M A; Harding, S E; Jumel, K; Solari, R; Ray, K P; Gay, N J

1999-09-07

In Drosophila, the Toll receptor signaling pathway is required for embryonic dorso-ventral patterning and at later developmental stages for innate immune responses. It is thought that dimerization of the receptor by binding of the ligand spätzle causes the formation of a postreceptor activation complex at the cytoplasmic surface of the membrane. Two components of this complex are the adaptor tube and protein kinase pelle. These proteins both have "death domains", protein interaction motifs found in a number of signaling pathways, particularly those involved in apoptotic cell death. It is thought that pelle is bound by tube during formation of the activation complexes, and that this interaction is mediated by the death domains. In this paper, we show using the yeast two-hybrid system that the wild-type tube and pelle death domains bind together. Mutant tube proteins which do not support signaling in the embryo are also unable to bind pelle in the 2-hybrid assay. We have purified proteins corresponding to the death domains of tube and pelle and show that these form corresponding heterodimeric complexes in vitro. Partial proteolysis reveals a smaller core consisting of the minimal death domain sequences. We have studied the tube/pelle interaction with the techniques of surface plasmon resonance, analytical ultracentrifugation and isothermal titration calorimetry. These measurements produce a value of K(d) for the complex of about 0.5 microM.

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

Phospho-ubiquitin-PARK2 complex as a marker for mitophagy defects.

PubMed

Callegari, Sylvie; Oeljeklaus, Silke; Warscheid, Bettina; Dennerlein, Sven; Thumm, Michael; Rehling, Peter; Dudek, Jan

2017-01-02

The E3 ubiquitin ligase PARK2 and the mitochondrial protein kinase PINK1 are required for the initiation of mitochondrial damage-induced mitophagy. Together, PARK2 and PINK1 generate a phospho-ubiquitin signal on outer mitochondrial membrane proteins that triggers recruitment of the autophagy machinery. This paper describes the detection of a defined 500-kDa phospho-ubiquitin-rich PARK2 complex that accumulates on mitochondria upon treatment with the membrane uncoupler CCCP. Formation of this complex is dependent on the presence of PINK1 and is absent in mutant forms of PARK2, whereby mitophagy is also arrested. These results signify a functional signaling complex that is essential for the progression of mitophagy. The visualization of the PARK2 signaling complex represents a novel marker for this critical step in mitophagy and can be used to monitor mitophagy progression in PARK2 mutants and to uncover additional upstream factors required for PARK2-mediated mitophagy signaling.

Fullerene Derived Molecular Electronic Devices

NASA Technical Reports Server (NTRS)

Menon, Madhu; Srivastava, Deepak; Saini, Subbash

1998-01-01

The carbon Nanotube junctions have recently emerged as excellent candidates for use as the building blocks in the formation of nanoscale electronic devices. While the simple joint of two dissimilar tubes can be generated by the introduction of a pair of heptagon-pentagon defects in an otherwise perfect hexagonal grapheme sheet, more complex joints require other mechanisms. In this work we explore structural and electronic properties of complex 3-point junctions of carbon nanotubes using a generalized tight-binding molecular-dynamics scheme.

The Regulation of Transcription in Memory Consolidation

PubMed Central

Alberini, Cristina M.; Kandel, Eric R.

2015-01-01

De novo transcription of DNA is a fundamental requirement for the formation of long-term memory. It is required during both consolidation and reconsolidation, the posttraining and postreactivation phases that change the state of the memory from a fragile into a stable and long-lasting form. Transcription generates both mRNAs that are translated into proteins, which are necessary for the growth of new synaptic connections, as well as noncoding RNA transcripts that have regulatory or effector roles in gene expression. The result is a cascade of events that ultimately leads to structural changes in the neurons that mediate long-term memory storage. The de novo transcription, critical for synaptic plasticity and memory formation, is orchestrated by chromatin and epigenetic modifications. The complexity of transcription regulation, its temporal progression, and the effectors produced all contribute to the flexibility and persistence of long-term memory formation. In this article, we provide an overview of the mechanisms contributing to this transcriptional regulation underlying long-term memory formation. PMID:25475090

Formation Control of the MAXIM L2 Libration Orbit Mission

NASA Technical Reports Server (NTRS)

Folta, David; Hartman, Kate; Howell, Kathleen; Marchand, Belinda

2004-01-01

The Micro-Arcsecond Imaging Mission (MAXIM), a proposed concept for the Structure and Evolution of the Universe (SEU) Black Hole Imaging mission, is designed to make a ten million-fold improvement in X-ray image clarity of celestial objects by providing better than 0.1 microarcsecond imaging. To achieve mission requirements, MAXIM will have to improve on pointing by orders of magnitude. This pointing requirement impacts the control and design of the formation. Currently the architecture is comprised of 25 spacecraft, which will form the sparse apertures of a grazing incidence X-ray interferometer covering the 0.3-10 keV bandpass. This configuration will deploy 24 spacecraft as optics modules and one as the detector. The formation must allow for long duration continuous science observations and also for reconfiguration that permits re-pointing of the formation. In this paper, we provide analysis and trades of several control efforts that are dependent upon the pointing requirements and the configuration and dimensions of the MAXIM formation. We emphasize the utilization of natural motions in the Lagrangian regions that minimize the control efforts and we address both continuous and discrete control via LQR and feedback linearization. Results provide control cost, configuration options, and capabilities as guidelines for the development of this complex mission.

Metadata requirements for results of diagnostic imaging procedures: a BIIF profile to support user applications

NASA Astrophysics Data System (ADS)

Brown, Nicholas J.; Lloyd, David S.; Reynolds, Melvin I.; Plummer, David L.

2002-05-01

A visible digital image is rendered from a set of digital image data. Medical digital image data can be stored as either: (a) pre-rendered format, corresponding to a photographic print, or (b) un-rendered format, corresponding to a photographic negative. The appropriate image data storage format and associated header data (metadata) required by a user of the results of a diagnostic procedure recorded electronically depends on the task(s) to be performed. The DICOM standard provides a rich set of metadata that supports the needs of complex applications. Many end user applications, such as simple report text viewing and display of a selected image, are not so demanding and generic image formats such as JPEG are sometimes used. However, these are lacking some basic identification requirements. In this paper we make specific proposals for minimal extensions to generic image metadata of value in various domains, which enable safe use in the case of two simple healthcare end user scenarios: (a) viewing of text and a selected JPEG image activated by a hyperlink and (b) viewing of one or more JPEG images together with superimposed text and graphics annotation using a file specified by a profile of the ISO/IEC Basic Image Interchange Format (BIIF).

Trimeric Association of Hox and TALE Homeodomain Proteins Mediates Hoxb2 Hindbrain Enhancer Activity

PubMed Central

Jacobs, Yakop; Schnabel, Catherine A.; Cleary, Michael L.

1999-01-01

Pbx/exd proteins modulate the DNA binding affinities and specificities of Hox proteins and contribute to the execution of Hox-dependent developmental programs in arthropods and vertebrates. Pbx proteins also stably heterodimerize and bind DNA with Meis and Pknox1-Prep1, additional members of the TALE (three-amino-acid loop extension) superclass of homeodomain proteins that function on common genetic pathways with a subset of Hox proteins. In this study, we demonstrated that Pbx and Meis bind DNA as heterotrimeric complexes with Hoxb1 on a genetically defined Hoxb2 enhancer, r4, that mediates the cross-regulatory transcriptional effects of Hoxb1 in vivo. The DNA binding specificity of the heterotrimeric complex for r4 is mediated by a Pbx-Hox site in conjunction with a distal Meis site, which we showed to be required for ternary complex formation and Meis-enhanced transcription. Formation of heterotrimeric complexes in which all three homeodomains bind their cognate DNA sites is topologically facilitated by the ability of Pbx and Meis to interact through their amino termini and bind DNA without stringent half-site orientation and spacing requirements. Furthermore, Meis site mutation in the Hoxb2 enhancer phenocopies Pbx-Hox site mutation to abrogate enhancer-directed expression of a reporter transgene in the murine embryonic hindbrain, demonstrating that DNA binding by all three proteins is required for trimer function in vivo. Our data provide in vitro and in vivo evidence for the combinatorial regulation of Hox and TALE protein functions that are mediated, in part, by their interdependent DNA binding activities as ternary complexes. As a consequence, Hoxb1 employs Pbx and Meis-related proteins, as a pair of essential cofactors in a higher-order molecular complex, to mediate its transcriptional effects on an endogenous Hox response element. PMID:10373562

An Elmo–Dock complex locally controls Rho GTPases and actin remodeling during cadherin-mediated adhesion

PubMed Central

Collins, Caitlin

2014-01-01

Cell–cell contact formation is a dynamic process requiring the coordination of cadherin-based cell–cell adhesion and integrin-based cell migration. A genome-wide RNA interference screen for proteins required specifically for cadherin-dependent cell–cell adhesion identified an Elmo–Dock complex. This was unexpected as Elmo–Dock complexes act downstream of integrin signaling as Rac guanine-nucleotide exchange factors. In this paper, we show that Elmo2 recruits Dock1 to initial cell–cell contacts in Madin–Darby canine kidney cells. At cell–cell contacts, both Elmo2 and Dock1 are essential for the rapid recruitment and spreading of E-cadherin, actin reorganization, localized Rac and Rho GTPase activities, and the development of strong cell–cell adhesion. Upon completion of cell–cell adhesion, Elmo2 and Dock1 no longer localize to cell–cell contacts and are not required subsequently for the maintenance of cell–cell adhesion. These studies show that Elmo–Dock complexes are involved in both integrin- and cadherin-based adhesions, which may help to coordinate the transition of cells from migration to strong cell–cell adhesion. PMID:25452388

Topoisomerase I (TopA) Is Recruited to ParB Complexes and Is Required for Proper Chromosome Organization during Streptomyces coelicolor Sporulation

PubMed Central

Szafran, Marcin; Skut, Patrycja; Ditkowski, Bartosz; Ginda, Katarzyna; Chandra, Govind; Zakrzewska-Czerwińska, Jolanta

2013-01-01

Streptomyces species are bacteria that resemble filamentous fungi in their hyphal mode of growth and sporulation. In Streptomyces coelicolor, the conversion of multigenomic aerial hyphae into chains of unigenomic spores requires synchronized septation accompanied by segregation of tens of chromosomes into prespore compartments. The chromosome segregation is dependent on ParB protein, which assembles into an array of nucleoprotein complexes in the aerial hyphae. Here, we report that nucleoprotein ParB complexes are bound in vitro and in vivo by topoisomerase I, TopA, which is the only topoisomerase I homolog found in S. coelicolor. TopA cannot be eliminated, and its depletion inhibits growth and blocks sporulation. Surprisingly, sporulation in the TopA-depleted strain could be partially restored by deletion of parB. Furthermore, the formation of regularly spaced ParB complexes, which is a prerequisite for proper chromosome segregation and septation during the development of aerial hyphae, has been found to depend on TopA. We hypothesize that TopA is recruited to ParB complexes during sporulation, and its activity is required to resolve segregating chromosomes. PMID:23913317

Science Competencies That Go Unassessed

ERIC Educational Resources Information Center

Gilmer, Penny J.; Sherdan, Danielle M.; Oosterhof, Albert; Rohani, Faranak; Rouby, Aaron

2011-01-01

Present large-scale assessments require the use of item formats, such as multiple choice, that can be administered and scored efficiently. This limits competencies that can be measured by these assessments. An alternative approach to large-scale assessments is being investigated that would include the use of complex performance assessments. As…

Two mechanisms for dissipation of excess light in monomeric and trimeric light-harvesting complexes

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

Dall'Osto, Luca; Cazzaniga, Stefano; Bressan, Mauro

Oxygenic photoautotrophs require mechanisms for rapidly matching the level of chlorophyll excited states from light harvesting with the rate of electron transport from water to carbon dioxide. These photoprotective reactions prevent formation of reactive excited states and photoinhibition. The fastest response to excess illumination is the so-called non-photochemical quenching which, in higher plants, requires the luminal pH sensor PsbS and other yet unidentified components of the photosystem II antenna. Both trimeric light-harvesting complex II (LHCII) and monomeric LHC proteins have been indicated as site(s) of the heat-dissipative reactions. Different mechanisms have been proposed: Energy transfer to a lutein quencher inmore » trimers, formation of a zeaxanthin radical cation in monomers. Here, we report on the construction of a mutant lacking all monomeric LHC proteins but retaining LHCII trimers. Its non-photochemical quenching induction rate was substantially slower with respect to the wild type. A carotenoid radical cation signal was detected in the wild type, although it was lost in the mutant. Here, we conclude that non-photochemical quenching is catalysed by two independent mechanisms, with the fastest activated response catalysed within monomeric LHC proteins depending on both zeaxanthin and lutein and on the formation of a radical cation. Trimeric LHCII was responsible for the slowly activated quenching component whereas inclusion in supercomplexes was not required. Finally, this latter activity does not depend on lutein nor on charge transfer events, whereas zeaxanthin was essential.« less

Hepatitis Delta Antigen Requires a Flexible Quasi-Double-Stranded RNA Structure To Bind and Condense Hepatitis Delta Virus RNA in a Ribonucleoprotein Complex

PubMed Central

Griffin, Brittany L.; Chasovskikh, Sergey; Dritschilo, Anatoly

2014-01-01

ABSTRACT The circular genome and antigenome RNAs of hepatitis delta virus (HDV) form characteristic unbranched, quasi-double-stranded RNA secondary structures in which short double-stranded helical segments are interspersed with internal loops and bulges. The ribonucleoprotein complexes (RNPs) formed by these RNAs with the virus-encoded protein hepatitis delta antigen (HDAg) perform essential roles in the viral life cycle, including viral replication and virion formation. Little is understood about the formation and structure of these complexes and how they function in these key processes. Here, the specific RNA features required for HDAg binding and the topology of the complexes formed were investigated. Selective 2′OH acylation analyzed by primer extension (SHAPE) applied to free and HDAg-bound HDV RNAs indicated that the characteristic secondary structure of the RNA is preserved when bound to HDAg. Notably, the analysis indicated that predicted unpaired positions in the RNA remained dynamic in the RNP. Analysis of the in vitro binding activity of RNAs in which internal loops and bulges were mutated and of synthetically designed RNAs demonstrated that the distinctive secondary structure, not the primary RNA sequence, is the major determinant of HDAg RNA binding specificity. Atomic force microscopy analysis of RNPs formed in vitro revealed complexes in which the HDV RNA is substantially condensed by bending or wrapping. Our results support a model in which the internal loops and bulges in HDV RNA contribute flexibility to the quasi-double-stranded structure that allows RNA bending and condensing by HDAg. IMPORTANCE RNA-protein complexes (RNPs) formed by the hepatitis delta virus RNAs and protein, HDAg, perform critical roles in virus replication. Neither the structures of these RNPs nor the RNA features required to form them have been characterized. HDV RNA is unusual in that it forms an unbranched quasi-double-stranded structure in which short base-paired segments are interspersed with internal loops and bulges. We analyzed the role of the HDV RNA sequence and secondary structure in the formation of a minimal RNP and visualized the structure of this RNP using atomic force microscopy. Our results indicate that HDAg does not recognize the primary sequence of the RNA; rather, the principle contribution of unpaired bases in HDV RNA to HDAg binding is to allow flexibility in the unbranched quasi-double-stranded RNA structure. Visualization of RNPs by atomic force microscopy indicated that the RNA is significantly bent or condensed in the complex. PMID:24741096

The pre-autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation.

PubMed

Suzuki, K; Kirisako, T; Kamada, Y; Mizushima, N; Noda, T; Ohsumi, Y

2001-11-01

Macroautophagy is a bulk degradation process induced by starvation in eukaryotic cells. In yeast, 15 Apg proteins coordinate the formation of autophagosomes. Several key reactions performed by these proteins have been described, but a comprehensive understanding of the overall network is still lacking. Based on Apg protein localization, we have identified a novel structure that functions in autophagosome formation. This pre-autophagosomal structure, containing at least five Apg proteins, i.e. Apg1p, Apg2p, Apg5p, Aut7p/Apg8p and Apg16p, is localized in the vicinity of the vacuole. Analysis of apg mutants revealed that the formation of both a phosphatidylethanolamine-conjugated Aut7p and an Apg12p- Apg5p conjugate is essential for the localization of Aut7p to the pre-autophagosomal structure. Vps30p/Apg6p and Apg14p, components of an autophagy- specific phosphatidylinositol 3-kinase complex, Apg9p and Apg16p are all required for the localization of Apg5p and Aut7p to the structure. The Apg1p protein kinase complex functions in the late stage of autophagosome formation. Here, we present the classification of Apg proteins into three groups that reflect each step of autophagosome formation.

The pre-autophagosomal structure organized by concerted functions of APG genes is essential for autophagosome formation

PubMed Central

Suzuki, Kuninori; Kirisako, Takayoshi; Kamada, Yoshiaki; Mizushima, Noboru; Noda, Takeshi; Ohsumi, Yoshinori

2001-01-01

Macroautophagy is a bulk degradation process induced by starvation in eukaryotic cells. In yeast, 15 Apg proteins coordinate the formation of autophagosomes. Several key reactions performed by these proteins have been described, but a comprehensive understanding of the overall network is still lacking. Based on Apg protein localization, we have identified a novel structure that functions in autophagosome formation. This pre-autophagosomal structure, containing at least five Apg proteins, i.e. Apg1p, Apg2p, Apg5p, Aut7p/Apg8p and Apg16p, is localized in the vicinity of the vacuole. Analysis of apg mutants revealed that the formation of both a phosphatidylethanolamine-conjugated Aut7p and an Apg12p– Apg5p conjugate is essential for the localization of Aut7p to the pre-autophagosomal structure. Vps30p/Apg6p and Apg14p, components of an autophagy- specific phosphatidylinositol 3-kinase complex, Apg9p and Apg16p are all required for the localization of Apg5p and Aut7p to the structure. The Apg1p protein kinase complex functions in the late stage of autophagosome formation. Here, we present the classification of Apg proteins into three groups that reflect each step of autophagosome formation. PMID:11689437

The human immunodeficiency virus type 1 long terminal repeat specifies two different transcription complexes, only one of which is regulated by Tat.

PubMed Central

Lu, X; Welsh, T M; Peterlin, B M

1993-01-01

The human immunodeficiency virus type 1 long terminal repeat sets up two different transcription complexes, which have been called processive and nonprocessive complexes. By mutating and substituting cis-acting sequences, we mapped elements of the human immunodeficiency virus long terminal repeat that are responsible for creating each transcription complex. Whereas processive complexes are efficiently assembled by upstream promoter elements in the absence of the TATA box, nonprocessive complexes absolutely require the TATA box. Moreover, the TATA box alone can set up these nonprocessive complexes, and nonprocessive but not processive complexes are trans activated by Tat. Finally, a strong DNA-binding site between the TATA box and trans-activation-responsive region interferes with either the assembly or movement of these nonprocessive complexes and diminishes the effects of Tat. Thus, Tat affects a critical step in the formation of elongation-competent transcription complexes. Images PMID:8445708

Evidence that Mediator is essential for Pol II transcription, but is not a required component of the preinitiation complex in vivo.

PubMed

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

2017-07-12

The Mediator complex has been described as a general transcription factor, but it is unclear if it is essential for Pol II transcription and/or is a required component of the preinitiation complex (PIC) in vivo. Here, we show that depletion of individual subunits, even those essential for cell growth, causes a general but only modest decrease in transcription. In contrast, simultaneous depletion of all Mediator modules causes a drastic decrease in transcription. Depletion of head or middle subunits, but not tail subunits, causes a downstream shift in the Pol II occupancy profile, suggesting that Mediator at the core promoter inhibits promoter escape. Interestingly, a functional PIC and Pol II transcription can occur when Mediator is not detected at core promoters. These results provide strong evidence that Mediator is essential for Pol II transcription and stimulates PIC formation, but it is not a required component of the PIC in vivo.

Nephrin Regulates Lamellipodia Formation by Assembling a Protein Complex That Includes Ship2, Filamin and Lamellipodin

PubMed Central

Venkatareddy, Madhusudan; Cook, Leslie; Abuarquob, Kamal; Verma, Rakesh; Garg, Puneet

2011-01-01

Actin dynamics has emerged at the forefront of podocyte biology. Slit diaphragm junctional adhesion protein Nephrin is necessary for development of the podocyte morphology and transduces phosphorylation-dependent signals that regulate cytoskeletal dynamics. The present study extends our understanding of Nephrin function by showing in cultured podocytes that Nephrin activation induced actin dynamics is necessary for lamellipodia formation. Upon activation Nephrin recruits and regulates a protein complex that includes Ship2 (SH2 domain containing 5′ inositol phosphatase), Filamin and Lamellipodin, proteins important in regulation of actin and focal adhesion dynamics, as well as lamellipodia formation. Using the previously described CD16-Nephrin clustering system, Nephrin ligation or activation resulted in phosphorylation of the actin crosslinking protein Filamin in a p21 activated kinase dependent manner. Nephrin activation in cell culture results in formation of lamellipodia, a process that requires specialized actin dynamics at the leading edge of the cell along with focal adhesion turnover. In the CD16-Nephrin clustering model, Nephrin ligation resulted in abnormal morphology of actin tails in human podocytes when Ship2, Filamin or Lamellipodin were individually knocked down. We also observed decreased lamellipodia formation and cell migration in these knock down cells. These data provide evidence that Nephrin not only initiates actin polymerization but also assembles a protein complex that is necessary to regulate the architecture of the generated actin filament network and focal adhesion dynamics. PMID:22194892

Focal Contacts as Mechanosensors

PubMed Central

Riveline, Daniel; Zamir, Eli; Balaban, Nathalie Q.; Schwarz, Ulrich S.; Ishizaki, Toshimasa; Narumiya, Shuh; Kam, Zvi; Geiger, Benjamin; Bershadsky, Alexander D.

2001-01-01

The transition of cell–matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II–driven contractility by a Rho target known as Rho-associated kinase (ROCK) was shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, we applied mechanical force to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling led to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein–tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppressed this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology protein mDia1 (Watanabe, N., T. Kato, A. Fujita, T. Ishizaki, and S. Narumiya. 1999. Nat. Cell Biol. 1:136–143), were sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced formation of the focal contacts still occurred in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force. PMID:11402062

Influence of Structure, Charge, and Concentration on the Pectin-Calcium-Surfactant Complexes.

PubMed

Joshi, Nidhi; Rawat, Kamla; Bohidar, H B

2016-05-12

Polymer-surfactant complex formation of pectin with different types of surfactants, cationic (cetyltrimethylammonium bromide, CTAB and dodecyl trimethylammonium bromide, DTAB), anionic (sodium dodecyl sulfate, SDS), and neutral (Triton X-100, TX-100), was investigated at room temperature in the presence and absence of cross-linker calcium chloride using light scattering, zeta potential, rheology, and UV-vis spectroscopic measurements where the surfactant concentration was maintained below their critical micellar concentration (CMC). Results indicated that the interaction of cationic surfactant with pectin in the presence and absence of calcium chloride was much stronger compared to anionic and neutral surfactants. The neutral surfactant showed identifiable interaction despite the absence of any charged headgroup, while anionic surfactant showed feeble or very weak interaction with the polymer. The pectin-CTAB or DTAB complex formation was attributed to associative electrostatic and hydrophobic interactions. On comparison between the cationic surfactants, it was found that CTAB interacts strongly with pectin because of its long hydrocarbon chain. The morphology of complexes formed exhibited random coil structures while at higher concentration of surfactant, rod-like or extended random coil structures were noticed. Thus, functional characteristics of the complex could be tuned by varying the type of surfactant (charge and structure) and its concentration. The differential network rigidity (pectin-CTAB versus pectin-DTAB gels) obtained from rheology measurements showed that addition of a very small amount of surfactant (concentration ≪ CMC) was required for enhancing network strength, while the presence of a large amount of surfactant resulted in the formation of fragile gels. No gel formation occurred when the surfactant concentration was close to their CMC values. Considering the importance of pectin in food and pharmaceutical industry, this study is relevant.

Inflammasome assembly in the chorioamniotic membranes during spontaneous labor at term.

PubMed

Gomez-Lopez, Nardhy; Romero, Roberto; Xu, Yi; Garcia-Flores, Valeria; Leng, Yaozhu; Panaitescu, Bogdan; Miller, Derek; Abrahams, Vikki M; Hassan, Sonia S

2017-05-01

Inflammasome activation requires two steps: priming and assembly of the multimeric complex. The second step includes assembly of the sensor molecule and adaptor protein ASC (an apoptosis-associated speck-like protein containing a CARD), which results in ASC speck formation and the recruitment of caspase (CASP)-1. Herein, we investigated whether there is inflammasome assembly in the chorioamniotic membranes and choriodecidual leukocytes from women who underwent spontaneous labor at term. Using in situ proximity ligation assays, ASC/CASP-1 complexes were determined in the chorioamniotic membranes from women who delivered at term without labor or underwent spontaneous labor at term with or without acute histologic chorioamnionitis (n=10-11 each). Also, ASC speck formation was determined by flow cytometry in the choriodecidual leukocytes isolated from women who delivered at term with or without spontaneous labor (n=9-12 each). (i) ASC/CASP-1 complexes were detected in the chorioamniotic membranes; (ii) ASC/CASP-1 complexes were greater in the chorioamniotic membranes from women who underwent spontaneous labor at term than in those without labor; (iii) ASC/CASP-1 complexes were even more abundant in the chorioamniotic membranes from women who underwent spontaneous labor at term with acute histologic chorioamnionitis than in those without this placental lesion; (iv) ASC speck formation was detected in the choriodecidual leukocytes; and (v) ASC speck formation was greater in the choriodecidual leukocytes isolated from women who underwent spontaneous labor at term than in those without labor. There is inflammasome assembly in the chorioamniotic membranes and choriodecidual leukocytes during spontaneous labor at term. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

Method and Apparatus for Automated Isolation of Nucleic Acids from Small Cell Samples

NASA Technical Reports Server (NTRS)

Sundaram, Shivshankar; Prabhakarpandian, Balabhaskar; Pant, Kapil; Wang, Yi

2014-01-01

RNA isolation is a ubiquitous need, driven by current emphasis on microarrays and miniaturization. With commercial systems requiring 100,000 to 1,000,000 cells for successful isolation, there is a growing need for a small-footprint, easy-to-use device that can harvest nucleic acids from much smaller cell samples (1,000 to 10,000 cells). The process of extraction of RNA from cell cultures is a complex, multi-step one, and requires timed, asynchronous operations with multiple reagents/buffers. An added complexity is the fragility of RNA (subject to degradation) and its reactivity to surface. A novel, microfluidics-based, integrated cartridge has been developed that can fully automate the complex process of RNA isolation (lyse, capture, and elute RNA) from small cell culture samples. On-cartridge cell lysis is achieved using either reagents or high-strength electric fields made possible by the miniaturized format. Traditionally, silica-based, porous-membrane formats have been used for RNA capture, requiring slow perfusion for effective capture. In this design, high efficiency capture/elution are achieved using a microsphere-based "microfluidized" format. Electrokinetic phenomena are harnessed to actively mix microspheres with the cell lysate and capture/elution buffer, providing important advantages in extraction efficiency, processing time, and operational flexibility. Successful RNA isolation was demonstrated using both suspension (HL-60) and adherent (BHK-21) cells. Novel features associated with this development are twofold. First, novel designs that execute needed processes with improved speed and efficiency were developed. These primarily encompass electric-field-driven lysis of cells. The configurations include electrode-containing constructs, or an "electrode-less" chip design, which is easy to fabricate and mitigates fouling at the electrode surface; and the "fluidized" extraction format based on electrokinetically assisted mixing and contacting of microbeads in a shape-optimized chamber. A secondary proprietary feature is in the particular layout integrating these components to perform the desired operation of RNA isolation. Apart from a novel functional capability, advantages of the innovation include reduced or eliminated use of toxic reagents, and operator-independent extraction of RNA.

Does stress remove the HDAC brakes for the formation and persistence of long-term memory?

PubMed

White, André O; Wood, Marcelo A

2014-07-01

It has been known for numerous decades that gene expression is required for long-lasting forms of memory. In the past decade, the study of epigenetic mechanisms in memory processes has revealed yet another layer of complexity in the regulation of gene expression. Epigenetic mechanisms do not only provide complexity in the protein regulatory complexes that control coordinate transcription for specific cell function, but the epigenome encodes critical information that integrates experience and cellular history for specific cell functions as well. Thus, epigenetic mechanisms provide a unique mechanism of gene expression regulation for memory processes. This may be why critical negative regulators of gene expression, such as histone deacetylases (HDACs), have powerful effects on the formation and persistence of memory. For example, HDAC inhibition has been shown to transform a subthreshold learning event into robust long-term memory and also generate a form of long-term memory that persists beyond the point at which normal long-term memory fails. A key question that is explored in this review, from a learning and memory perspective, is whether stress-dependent signaling drives the formation and persistence of long-term memory via HDAC-dependent mechanisms. Copyright © 2013 Elsevier Inc. All rights reserved.

Does stress remove the HDAC brakes for the formation and persistence of long-term memory?

PubMed Central

White, André O.; Wood, Marcelo A.

2013-01-01

It has been known for numerous decades that gene expression is required for long-lasting forms of memory. In the past decade, the study of epigenetic mechanisms in memory processes has revealed yet another layer of complexity in the regulation of gene expression. Epigenetic mechanisms do not only provide complexity in the protein regulatory complexes that control coordinate transcription for specific cell function, but the epigenome encodes critical information that integrates experience and cellular history for specific cell functions as well. Thus, epigenetic mechanisms provide a unique mechanism of gene expression regulation for memory processes. This may be why critical negative regulators of gene expression, such as histone deacetylases (HDACs), have powerful effects on the formation and persistence of memory. For example, HDAC inhibition has been shown to transform a subthreshold learning event into robust long-term memory and also generate a form of long-term memory that persists beyond the point at which normal long-term memory fails. A key question that is explored in this review, from a learning and memory perspective, is whether stress-dependent signaling drives the formation and persistence of long-term memory via HDAC-dependent mechanisms. PMID:24149059

N-terminal truncations in the FhlA protein result in formate- and MoeA-independent expression of the hyc (formate hydrogenlyase) operon of Escherichia coli.

PubMed

Self, W T; Hasona, A; Shanmugam, K T

2001-11-01

The formate hydrogenlyase complex of Escherichia coli catalyses the cleavage of formate to CO2 and H2 and consists of a molybdoenzyme formate dehydrogenase-H, hydrogenase 3 and intermediate electron carriers. The structural genes of this enzyme complex are activated by the FhlA protein in the presence of both formate and molybdate; ModE-Mo serves as a secondary activator. Mutational analysis of the FhlA protein established that the unique N-terminal region of this protein was responsible for formate- and molybdenum-dependent transcriptional control of the hyc operon. Analysis of the N-terminal sequence of the FhlA protein revealed a unique motif (amino acids 7-37), which is also found in ATPases associated with several members of the ABC-type transporter family. A deletion derivative of FhlA lacking these amino acids (FhlA9-2) failed to activate the hyc operon in vivo, although the FhlA9-2 did bind to hyc promoter DNA in vitro. The ATPase activity of the FhlA9-2-DNA-formate complex was at least three times higher than that of the native protein-DNA-formate complex, and this degree of activity was achieved at a lower formate level. Extending the deletion to amino acid 117 (FhlA167) not only reversed the FhlA(-) phenotype of FhlA9-2, but also led to both molybdenum- and formate-independence. Deleting the entire N-terminal domain (between amino acids 5 and 374 of the 692 amino acid protein) also led to an effector-independent transcriptional activator (FhlA165), which had a twofold higher level of hyc operon expression than the native protein. Both FhlA165 and FhlA167 still required ModE-Mo as a secondary activator for an optimal level of hyc-lac expression. The FhlA165 protein also had a twofold higher affinity to hyc promoter DNA than the native FhlA protein, while the FhlA167 protein had a significantly lower affinity for hyc promoter DNA in vitro. Although the ATPase activity of the native protein was increased by formate, the ATPase activity of neither FhlA165 or FhlA167 responded to formate. Removal of the first 117 amino acids of the FhlA protein appears to result in a constitutive, effector-independent activation of transcription of the genes encoding the components of the formate hydrogenlyase complex. The sequence similarity to ABC-ATPases, combined with the properties of the FhlA deletion proteins, led to the proposal that the N-terminal region of the native FhlA protein interacts with formate transport proteins, both as a formate transport facilitator and as a cytoplasmic acceptor.

Bicarbonate requirement for the water-oxidizing complex of photosystem II.

PubMed

Klimov, V V; Baranov, S V

2001-01-05

It is well established that bicarbonate stimulates electron transfer between the primary and secondary electron acceptors, Q(A) and Q(B), in formate-inhibited photosystem II; the non-heme Fe between Q(A) and Q(B) plays an essential role in the bicarbonate binding. Strong evidence of a bicarbonate requirement for the water-oxidizing complex (WOC), both O2 evolving and assembling from apo-WOC and Mn2+, of photosystem II (PSII) preparations has been presented in a number of publications during the last 5 years. The following explanations for the involvement of bicarbonate in the events on the donor side of PSII are considered: (1) bicarbonate serves as an electron donor (alternative to water or as a way of involvement of water molecules in the oxidative reactions) to the Mn-containing O2 center; (2) bicarbonate facilitates reassembly of the WOC from apo-WOC and Mn2+ due to formation of the complexes MnHCO3+ and Mn(HCO3)2 leading to an easier oxidation of Mn2+ with PSII; (3) bicarbonate is an integral component of the WOC essential for its function and stability; it may be considered a direct ligand to the Mn cluster; (4) the WOC is stabilized by bicarbonate through its binding to other components of PSII.

β-Subunit of the Ostα-Ostβ Organic Solute Transporter Is Required Not Only for Heterodimerization and Trafficking but Also for Function*

PubMed Central

Christian, Whitney V.; Li, Na; Hinkle, Patricia M.; Ballatori, Nazzareno

2012-01-01

The organic solute transporter, Ost/Slc51, is composed of two distinct proteins that must heterodimerize to generate transport activity, but the role of the individual subunits in mediating transport activity is unknown. The present study identified regions in Ostβ required for heterodimerization with Ostα, trafficking of the Ostα-Ostβ complex to the plasma membrane, and bile acid transport activity in HEK293 cells. Bimolecular fluorescence complementation analysis revealed that a 25-amino acid peptide containing the Ostβ transmembrane (TM) domain heterodimerized with Ostα, although the resulting complex failed to reach the plasma membrane and generate cellular [3H]taurocholate transport activity. Deletion of the single TM domain of Ostβ abolished interaction with Ostα, demonstrating that the TM segment is necessary and sufficient for formation of a heteromeric complex with Ostα. Mutation of the highly conserved tryptophan-asparagine sequence within the TM domain of Ostβ to alanines did not prevent cell surface trafficking, but abolished transport activity. Removal of the N-terminal 27 amino acids of Ostβ resulted in a transporter complex that reached the plasma membrane and exhibited transport activity at 30 °C. Complete deletion of the C terminus of Ostβ abolished [3H]taurocholate transport activity, but reinsertion of two native arginines immediately C-terminal to the TM domain rescued this defect. These positively charged residues establish the correct Nexo/Ccyt topology of the peptide, in accordance with the positive inside rule. Together, the results demonstrate that Ostβ is required for both proper trafficking of Ostα and formation of the functional transport unit, and identify specific residues of Ostβ critical for these processes. PMID:22535958

From path models to commands during additive printing of large-scale architectural designs

NASA Astrophysics Data System (ADS)

Chepchurov, M. S.; Zhukov, E. M.; Yakovlev, E. A.; Matveykin, V. G.

2018-05-01

The article considers the problem of automation of the formation of large complex parts, products and structures, especially for unique or small-batch objects produced by a method of additive technology [1]. Results of scientific research in search for the optimal design of a robotic complex, its modes of operation (work), structure of its control helped to impose the technical requirements on the technological process for manufacturing and design installation of the robotic complex. Research on virtual models of the robotic complexes allowed defining the main directions of design improvements and the main goal (purpose) of testing of the the manufactured prototype: checking the positioning accuracy of the working part.

Evaluation of Cu(i) binding to the E2 domain of the amyloid precursor protein - a lesson in quantification of metal binding to proteins via ligand competition.

PubMed

Young, Tessa R; Wedd, Anthony G; Xiao, Zhiguang

2018-01-24

The extracellular domain E2 of the amyloid precursor protein (APP) features a His-rich metal-binding site (denoted as the M1 site). In conjunction with surrounding basic residues, the site participates in interactions with components of the extracellular matrix including heparins, a class of negatively charged polysaccharide molecules of varying length. This work studied the chemistry of Cu(i) binding to APP E2 with the probe ligands Bcs, Bca, Fz and Fs. APP E2 forms a stable Cu(i)-mediated ternary complex with each of these anionic ligands. The complex with Bca was selected for isolation and characterization and was demonstrated, by native ESI-MS analysis, to have the stoichiometry E2 : Cu(i) : Bca = 1 : 1 : 1. Formation of these ternary complexes is specific for the APP E2 domain and requires Cu(i) coordination to the M1 site. Mutation of the M1 site was consistent with the His ligands being part of the E2 ligand set. It is likely that interactions between the negatively charged probe ligands and a positively charged patch on the surface of APP E2 are one aspect of the generation of the stable ternary complexes. Their formation prevented meaningful quantification of the affinity of Cu(i) binding to the M1 site with these probe ligands. However, the ternary complexes are disrupted by heparin, allowing reliable determination of a picomolar Cu(i) affinity for the E2/heparin complex with the Fz or Bca probe ligands. This is the first documented example of the formation of stable ternary complexes between a Cu(i) binding protein and a probe ligand. The ready disruption of the complexes by heparin identified clear 'tell-tale' signs for diagnosis of ternary complex formation and allowed a systematic review of conditions and criteria for reliable determination of affinities for metal binding via ligand competition. This study also provides new insights into a potential correlation of APP functions regulated by copper binding and heparin interaction.

New insights into the mechanism of interaction between CO2 and polymers from thermodynamic parameters obtained by in situ ATR-FTIR spectroscopy.

PubMed

Gabrienko, Anton A; Ewing, Andrew V; Chibiryaev, Andrey M; Agafontsev, Alexander M; Dubkov, Konstantin A; Kazarian, Sergei G

2016-03-07

This work reports new physical insights of the thermodynamic parameters and mechanisms of possible interactions occurring in polymers subjected to high-pressure CO2. ATR-FTIR spectroscopy has been used in situ to determine the thermodynamic parameters of the intermolecular interactions between CO2 and different functional groups of the polymers capable of specific interactions with sorbed CO2 molecules. Based on the measured ATR-FTIR spectra of the polymer samples subjected to high-pressure CO2 (30 bar) at different temperatures (300-340 K), it was possible to characterize polymer-polymer and CO2-polymer interactions. Particularly, the enthalpy and entropy of the formation of the specific non-covalent complexes between CO2 and the hydroxy (-OH), carbonyl (C[double bond, length as m-dash]O) and hydroxyimino ([double bond, length as m-dash]N-OH) functional groups of the polymer samples have been measured. Furthermore, the obtained spectroscopic results have provided an opportunity for the structure of these complexes to be proposed. An interesting phenomenon regarding the behavior of CO2/polymer systems has also been observed. It has been found that only for the polyketone, the value of enthalpy was negative indicating an exothermic process during the formation of the CO2-polymer non-covalent complexes. Conversely, for the polyoxime and polyalcohol samples there is a positive enthalpy determined. This is a result of the initial polymer-polymer interactions requiring more energy to break than is released during the formation of the CO2-polymer complex. The effect of increasing temperature to facilitate the breaking of the polymer-polymer interactions has also been observed. Hence, a mechanism for the formation of CO2-polymer complexes was suggested based on these results, which occurs via a two-step process: (1) the breaking of the existing polymer-polymer interactions followed by (2) the formation of new CO2-polymer non-covalent interactions.

Suppression of Akt-mediated HDAC3 expression and CDK2 T39 phosphorylation by a bichalcone analog contributes to S phase retardation of cancer cells.

PubMed

Hung, Kuang-Chen; Lin, Meng-Liang; Hsu, Shih-Wei; Lee, Chuan-Chun; Huang, Ren-Yu; Wu, Tian-Shung; Chen, Shih-Shun

2018-06-15

Targeting cell cycle regulators has been a suggested mechanism for therapeutic cancer strategies. We report here that the bichalcone analog TSWU-CD4 induces S phase arrest of human cancer cells by inhibiting the formation of cyclin A-phospho (p)-cyclin-dependent kinase 2 (CDK2, threonine [Thr] 39) complexes, independent of mutant p53 expression. Ectopic expression of CDK2 (T39E), which mimics phosphorylation of the Thr 39 residue of CDK2, partially rescues the cells from TSWU-CD4-induced S phase arrest, whereas phosphorylation-deficient CDK2 (T39A) expression regulates cell growth with significant S phase arrest and enhances TSWU-CD4-triggered S phase arrest. Decreased histone deacetylase 3 (HDAC3) expression after TSWU-CD4 treatment was demonstrated, and TSWU-CD4 induced S phase arrest and inhibitory effects on cyclin A expression and CDK2 Thr 39 phosphorylation, while cyclin A-p-CDK2 (Thr 39) complex formation was suppressed by ectopic wild-type HDAC3 expression. The co-transfection of CDK2 (T39E) along with HDAC3 completely restored cyclin A expression, Thr 39-phosphorylated CDK2, cyclin A-p-CDK2 (Thr 39) complex formation, and the S phase population to normal levels. Protein kinase B (Akt) inactivation was required for TSWU-CD4-induced S phase cell cycle arrest, because constitutively active Akt1 blocks the induction of S phase arrest and the suppression of cyclin A and HDAC3 expression, CDK2 Thr 39 phosphorylation, and cyclin A-p-CDK2 (Thr 39) complex formation by TSWU-CD4. Taken together, our results indicate that TSWU-CD4 induces S phase arrest by inhibiting Akt-mediated HDAC3 expression and CDK2 Thr 39 phosphorylation to suppress the formation of cyclin A-p-CDK2 (Thr 39) complexes. Copyright © 2018 Elsevier B.V. All rights reserved.

Intrinsic disorder in the partitioning protein KorB persists after co-operative complex formation with operator DNA and KorA.

PubMed

Hyde, Eva I; Callow, Philip; Rajasekar, Karthik V; Timmins, Peter; Patel, Trushar R; Siligardi, Giuliano; Hussain, Rohanah; White, Scott A; Thomas, Christopher M; Scott, David J

2017-08-30

The ParB protein, KorB, from the RK2 plasmid is required for DNA partitioning and transcriptional repression. It acts co-operatively with other proteins, including the repressor KorA. Like many multifunctional proteins, KorB contains regions of intrinsically disordered structure, existing in a large ensemble of interconverting conformations. Using NMR spectroscopy, circular dichroism and small-angle neutron scattering, we studied KorB selectively within its binary complexes with KorA and DNA, and within the ternary KorA/KorB/DNA complex. The bound KorB protein remains disordered with a mobile C-terminal domain and no changes in the secondary structure, but increases in the radius of gyration on complex formation. Comparison of wild-type KorB with an N-terminal deletion mutant allows a model of the ensemble average distances between the domains when bound to DNA. We propose that the positive co-operativity between KorB, KorA and DNA results from conformational restriction of KorB on binding each partner, while maintaining disorder. © 2017 The Author(s).

Structures of RNA Polymerase Closed and Intermediate Complexes Reveal Mechanisms of DNA Opening and Transcription Initiation.

PubMed

Glyde, Robert; Ye, Fuzhou; Darbari, Vidya Chandran; Zhang, Nan; Buck, Martin; Zhang, Xiaodong

2017-07-06

Gene transcription is carried out by RNA polymerases (RNAPs). For transcription to occur, the closed promoter complex (RPc), where DNA is double stranded, must isomerize into an open promoter complex (RPo), where the DNA is melted out into a transcription bubble and the single-stranded template DNA is delivered to the RNAP active site. Using a bacterial RNAP containing the alternative σ 54 factor and cryoelectron microscopy, we determined structures of RPc and the activator-bound intermediate complex en route to RPo at 3.8 and 5.8 Å. Our structures show how RNAP-σ 54 interacts with promoter DNA to initiate the DNA distortions required for transcription bubble formation, and how the activator interacts with RPc, leading to significant conformational changes in RNAP and σ 54 that promote RPo formation. We propose that DNA melting is an active process initiated in RPc and that the RNAP conformations of intermediates are significantly different from that of RPc and RPo. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Characterization of Cyclodextrin/Volatile Inclusion Complexes: A Review.

PubMed

Kfoury, Miriana; Landy, David; Fourmentin, Sophie

2018-05-17

Cyclodextrins (CDs) are a family of cyclic oligosaccharides that constitute one of the most widely used molecular hosts in supramolecular chemistry. Encapsulation in the hydrophobic cavity of CDs positively affects the physical and chemical characteristics of the guests upon the formation of inclusion complexes. Such a property is interestingly employed to retain volatile guests and reduce their volatility. Within this scope, the starting crucial point for a suitable and careful characterization of an inclusion complex is to assess the value of the formation constant (K f ), also called stability or binding constant. This task requires the application of the appropriate analytical method and technique. Thus, the aim of the present paper is to give a general overview of the main analytical tools used for the determination of K f values for CD/volatile inclusion complexes. This review emphasizes on the advantages, inconvenients and limits of each applied method. A special attention is also dedicated to the improvement of the current methods and to the development of new techniques. Further, the applicability of each technique is illustrated by a summary of data obtained from the literature.

Casein Kinase 1 and Phosphorylation of Cohesin Subunit Rec11 (SA3) Promote Meiotic Recombination through Linear Element Formation.

PubMed

Phadnis, Naina; Cipak, Lubos; Polakova, Silvia; Hyppa, Randy W; Cipakova, Ingrid; Anrather, Dorothea; Karvaiova, Lucia; Mechtler, Karl; Smith, Gerald R; Gregan, Juraj

2015-05-01

Proper meiotic chromosome segregation, essential for sexual reproduction, requires timely formation and removal of sister chromatid cohesion and crossing-over between homologs. Early in meiosis cohesins hold sisters together and also promote formation of DNA double-strand breaks, obligate precursors to crossovers. Later, cohesin cleavage allows chromosome segregation. We show that in fission yeast redundant casein kinase 1 homologs, Hhp1 and Hhp2, previously shown to regulate segregation via phosphorylation of the Rec8 cohesin subunit, are also required for high-level meiotic DNA breakage and recombination. Unexpectedly, these kinases also mediate phosphorylation of a different meiosis-specific cohesin subunit Rec11. This phosphorylation in turn leads to loading of linear element proteins Rec10 and Rec27, related to synaptonemal complex proteins of other species, and thereby promotes DNA breakage and recombination. Our results provide novel insights into the regulation of chromosomal features required for crossing-over and successful reproduction. The mammalian functional homolog of Rec11 (STAG3) is also phosphorylated during meiosis and appears to be required for fertility, indicating wide conservation of the meiotic events reported here.

Mapping of interaction domains between human repair proteins ERCC1 and XPF.

PubMed

de Laat, W L; Sijbers, A M; Odijk, H; Jaspers, N G; Hoeijmakers, J H

1998-09-15

ERCC1-XPF is a heterodimeric protein complexinvolved in nucleotide excision repair and recombinational processes. Like its homologous complex in Saccharomyces cerevisiae , Rad10-Rad1, it acts as a structure-specific DNA endonuclease, cleaving at duplex-single-stranded DNA junctions. In repair, ERCC1-XPF and Rad10-Rad1 make an incision on the the 5'-side of the lesion. No humans with a defect in the ERCC1 subunit of this protein complex have been identified and ERCC1-deficient mice suffer from severe developmental problems and signs of premature aging on top of a repair-deficient phenotype. Xeroderma pigmentosum group F patients carry mutations in the XPF subunit and generally show the clinical symptoms of mild DNA repair deficiency. All XP-F patients examined demonstrate reduced levels of XPF and ERCC1 protein, suggesting that proper complex formation is required for stability of the two proteins. To better understand the molecular and clinical consequences of mutations in the ERCC1-XPF complex, we decided to map the interaction domains between the two subunits. The XPF-binding domain comprises C-terminal residues 224-297 of ERCC1. Intriguingly, this domain resides outside the region of homology with its yeast Rad10 counterpart. The ERCC1-binding domain in XPF maps to C-terminal residues 814-905. ERCC1-XPF complex formation is established by a direct interaction between these two binding domains. A mutation from an XP-F patient that alters the ERCC1-binding domain in XPF indeed affects complex formation with ERCC1.

Mapping of interaction domains between human repair proteins ERCC1 and XPF.

PubMed Central

de Laat, W L; Sijbers, A M; Odijk, H; Jaspers, N G; Hoeijmakers, J H

1998-01-01

ERCC1-XPF is a heterodimeric protein complexinvolved in nucleotide excision repair and recombinational processes. Like its homologous complex in Saccharomyces cerevisiae , Rad10-Rad1, it acts as a structure-specific DNA endonuclease, cleaving at duplex-single-stranded DNA junctions. In repair, ERCC1-XPF and Rad10-Rad1 make an incision on the the 5'-side of the lesion. No humans with a defect in the ERCC1 subunit of this protein complex have been identified and ERCC1-deficient mice suffer from severe developmental problems and signs of premature aging on top of a repair-deficient phenotype. Xeroderma pigmentosum group F patients carry mutations in the XPF subunit and generally show the clinical symptoms of mild DNA repair deficiency. All XP-F patients examined demonstrate reduced levels of XPF and ERCC1 protein, suggesting that proper complex formation is required for stability of the two proteins. To better understand the molecular and clinical consequences of mutations in the ERCC1-XPF complex, we decided to map the interaction domains between the two subunits. The XPF-binding domain comprises C-terminal residues 224-297 of ERCC1. Intriguingly, this domain resides outside the region of homology with its yeast Rad10 counterpart. The ERCC1-binding domain in XPF maps to C-terminal residues 814-905. ERCC1-XPF complex formation is established by a direct interaction between these two binding domains. A mutation from an XP-F patient that alters the ERCC1-binding domain in XPF indeed affects complex formation with ERCC1. PMID:9722633

Charge-transfer complexes of 2,3-dichloro-5,6-dicyano-1,4-benzoquinone with amino molecules in polar solvents

NASA Astrophysics Data System (ADS)

Berto, Silvia; Chiavazza, Enrico; Ribotta, Valentina; Daniele, Pier Giuseppe; Barolo, Claudia; Giacomino, Agnese; Vione, Davide; Malandrino, Mery

2015-10-01

The charge-transfer complexes have scientific relevance because this type of molecular interaction is at the basis of the activity of pharmacological compounds and because the absorption bands of the complexes can be used for the quantification of electron donor molecules. This work aims to assess the stability of the charge-transfer complexes between the electron acceptor 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) and two drugs, procaine and atenolol, in acetonitrile and ethanol. The stability of DDQ in solution and the time required to obtain the maximum complex formation were evaluated. The stoichiometry and the stability of the complexes were determined, respectively, by Job's plot method and by the elaboration of UV-vis titrations data. The latter task was carried out by using the non-linear global analysis approach to determine the equilibrium constants. This approach to data elaboration allowed us to overcome the disadvantages of the classical linear-regression method, to obtain reliable values of the association constants and to calculate the entire spectra of the complexes. NMR spectra were recorded to identify the portion of the donor molecule that was involved in the interaction. The data support the participation of the aliphatic amino groups in complex formation and exclude the involvement of the aromatic amine present in the procaine molecule.

Gene duplication and promoter mutation expand the range csgD-dependent biofilm responses in a STEC population

USDA-ARS?s Scientific Manuscript database

Expression of Escherichia coli major biofilm components, curli fimbriae and cellulose, require the CsgD transcription factor. A complex regulatory network allows environmental control of csgD transcription and biofilm formation. However, most clinical serotype O157:H7 strains contain prophage inser...

Electrostatic Interactions between Elongated Monomers Drive Filamentation of Drosophila Shrub, a Metazoan ESCRT-III Protein.

PubMed

McMillan, Brian J; Tibbe, Christine; Jeon, Hyesung; Drabek, Andrew A; Klein, Thomas; Blacklow, Stephen C

2016-08-02

The endosomal sorting complex required for transport (ESCRT) is a conserved protein complex that facilitates budding and fission of membranes. It executes a key step in many cellular events, including cytokinesis and multi-vesicular body formation. The ESCRT-III protein Shrub in flies, or its homologs in yeast (Snf7) or humans (CHMP4B), is a critical polymerizing component of ESCRT-III needed to effect membrane fission. We report the structural basis for polymerization of Shrub and define a minimal region required for filament formation. The X-ray structure of the Shrub core shows that individual monomers in the lattice interact in a staggered arrangement using complementary electrostatic surfaces. Mutations that disrupt interface salt bridges interfere with Shrub polymerization and function. Despite substantial sequence divergence and differences in packing interactions, the arrangement of Shrub subunits in the polymer resembles that of Snf7 and other family homologs, suggesting that this intermolecular packing mechanism is shared among ESCRT-III proteins. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

ATF7IP-Mediated Stabilization of the Histone Methyltransferase SETDB1 Is Essential for Heterochromatin Formation by the HUSH Complex.

PubMed

Timms, Richard T; Tchasovnikarova, Iva A; Antrobus, Robin; Dougan, Gordon; Lehner, Paul J

2016-10-11

The histone methyltransferase SETDB1 plays a central role in repressive chromatin processes, but the functional requirement for its binding partner ATF7IP has remained enigmatic. Here, we show that ATF7IP is essential for SETDB1 stability: nuclear SETDB1 protein is degraded by the proteasome upon ablation of ATF7IP. As a result, ATF7IP is critical for repression that requires H3K9 trimethylation by SETDB1, including transgene silencing by the HUSH complex. Furthermore, we show that loss of ATF7IP phenocopies loss of SETDB1 in genome-wide assays. ATF7IP and SETDB1 knockout cells exhibit near-identical defects in the global deposition of H3K9me3, which results in similar dysregulation of the transcriptome. Overall, these data identify a critical functional role for ATF7IP in heterochromatin formation by regulating SETDB1 abundance in the nucleus. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Double-stranded DNA translocase activity of transcription factor TFIIH and the mechanism of RNA polymerase II open complex formation.

PubMed

Fishburn, James; Tomko, Eric; Galburt, Eric; Hahn, Steven

2015-03-31

Formation of the RNA polymerase II (Pol II) open complex (OC) requires DNA unwinding mediated by the transcription factor TFIIH helicase-related subunit XPB/Ssl2. Because XPB/Ssl2 binds DNA downstream from the location of DNA unwinding, it cannot function using a conventional helicase mechanism. Here we show that yeast TFIIH contains an Ssl2-dependent double-stranded DNA translocase activity. Ssl2 tracks along one DNA strand in the 5' → 3' direction, implying it uses the nontemplate promoter strand to reel downstream DNA into the Pol II cleft, creating torsional strain and leading to DNA unwinding. Analysis of the Ssl2 and DNA-dependent ATPase activity of TFIIH suggests that Ssl2 has a processivity of approximately one DNA turn, consistent with the length of DNA unwound during transcription initiation. Our results can explain why maintaining the OC requires continuous ATP hydrolysis and the function of TFIIH in promoter escape. Our results also suggest that XPB/Ssl2 uses this translocase mechanism during DNA repair rather than physically wedging open damaged DNA.

Huntingtin Is Required for Epithelial Polarity through RAB11A-Mediated Apical Trafficking of PAR3-aPKC

PubMed Central

Elias, Salah; McGuire, John Russel; Yu, Hua; Humbert, Sandrine

2015-01-01

The establishment of apical-basolateral polarity is important for both normal development and disease, for example, during tumorigenesis and metastasis. During this process, polarity complexes are targeted to the apical surface by a RAB11A-dependent mechanism. Huntingtin (HTT), the protein that is mutated in Huntington disease, acts as a scaffold for molecular motors and promotes microtubule-based dynamics. Here, we investigated the role of HTT in apical polarity during the morphogenesis of the mouse mammary epithelium. We found that the depletion of HTT from luminal cells in vivo alters mouse ductal morphogenesis and lumen formation. HTT is required for the apical localization of PAR3-aPKC during epithelial morphogenesis in virgin, pregnant, and lactating mice. We show that HTT forms a complex with PAR3, aPKC, and RAB11A and ensures the microtubule-dependent apical vesicular translocation of PAR3-aPKC through RAB11A. We thus propose that HTT regulates polarized vesicular transport, lumen formation and mammary epithelial morphogenesis. PMID:25942483

The Ferredoxin-Like Proteins HydN and YsaA Enhance Redox Dye-Linked Activity of the Formate Dehydrogenase H Component of the Formate Hydrogenlyase Complex.

PubMed

Pinske, Constanze

2018-01-01

Formate dehydrogenase H (FDH-H) and [NiFe]-hydrogenase 3 (Hyd-3) form the catalytic components of the hydrogen-producing formate hydrogenlyase (FHL) complex, which disproportionates formate to H 2 and CO 2 during mixed acid fermentation in enterobacteria. FHL comprises minimally seven proteins and little is understood about how this complex is assembled. Early studies identified a ferredoxin-like protein, HydN, as being involved in FDH-H assembly into the FHL complex. In order to understand how FDH-H and its small subunit HycB, which is also a ferredoxin-like protein, attach to the FHL complex, the possible roles of HydN and its paralogue, YsaA, in FHL complex stability and assembly were investigated. Deletion of the hycB gene reduced redox dye-mediated FDH-H activity to approximately 10%, abolished FHL-dependent H 2 -production, and reduced Hyd-3 activity. These data are consistent with HycB being an essential electron transfer component of the FHL complex. The FDH-H activity of the hydN and the ysaA deletion strains was reduced to 59 and 57% of the parental, while the double deletion reduced activity of FDH-H to 28% and the triple deletion with hycB to 1%. Remarkably, and in contrast to the hycB deletion, the absence of HydN and YsaA was without significant effect on FHL-dependent H 2 -production or total Hyd-3 activity; FDH-H protein levels were also unaltered. This is the first description of a phenotype for the E. coli ysaA deletion strain and identifies it as a novel factor required for optimal redox dye-linked FDH-H activity. A ysaA deletion strain could be complemented for FDH-H activity by hydN and ysaA , but the hydN deletion strain could not be complemented. Introduction of these plasmids did not affect H 2 production. Bacterial two-hybrid interactions showed that YsaA, HydN, and HycB interact with each other and with the FDH-H protein. Further novel anaerobic cross-interactions of 10 ferredoxin-like proteins in E. coli were also discovered and described. Together, these data indicate that FDH-H activity measured with the redox dye benzyl viologen is the sum of the FDH-H protein interacting with three independent small subunits and suggest that FDH-H can associate with different redox-protein complexes in the anaerobic cell to supply electrons from formate oxidation.

Identification of amino acids in the transmembrane and juxtamembrane domains of the platelet-derived growth factor receptor required for productive interaction with the bovine papillomavirus E5 protein.

PubMed

Petti, L M; Reddy, V; Smith, S O; DiMaio, D

1997-10-01

The bovine papillomavirus E5 protein forms a stable complex with the cellular platelet-derived growth factor (PDGF) beta receptor, resulting in receptor activation and cell transformation. Amino acids in both the putative transmembrane domain and extracytoplasmic carboxyl-terminal domain of the E5 protein appear important for PDGF receptor binding and activation. Previous analysis indicated that the transmembrane domain of the receptor was also required for complex formation and receptor activation. Here we analyzed receptor chimeras and point mutants to identify specific amino acids in the PDGF beta receptor required for productive interaction with the E5 protein. These receptor mutants were analyzed in murine Ba/F3 cells, which do not express endogenous receptor. Our results confirmed the importance of the transmembrane domain of the receptor for complex formation, receptor tyrosine phosphorylation, and mitogenic signaling in response to the E5 protein and established that the threonine residue in this domain is required for these activities. In addition, a positive charge in the extracellular juxtamembrane domain of the receptor was required for E5 interaction and signaling, whereas replacement of the wild-type lysine with either a neutral or acidic amino acid inhibited E5-induced receptor activation and transformation. All of the receptor mutants defective for activation by the E5 protein responded to acute treatment with PDGF and to stable expression of v-Sis, a form of PDGF. The required juxtamembrane lysine and transmembrane threonine are predicted to align precisely on the same face of an alpha helix packed in a left-handed coiled-coil geometry. These results establish that the E5 protein and v-Sis recognize distinct binding sites on the PDGF beta receptor and further clarify the nature of the interaction between the viral transforming protein and its cellular target.

Origin Licensing Requires ATP Binding and Hydrolysis by the MCM Replicative Helicase

PubMed Central

Coster, Gideon; Frigola, Jordi; Beuron, Fabienne; Morris, Edward P.; Diffley, John F.X.

2014-01-01

Summary Loading of the six related Minichromosome Maintenance (MCM) proteins as head-to-head double hexamers during DNA replication origin licensing is crucial for ensuring once-per-cell-cycle DNA replication in eukaryotic cells. Assembly of these prereplicative complexes (pre-RCs) requires the Origin Recognition Complex (ORC), Cdc6, and Cdt1. ORC, Cdc6, and MCM are members of the AAA+ family of ATPases, and pre-RC assembly requires ATP hydrolysis. Here we show that ORC and Cdc6 mutants defective in ATP hydrolysis are competent for origin licensing. However, ATP hydrolysis by Cdc6 is required to release nonproductive licensing intermediates. We show that ATP binding stabilizes the wild-type MCM hexamer. Moreover, by analyzing MCM containing mutant subunits, we show that ATP binding and hydrolysis by MCM are required for Cdt1 release and double hexamer formation. This work alters our view of how ATP is used by licensing factors to assemble pre-RCs. PMID:25087873

Absorption spectroscopic and FTIR studies on EDA complexes between TNT (2,4,6-trinitrotoluene) with amines in DMSO and determination of the vertical electron affinity of TNT.

PubMed

Sharma, S P; Lahiri, S C

2008-06-01

TNT (2,4,6-trinitrotoluene) formed deep red 1:1 CT complexes with chromogenic agents like isopropylamine, ethylenediamine, bis(3-aminopropyl)amine and tetraethylenepentamine in DMSO. The complexes were also observed in solvents like methanol, acetone, etc. when the amines were present in large excess. The isopropylamine, complex showed three absorption peaks (at 378, 532 and 629 nm) whereas higher amines showed four peaks (at 370, 463, 532 and 629 nm). The peak at 463 nm vanished rapidly. The peak of the complexes near 530 nm required about 8-10 min to develop and the complexes were stable for about an hour but the peak slowly shifted towards 500 nm and the complexes were found to be stable for more than 24 h. The evidence of complex formation was obtained from distinct spots in HPTLC plates and from the shifts in frequencies and formation of new peaks in FTIR spectra. The peaks near 460 nm (transient) and 530 nm may be due to Janovsky reaction but could not be established. The extinction coefficients of the complexes were determined directly which enabled the accurate determination of the association constants KDA with TNT and amines in stoichiometric ratios. The results were verified using iterative method. The quantification of TNT was made using epsilon value of the complex with ethylenediamine. The vertical electron affinity (EA) of TNT was calculated using the method suggested by Mulliken.

Formation Control of the MAXIM L2 Libration Orbit Mission

NASA Technical Reports Server (NTRS)

Folta, David; Hartman, Kate; Howell, Kathleen; Marchand, Belinda

2004-01-01

The Micro-Arcsecond X-ray Imaging Mission (MAXIM), a proposed concept for the Structure and Evolution of the Universe (SEU) Black Hole Imager mission, is designed to make a ten million-fold improvement in X-ray image clarity of celestial objects by providing better than 0.1 micro-arcsecond imaging. Currently the mission architecture comprises 25 spacecraft, 24 as optics modules and one as the detector, which will form sparse sub-apertures of a grazing incidence X-ray interferometer covering the 0.3-10 keV bandpass. This formation must allow for long duration continuous science observations and also for reconfiguration that permits re-pointing of the formation. To achieve these mission goals, the formation is required to cooperatively point at desired targets. Once pointed, the individual elements of the MAXIM formation must remain stable, maintaining their relative positions and attitudes below a critical threshold. These pointing and formation stability requirements impact the control and design of the formation. In this paper, we provide analysis of control efforts that are dependent upon the stability and the configuration and dimensions of the MAXIM formation. We emphasize the utilization of natural motions in the Lagrangian regions to minimize the control efforts and we address continuous control via input feedback linearization (IFL). Results provide control cost, configuration options, and capabilities as guidelines for the development of this complex mission.

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

JAMI: a Java library for molecular interactions and data interoperability.

PubMed

Sivade Dumousseau, M; Koch, M; Shrivastava, A; Alonso-López, D; De Las Rivas, J; Del-Toro, N; Combe, C W; Meldal, B H M; Heimbach, J; Rappsilber, J; Sullivan, J; Yehudi, Y; Orchard, S

2018-04-11

A number of different molecular interactions data download formats now exist, designed to allow access to these valuable data by diverse user groups. These formats include the PSI-XML and MITAB standard interchange formats developed by Molecular Interaction workgroup of the HUPO-PSI in addition to other, use-specific downloads produced by other resources. The onus is currently on the user to ensure that a piece of software is capable of read/writing all necessary versions of each format. This problem may increase, as data providers strive to meet ever more sophisticated user demands and data types. A collaboration between EMBL-EBI and the University of Cambridge has produced JAMI, a single library to unify standard molecular interaction data formats such as PSI-MI XML and PSI-MITAB. The JAMI free, open-source library enables the development of molecular interaction computational tools and pipelines without the need to produce different versions of software to read different versions of the data formats. Software and tools developed on top of the JAMI framework are able to integrate and support both PSI-MI XML and PSI-MITAB. The use of JAMI avoids the requirement to chain conversions between formats in order to reach a desired output format and prevents code and unit test duplication as the code becomes more modular. JAMI's model interfaces are abstracted from the underlying format, hiding the complexity and requirements of each data format from developers using JAMI as a library.

Preferential pathways in complex fracture systems and their influence on large scale transport

NASA Astrophysics Data System (ADS)

Willmann, M.; Mañé, R.; Tyukhova, A.

2017-12-01

Many subsurface applications in complex fracture systems require large-scale predictions. Precise predictions are difficult because of the existence of preferential pathways at different scales. The intrinsic complexity of fracture systems increases within fractured sedimentary formations, because also the coupling of fractures and matrix has to be taken into account. This interplay of fracture system and the sedimentary matrix is strongly controlled by the actual fracture aperture of an individual fracture. And an effective aperture cannot be easily be determined because of the preferential pathways along the fracture plane. We investigate the influence of these preferential pathways on large scale solute transport and upscale the aperture. By explicitly modeling flow and particle tracking in individual fractures, we develop a new effective transport aperture, which is weighted by the aperture along the preferential paths, a Lagrangian aperture. We show that this new aperture is consistently larger than existing definitions of effective flow and transport apertures. Finally, we apply our results to a fractured sedimentary formation in Northern Switzerland.

Mediator structure and rearrangements required for holoenzyme formation.

PubMed

Tsai, Kuang-Lei; Yu, Xiaodi; Gopalan, Sneha; Chao, Ti-Chun; Zhang, Ying; Florens, Laurence; Washburn, Michael P; Murakami, Kenji; Conaway, Ronald C; Conaway, Joan W; Asturias, Francisco J

2017-04-13

The conserved Mediator co-activator complex has an essential role in the regulation of RNA polymerase II transcription in all eukaryotes. Understanding the structure and interactions of Mediator is crucial for determining how the complex influences transcription initiation and conveys regulatory information to the basal transcription machinery. Here we present a 4.4 Å resolution cryo-electron microscopy map of Schizosaccharomyces pombe Mediator in which conserved Mediator subunits are individually resolved. The essential Med14 subunit works as a central backbone that connects the Mediator head, middle and tail modules. Comparison with a 7.8 Å resolution cryo-electron microscopy map of a Mediator-RNA polymerase II holoenzyme reveals that changes in the structure of Med14 facilitate a large-scale Mediator rearrangement that is essential for holoenzyme formation. Our study suggests that access to different conformations and crosstalk between structural elements are essential for the Mediator regulation mechanism, and could explain the capacity of the complex to integrate multiple regulatory signals.

A Survey of Complex Object Technologies for Digital Libraries

NASA Technical Reports Server (NTRS)

Nelson, Michael L.; Argue, Brad; Efron, Miles; Denn, Sheila; Pattuelli, Maria Cristina

2001-01-01

Many early web-based digital libraries (DLs) had implicit assumptions reflected in their architecture that the unit of focus in the DL (frequently "reports" or "e-prints") would only be manifested in a single, or at most a few, common file formats such as PDF or PostScript. DLs have now matured to the point where their contents are commonly no longer simple files. Complex objects in DLs have emerged from in response to various requirements, including: simple aggregation of formats and supporting files, bundling additional information to aid digital preservation, creating opaque digital objects for e-commerce applications, and the incorporation of dynamic services with the traditional data files. We examine a representative (but not necessarily exhaustive) number of current and recent historical web-based complex object technologies and projects that are applicable to DLs: Aurora, Buckets, ComMentor, Cryptolopes, Digibox, Document Management Alliance, FEDORA, Kahn-Wilensky Framework Digital Objects, Metadata Encoding & Transmission Standard, Multivalent Documents, Open eBooks, VERS Encapsulated Objects, and the Warwick Framework.

Strategies in Interventional Radiology: Formation of an Interdisciplinary Center of Vascular Anomalies - Chances and Challenges for Effective and Efficient Patient Management.

PubMed

Sadick, Maliha; Dally, Franz Josef; Schönberg, Stefan O; Stroszczynski, Christian; Wohlgemuth, Walter A

2017-10-01

Background  Radiology is an interdisciplinary field dedicated to the diagnosis and treatment of numerous diseases and is involved in the development of multimodal treatment concepts. Method  Interdisciplinary case management, a broad spectrum of diagnostic imaging facilities and dedicated endovascular radiological treatment options are valuable tools that allow radiology to set up an interdisciplinary center for vascular anomalies. Results  Image-based diagnosis combined with endovascular treatment options is an essential tool for the treatment of patients with highly complex vascular diseases. These vascular anomalies can affect numerous parts of the body so that a multidisciplinary treatment approach is required for optimal patient care. Conclusion  This paper discusses the possibilities and challenges regarding effective and efficient patient management in connection with the formation of an interdisciplinary center for vascular anomalies with strengthening of the clinical role of radiologists. Key points   · Vascular anomalies, which include vascular tumors and malformations, are complex to diagnose and treat.. · There are far more patients with vascular anomalies requiring therapy than interdisciplinary centers for vascular anomalies - there is currently a shortage of dedicated interdisciplinary centers for vascular anomalies in Germany that can provide dedicated care for affected patients.. · Radiology includes a broad spectrum of diagnostic and minimally invasive therapeutic tools which allow the formation of an interdisciplinary center for vascular anomalies for effective, efficient and comprehensive patient management.. Citation Format · Sadick M, Dally FJ, Schönberg SO et al. Strategies in Interventional Radiology: Formation of an Interdisciplinary Center of Vascular Anomalies - Chances and Challenges for Effective and Efficient Patient Management. Fortschr Röntgenstr 2017; 189: 957 - 966. © Georg Thieme Verlag KG Stuttgart · New York.

DNA Polymerase α Subunit Residues and Interactions Required for Efficient Initiation Complex Formation Identified by a Genetic Selection.

PubMed

Lindow, Janet C; Dohrmann, Paul R; McHenry, Charles S

2015-07-03

Biophysical and structural studies have defined many of the interactions that occur between individual components or subassemblies of the bacterial replicase, DNA polymerase III holoenzyme (Pol III HE). Here, we extended our knowledge of residues and interactions that are important for the first step of the replicase reaction: the ATP-dependent formation of an initiation complex between the Pol III HE and primed DNA. We exploited a genetic selection using a dominant negative variant of the polymerase catalytic subunit that can effectively compete with wild-type Pol III α and form initiation complexes, but cannot elongate. Suppression of the dominant negative phenotype was achieved by secondary mutations that were ineffective in initiation complex formation. The corresponding proteins were purified and characterized. One class of mutant mapped to the PHP domain of Pol III α, ablating interaction with the ϵ proofreading subunit and distorting the polymerase active site in the adjacent polymerase domain. Another class of mutation, found near the C terminus, interfered with τ binding. A third class mapped within the known β-binding domain, decreasing interaction with the β2 processivity factor. Surprisingly, mutations within the β binding domain also ablated interaction with τ, suggesting a larger τ binding site than previously recognized. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Stabilization and prolonged reactivity of aqueous-phase ozone with cyclodextrin

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

Dettmer, Adam; Ball, Raymond; Boving, Thomas B.

Recalcitrant organic groundwater contaminants, such as 1,4-dioxane, may require strong oxidants for complete mineralization. However, their efficacy for in-situ chemical oxidation (ISCO) is limited by oxidant decay and reactivity. Hydroxypropyl-β-cyclodextrin (HPβCD) was examined for its ability to stabilize aqueous-phase ozone (O3) and prolong oxidation potential through inclusion complex formation. Partial transformation of HPβCD by O3 was observed. However, HPβCD proved to be sufficiently recalcitrant, because it was only partially degraded in the presence of O3. The formation of a HPβCD:O3 clathrate complex was observed, which stabilized decay of O3. The presence of HPβCD increased the O3 half-life linearly with increasingmore » HPβCD:O3 molar ratio. The O3 half-life in solutions increased by as much as 40-fold relative to HPβCD-free O3 solutions. Observed O3 release from HPβCD and indigo oxidation confirmed that the formation of the inclusion complex is reversible. This proof-of-concept study demonstrates that HPβCD can complex O3 while preserving its reactivity. These results suggest that the use of clathrate stabilizers, such as HPβCD, can support the development of a facilitated-transport enabled ISCO for the O3treatment of groundwater contaminated with recalcitrant compounds.« less

Intermediates in monensin biosynthesis: A late step in biosynthesis of the polyether ionophore monensin is crucial for the integrity of cation binding.

PubMed

Hüttel, Wolfgang; Spencer, Jonathan B; Leadlay, Peter F

2014-01-01

Polyether antibiotics such as monensin are biosynthesised via a cascade of directed ring expansions operating on a putative polyepoxide precursor. The resulting structures containing fused cyclic ethers and a lipophilic backbone can form strong ionophoric complexes with certain metal cations. In this work, we demonstrate for monensin biosynthesis that, as well as ether formation, a late-stage hydroxylation step is crucial for the correct formation of the sodium monensin complex. We have investigated the last two steps in monensin biosynthesis, namely hydroxylation catalysed by the P450 monooxygenase MonD and O-methylation catalysed by the methyl-transferase MonE. The corresponding genes were deleted in-frame in a monensin-overproducing strain of Streptomyces cinnamonensis. The mutants produced the expected monensin derivatives in excellent yields (ΔmonD: 1.13 g L(-1) dehydroxymonensin; ΔmonE: 0.50 g L(-1) demethylmonensin; and double mutant ΔmonDΔmonE: 0.34 g L(-1) dehydroxydemethylmonensin). Single crystals were obtained from purified fractions of dehydroxymonensin and demethylmonensin. X-ray structure analysis revealed that the conformation of sodium dimethylmonensin is very similar to that of sodium monensin. In contrast, the coordination of the sodium ion is significantly different in the sodium dehydroxymonensin complex. This shows that the final constitution of the sodium monensin complex requires this tailoring step as well as polyether formation.

Single site mutations in the hetero-oligomeric Mrp antiporter from alkaliphilic Bacillus pseudofirmus OF4 that affect Na+/H+ antiport activity, sodium exclusion, individual Mrp protein levels, or Mrp complex formation.

PubMed

Morino, Masato; Natsui, Shinsuke; Ono, Tomohiro; Swartz, Talia H; Krulwich, Terry A; Ito, Masahiro

2010-10-01

Mrp systems are widely distributed and structurally complex cation/proton antiporters. Antiport activity requires hetero-oligomeric complexes of all six or seven hydrophobic Mrp proteins (MrpA-MrpG). Here, a panel of site-directed mutants in conserved or proposed motif residues was made in the Mrp Na(+)(Li(+))/H(+) antiporter from an alkaliphilic Bacillus. The mutant operons were expressed in antiporter-deficient Escherichia coli KNabc and assessed for antiport properties, support of sodium resistance, membrane levels of each Mrp protein, and presence of monomeric and dimeric Mrp complexes. Antiport did not depend on a VFF motif or a conserved tyrosine pair, but a role for a conserved histidine in a potential quinone binding site of MrpA was supported. The importance of several acidic residues for antiport was confirmed, and the importance of additional residues was demonstrated (e.g. three lysine residues conserved across MrpA, MrpD, and membrane-bound respiratory Complex I subunits (NuoL/M/N)). The results extended indications that MrpE is required for normal membrane levels of other Mrp proteins and for complex formation. Moreover, mutations in several other Mrp proteins lead to greatly reduced membrane levels of MrpE. Thus, changes in either of the two Mrp modules, MrpA-MrpD and MrpE-MrpG, influence the other. Two mutants, MrpB-P37G and MrpC-Q70A, showed a normal phenotype but lacked the MrpA-MrpG monomeric complex while retaining the dimeric hetero-oligomeric complex. Finally, MrpG-P81A and MrpG-P81G mutants exhibited no antiport activity but supported sodium resistance and a low [Na(+)](in). Such mutants could be used to screen hypothesized but uncharacterized sodium efflux functions of Mrp apart from Na(+) (Li(+))/H(+) antiport.

A Toxoplasma MORN1 Null Mutant Undergoes Repeated Divisions but Is Defective in Basal Assembly, Apicoplast Division and Cytokinesis

PubMed Central

Lorestani, Alexander; Sheiner, Lilach; Yang, Kevin; Robertson, Seth D.; Sahoo, Nivedita; Brooks, Carrie F.; Ferguson, David J. P.; Striepen, Boris; Gubbels, Marc-Jan

2010-01-01

The membrane occupation and recognition nexus protein 1 (MORN1) is highly conserved among apicomplexan parasites and is associated with several structures that have a role in cell division. Here we dissected the role of MORN1 using the relatively simple budding process of Toxoplasma gondii as a model. Ablation of MORN1 in a conditional null mutant resulted in pronounced defects suggesting a central role for MORN1 in apicoplast segregation and in daughter cell budding. Lack of MORN1 resulted in double-headed parasites. These Janus-headed parasites form two complete apical complexes but fail to assemble a basal complex. Moreover, these parasites were capable of undergoing several more budding rounds resulting in the formation of up to 16-headed parasites conjoined at the basal end. Despite this segregation defect, the mother's cytoskeleton was completely disassembled in every budding round. Overall this argues that successful completion of the budding is not required for cell cycle progression. None of the known basal complex components, including a set of recently identified inner membrane complex (IMC) proteins, localized correctly in these multi-headed parasites. These data suggest that MORN1 is essential for assembly of the basal complex, and that lack of the basal complex abolishes the contractile capacity assigned to the basal complex late in daughter formation. Consistent with this hypothesis we observe that MORN1 mutants fail to efficiently constrict and divide the apicoplast. We used the null background provided by the mutant to dissect the function of subdomains of the MORN1 protein. This demonstrated that deletion of a single MORN domain already prevented the function of MORN1 whereas a critical role for the short linker between MORN domains 6 and 7 was identified. In conclusion, MORN1 is required for basal complex assembly and loss of MORN1 results in defects in apicoplast division and daughter segregation. PMID:20808817

Sequence-dependent DNA flexibility mediates DNase I cleavage.

PubMed

Heddi, Brahim; Abi-Ghanem, Josephine; Lavigne, Marc; Hartmann, Brigitte

2010-01-08

Understanding the preference of nonspecific proteins for certain DNA structural features requires an accurate description of the properties of free DNA, especially regarding their possible predisposition to adopt a conformation that favors the formation of a complex. Exploiting previous exhaustive NMR studies performed on free DNA oligomers, we investigated the molecular basis of DNase I sensitivity under conditions where DNase I binding limits the probability of cleavage. We showed that cleavage intensity was correlated with adjacent 3' phosphate linkage flexibility, monitored by (31)P chemical shifts. Examining NMR-refined DNA structures highlighted that sequence-dependent flexible phosphates were associated with large minor groove variations that may promote the affinity of DNase I, according to relevant DNA-protein complexes. In sum, this work demonstrates that specificity in DNA-DNase I interaction is mediated by DNA flexibility, which influences the induced-fit transitions required to form productive complexes.

WAVE2 Regulates High-Affinity Integrin Binding by Recruiting Vinculin and Talin to the Immunological Synapse▿

PubMed Central

Nolz, Jeffrey C.; Medeiros, Ricardo B.; Mitchell, Jason S.; Zhu, Peimin; Freedman, Bruce D.; Shimizu, Yoji; Billadeau, Daniel D.

2007-01-01

T-cell-receptor (TCR)-mediated integrin activation is required for T-cell-antigen-presenting cell conjugation and adhesion to extracellular matrix components. While it has been demonstrated that the actin cytoskeleton and its regulators play an essential role in this process, no mechanism has been established which directly links TCR-induced actin polymerization to the activation of integrins. Here, we demonstrate that TCR stimulation results in WAVE2-ARP2/3-dependent F-actin nucleation and the formation of a complex containing WAVE2, ARP2/3, vinculin, and talin. The verprolin-connecting-acidic (VCA) domain of WAVE2 mediates the formation of the ARP2/3-vinculin-talin signaling complex and talin recruitment to the immunological synapse (IS). Interestingly, although vinculin is not required for F-actin or integrin accumulation at the IS, it is required for the recruitment of talin. In addition, RNA interference of either WAVE2 or vinculin inhibits activation-dependent induction of high-affinity integrin binding to VCAM-1. Overall, these findings demonstrate a mechanism in which signals from the TCR produce WAVE2-ARP2/3-mediated de novo actin polymerization, leading to integrin clustering and high-affinity binding through the recruitment of vinculin and talin. PMID:17591693

WAVE2 regulates high-affinity integrin binding by recruiting vinculin and talin to the immunological synapse.

PubMed

Nolz, Jeffrey C; Medeiros, Ricardo B; Mitchell, Jason S; Zhu, Peimin; Freedman, Bruce D; Shimizu, Yoji; Billadeau, Daniel D

2007-09-01

T-cell-receptor (TCR)-mediated integrin activation is required for T-cell-antigen-presenting cell conjugation and adhesion to extracellular matrix components. While it has been demonstrated that the actin cytoskeleton and its regulators play an essential role in this process, no mechanism has been established which directly links TCR-induced actin polymerization to the activation of integrins. Here, we demonstrate that TCR stimulation results in WAVE2-ARP2/3-dependent F-actin nucleation and the formation of a complex containing WAVE2, ARP2/3, vinculin, and talin. The verprolin-connecting-acidic (VCA) domain of WAVE2 mediates the formation of the ARP2/3-vinculin-talin signaling complex and talin recruitment to the immunological synapse (IS). Interestingly, although vinculin is not required for F-actin or integrin accumulation at the IS, it is required for the recruitment of talin. In addition, RNA interference of either WAVE2 or vinculin inhibits activation-dependent induction of high-affinity integrin binding to VCAM-1. Overall, these findings demonstrate a mechanism in which signals from the TCR produce WAVE2-ARP2/3-mediated de novo actin polymerization, leading to integrin clustering and high-affinity binding through the recruitment of vinculin and talin.

Requirement of kinesin-mediated membrane transport of WAVE2 along microtubules for lamellipodia formation promoted by hepatocyte growth factor.

PubMed

Takahashi, Kazuhide; Suzuki, Katsuo

2008-07-01

Lamellipodia formation necessary for epithelial cell migration and invasion is accomplished by rearrangement of the actin cytoskeleton at the leading edge through membrane transport of WAVE2. However, how WAVE2 is transported to the cell periphery where lamellipodia are formed remains to be established. We report here that hepatocyte growth factor (HGF) promoted lamellipodia formation and intracellular transport of WAVE2 to the cell periphery, depending on Rac1 activity, in MDA-MB-231 human breast cancer cells. Immunoblot analyses indicating the coimmunoprecipitation of WAVE2 with kinesin heavy chain KIF5B, one of the motor proteins, and IQGAP1 suggest that KIF5B and IQGAP1 formed a complex with WAVE2 in serum-starved cells and increased in their amount after HGF stimulation. Both downregulation of KIF5B by the small interfering RNA and depolymerization of microtubules with nocodazole abrogated the HGF-induced lamellipodia formation and WAVE2 transport. Therefore, we propose here that the promotion of lamellipodia formation by HGF in MDA-MB-231 cells is Rac1-dependent and requires KIF5B-mediated transport of WAVE2 and IQGAP1 to the cell periphery along microtubules.

Roles of the nuclear lamina in stable nuclear association and assembly of a herpesviral transactivator complex on viral immediate-early genes.

PubMed

Silva, Lindsey; Oh, Hyung Suk; Chang, Lynne; Yan, Zhipeng; Triezenberg, Steven J; Knipe, David M

2012-01-01

Little is known about the mechanisms of gene targeting within the nucleus and its effect on gene expression, but most studies have concluded that genes located near the nuclear periphery are silenced by heterochromatin. In contrast, we found that early herpes simplex virus (HSV) genome complexes localize near the nuclear lamina and that this localization is associated with reduced heterochromatin on the viral genome and increased viral immediate-early (IE) gene transcription. In this study, we examined the mechanism of this effect and found that input virion transactivator protein, virion protein 16 (VP16), targets sites adjacent to the nuclear lamina and is required for targeting of the HSV genome to the nuclear lamina, exclusion of heterochromatin from viral replication compartments, and reduction of heterochromatin on the viral genome. Because cells infected with the VP16 mutant virus in1814 showed a phenotype similar to that of lamin A/C(-/-) cells infected with wild-type virus, we hypothesized that the nuclear lamina is required for VP16 activator complex formation. In lamin A/C(-/-) mouse embryo fibroblasts, VP16 and Oct-1 showed reduced association with the viral IE gene promoters, the levels of VP16 and HCF-1 stably associated with the nucleus were lower than in wild-type cells, and the association of VP16 with HCF-1 was also greatly reduced. These results show that the nuclear lamina is required for stable nuclear localization and formation of the VP16 activator complex and provide evidence for the nuclear lamina being the site of assembly of the VP16 activator complex. The targeting of chromosomes in the cell nucleus is thought to be important in the regulation of expression of genes on the chromosomes. The major documented effect of intranuclear targeting has been silencing of chromosomes at sites near the nuclear periphery. In this study, we show that targeting of the herpes simplex virus DNA genome to the nuclear periphery promotes formation of transcriptional activator complexes on the viral genome, demonstrating that the nuclear periphery also has sites for activation of transcription. These results highlight the importance of the nuclear lamina, the structure that lines the inner nuclear membrane, in both transcriptional activation and repression. Future studies defining the molecular structures of these two types of nuclear sites should define new levels of gene regulation.

Immune complex-induced human monocyte procoagulant activity. I. a rapid unidirectional lymphocyte-instructed pathway.

PubMed

Schwartz, B S; Edgington, T S

1981-09-01

It has previously been described that soluble antigen:antibody complexes in antigen excess can induce an increase in the procoagulant activity of human peripheral blood mononuclear cells. It has been proposed that this response may explain the presence of fibrin in immune complex-mediated tissue lesions. In the present study we define cellular participants and their roles in the procoagulant response to soluble immune complexes. Monocytes were shown by cell fractionation and by a direct cytologic assay to be the cell of origin of the procoagulant activity; and virtually all monocytes were able to participate in the response. Monocytes, however, required the presence of lymphocytes to respond. The procoagulant response required cell cooperation, and this collaborative interaction between lymphocytes and monocytes appeared to be unidirectional. Lymphocytes once triggered by immune complexes induced monocytes to synthesize the procoagulant product. Intact viable lymphocytes were required to present instructions to monocytes; no soluble mediator could be found to subserve this function. Indeed, all that appeared necessary to induce monocytes to produce procoagulant activity was an encounter with lymphocytes that had previously been in contact with soluble immune complexes. The optimum cellular ratio for this interaction was four lymphocytes per monocyte, about half the ratio in peripheral blood. The procoagulant response was rapid, reaching a maximum within 6 h after exposure to antigen:antibody complexes. The procoagulant activity was consistent with tissue factor because Factors VII and X and prothrombin were required for clotting of fibrinogen. WE propose that this pathway differs from a number of others involving cells of the immune system. Elucidation of the pathway may clarify the role of this lymphocyte-instructed monocyte response in the Shwartzman phenomenon and other thrombohemorrhagic events associated with immune cell function and the formation of immune complexes.

PCI: A PATRAN-NASTRAN model translator

NASA Technical Reports Server (NTRS)

Sheerer, T. J.

1990-01-01

The amount of programming required to develop a PATRAN-NASTRAN translator was surprisingly small. The approach taken produced a highly flexible translator comparable with the PATNAS translator and superior to the PATCOS translator. The coding required varied from around ten lines for a shell element to around thirty for a bar element, and the time required to add a feature to the program is typically less than an hour. The use of a lookup table for element names makes the translator also applicable to other versions of NASTRAN. The saving in time as a result of using PDA's Gateway utilities was considerable. During the writing of the program it became apparent that, with a somewhat more complex structure, it would be possible to extend the element data file to contain all data required to define the translation from PATRAN to NASTRAN by mapping of data between formats. Similar data files on property, material and grid formats would produce a completely universal translator from PATRAN to any FEA program, or indeed any CAE system.

Molecular requirements for actin-based lamella formation in Drosophila S2 cells

PubMed Central

Rogers, Stephen L.; Wiedemann, Ursula; Stuurman, Nico; Vale, Ronald D.

2003-01-01

Cell migration occurs through the protrusion of the actin-enriched lamella. Here, we investigated the effects of RNAi depletion of ∼90 proteins implicated in actin function on lamella formation in Drosophila S2 cells. Similar to in vitro reconstitution studies of actin-based Listeria movement, we find that lamellae formation requires a relatively small set of proteins that participate in actin nucleation (Arp2/3 and SCAR), barbed end capping (capping protein), filament depolymerization (cofilin and Aip1), and actin monomer binding (profilin and cyclase-associated protein). Lamellae are initiated by parallel and partially redundant signaling pathways involving Rac GTPases and the adaptor protein Nck, which stimulate SCAR, an Arp2/3 activator. We also show that RNAi of three proteins (kette, Abi, and Sra-1) known to copurify with and inhibit SCAR in vitro leads to SCAR degradation, revealing a novel function of this protein complex in SCAR stability. Our results have identified an essential set of proteins involved in actin dynamics during lamella formation in Drosophila S2 cells. PMID:12975351

Mouse HFM1/Mer3 Is Required for Crossover Formation and Complete Synapsis of Homologous Chromosomes during Meiosis

PubMed Central

Guiraldelli, Michel F.; Eyster, Craig; Wilkerson, Joseph L.; Dresser, Michael E.; Pezza, Roberto J.

2013-01-01

Faithful chromosome segregation during meiosis requires that homologous chromosomes associate and recombine. Chiasmata, the cytological manifestation of recombination, provide the physical link that holds the homologs together as a pair, facilitating their orientation on the spindle at meiosis I. Formation of most crossover (CO) events requires the assistance of a group of proteins collectively known as ZMM. HFM1/Mer3 is in this group of proteins and is required for normal progression of homologous recombination and proper synapsis between homologous chromosomes in a number of model organisms. Our work is the first study in mammals showing the in vivo function of mouse HFM1. Cytological observations suggest that initial steps of recombination are largely normal in a majority of Hfm1−/− spermatocytes. Intermediate and late stages of recombination appear aberrant, as chromosomal localization of MSH4 is altered and formation of MLH1foci is drastically reduced. In agreement, chiasma formation is reduced, and cells arrest with subsequent apoptosis at diakinesis. Our results indicate that deletion of Hfm1 leads to the elimination of a major fraction but not all COs. Formation of chromosome axial elements and homologous pairing is apparently normal, and Hfm1−/− spermatocytes progress to the end of prophase I without apparent developmental delay or apoptosis. However, synapsis is altered with components of the central region of the synaptonemal complex frequently failing to extend the full length of the chromosome axes. We propose that initial steps of recombination are sufficient to support homology recognition, pairing, and initial chromosome synapsis and that HFM1 is required to form normal numbers of COs and to complete synapsis. PMID:23555294

Geobase Information System Impacts on Space Image Formats

NASA Technical Reports Server (NTRS)

Simonett, D. S. (Editor); Smith, T. R. (Editor); Tobler, W. (Editor); Marks, D. G. (Editor); Frew, J. E. (Editor); Dozier, J. C. (Editor)

1978-01-01

As Geobase Information Systems increase in number, size and complexity, the format compatability of satellite remote sensing data becomes increasingly more important. Because of the vast and continually increasing quantity of data available from remote sensing systems the utility of these data is increasingly dependent on the degree to which their formats facilitate, or hinder, their incorporation into Geobase Information Systems. To merge satellite data into a geobase system requires that they both have a compatible geographic referencing system. Greater acceptance of satellite data by the user community will be facilitated if the data are in a form which most readily corresponds to existing geobase data structures. The conference addressed a number of specific topics and made recommendations.

Regulation of Botulinum Neurotoxin Synthesis and Toxin Complex Formation by Arginine and Glucose in Clostridium botulinum ATCC 3502.

PubMed

Fredrick, Chase M; Lin, Guangyun; Johnson, Eric A

2017-07-01

Botulinum neurotoxin (BoNT), produced by neurotoxigenic clostridia, is the most potent biological toxin known and the causative agent of the paralytic disease botulism. The nutritional, environmental, and genetic regulation of BoNT synthesis, activation, stability, and toxin complex (TC) formation is not well studied. Previous studies indicated that growth and BoNT formation were affected by arginine and glucose in Clostridium botulinum types A and B. In the present study, C. botulinum ATCC 3502 was grown in toxin production medium (TPM) with different levels of arginine and glucose and of three products of arginine metabolism, citrulline, proline, and ornithine. Cultures were analyzed for growth (optical density at 600 nm [OD 600 ]), spore formation, and BoNT and TC formation by Western blotting and immunoprecipitation and for BoNT activity by mouse bioassay. A high level of arginine (20 g/liter) repressed BoNT production approximately 1,000-fold, enhanced growth, slowed lysis, and reduced endospore production by greater than 1,000-fold. Similar effects on toxin production were seen with equivalent levels of citrulline but not ornithine or proline. In TPM lacking glucose, levels of formation of BoNT/A1 and TC were significantly decreased, and extracellular BoNT and TC proteins were partially inactivated after the first day of culture. An understanding of the regulation of C. botulinum growth and BoNT and TC formation should be valuable in defining requirements for BoNT formation in foods and clinical samples, improving the quality of BoNT for pharmaceutical preparations, and elucidating the biological functions of BoNTs for the bacterium. IMPORTANCE Botulinum neurotoxin (BoNT) is a major food safety and bioterrorism concern and is also an important pharmaceutical, and yet the regulation of its synthesis, activation, and stability in culture media, foods, and clinical samples is not well understood. This paper provides insights into the effects of critical nutrients on growth, lysis, spore formation, BoNT and TC production, and stability of BoNTs of C. botulinum We show that for C. botulinum ATCC 3502 cultured in a complex medium, a high level of arginine repressed BoNT expression by ca. 1,000-fold and also strongly reduced sporulation. Arginine stimulated growth and compensated for a lack of glucose. BoNT and toxin complex proteins were partially inactivated in a complex medium lacking glucose. This work should aid in optimizing BoNT production for pharmaceutical uses, and furthermore, an understanding of the nutritional regulation of growth and BoNT formation may provide insights into growth and BoNT formation in foods and clinical samples and into the enigmatic function of BoNTs in nature. Copyright © 2017 American Society for Microbiology.

The ribosome-associated complex antagonizes prion formation in yeast.

PubMed

Amor, Alvaro J; Castanzo, Dominic T; Delany, Sean P; Selechnik, Daniel M; van Ooy, Alex; Cameron, Dale M

2015-01-01

The number of known fungal proteins capable of switching between alternative stable conformations is steadily increasing, suggesting that a prion-like mechanism may be broadly utilized as a means to propagate altered cellular states. To gain insight into the mechanisms by which cells regulate prion formation and toxicity we examined the role of the yeast ribosome-associated complex (RAC) in modulating both the formation of the [PSI(+)] prion - an alternative conformer of Sup35 protein - and the toxicity of aggregation-prone polypeptides. The Hsp40 RAC chaperone Zuo1 anchors the RAC to ribosomes and stimulates the ATPase activity of the Hsp70 chaperone Ssb. We found that cells lacking Zuo1 are sensitive to over-expression of some aggregation-prone proteins, including the Sup35 prion domain, suggesting that co-translational protein misfolding increases in Δzuo1 strains. Consistent with this finding, Δzuo1 cells exhibit higher frequencies of spontaneous and induced prion formation. Cells expressing mutant forms of Zuo1 lacking either a C-terminal charged region required for ribosome association, or the J-domain responsible for Ssb ATPase stimulation, exhibit similarly high frequencies of prion formation. Our findings are consistent with a role for the RAC in chaperoning nascent Sup35 to regulate folding of the N-terminal prion domain as it emerges from the ribosome.

The two sides of the coin: Similarities and differences in the pathomechanisms of fistulas and stricture formations in irritable bowel disease.

PubMed

Scharl, Michael; Bruckner, Ramona S; Rogler, Gerhard

2016-08-01

Fistulas and fibrosis or strictures represent frequent complications in irritable bowel disease (IBD) patients. To date, treatment options for fistulas are limited and surgery is often required. Similarly, no preventive treatment for fibrosis and stricture formation has been established. Frequently, stricture formation and fibrosis precede fistula formation, indicating that both processes may be connected or interrelated. Knowledge about the pathology of both processes is limited. A crucial role for the epithelial-to-mesenchymal transition (EMT) in fistula development has been demonstrated. Of note, EMT also plays a major role in the pathogenesis of fibrosis in many organs, and most likely also plays that role in the intestine. In addition, aberrant matrix remodeling, as well as soluble factors such as tumor necrosis factor (TNF), interleukin 13 (IL-13) and tumor growth factor beta (TGFβ) were involved, both in the onset of the fistula and fibrosis formation. Both fistulas and fibrosis may occur due to deregulated wound healing mechanisms from chronic and severe intestinal inflammation; however, further research is required to obtain a better understanding of the complex pathophysiology of fistula and intestinal fibrosis formation, to allow the development of new and more effective preventive treatment options for those important disease complications.

Rac1 Protein Signaling Is Required for DNA Damage Response Stimulated by Topoisomerase II Poisons*

PubMed Central

Huelsenbeck, Stefanie C.; Schorr, Anne; Roos, Wynand P.; Huelsenbeck, Johannes; Henninger, Christian; Kaina, Bernd; Fritz, Gerhard

2012-01-01

To investigate the potency of the topoisomerase II (topo II) poisons doxorubicin and etoposide to stimulate the DNA damage response (DDR), S139 phosphorylation of histone H2AX (γH2AX) was analyzed using rat cardiomyoblast cells (H9c2). Etoposide caused a dose-dependent increase in the γH2AX level as shown by Western blotting. By contrast, the doxorubicin response was bell-shaped with high doses failing to increase H2AX phosphorylation. Identical results were obtained by immunohistochemical analysis of γH2AX focus formation, comet assay-based DNA strand break analysis, and measuring the formation of the topo II-DNA cleavable complex. At low dose, doxorubicin activated ataxia telangiectasia mutated (ATM) but not ATM and Rad3-related (ATR). Both the lipid-lowering drug lovastatin and the Rac1-specific inhibitor NSC23766 attenuated doxorubicin- and etoposide-stimulated H2AX phosphorylation, induction of DNA strand breaks, and topo II-DNA complex formation. Lovastatin and NSC23766 acted in an additive manner. They did not attenuate doxorubicin-induced increase in p-ATM and p-Chk2 levels. DDR stimulated by topo II poisons was partially blocked by inhibition of type I p21-associated kinases. DDR evoked by the topoisomerase I poison topotecan remained unaffected by lovastatin. The data show that the mechanisms involved in DDR stimulated by topo II poisons are agent-specific with anthracyclines lacking DDR-stimulating activity at high doses. Pharmacological inhibition of Rac1 signaling counteracts doxorubicin- and etoposide-stimulated DDR by disabling the formation of the topo II-DNA cleavable complex. Based on the data we suggest that Rac1-regulated mechanisms are required for DNA damage induction and subsequent activation of the DDR following treatment with topo II but not topo I poisons. PMID:23012366

Rac1 protein signaling is required for DNA damage response stimulated by topoisomerase II poisons.

PubMed

Huelsenbeck, Stefanie C; Schorr, Anne; Roos, Wynand P; Huelsenbeck, Johannes; Henninger, Christian; Kaina, Bernd; Fritz, Gerhard

2012-11-09

To investigate the potency of the topoisomerase II (topo II) poisons doxorubicin and etoposide to stimulate the DNA damage response (DDR), S139 phosphorylation of histone H2AX (γH2AX) was analyzed using rat cardiomyoblast cells (H9c2). Etoposide caused a dose-dependent increase in the γH2AX level as shown by Western blotting. By contrast, the doxorubicin response was bell-shaped with high doses failing to increase H2AX phosphorylation. Identical results were obtained by immunohistochemical analysis of γH2AX focus formation, comet assay-based DNA strand break analysis, and measuring the formation of the topo II-DNA cleavable complex. At low dose, doxorubicin activated ataxia telangiectasia mutated (ATM) but not ATM and Rad3-related (ATR). Both the lipid-lowering drug lovastatin and the Rac1-specific inhibitor NSC23766 attenuated doxorubicin- and etoposide-stimulated H2AX phosphorylation, induction of DNA strand breaks, and topo II-DNA complex formation. Lovastatin and NSC23766 acted in an additive manner. They did not attenuate doxorubicin-induced increase in p-ATM and p-Chk2 levels. DDR stimulated by topo II poisons was partially blocked by inhibition of type I p21-associated kinases. DDR evoked by the topoisomerase I poison topotecan remained unaffected by lovastatin. The data show that the mechanisms involved in DDR stimulated by topo II poisons are agent-specific with anthracyclines lacking DDR-stimulating activity at high doses. Pharmacological inhibition of Rac1 signaling counteracts doxorubicin- and etoposide-stimulated DDR by disabling the formation of the topo II-DNA cleavable complex. Based on the data we suggest that Rac1-regulated mechanisms are required for DNA damage induction and subsequent activation of the DDR following treatment with topo II but not topo I poisons.

Scientific Workflow Management in Proteomics

PubMed Central

de Bruin, Jeroen S.; Deelder, André M.; Palmblad, Magnus

2012-01-01

Data processing in proteomics can be a challenging endeavor, requiring extensive knowledge of many different software packages, all with different algorithms, data format requirements, and user interfaces. In this article we describe the integration of a number of existing programs and tools in Taverna Workbench, a scientific workflow manager currently being developed in the bioinformatics community. We demonstrate how a workflow manager provides a single, visually clear and intuitive interface to complex data analysis tasks in proteomics, from raw mass spectrometry data to protein identifications and beyond. PMID:22411703

eF-seek: prediction of the functional sites of proteins by searching for similar electrostatic potential and molecular surface shape.

PubMed

Kinoshita, Kengo; Murakami, Yoichi; Nakamura, Haruki

2007-07-01

We have developed a method to predict ligand-binding sites in a new protein structure by searching for similar binding sites in the Protein Data Bank (PDB). The similarities are measured according to the shapes of the molecular surfaces and their electrostatic potentials. A new web server, eF-seek, provides an interface to our search method. It simply requires a coordinate file in the PDB format, and generates a prediction result as a virtual complex structure, with the putative ligands in a PDB format file as the output. In addition, the predicted interacting interface is displayed to facilitate the examination of the virtual complex structure on our own applet viewer with the web browser (URL: http://eF-site.hgc.jp/eF-seek).

Capturing tumor complexity in vitro: Comparative analysis of 2D and 3D tumor models for drug discovery.

PubMed

Stock, Kristin; Estrada, Marta F; Vidic, Suzana; Gjerde, Kjersti; Rudisch, Albin; Santo, Vítor E; Barbier, Michaël; Blom, Sami; Arundkar, Sharath C; Selvam, Irwin; Osswald, Annika; Stein, Yan; Gruenewald, Sylvia; Brito, Catarina; van Weerden, Wytske; Rotter, Varda; Boghaert, Erwin; Oren, Moshe; Sommergruber, Wolfgang; Chong, Yolanda; de Hoogt, Ronald; Graeser, Ralph

2016-07-01

Two-dimensional (2D) cell cultures growing on plastic do not recapitulate the three dimensional (3D) architecture and complexity of human tumors. More representative models are required for drug discovery and validation. Here, 2D culture and 3D mono- and stromal co-culture models of increasing complexity have been established and cross-comparisons made using three standard cell carcinoma lines: MCF7, LNCaP, NCI-H1437. Fluorescence-based growth curves, 3D image analysis, immunohistochemistry and treatment responses showed that end points differed according to cell type, stromal co-culture and culture format. The adaptable methodologies described here should guide the choice of appropriate simple and complex in vitro models.

Capturing tumor complexity in vitro: Comparative analysis of 2D and 3D tumor models for drug discovery

PubMed Central

Stock, Kristin; Estrada, Marta F.; Vidic, Suzana; Gjerde, Kjersti; Rudisch, Albin; Santo, Vítor E.; Barbier, Michaël; Blom, Sami; Arundkar, Sharath C.; Selvam, Irwin; Osswald, Annika; Stein, Yan; Gruenewald, Sylvia; Brito, Catarina; van Weerden, Wytske; Rotter, Varda; Boghaert, Erwin; Oren, Moshe; Sommergruber, Wolfgang; Chong, Yolanda; de Hoogt, Ronald; Graeser, Ralph

2016-01-01

Two-dimensional (2D) cell cultures growing on plastic do not recapitulate the three dimensional (3D) architecture and complexity of human tumors. More representative models are required for drug discovery and validation. Here, 2D culture and 3D mono- and stromal co-culture models of increasing complexity have been established and cross-comparisons made using three standard cell carcinoma lines: MCF7, LNCaP, NCI-H1437. Fluorescence-based growth curves, 3D image analysis, immunohistochemistry and treatment responses showed that end points differed according to cell type, stromal co-culture and culture format. The adaptable methodologies described here should guide the choice of appropriate simple and complex in vitro models. PMID:27364600

Flavivirus Replication Complex Assembly Revealed by DNAJC14 Functional Mapping

PubMed Central

Yi, Zhigang; Yuan, Zhenghong; Rice, Charles M.

2012-01-01

DNAJC14 is an Hsp40 family member that broadly modulates flavivirus replication. The mechanism by which DNAJC14 stoichiometrically participates in flavivirus replication complex (RC) formation is unknown; both reduced and elevated levels result in replication inhibition. Using yellow fever virus (YFV), we demonstrate that DNAJC14 redistributes and clusters with YFV nonstructural proteins via a transmembrane domain and a newly identified membrane-binding domain (MBD), which both mediate targeting to detergent-resistant membranes. Furthermore, the RC and DNAJC14 reside as part of a protein interaction network that remains after 1% Triton solubilization. Mutagenesis studies demonstrate that entry into this protein interaction network requires the DNAJC14 C-terminal self-interaction domain. Fusion of the DNAJC14 MBD and self-interaction domain with another Hsp40 family protein is sufficient to confer YFV-inhibitory activity. Our findings support a novel model of DNAJC14 action that includes specific membrane targeting of both DNAJC14 and YFV replication proteins, the formation of protein interactions, and a microdomain-specific chaperone event leading to RC formation. This process alters the properties of the RC membrane and results in the formation of a protein scaffold that maintains the RC. PMID:22915803

Identification of a novel mitochondrial complex I assembly factor ACDH-12 in Caenorhabditis elegans.

PubMed

Chuaijit, Sirithip; Boonyatistan, Worawit; Boonchuay, Pichsinee; Metheetrairut, Chanatip; Suthammarak, Wichit

2018-03-11

Assembly of complex I of the mitochondrial respiratory chain (MRC) requires not only structural subunits for electron transport, but also assembly factors. In the nematode Caenorhabditis elegans, NUAF-1 and NUAF-3 are the only two assembly factors that have been characterized. In this study, we identify ACDH-12 as an assembly factor of the respiratory complex I. We demonstrate for the first time that a deficiency of ACDH-12 affects the formation and function of complex I. RNAi knockdown of acdh-12 also shortens lifespan and decreases fecundity. Although ACDH-12 has long been recognized as a very long-chain acyl-CoA dehydrogenase (VLCAD), the knockdown nematodes did not exhibit any change in body fat content. We suggested that in Caenorhabditis elegans, ACDH-12 is required for the assembly of the respiratory complex I, but may not be crucial to fatty acid oxidation. Interestingly, sequence analysis shows high homology between ACDH-12 and the human ACAD9, a protein that has initially been identified as a VLCAD, but later found to also be involved in the assembly of complex I in human. Copyright © 2018 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

Polymer dual ring resonators for label-free optical biosensing using microfluidics.

PubMed

Salleh, Muhammad H M; Glidle, Andrew; Sorel, Marc; Reboud, Julien; Cooper, Jonathan M

2013-04-18

We demonstrate a polymer resonator microfluidic biosensor that overcomes the complex manufacturing procedures required to fabricate traditional devices. In this new format, we show that a gapless light coupling photonic configuration, fabricated in SU8 polymer, can achieve high sensitivity, label-free chemical sensing in solution and high sensitivity biological sensing, at visible wavelengths.

Physically absorbable reagents-collectors in elementary flotation

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

S.A. Kondrat'ev; I.G. Bochkarev

2007-09-15

Based on the reviewed researches held at the Institute of Mining, Siberian Branch, Russian Academy of Sciences, the effect of physically absorbable reagents-collectors on formation of a flotation complex and its stability in turbulent pulp flows in flotation machines of basic types is considered. The basic requirements for physically absorbable reagents-collectors at different flotation stages are established.

Photosynthesis-dependent formation of convoluted plasma membrane domains in Chara internodal cells is independent of chloroplast position.

PubMed

Foissner, Ilse; Sommer, Aniela; Hoeftberger, Margit

2015-07-01

The characean green alga Chara australis forms complex plasma membrane convolutions called charasomes when exposed to light. Charasomes are involved in local acidification of the surrounding medium which facilitates carbon uptake required for photosynthesis. They have hitherto been only described in the internodal cells and in close contact with the stationary chloroplasts. Here, we show that charasomes are not only present in the internodal cells of the main axis, side branches, and branchlets but that the plasma membranes of chloroplast-containing nodal cells, protonemata, and rhizoids are also able to invaginate into complex domains. Removal of chloroplasts by local irradiation with intense light revealed that charasomes can develop at chloroplast-free "windows" and that the resulting pH banding pattern is independent of chloroplast or window position. Charasomes were not detected along cell walls containing functional plasmodesmata. However, charasomes formed next to a smooth wound wall which was deposited onto the plasmodesmata-containing wall when the neighboring cell was damaged. In contrast, charasomes were rarely found at uneven, bulged wound walls which protrude into the streaming endoplasm and which were induced by ligation or puncturing. The results of this study show that charasome formation, although dependent on photosynthesis, does not require intimate contact with chloroplasts. Our data suggest further that the presence of plasmodesmata inhibits charasome formation and/or that exposure to the outer medium is a prerequisite for charasome formation. Finally, we hypothesize that the absence of charasomes at bulged wound walls is due to the disturbance of uniform laminar mass streaming.

Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent mechanism.

PubMed

Riveline, D; Zamir, E; Balaban, N Q; Schwarz, U S; Ishizaki, T; Narumiya, S; Kam, Z; Geiger, B; Bershadsky, A D

2001-06-11

The transition of cell-matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II-driven contractility by a Rho target known as Rho-associated kinase (ROCK) was shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, we applied mechanical force to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling led to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein-tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppressed this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology protein mDia1 (Watanabe, N., T. Kato, A. Fujita, T. Ishizaki, and S. Narumiya. 1999. Nat. Cell Biol. 1:136-143), were sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced formation of the focal contacts still occurred in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force.

ESI-MS of Cucurbituril Complexes Under Negative Polarity.

PubMed

Rodrigues, Maria A A; Mendes, Débora C; Ramamurthy, Vaidhyanathan; Da Silva, José P

2017-11-01

Electrospray ionization mass spectrometry (ESI-MS) is a powerful tool to study host-guest supramolecular interactions. ESI-MS can be used for detailed gas-phase reactivity studies, to clarify the structure, or simply to verify the formation of complexes. Depending on the structure of the host and of the guest, negative and/or positive ESI are used. Here we report the unexpected formation of host-guest complexes between cucurbit[n]urils (n = 7, 8, CB[n]) and amine, styryl pyridine, and styryl pyridine dimer cations, under negative ESI. Non-complexed CB[n] form double charged halide (Br - , Cl - , F - ) adducts. Under negative ESI, halide ions interact with CB[n] outer surface hydrogen atoms. One to one host-guest complexes (1:1) of CB[n] with positive charged guests were also observed as single and double charged ions under negative ESI. The positive charge of guests is neutralized by ion-pairing with halide anions. Depending on the number of positive charges guests retain in the gas phase, one or two additional halide ions are required for neutralization. Complexes 1:2 of CB[8] with styryl pyridines retain two halide ions in the gas phase, one per guest. Styryl pyridine dimers form 1:1 complexes possessing a single extra halide ion and therefore a single positive charge. Negative ESI is sensitive to small structural differences between complexes, distinguishing between 1:2 complexes of styryl pyridine-CB[8] and corresponding 1:1 complexes with the dimer. Negative ESI gives simpler spectra than positive ESI and allows the determination of guest charge state of CB[n] complexes in the gas phase. Graphical Abstract ᅟ.

Influence of dose rate on the induction of simple and complex chromosome exchanges by gamma rays.

PubMed

Loucas, Bradford D; Eberle, Richard; Bailey, Susan M; Cornforth, Michael N

2004-10-01

Single-color painting of whole chromosomes, or protocols in which only a few chromosomes are distinctively painted, will always fail to detect a proportion of complex exchanges because they frequently produce pseudosimple painting patterns that are indistinguishable from those produced by bona fide simple exchanges. When 24-color multi-fluor FISH (mFISH) was employed for the purpose of distinguishing (truly) simple from pseudosimple exchanges, it was confirmed that the acute low-LET radiation dose-response relationship for simple exchanges lacked significant upward curvature. This result has been interpreted to indicate that the formation of simple exchanges requires only one chromosome locus be damaged (e.g. broken) by radiation to initiate an exchange-not two, as classical cytogenetic theory maintains. Because a one-lesion mechanism implies single-track action, it follows that the production of simple exchanges should not be influenced by changes in dose rate. To examine this prediction, we irradiated noncycling primary human fibroblasts with graded doses of (137)Cs gamma rays at an acute dose rate of 1.10 Gy/min and compared, using mFISH, the yield of simple exchanges to that observed after exposure to the same radiation delivered at a chronic dose rate of 0.08 cGy/min. The shape of the dose response was found to be quasi-linear for both dose rates, but, counter to providing support for a one-lesion mechanism, the yield of simple aberrations was greatly reduced by protracted exposure. Although chronic doses were delivered at rates low enough to produce damage exclusively by single-track action, this did not altogether eliminate the formation of complex aberrations, an analysis of which leads to the conclusion that a single track of low-LET radiation is capable of inducing complex exchanges requiring up to four proximate breaks for their formation. For acute exposures, the ratio of simple reciprocal translocations to simple dicentrics was near unity.

Oxidative Stress and the Homeodynamics of Iron Metabolism

PubMed Central

Bresgen, Nikolaus; Eckl, Peter M.

2015-01-01

Iron and oxygen share a delicate partnership since both are indispensable for survival, but if the partnership becomes inadequate, this may rapidly terminate life. Virtually all cell components are directly or indirectly affected by cellular iron metabolism, which represents a complex, redox-based machinery that is controlled by, and essential to, metabolic requirements. Under conditions of increased oxidative stress—i.e., enhanced formation of reactive oxygen species (ROS)—however, this machinery may turn into a potential threat, the continued requirement for iron promoting adverse reactions such as the iron/H2O2-based formation of hydroxyl radicals, which exacerbate the initial pro-oxidant condition. This review will discuss the multifaceted homeodynamics of cellular iron management under normal conditions as well as in the context of oxidative stress. PMID:25970586

Interactions of chloride and formate at the donor and the acceptor side of photosystem II.

PubMed

Jajoo, Anjana; Bharti, Sudhakar; Kawamori, Asako

2005-02-01

Chloride is required for the maximum activity of the oxygen evolving complex (OEC) while formate inhibits the function of OEC. On the basis of the measurements of oxygen evolution rates and the S(2) state multiline EPR signal, an interaction between the action of chloride and formate at the donor side of PS II has been suggested. Moreover, the Fe(2)+Q-A EPR signals were measured to investigate a common binding site of both these anions at the PS II acceptor side. Other monovalent anions like bromide, nitrate etc. could influence the effects of formate to a small extent at the donor side of PS II, but not significantly at the acceptor side of PS II. The results presented in this paper clearly suggest a competitive binding of formate and chloride at the PS II acceptor side.

Reaction mechanism of molybdoenzyme formate dehydrogenase.

PubMed

Leopoldini, Monica; Chiodo, Sandro G; Toscano, Marirosa; Russo, Nino

2008-01-01

Formate dehydrogenase is a molybdoenzyme of the anaerobic formate hydrogen lyase complex of the Escherichia coli microorganism that catalyzes the oxidation of formate to carbon dioxide. The two proposed mechanisms of reaction, which differ in the occurrence of a direct coordination or not of a SeCys residue to the molybdenum metal during catalysis were analyzed at the density functional level in both vacuum and protein environments. Some DF functionals, in addition to the very popular B3LYP one, were employed to compute barrier heights. Results revealed the role played by the SeCys residue in performing the abstraction of the proton from the formate substrate. The computation of the energetic profiles for both mechanisms indicated that the reaction barriers are higher when the selenium is directly coordinated to the metal, whereas less energy is required when SeCys is not a ligand at the molybdenum site.

Escherichia coli biofilms have an organized and complex extracellular matrix structure.

PubMed

Hung, Chia; Zhou, Yizhou; Pinkner, Jerome S; Dodson, Karen W; Crowley, Jan R; Heuser, John; Chapman, Matthew R; Hadjifrangiskou, Maria; Henderson, Jeffrey P; Hultgren, Scott J

2013-09-10

Bacterial biofilms are ubiquitous in nature, and their resilience is derived in part from a complex extracellular matrix that can be tailored to meet environmental demands. Although common developmental stages leading to biofilm formation have been described, how the extracellular components are organized to allow three-dimensional biofilm development is not well understood. Here we show that uropathogenic Escherichia coli (UPEC) strains produce a biofilm with a highly ordered and complex extracellular matrix (ECM). We used electron microscopy (EM) techniques to image floating biofilms (pellicles) formed by UPEC. EM revealed intricately constructed substructures within the ECM that encase individual, spatially segregated bacteria with a distinctive morphology. Mutational and biochemical analyses of these biofilms confirmed curli as a major matrix component and revealed important roles for cellulose, flagella, and type 1 pili in pellicle integrity and ECM infrastructure. Collectively, the findings of this study elucidated that UPEC pellicles have a highly organized ultrastructure that varies spatially across the multicellular community. Bacteria can form biofilms in diverse niches, including abiotic surfaces, living cells, and at the air-liquid interface of liquid media. Encasing these cellular communities is a self-produced extracellular matrix (ECM) that can be composed of proteins, polysaccharides, and nucleic acids. The ECM protects biofilm bacteria from environmental insults and also makes the dissolution of biofilms very challenging. As a result, formation of biofilms within humans (during infection) or on industrial material (such as water pipes) has detrimental and costly effects. In order to combat bacterial biofilms, a better understanding of components required for biofilm formation and the ECM is required. This study defined the ECM composition and architecture of floating pellicle biofilms formed by Escherichia coli.

Beclin 1 Is Required for Neuron Viability and Regulates Endosome Pathways via the UVRAG-VPS34 Complex

PubMed Central

Wold, Mitchell S.; Gong, Shiaoching; Phillips, Greg R.; Dou, Zhixun; Zhao, Yanxiang; Heintz, Nathaniel; Zong, Wei-Xing; Yue, Zhenyu

2014-01-01

Deficiency of autophagy protein beclin 1 is implicated in tumorigenesis and neurodegenerative diseases, but the molecular mechanism remains elusive. Previous studies showed that Beclin 1 coordinates the assembly of multiple VPS34 complexes whose distinct phosphatidylinositol 3-kinase III (PI3K-III) lipid kinase activities regulate autophagy at different steps. Recent evidence suggests a function of beclin 1 in regulating multiple VPS34-mediated trafficking pathways beyond autophagy; however, the precise role of beclin 1 in autophagy-independent cellular functions remains poorly understood. Herein we report that beclin 1 regulates endocytosis, in addition to autophagy, and is required for neuron viability in vivo. We find that neuronal beclin 1 associates with endosomes and regulates EEA1/early endosome localization and late endosome formation. Beclin 1 maintains proper cellular phosphatidylinositol 3-phosphate (PI(3)P) distribution and total levels, and loss of beclin 1 causes a disruption of active Rab5 GTPase-associated endosome formation and impairment of endosome maturation, likely due to a failure of Rab5 to recruit VPS34. Furthermore, we find that Beclin 1 deficiency causes complete loss of the UVRAG-VPS34 complex and associated lipid kinase activity. Interestingly, beclin 1 deficiency impairs p40phox-linked endosome formation, which is rescued by overexpressed UVRAG or beclin 1, but not by a coiled-coil domain-truncated beclin 1 (a UVRAG-binding mutant), Atg14L or RUBICON. Thus, our study reveals the essential role for beclin 1 in neuron survival involving multiple membrane trafficking pathways including endocytosis and autophagy, and suggests that the UVRAG-beclin 1 interaction underlies beclin 1's function in endocytosis. PMID:25275521

Subunits of the Snf1 kinase heterotrimer show interdependence for association and activity.

PubMed

Elbing, Karin; Rubenstein, Eric M; McCartney, Rhonda R; Schmidt, Martin C

2006-09-08

The Snf1 kinase and its mammalian orthologue, the AMP-activated protein kinase (AMPK), function as heterotrimers composed of a catalytic alpha-subunit and two non-catalytic subunits, beta and gamma. The beta-subunit is thought to hold the complex together and control subcellular localization whereas the gamma-subunit plays a regulatory role by binding to and blocking the function of an auto-inhibitory domain (AID) present in the alpha-subunit. In addition, catalytic activity requires phosphorylation by a distinct upstream kinase. In yeast, any one of three Snf1-activating kinases, Sak1, Tos3, or Elm1, can fulfill this role. We have previously shown that Sak1 is the only Snf1-activating kinase that forms a stable complex with Snf1. Here we show that the formation of the Sak1.Snf1 complex requires the beta- and gamma-subunits in vivo. However, formation of the Sak1.Snf1 complex is not necessary for glucose-regulated phosphorylation of the Snf1 activation loop. Snf1 kinase purified from cells lacking the beta-subunits do not contain any gamma-subunit, indicating that the Snf1 kinase does not form a stable alphagamma dimer in vivo. In vitro kinase assays using purified full-length and truncated Snf1 proteins demonstrate that the kinase domain, which lacks the AID, is significantly more active than the full-length Snf1 protein. Addition of purified beta- and gamma-subunits could stimulate the kinase activity of the full-length alpha-subunit but only when all three subunits were present, suggesting an interdependence of all three subunits for assembly of a functional complex.

Ran1 functions to control the Cdc10/Sct1 complex through Puc1.

PubMed Central

Caligiuri, M; Connolly, T; Beach, D

1997-01-01

We have undertaken a biochemical analysis of the regulation of the G1/S-phase transition and commitment to the cell cycle in the fission yeast Schizosaccharomyces pombe. The execution of Start requires the activity of the Cdc2 protein kinase and the Sct1/Cdc10 transcription complex. Progression through G1 also requires the Ran1 protein kinase whose inactivation leads to activation of the meiotic pathway under conditions normally inhibitory to this process. We have found that in addition to Cdc2, Sct1/Cdc10 complex formation requires Ran1. We demonstrate that the Puc1 cyclin associates with Ran1 and Cdc10 in vivo and that the Ran1 protein kinase functions to control the association between Puc1 and Cdc10. In addition, we present evidence that the phosphorylation state of Cdc10 is altered upon inactivation of Ran1. These results provide biochemical evidence that demonstrate one mechanism by which the Ran1 protein kinase serves to control cell fate through Cdc10 and Puc1. Images PMID:9201720

Evidence that Mediator is essential for Pol II transcription, but is not a required component of the preinitiation complex in vivo

PubMed Central

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

2017-01-01

The Mediator complex has been described as a general transcription factor, but it is unclear if it is essential for Pol II transcription and/or is a required component of the preinitiation complex (PIC) in vivo. Here, we show that depletion of individual subunits, even those essential for cell growth, causes a general but only modest decrease in transcription. In contrast, simultaneous depletion of all Mediator modules causes a drastic decrease in transcription. Depletion of head or middle subunits, but not tail subunits, causes a downstream shift in the Pol II occupancy profile, suggesting that Mediator at the core promoter inhibits promoter escape. Interestingly, a functional PIC and Pol II transcription can occur when Mediator is not detected at core promoters. These results provide strong evidence that Mediator is essential for Pol II transcription and stimulates PIC formation, but it is not a required component of the PIC in vivo. DOI: http://dx.doi.org/10.7554/eLife.28447.001 PMID:28699889

Monte Carlo Simulations of the Formation Flying Dynamics for the Magnetospheric Multiscale (MMS) Mission

NASA Technical Reports Server (NTRS)

Schiff, Conrad; Dove, Edwin

2011-01-01

The MMS mission is an ambitious space physics mission that will fly 4 spacecraft in a tetrahedron formation in a series of highly elliptical orbits in order to study magnetic reconnection in the Earth's magnetosphere. The mission design is comprised of a combination of deterministic orbit adjust and random maintenance maneuvers distributed over the 2.5 year mission life. Formal verification of the requirements is achieved by analysis through the use of the End-to-End (ETE) code, which is a modular simulation of the maneuver operations over the entire mission duration. Error models for navigation accuracy (knowledge) and maneuver execution (control) are incorporated to realistically simulate the possible maneuver scenarios that might be realized These error models, coupled with the complex formation flying physics, lead to non-trivial effects that must be taken into account by the ETE automation. Using the ETE code, the MMS Flight Dynamics team was able to demonstrate that the current mission design satisfies the mission requirements.

Nuclear Condensation during Mouse Erythropoiesis Requires Caspase-3-Mediated Nuclear Opening.

PubMed

Zhao, Baobing; Mei, Yang; Schipma, Matthew J; Roth, Eric Wayne; Bleher, Reiner; Rappoport, Joshua Z; Wickrema, Amittha; Yang, Jing; Ji, Peng

2016-03-07

Mammalian erythropoiesis involves chromatin condensation that is initiated in the early stage of terminal differentiation. The mechanisms of chromatin condensation during erythropoiesis are unclear. Here, we show that the mouse erythroblast forms large, transient, and recurrent nuclear openings that coincide with the condensation process. The opening lacks nuclear lamina, nuclear pore complexes, and nuclear membrane, but it is distinct from nuclear envelope changes that occur during apoptosis and mitosis. A fraction of the major histones are released from the nuclear opening and degraded in the cytoplasm. We demonstrate that caspase-3 is required for the nuclear opening formation throughout terminal erythropoiesis. Loss of caspase-3 or ectopic expression of a caspase-3 non-cleavable lamin B mutant blocks nuclear opening formation, histone release, chromatin condensation, and terminal erythroid differentiation. We conclude that caspase-3-mediated nuclear opening formation accompanied by histone release from the opening is a critical step toward chromatin condensation during erythropoiesis in mice. Copyright © 2016 Elsevier Inc. All rights reserved.

Nuclear condensation during mouse erythropoiesis requires caspase-3-mediated nuclear opening

PubMed Central

Zhao, Baobing; Mei, Yang; Schipma, Matthew J; Roth, Eric Wayne; Bleher, Reiner; Rappoport, Joshua Z.; Wickrema, Amittha; Yang, Jing; Ji, Peng

2016-01-01

SUMMARY Mammalian erythropoiesis involves chromatin condensation that is initiated in the early stage of terminal differentiation. The mechanisms of chromatin condensation during erythropoiesis are unclear. Here, we show that the mouse erythroblast forms large, transient, and recurrent nuclear openings that coincide with the condensation process. The opening lacks nuclear lamina, nuclear pore complexes, and nuclear membrane, but it is distinct from nuclear envelope changes that occur during apoptosis and mitosis. A fraction of the major histones are released from the nuclear opening and degraded in the cytoplasm. We demonstrate that caspase-3 is required for the nuclear opening formation throughout terminal erythropoiesis. Loss of caspase-3 or ectopic expression of a caspase-3 non-cleavable lamin B mutant blocks nuclear opening formation, histone release, chromatin condensation, and terminal erythroid differentiation. We conclude that caspase-3-mediated nuclear opening formation accompanied by histone release from the opening is a critical step towards chromatin condensation during erythropoiesis in mice. PMID:26954545

Cellular v-ATPase is required for virion assembly compartment formation in human cytomegalovirus infection

PubMed Central

Pavelin, Jonathan; McCormick, Dominique; Chiweshe, Stephen; Ramachandran, Saranya; Lin, Yao-Tang

2017-01-01

Successful generation of virions from infected cells is a complex process requiring orchestrated regulation of host and viral genes. Cells infected with human cytomegalovirus (HCMV) undergo a dramatic reorganization of membrane organelles resulting in the formation of the virion assembly compartment, a process that is not fully understood. Here we show that acidification of vacuoles by the cellular v-ATPase is a crucial step in the formation of the virion assembly compartment and disruption of acidification results in mis-localization of virion components and a profound reduction in infectious virus levels. In addition, knockdown of ATP6V0C blocks the increase in nuclear size, normally associated with HCMV infection. Inhibition of the v-ATPase does not affect intracellular levels of viral DNA synthesis or gene expression, consistent with a defect in assembly and egress. These studies identify a novel host factor involved in virion production and a potential target for antiviral therapy. PMID:29093211

The interaction of insulin with phospholipids

PubMed Central

Perry, M. C.; Tampion, W.; Lucy, J. A.

1971-01-01

1. A simple two-phase chloroform–aqueous buffer system was used to investigate the interaction of insulin with phospholipids and other amphipathic substances. 2. The distribution of 125I-labelled insulin in this system was determined after incubation at 37°C. Phosphatidic acid, dicetylphosphoric acid and, to a lesser extent, phosphatidylcholine and cetyltrimethylammonium bromide solubilized 125I-labelled insulin in the chloroform phase, indicating the formation of chloroform-soluble insulin–phospholipid or insulin–amphipath complexes. Phosphatidylethanolamine, sphingomyelin, cholesterol, stearylamine and Triton X-100 were without effect. 3. Formation of insulin–phospholipid complex was confirmed by paper chromatography. 4. The two-phase system was adapted to act as a simple functional system with which to investigate possible effects of insulin on the structural and functional properties of phospholipid micelles in chloroform, by using the distribution of [14C]glucose between the two phases as a monitor of phospholipid–insulin interactions. The ability of phospholipids to solubilize [14C]glucose in chloroform increased in the order phosphatidylcholine

Formation of HDL-like complexes from apolipoprotein A-I(M) and DMPC.

PubMed

Suurkuusk, M; Singh, S K

2000-01-20

Conditions for the preparation of reconstituted high density lipoproteins (HDLs) by incubation of the synthetic lipid dimyristoylphosphatidylcholine (DMPC) and recombinant apolipoprotein A-I(M) have been investigated as a function of ratio of incubation lipid to protein, incubation temperature and the lipid form (multilamellar (MLV) or small unilamellar (SUV) vesicles). The size distributions of the resultant lipid-protein complex particles from various incubations have been evaluated by native gel electrophoresis. Structural changes of the protein after incorporation into these complex particles have been estimated by CD. Thermal characteristics of the particles has been examined by DSC and correlated with CD results. Titration calorimetry has been used to obtain interaction parameters based on a simplified binding model. It is hypothesized that the major enthalpic step in the production of rHDLs is the primary association step between protein and lipid vesicles. It has been shown that by raising the temperature and incubation ratio, the formation of rHDL particles can be directed towards smaller size and a narrower size distribution. The results have been described on the basis of a model where formation of discoidal particles requires prior saturation of vesicle surface area by adsorbed protein, thus explaining differences between particles formed from MLVs and SUVs.

Radio data archiving system

NASA Astrophysics Data System (ADS)

Knapic, C.; Zanichelli, A.; Dovgan, E.; Nanni, M.; Stagni, M.; Righini, S.; Sponza, M.; Bedosti, F.; Orlati, A.; Smareglia, R.

2016-07-01

Radio Astronomical Data models are becoming very complex since the huge possible range of instrumental configurations available with the modern Radio Telescopes. What in the past was the last frontiers of data formats in terms of efficiency and flexibility is now evolving with new strategies and methodologies enabling the persistence of a very complex, hierarchical and multi-purpose information. Such an evolution of data models and data formats require new data archiving techniques in order to guarantee data preservation following the directives of Open Archival Information System and the International Virtual Observatory Alliance for data sharing and publication. Currently, various formats (FITS, MBFITS, VLBI's XML description files and ancillary files) of data acquired with the Medicina and Noto Radio Telescopes can be stored and handled by a common Radio Archive, that is planned to be released to the (inter)national community by the end of 2016. This state-of-the-art archiving system for radio astronomical data aims at delegating as much as possible to the software setting how and where the descriptors (metadata) are saved, while the users perform user-friendly queries translated by the web interface into complex interrogations on the database to retrieve data. In such a way, the Archive is ready to be Virtual Observatory compliant and as much as possible user-friendly.

Comparison of colorimetry and electrothermal atomic absorption spectroscopy for the quantification of non-transferrin bound iron in human sera.

PubMed

Jittangprasert, Piyada; Wilairat, Prapin; Pootrakul, Pensri

2004-12-01

This paper describes a comparison of two analytical techniques, one employing bathophenanthrolinedisulfonate (BPT), a most commonly-used reagent for Fe (II) determination, as chromogen and an electrothermal atomic absorption spectroscopy (ETAAS) for the quantification of non-transferrin bound iron (NTBI) in sera from thalassemic patients. Nitrilotriacetic acid (NTA) was employed as the ligand for binding iron from low molecular weight iron complexes present in the serum but without removing iron from the transferrin protein. After ultrafiltration the Fe (III)-NTA complex was then quantified by both methods. Kinetic study of the rate of the Fe (II)-BPT complex formation for various excess amounts of NTA ligand was also carried out. The kinetic data show that a minimum time duration (> 60 minutes) is necessary for complete complex formation when large excess of NTA is used. Calibration curves given by colorimetric and ETAAS methods were linear over the range of 0.15-20 microM iron (III). The colorimetric and ETAAS methods exhibited detection limit (3sigma) of 0.13 and 0.14 microM, respectively. The NTBI concentrations from 55 thalassemic serum samples measured employing BPT as chromogen were statistically compared with the results determined by ETAAS. No significant disagreement at 95% confidence level was observed. It is, therefore, possible to select any one of these two techniques for determination of NTBI in serum samples of thalassemic patients. However, the colorimetric procedure requires a longer analysis time because of a slow rate of exchange of NTA ligand with BPT, leading to the slow rate of formation of the colored complex.

Heat-induced formation of a specific binding site for self-assembled Congo Red in the V domain of immunoglobulin L chain lambda.

PubMed

Piekarska, B; Konieczny, L; Rybarska, J; Stopa, B; Zemanek, G; Szneler, E; Król, M; Nowak, M; Roterman, I

2001-11-01

Moderate heating (40-50 degrees C) of immunoglobulins makes them accessible for binding with Congo Red and some related highly associated dyes. The binding is specific and involves supramolecular dye ligands presenting ribbon-like micellar bodies. The L chain lambda dimer, which upon heating disclosed the same binding requirement with respect to supramolecular dye ligands, was used in this work to identify the site of their attachment. Two clearly defined dye-protein (L lambda chain) complexes arise upon heating, here called complex I and complex II. The first is formed at low temperatures (up to 40-45 degrees C) and hence by a still native protein, while the formation of the second one is associated with domain melting above 55 degrees C. They contain 4 and 8 dye molecules bound per L chain monomer, respectively. Complex I also forms efficiently at high dye concentration even at ambient temperature. Complex I and its formation was the object of the present studies. Three structural events that could make the protein accessible to penetration by the large dye ligand were considered to occur in L chains upon heating: local polypeptide chain destabilization, VL-VL domain incoherence, and protein melting. Of these three possibilities, local low-energy structural alteration was found to correlate best with the formation of complex I. It was identified as decreased packing stability of the N-terminal polypeptide chain fragment, which as a result made the V domain accessible for dye penetration. The 19-amino acid N-terminal fragment becomes susceptible to proteolytic cleavage after being replaced by the dye at its packing locus. Its splitting from the dye-protein complex was proved by amino acid sequence analysis. The emptied packing locus, which becomes the site that holds the dye, is bordered by strands of amino acids numbered 74-80 and 105-110, as shown by model analysis. The character of the temperature-induced local polypeptide chain destabilization and its possible role in intramolecular antibody signaling is discussed. Copyright 2001 John Wiley & Sons, Inc.

SWIFT MODELLER: a Java based GUI for molecular modeling.

PubMed

Mathur, Abhinav; Shankaracharya; Vidyarthi, Ambarish S

2011-10-01

MODELLER is command line argument based software which requires tedious formatting of inputs and writing of Python scripts which most people are not comfortable with. Also the visualization of output becomes cumbersome due to verbose files. This makes the whole software protocol very complex and requires extensive study of MODELLER manuals and tutorials. Here we describe SWIFT MODELLER, a GUI that automates formatting, scripting and data extraction processes and present it in an interactive way making MODELLER much easier to use than before. The screens in SWIFT MODELLER are designed keeping homology modeling in mind and their flow is a depiction of its steps. It eliminates the formatting of inputs, scripting processes and analysis of verbose output files through automation and makes pasting of the target sequence as the only prerequisite. Jmol (3D structure visualization tool) has been integrated into the GUI which opens and demonstrates the protein data bank files created by the MODELLER software. All files required and created by the software are saved in a folder named after the work instance's date and time of execution. SWIFT MODELLER lowers the skill level required for the software through automation of many of the steps in the original software protocol, thus saving an enormous amount of time per instance and making MODELLER very easy to work with.

Simulations of photochemical smog formation in complex urban areas

NASA Astrophysics Data System (ADS)

Muilwijk, C.; Schrijvers, P. J. C.; Wuerz, S.; Kenjereš, S.

2016-12-01

In the present study we numerically investigated the dispersion of photochemical reactive pollutants in complex urban areas by applying an integrated Computational Fluid Dynamics (CFD) and Computational Reaction Dynamics (CRD) approach. To model chemical reactions involved in smog generation, the Generic Reaction Set (GRS) approach is used. The GRS model was selected since it does not require detailed modeling of a large set of reactive components. Smog formation is modeled first in the case of an intensive traffic emission, subjected to low to moderate wind conditions in an idealized two-dimensional street canyon with a building aspect ratio (height/width) of one. It is found that Reactive Organic Components (ROC) play an important role in the chemistry of smog formation. In contrast to the NOx/O3 photochemical steady state model that predicts a depletion of the (ground level) ozone, the GRS model predicts generation of ozone. Secondly, the effect of direct sunlight and shadow within the street canyon on the chemical reaction dynamics is investigated for three characteristic solar angles (morning, midday and afternoon). Large differences of up to one order of magnitude are found in the ozone production for different solar angles. As a proof of concept for real urban areas, the integrated CFD/CRD approach is applied for a real scale (1 × 1 km2) complex urban area (a district of the city of Rotterdam, The Netherlands) with high traffic emissions. The predicted pollutant concentration levels give realistic values that correspond to moderate to heavy smog. It is concluded that the integrated CFD/CRD method with the GRS model of chemical reactions is both accurate and numerically robust, and can be used for modeling of smog formation in complex urban areas.

Comparative analysis of the ternary complex factors Elk-1, SAP-1a and SAP-2 (ERP/NET).

PubMed Central

Price, M A; Rogers, A E; Treisman, R

1995-01-01

A transcription factor ternary complex composed of Serum Response Factor (SRF) and Ternary Complex Factor (TCF) mediates the response of the c-fos Serum Response Element (SRE) to growth factors and mitogens. Three Ets domain proteins, Elk-1, SAP-1 and ERP/NET, have been reported to have the properties of TCF. Here we compare Elk-1 and SAP-1a with the human ERP/NET homologue SAP-2. All three TCF RNAs are ubiquitously expressed at similar relative levels. All three proteins contain conserved regions that interact with SRF and the c-fos SRE with comparable efficiency, but in vitro complex formation by SAP-2 is strongly inhibited by its C-terminal sequences. Similarly, only Elk-1 and SAP-1a efficiently bind the c-fos SRE in vivo; ternary complex formation by SAP-2 is weak and is substantially unaffected by serum stimulation or v-ras co-expression. All three TCFs contain C-terminal transcriptional activation domains that are phosphorylated following growth factor stimulation. Activation requires conserved S/T-P motifs found in all the TCF family members. Each TCF activation domain can be phosphorylated in vitro by partially purified ERK2, and ERK activation in vivo is sufficient to potentiate transcriptional activation. Images PMID:7540136

Comparative analysis of the ternary complex factors Elk-1, SAP-1a and SAP-2 (ERP/NET).

PubMed

Price, M A; Rogers, A E; Treisman, R

1995-06-01

A transcription factor ternary complex composed of Serum Response Factor (SRF) and Ternary Complex Factor (TCF) mediates the response of the c-fos Serum Response Element (SRE) to growth factors and mitogens. Three Ets domain proteins, Elk-1, SAP-1 and ERP/NET, have been reported to have the properties of TCF. Here we compare Elk-1 and SAP-1a with the human ERP/NET homologue SAP-2. All three TCF RNAs are ubiquitously expressed at similar relative levels. All three proteins contain conserved regions that interact with SRF and the c-fos SRE with comparable efficiency, but in vitro complex formation by SAP-2 is strongly inhibited by its C-terminal sequences. Similarly, only Elk-1 and SAP-1a efficiently bind the c-fos SRE in vivo; ternary complex formation by SAP-2 is weak and is substantially unaffected by serum stimulation or v-ras co-expression. All three TCFs contain C-terminal transcriptional activation domains that are phosphorylated following growth factor stimulation. Activation requires conserved S/T-P motifs found in all the TCF family members. Each TCF activation domain can be phosphorylated in vitro by partially purified ERK2, and ERK activation in vivo is sufficient to potentiate transcriptional activation.

Positive regulation of meiotic DNA double-strand break formation by activation of the DNA damage checkpoint kinase Mec1(ATR).

PubMed

Gray, Stephen; Allison, Rachal M; Garcia, Valerie; Goldman, Alastair S H; Neale, Matthew J

2013-07-31

During meiosis, formation and repair of programmed DNA double-strand breaks (DSBs) create genetic exchange between homologous chromosomes-a process that is critical for reductional meiotic chromosome segregation and the production of genetically diverse sexually reproducing populations. Meiotic DSB formation is a complex process, requiring numerous proteins, of which Spo11 is the evolutionarily conserved catalytic subunit. Precisely how Spo11 and its accessory proteins function or are regulated is unclear. Here, we use Saccharomyces cerevisiae to reveal that meiotic DSB formation is modulated by the Mec1(ATR) branch of the DNA damage signalling cascade, promoting DSB formation when Spo11-mediated catalysis is compromised. Activation of the positive feedback pathway correlates with the formation of single-stranded DNA (ssDNA) recombination intermediates and activation of the downstream kinase, Mek1. We show that the requirement for checkpoint activation can be rescued by prolonging meiotic prophase by deleting the NDT80 transcription factor, and that even transient prophase arrest caused by Ndt80 depletion is sufficient to restore meiotic spore viability in checkpoint mutants. Our observations are unexpected given recent reports that the complementary kinase pathway Tel1(ATM) acts to inhibit DSB formation. We propose that such antagonistic regulation of DSB formation by Mec1 and Tel1 creates a regulatory mechanism, where the absolute frequency of DSBs is maintained at a level optimal for genetic exchange and efficient chromosome segregation.

WAVE2 is regulated by multiple phosphorylation events within its VCA domain

PubMed Central

Pocha, Shirin M; Cory, Giles O

2009-01-01

The (Wiskott-Aldrich Syndrome Protein)-family verprolin homologous protein (WAVE) family of proteins occupies a pivotal position in the cell, converting extracellular signals into the formation of branched filamentous (F) actin structures. WAVE proteins contain a verprolin central acidic (VCA) domain at their C-terminus, responsible for binding to and activating the Arp2/3 complex, which in-turn nucleates the formation of new actin filaments. Here we identify five Casein Kinase 2 (CK2) phosphorylation sites within the VCA domain of WAVE2, serines 482, 484, 488, 489, and 497. Phosphorylation of these sites is required for a high affinity interaction with the Arp2/3 complex. Phosphorylation of ser 482 and 484 specifically inhibits the activation of the Arp2/3 complex by the WAVE2 VCA domain, but has no effect on the affinity for the Arp2/3 complex when the other phosphorylation sites are occupied. We demonstrate phosphorylation of all five sites on endogenous WAVE2 and show that their mutation to non-phosphorylatable alanine residues inhibits WAVE2 function in vivo, inhibiting cell ruffling and disrupting the integrity of the leading edge of migrating cells. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. PMID:19012317

WAVE2 is regulated by multiple phosphorylation events within its VCA domain.

PubMed

Pocha, Shirin M; Cory, Giles O

2009-01-01

The (Wiskott-Aldrich Syndrome Protein)-family verprolin homologous protein (WAVE) family of proteins occupies a pivotal position in the cell, converting extracellular signals into the formation of branched filamentous (F) actin structures. WAVE proteins contain a verprolin central acidic (VCA) domain at their C-terminus, responsible for binding to and activating the Arp2/3 complex, which in-turn nucleates the formation of new actin filaments. Here we identify five Casein Kinase 2 (CK2) phosphorylation sites within the VCA domain of WAVE2, serines 482, 484, 488, 489, and 497. Phosphorylation of these sites is required for a high affinity interaction with the Arp2/3 complex. Phosphorylation of ser 482 and 484 specifically inhibits the activation of the Arp2/3 complex by the WAVE2 VCA domain, but has no effect on the affinity for the Arp2/3 complex when the other phosphorylation sites are occupied. We demonstrate phosphorylation of all five sites on endogenous WAVE2 and show that their mutation to non-phosphorylatable alanine residues inhibits WAVE2 function in vivo, inhibiting cell ruffling and disrupting the integrity of the leading edge of migrating cells. (c) 2008 Wiley-Liss, Inc.

A model complex of a possible intermediate in the mechanism of action of peptide deformylase: first example of an (N2S)zinc(II)-formate complex.

PubMed

Chang, S C; Sommer, R D; Rheingold, A L; Goldberg, D P

2001-11-21

The synthesis and crystallographic characterization of a new (N2S)zinc-alkyl complex and (N2S)zinc-formate complex is described; the bonding mode of the formate complex has implications for the mechanism of action of the enzyme peptide deformylase.

Establishing an unusual cell type: How to make a dikaryon

PubMed Central

Kruzel, Emilia K.; Hull, Christina M.

2010-01-01

Summary The dikaryons of basidiomycete fungi represent an unusual cell type required for complete sexual development. Dikaryon formation occurs via the activities of cell type-specific homeodomain transcription factors, which form regulatory complexes to establish the dikaryotic state. Decades of classical genetic and cell biological studies in mushrooms have provided a foundation for more recent molecular studies in the pathogenic species Ustilago maydis and Cryptococcus neoformans. Studies in these systems have revealed novel mechanisms of regulation that function downstream of classic homeodomain complexes to ensure that dikaryons are established and propagated. Comparisons of these dikaryon-specific networks promise to reveal the nature of regulatory network evolution and the adaptations responsible for driving complex eukaryotic development. PMID:21036099

Recordkeeping alters economic history by promoting reciprocity

PubMed Central

Basu, Sudipta; Dickhaut, John; Hecht, Gary; Towry, Kristy; Waymire, Gregory

2009-01-01

We experimentally demonstrate a causal link between recordkeeping and reciprocal exchange. Recordkeeping improves memory of past interactions in a complex exchange environment, which promotes reputation formation and decision coordination. Economies with recordkeeping exhibit a beneficially altered economic history where the risks of exchanging with strangers are substantially lessened. Our findings are consistent with prior assertions that complex and extensive reciprocity requires sophisticated memory to store information on past transactions. We offer insights on this research by scientifically demonstrating that reciprocity can be facilitated by information storage external to the brain. This is consistent with the archaeological record, which suggests that prehistoric transaction records and the invention of writing for recordkeeping were linked to increased complexity in human interaction. PMID:19147843

Single Molecule Force Measurement for Protein Synthesis on the Ribosome

NASA Astrophysics Data System (ADS)

Uemura, Sotaro

2008-04-01

The ribosome is a molecular machine that translates the genetic code described on the messenger RNA (mRNA) into an amino acid sequence through repetitive cycles of transfer RNA (tRNA) selection, peptide bond formation and translocation. Although the detailed interactions between the translation components have been revealed by extensive structural and biochemical studies, it is not known how the precise regulation of macromolecular movements required at each stage of translation is achieved. Here we demonstrate an optical tweezer assay to measure the rupture force between a single ribosome complex and mRNA. The rupture force was compared between ribosome complexes assembled on an mRNA with and without a strong Shine-Dalgarno (SD) sequence. The removal of the SD sequence significantly reduced the rupture force, indicating that the SD interactions contribute significantly to the stability of the ribosomal complex on the mRNA in a pre-peptidyl transfer state. In contrast, the post-peptidyl transfer state weakened the rupture force as compared to the complex in a pre-peptidyl transfer state and it was the same for both the SD-containing and SD-deficient mRNAs. The results suggest that formation of the first peptide bond destabilizes the SD interaction, resulting in the weakening of the force with which the ribosome grips an mRNA. This might be an important requirement to facilitate movement of the ribosome along mRNA during the first translocation step. In this article, we discuss about the above new results including the introduction of the ribosome translation mechanism and the optical tweezer method.

Biospecimen Complexity-the Next Challenge for Cancer Research Biobanks?

PubMed

Watson, Peter H

2017-02-15

Purpose: Biospecimens (e.g., tissues, bloods, fluids) are critical for translational cancer research to generate the necessary knowledge to guide implementation of precision medicine. Rising demand and the need for higher quality biospecimens are already evident. Experimental Design: The recent increase in requirement for biospecimen complexity in terms of linked biospecimen types, multiple preservation formats, and longitudinal data was explored by assessing trends in cancer research publications from 2000 to 2014. Results: A PubMed search shows that there has been an increase in both raw numbers and the relative proportion (adjusted for total numbers of articles in each period) of the subgroups of articles typically associated with the use of biospecimens and both dense treatment and/or outcomes data and multiple biospecimen formats. Conclusions: Increasing biospecimen complexity is a largely unrecognized and new pressure on cancer research biobanks. New approaches to cancer biospecimen resources are needed such as the implementation of more efficient and dynamic consent mechanisms, stronger participant involvement in biobank governance, development of requirements for registration of collections, and models to establish stock targets for biobanks. In particular, the latter two approaches would enable funders to establish a better balance between biospecimen supply and research demand, reduce expenditure on duplicate collections, and encourage increased efficiency of biobanks to respond to the research need for more complex cases. This in turn would also enable biobanks to focus more on quality and standardization that are surely factors in the even more important arena of research reproducibility. Clin Cancer Res; 23(4); 894-8. ©2016 AACR . ©2016 American Association for Cancer Research.

Presynaptic dystroglycan-pikachurin complex regulates the proper synaptic connection between retinal photoreceptor and bipolar cells.

PubMed

Omori, Yoshihiro; Araki, Fumiyuki; Chaya, Taro; Kajimura, Naoko; Irie, Shoichi; Terada, Koji; Muranishi, Yuki; Tsujii, Toshinori; Ueno, Shinji; Koyasu, Toshiyuki; Tamaki, Yasuhiro; Kondo, Mineo; Amano, Shiro; Furukawa, Takahisa

2012-05-02

Dystroglycan (DG) is a key component of the dystrophin-glycoprotein complex (DGC) at the neuromuscular junction postsynapse. In the mouse retina, the DGC is localized at the presynapse of photoreceptor cells, however, the function of presynaptic DGC is poorly understood. Here, we developed and analyzed retinal photoreceptor-specific DG conditional knock-out (DG CKO) mice. We found that the DG CKO retina showed a reduced amplitude and a prolonged implicit time of the ERG b-wave. Electron microscopic analysis revealed that bipolar dendrite invagination into the photoreceptor terminus is perturbed in the DG CKO retina. In the DG CKO retina, pikachurin, a DG ligand in the retina, is markedly decreased at photoreceptor synapses. Interestingly, in the Pikachurin(-/-) retina, the DG signal at the ribbon synaptic terminus was severely reduced, suggesting that pikachurin is required for the presynaptic accumulation of DG at the photoreceptor synaptic terminus, and conversely DG is required for pikachurin accumulation. Furthermore, we found that overexpression of pikachurin induces formation and clustering of a DG-pikachurin complex on the cell surface. The Laminin G repeats of pikachurin, which are critical for its oligomerization and interaction with DG, were essential for the clustering of the DG-pikachurin complex as well. These results suggest that oligomerization of pikachurin and its interaction with DG causes DG assembly on the synapse surface of the photoreceptor synaptic terminals. Our results reveal that the presynaptic interaction of pikachurin with DG at photoreceptor terminals is essential for both the formation of proper photoreceptor ribbon synaptic structures and normal retinal electrophysiology.

Nanoaggregation of inclusion complexes of glibenclamide with cyclodextrins.

PubMed

Lucio, David; Irache, Juan Manuel; Font, María; Martínez-Ohárriz, María Cristina

2017-03-15

Glibenclamide is a sulfonylurea used for the oral treatment of type II diabetes mellitus. This drug shows low bioavailability as consequence of its low solubility. In order to solve this problem, the interaction with cyclodextrin has been proposed. This study tries to provide an explanation about the processes involved in the formation of GB-βCDs complexes, which have been interpreted in different ways by several authors. Among native cyclodextrins, βCD presents the most appropriate cavity to host glibenclamide molecules showing A L solubility diagrams (K 1:1 ≈1700M -1 ). However, [Formula: see text] solubility profiles were found for βCD derivatives, highlighting the coexistence of several phenomena involved in the drug solubility enhancement. At low CD concentration, the formation of inclusion complexes can be studied and the stability constants can be calculated (K 1:1 ≈1400M -1 ). Whereas at high CD concentration, the enhancement of GB solubility would be mainly attributed to the formation of nanoaggregates of CD and GB-CD complexes (sizes between 100 and 300nm). The inclusion mode into βCD occurs through the cyclohexyl ring of GB, adopting a semi-folded conformation which maximizes the hydrogen bond network. As consequence of all these phenomena, a 150-fold enhancement of drug solubility has been achieved using β-cyclodextrin derivatives. Thus, its use has proven to be an interesting tool to improve the oral administration of glibenclamide in accordance with dosage bulk and dose/solubility ratio requirements. Copyright © 2017 Elsevier B.V. All rights reserved.

Intermediates in monensin biosynthesis: A late step in biosynthesis of the polyether ionophore monensin is crucial for the integrity of cation binding

PubMed Central

Spencer, Jonathan B; Leadlay, Peter F

2014-01-01

Summary Polyether antibiotics such as monensin are biosynthesised via a cascade of directed ring expansions operating on a putative polyepoxide precursor. The resulting structures containing fused cyclic ethers and a lipophilic backbone can form strong ionophoric complexes with certain metal cations. In this work, we demonstrate for monensin biosynthesis that, as well as ether formation, a late-stage hydroxylation step is crucial for the correct formation of the sodium monensin complex. We have investigated the last two steps in monensin biosynthesis, namely hydroxylation catalysed by the P450 monooxygenase MonD and O-methylation catalysed by the methyl-transferase MonE. The corresponding genes were deleted in-frame in a monensin-overproducing strain of Streptomyces cinnamonensis. The mutants produced the expected monensin derivatives in excellent yields (ΔmonD: 1.13 g L−1 dehydroxymonensin; ΔmonE: 0.50 g L−1 demethylmonensin; and double mutant ΔmonDΔmonE: 0.34 g L−1 dehydroxydemethylmonensin). Single crystals were obtained from purified fractions of dehydroxymonensin and demethylmonensin. X-ray structure analysis revealed that the conformation of sodium dimethylmonensin is very similar to that of sodium monensin. In contrast, the coordination of the sodium ion is significantly different in the sodium dehydroxymonensin complex. This shows that the final constitution of the sodium monensin complex requires this tailoring step as well as polyether formation. PMID:24605157

3D Structure and Interaction of p24β and p24δ Golgi Dynamics Domains: Implication for p24 Complex Formation and Cargo Transport.

PubMed

Nagae, Masamichi; Hirata, Tetsuya; Morita-Matsumoto, Kana; Theiler, Romina; Fujita, Morihisa; Kinoshita, Taroh; Yamaguchi, Yoshiki

2016-10-09

The p24 family consists of four subfamilies (p24α, p24β, p24γ, and p24δ), and the proteins are thought to form hetero-oligomeric complexes for efficient transport of cargo proteins from the endoplasmic reticulum to the Golgi apparatus. The proteins possess a conserved luminal Golgi dynamics (GOLD) domain, whose functions are largely unknown. Here, we present structural and biochemical studies of p24β1 and p24δ1 GOLD domains. Use of GOLD domain-deleted mutants revealed that the GOLD domain of p24δ1 is required for proper p24 hetero-oligomeric complex formation and efficient transport of GPI-anchored proteins. The p24β1 and p24δ1 GOLD domains share a common β-sandwich fold with a characteristic intrasheet disulfide bond. The GOLD domain of p24δ1 crystallized as dimers, allowing the analysis of a homophilic interaction site. Surface plasmon resonance and solution NMR analyses revealed that p24β1 and p24δ1 GOLD domains interact weakly (K d = ~10 -4 M). Bi-protein titration provided interaction site maps. We propose that the heterophilic interaction of p24 GOLD domains contributes to the formation of the p24 hetero-oligomeric complex and to efficient cargo transport. Copyright © 2016 Elsevier Ltd. All rights reserved.

FLO11 expression and lipid biosynthesis are required for air-liquid biofilm formation in a Saccharomyces cerevisiae flor strain.

PubMed

Zara, Giacomo; Goffrini, Paola; Lodi, Tiziana; Zara, Severino; Mannazzu, Ilaria; Budroni, Marilena

2012-11-01

Air-liquid biofilm formation is largely dependent on Flo11p and seems related to cell lipid content and composition. Here, it is shown that in the presence of cerulenin, a known inhibitor of the fatty acid synthase complex, biofilm formation is inhibited together with FLO11 transcription in a flor strain of Saccharomyces cerevisiae, while the administration of saturated fatty acids to cerulenin-containing medium restores biofilm formation and FLO11 transcription. It is also shown that, in biofilm cells, the FLO11 transcription is accompanied by the transcription of ACC1, ACS1 and INO1 key genes in lipid biosynthesis and that biofilm formation is affected by the lack of inositol in flor medium. These results are compatible with the hypothesis that the air-liquid biofilm formation depends on FLO11 transcription levels as well as on fatty acids biosynthesis. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Experimental study of PAM-4, CAP-16, and DMT for 100 Gb/s short reach optical transmission systems.

PubMed

Zhong, Kangping; Zhou, Xian; Gui, Tao; Tao, Li; Gao, Yuliang; Chen, Wei; Man, Jiangwei; Zeng, Li; Lau, Alan Pak Tao; Lu, Chao

2015-01-26

Advanced modulation formats combined with digital signal processing and direct detection is a promising way to realize high capacity, low cost and power efficient short reach optical transmission system. In this paper, we present a detailed investigation on the performance of three advanced modulation formats for 100 Gb/s short reach transmission system. They are PAM-4, CAP-16 and DMT. The detailed digital signal processing required for each modulation format is presented. Comprehensive simulations are carried out to evaluate the performance of each modulation format in terms of received optical power, transmitter bandwidth, relative intensity noise and thermal noise. The performance of each modulation format is also experimentally studied. To the best of our knowledge, we report the first demonstration of a 112 Gb/s transmission over 10km of SSMF employing single band CAP-16 with EML. Finally, a comparison of computational complexity of DSP for the three formats is presented.

Effects of expected-value information and display format on recognition of aircraft subsystem abnormalities

NASA Technical Reports Server (NTRS)

Palmer, Michael T.; Abbott, Kathy H.

1994-01-01

This study identifies improved methods to present system parameter information for detecting abnormal conditions and to identify system status. Two workstation experiments were conducted. The first experiment determined if including expected-value-range information in traditional parameter display formats affected subject performance. The second experiment determined if using a nontraditional parameter display format, which presented relative deviation from expected value, was better than traditional formats with expected-value ranges included. The inclusion of expected-value-range information onto traditional parameter formats was found to have essentially no effect. However, subjective results indicated support for including this information. The nontraditional column deviation parameter display format resulted in significantly fewer errors compared with traditional formats with expected-value-ranges included. In addition, error rates for the column deviation parameter display format remained stable as the scenario complexity increased, whereas error rates for the traditional parameter display formats with expected-value ranges increased. Subjective results also indicated that the subjects preferred this new format and thought that their performance was better with it. The column deviation parameter display format is recommended for display applications that require rapid recognition of out-of-tolerance conditions, especially for a large number of parameters.

Formation of the acrosome complex in the bush cricket Gampsocleis gratiosa (Orthoptera: Tettigoniidae).

PubMed

Su, Cai Xia; Chen, Jie; Shi, Fu Ming; Guo, Ming Shen; Chang, Yan Lin

2017-07-01

The acrosome complex plays an indispensable role in the normal function of mature spermatozoa. However, the dynamic process of acrosome complex formation in insect remains poorly understood. Gampsocleis gratiosa Brunner von Wattenwyl possesses the typical characteristic of insect sperms, which is tractable in terms of size, and therefore was selected for the acrosome formation study in this report. The results show that acrosome formation can be divided into six phases: round, rotating, rhombic, cylindrical, transforming and mature phase, based on the morphological dynamics of acrosome complex and nucleus. In addition, the cytoskeleton plays a critical role in the process of acrosome formation. The results from this study indicate that: (1) glycoprotein is the major component of the acrosome proper; (2) the microfilament is one element of the acrosome complex, and may mediate the morphologic change of the acrosome complex; (3) the microtubules might also shape the nucleus and acrosome complex during the acrosome formation. Copyright © 2017 Elsevier Ltd. All rights reserved.

Automated Processing of Plasma Samples for Lipoprotein Separation by Rate-Zonal Ultracentrifugation.

PubMed

Peters, Carl N; Evans, Iain E J

2016-12-01

Plasma lipoproteins are the primary means of lipid transport among tissues. Defining alterations in lipid metabolism is critical to our understanding of disease processes. However, lipoprotein measurement is limited to specialized centers. Preparation for ultracentrifugation involves the formation of complex density gradients that is both laborious and subject to handling errors. We created a fully automated device capable of forming the required gradient. The design has been made freely available for download by the authors. It is inexpensive relative to commercial density gradient formers, which generally create linear gradients unsuitable for rate-zonal ultracentrifugation. The design can easily be modified to suit user requirements and any potential future improvements. Evaluation of the device showed reliable peristaltic pump accuracy and precision for fluid delivery. We also demonstrate accurate fluid layering with reduced mixing at the gradient layers when compared to usual practice by experienced laboratory personnel. Reduction in layer mixing is of critical importance, as it is crucial for reliable lipoprotein separation. The automated device significantly reduces laboratory staff input and reduces the likelihood of error. Overall, this device creates a simple and effective solution to formation of complex density gradients. © 2015 Society for Laboratory Automation and Screening.

Antimicrobial enzymes: an emerging strategy to fight microbes and microbial biofilms.

PubMed

Thallinger, Barbara; Prasetyo, Endry N; Nyanhongo, Gibson S; Guebitz, Georg M

2013-01-01

With the increasing prevalence of antibiotic resistance, antimicrobial enzymes aimed at the disruption of bacterial cellular machinery and biofilm formation are under intense investigation. Several enzyme-based products have already been commercialized for application in the healthcare, food and biomedical industries. Successful removal of complex biofilms requires the use of multi-enzyme formulations that contain enzymes capable of degrading microbial DNA, polysaccharides, proteins and quorum-sensing molecules. The inclusion of anti-quorum sensing enzymes prevents biofilm reformation. The development of effective complex enzyme formulations is urgently needed to deal with the problems associated with biofilm formation in manufacturing, environmental protection and healthcare settings. Nevertheless, advances in synthetic biology, enzyme engineering and whole DNA-Sequencing technologies show great potential to facilitate the development of more effective antimicrobial and anti-biofilm enzymes. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Probing the role of intercalating protein sidechains for kink formation in DNA

PubMed Central

Sandmann, Achim

2018-01-01

Protein binding can induce DNA kinks, which are for example important to enhance the specificity of the interaction and to facilitate the assembly of multi protein complexes. The respective proteins frequently exhibit amino acid sidechains that intercalate between the DNA base steps at the site of the kink. However, on a molecular level there is only little information available about the role of individual sidechains for kink formation. To unravel structural principles of protein-induced DNA kinking we have performed molecular dynamics (MD) simulations of five complexes that varied in their architecture, function, and identity of intercalated residues. Simulations were performed for the DNA complexes of wildtype proteins (Sac7d, Sox-4, CcpA, TFAM, TBP) and for mutants, in which the intercalating residues were individually or combined replaced by alanine. The work revealed that for systems with multiple intercalated residues, not all of them are necessarily required for kink formation. In some complexes (Sox-4, TBP), one of the residues proved to be essential for kink formation, whereas the second residue has only a very small effect on the magnitude of the kink. In other systems (e.g. Sac7d) each of the intercalated residues proved to be individually capable of conferring a strong kink suggesting a partially redundant role of the intercalating residues. Mutation of the key residues responsible for kinking either resulted in stable complexes with reduced kink angles or caused conformational instability as evidenced by a shift of the kink to an adjacent base step. Thus, MD simulations can help to identify the role of individual inserted residues for kinking, which is not readily apparent from an inspection of the static structures. This information might be helpful for understanding protein-DNA interactions in more detail and for designing proteins with altered DNA binding properties in the future. PMID:29432448

Probing the role of intercalating protein sidechains for kink formation in DNA.

PubMed

Sandmann, Achim; Sticht, Heinrich

2018-01-01

Protein binding can induce DNA kinks, which are for example important to enhance the specificity of the interaction and to facilitate the assembly of multi protein complexes. The respective proteins frequently exhibit amino acid sidechains that intercalate between the DNA base steps at the site of the kink. However, on a molecular level there is only little information available about the role of individual sidechains for kink formation. To unravel structural principles of protein-induced DNA kinking we have performed molecular dynamics (MD) simulations of five complexes that varied in their architecture, function, and identity of intercalated residues. Simulations were performed for the DNA complexes of wildtype proteins (Sac7d, Sox-4, CcpA, TFAM, TBP) and for mutants, in which the intercalating residues were individually or combined replaced by alanine. The work revealed that for systems with multiple intercalated residues, not all of them are necessarily required for kink formation. In some complexes (Sox-4, TBP), one of the residues proved to be essential for kink formation, whereas the second residue has only a very small effect on the magnitude of the kink. In other systems (e.g. Sac7d) each of the intercalated residues proved to be individually capable of conferring a strong kink suggesting a partially redundant role of the intercalating residues. Mutation of the key residues responsible for kinking either resulted in stable complexes with reduced kink angles or caused conformational instability as evidenced by a shift of the kink to an adjacent base step. Thus, MD simulations can help to identify the role of individual inserted residues for kinking, which is not readily apparent from an inspection of the static structures. This information might be helpful for understanding protein-DNA interactions in more detail and for designing proteins with altered DNA binding properties in the future.

Modular "Click" Preparation of Bifunctional Polymeric Heterometallic Catalysts.

PubMed

Wang, Wenlong; Zhao, Liyuan; Lv, Hui; Zhang, Guodong; Xia, Chungu; Hahn, F Ekkehardt; Li, Fuwei

2016-06-27

Heterobimetallic molecular complexes or strictly alternating metallated polymers are obtained by a click reaction between mononuclear metal complexes (secondary building units, SBUs) bearing NHCs functionalized with either p-azidophenyl or p-ethynylphenyl wingtips. With a copper-NHC complex as SBU the formation of molecular or polymeric compounds did not require any additives as the copper complex catalyzes the click reaction. Transmetallation from heterobimetallic Cu/Ag derivatives to Cu/Pd derivatives was achieved. The linker between the SBUs (flexible or rigid) influences the catalytic activity of the heterobimetallic compounds. The polymer with alternating copper-NHC and silver-NHC units and a flexible methylene-triazole bridge between them shows the highest activity in the catalytic alkynylation of trifluoromethyl ketones to give fluorinated propargylic alcohols. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Supercritical carbon dioxide processing of active pharmaceutical ingredients for polymorphic control and for complex formation.

PubMed

Moribe, Kunikazu; Tozuka, Yuichi; Yamamoto, Keiji

2008-02-14

Supercritical fluid technique have been exploited in extraction, separation and crystallization processes. In the field of pharmaceutics, supercritical carbon dioxide (scCO(2)) has been used for the purpose of micronization, polymorphic control, and preparation of solid dispersion and complexes. Particle design of active pharmaceutical ingredients is important to make the solid dosage forms with suitable physicochemical properties. Control of the characteristic properties of particles, such as size, shape, crystal structure and morphology is required to optimize the formulation. For solubility enhancement of poorly water-soluble drugs, preparation of the solid dispersion or the complexation with proper drugs or excipients should be a promising approach. This review focuses on aspects of polymorphic control and complexation behavior of active pharmaceutical ingredients by scCO(2) processing.

Multicopper Oxidase Involvement in Both Mn(II) and Mn(III) Oxidation during Bacterial Formation of MnO2

PubMed Central

Soldatova, Alexandra V.; Butterfield, Cristina; Oyerinde, Oyeyemi F.; Tebo, Bradley M.; Spiro, Thomas G.

2013-01-01

Global cycling of environmental manganese requires catalysis by bacteria and fungi for MnO2 formation, since abiotic Mn(II) oxidation is slow under ambient conditions. Genetic evidence from several bacteria implicates multicopper oxidases (MCOs) as being required for MnO2 formation. However, MCOs catalyze one-electron oxidations, whereas conversion of Mn(II) to MnO2 is a two-electron process. Trapping experiments with pyrophosphate (PP), a Mn(III) chelator, have demonstrated that Mn(III) is an intermediate in Mn(II) oxidation when mediated by exosporium from the Mn-oxidizing bacterium Bacillus SG-1. The reaction of Mn(II) depends on O2 and is inhibited by azide, consistent with MCO catalysis. We show that the subsequent conversion of Mn(III) to MnO2 also depends on O2 and is inhibited by azide. Thus, both oxidation steps appear to be MCO-mediated, likely by the same enzyme, indicated by genetic evidence to be the MnxG gene product. We propose a model of how the manganese oxidase active site may be organized to couple successive electron transfers to the formation of polynuclear Mn(IV) complexes as precursors to MnO2 formation. PMID:22892957

Genetic Dissection of Aversive Associative Olfactory Learning and Memory in Drosophila Larvae

PubMed Central

Widmann, Annekathrin; Artinger, Marc; Biesinger, Lukas; Boepple, Kathrin; Schlechter, Jana; Selcho, Mareike; Thum, Andreas S.

2016-01-01

Memory formation is a highly complex and dynamic process. It consists of different phases, which depend on various neuronal and molecular mechanisms. In adult Drosophila it was shown that memory formation after aversive Pavlovian conditioning includes—besides other forms—a labile short-term component that consolidates within hours to a longer-lasting memory. Accordingly, memory formation requires the timely controlled action of different neuronal circuits, neurotransmitters, neuromodulators and molecules that were initially identified by classical forward genetic approaches. Compared to adult Drosophila, memory formation was only sporadically analyzed at its larval stage. Here we deconstruct the larval mnemonic organization after aversive olfactory conditioning. We show that after odor-high salt conditioning larvae form two parallel memory phases; a short lasting component that depends on cyclic adenosine 3’5’-monophosphate (cAMP) signaling and synapsin gene function. In addition, we show for the first time for Drosophila larvae an anesthesia resistant component, which relies on radish and bruchpilot gene function, protein kinase C activity, requires presynaptic output of mushroom body Kenyon cells and dopamine function. Given the numerical simplicity of the larval nervous system this work offers a unique prospect for studying memory formation of defined specifications, at full-brain scope with single-cell, and single-synapse resolution. PMID:27768692

Genetic Dissection of Aversive Associative Olfactory Learning and Memory in Drosophila Larvae.

PubMed

Widmann, Annekathrin; Artinger, Marc; Biesinger, Lukas; Boepple, Kathrin; Peters, Christina; Schlechter, Jana; Selcho, Mareike; Thum, Andreas S

2016-10-01

Memory formation is a highly complex and dynamic process. It consists of different phases, which depend on various neuronal and molecular mechanisms. In adult Drosophila it was shown that memory formation after aversive Pavlovian conditioning includes-besides other forms-a labile short-term component that consolidates within hours to a longer-lasting memory. Accordingly, memory formation requires the timely controlled action of different neuronal circuits, neurotransmitters, neuromodulators and molecules that were initially identified by classical forward genetic approaches. Compared to adult Drosophila, memory formation was only sporadically analyzed at its larval stage. Here we deconstruct the larval mnemonic organization after aversive olfactory conditioning. We show that after odor-high salt conditioning larvae form two parallel memory phases; a short lasting component that depends on cyclic adenosine 3'5'-monophosphate (cAMP) signaling and synapsin gene function. In addition, we show for the first time for Drosophila larvae an anesthesia resistant component, which relies on radish and bruchpilot gene function, protein kinase C activity, requires presynaptic output of mushroom body Kenyon cells and dopamine function. Given the numerical simplicity of the larval nervous system this work offers a unique prospect for studying memory formation of defined specifications, at full-brain scope with single-cell, and single-synapse resolution.

Atg6/UVRAG/Vps34-Containing Lipid Kinase Complex Is Required for Receptor Downregulation through Endolysosomal Degradation and Epithelial Polarity during Drosophila Wing Development

PubMed Central

Szatmári, Zsuzsanna; Sass, Miklós

2014-01-01

Atg6 (Beclin 1 in mammals) is a core component of the Vps34 PI3K (III) complex, which promotes multiple vesicle trafficking pathways. Atg6 and Vps34 form two distinct PI3K (III) complexes in yeast and mammalian cells, either with Atg14 or with UVRAG. The functions of these two complexes are not entirely clear, as both Atg14 and UVRAG have been suggested to regulate both endocytosis and autophagy. In this study, we performed a microscopic analysis of UVRAG, Atg14, or Atg6 loss-of-function cells in the developing Drosophila wing. Both autophagy and endocytosis are seriously impaired and defective endolysosomes accumulate upon loss of Atg6. We show that Atg6 is required for the downregulation of Notch and Wingless signaling pathways; thus it is essential for normal wing development. Moreover, the loss of Atg6 impairs cell polarity. Atg14 depletion results in autophagy defects with no effect on endocytosis or cell polarity, while the silencing of UVRAG phenocopies all but the autophagy defect of Atg6 depleted cells. Thus, our results indicate that the UVRAG-containing PI3K (III) complex is required for receptor downregulation through endolysosomal degradation and for the establishment of proper cell polarity in the developing wing, while the Atg14-containing complex is involved in autophagosome formation. PMID:25006588

Cofilin, But Not Profilin, Is Required for Myosin-I-Induced Actin Polymerization and the Endocytic Uptake in Yeast

PubMed Central

Idrissi, Fatima-Zahra; Wolf, Bianka L.; Geli, M. Isabel

2002-01-01

Mutations in the budding yeast myosins-I (MYO3 and MYO5) cause defects in the actin cytoskeleton and in the endocytic uptake. Robust evidence also indicates that these proteins induce Arp2/3-dependent actin polymerization. Consistently, we have recently demonstrated, using fluorescence microscopy, that Myo5p is able to induce cytosol-dependent actin polymerization on the surface of Sepharose beads. Strikingly, we now observed that, at short incubation times, Myo5p induced the formation of actin foci that resembled the yeast cortical actin patches, a plasma membrane-associated structure that might be involved in the endocytic uptake. Analysis of the machinery required for the formation of the Myo5p-induced actin patches in vitro demonstrated that the Arp2/3 complex was necessary but not sufficient in the assay. In addition, we found that cofilin was directly involved in the process. Strikingly though, the cofilin requirement seemed to be independent of its ability to disassemble actin filaments and profilin, a protein that closely cooperates with cofilin to maintain a rapid actin filament turnover, was not needed in the assay. In agreement with these observations, we found that like the Arp2/3 complex and the myosins-I, cofilin was essential for the endocytic uptake in vivo, whereas profilin was dispensable. PMID:12429847

Neutralized adenovirus-immune complexes can mediate effective gene transfer via an Fc receptor-dependent infection pathway.

PubMed

Leopold, Philip L; Wendland, Rebecca L; Vincent, Theresa; Crystal, Ronald G

2006-10-01

Neutralization of adenovirus (Ad) by anti-Ad neutralizing antibodies in serum involves formation of Ad-immune complexes that prevent the virus from interacting with target cells. We hypothesized that Ad-immune complexes likely contain viable Ad vectors which, although no longer capable of gaining access to receptors on target cells, may be able to express transgenes in cells bearing Fc receptors for immunoglobulins, i.e., that antibody-based "neutralization" of Ad vectors may be circumvented by the Fc receptor pathway. To test this hypothesis, we expressed the Fcgamma receptor IIA (FcgammaR) in A549 lung epithelial cells or human dermal fibroblasts and evaluated gene transfer in the presence of human neutralizing anti-Ad serum. FcgammaR-expressing cells bound and internalized copious amounts of Ad, with a distinct population of internalized Ad trafficking to the nucleus. The dose-response curves for inhibition of gene transfer revealed that FcgammaR-expressing cells required a more-than-10-fold higher concentration of anti-Ad serum to achieve 50% inhibition of Ad-encoded beta-galactosidase expression compared with non-FcgammaR-expressing cells. The discrepancy between neutralization of Ad during infection of FcgammaR-expressing cells and neutralization of Ad during infection of non-FcgammaR-expressing cells occurred with either heat-inactivated or non-heat-inactivated sera, was blocked by addition of purified Fc domain protein, and did not require the cytoplasmic domain of FcgammaR, suggesting that immune complex internalization proceeded via endocytosis rather than phagocytosis. FcgammaR-mediated infection by Ad-immune complexes did not require expression of the coxsackie virus-Ad receptor (CAR) since similar data were obtained when CAR-deficient human dermal fibroblasts were engineered to express FcgammaR. However, interaction of the Ad penton base with cell surface integrins contributed to the difference in neutralization between FcgammaR-expressing and non-FcgammaR-expressing cells. The data indicate that complexes formed from Ad and anti-Ad neutralizing antibodies, while compromised with respect to infection of non-FcgammaR-expressing target cells, maintain the potential to transfer genes to FcgammaR-expressing cells, with consequent expression of the transgene. The formation of Ad-immune complexes that can target viable virus to antigen-presenting cells may account for the success of Ad-based vaccines administered in the presence of low levels of neutralizing anti-Ad antibody.

IRON AND FREE RADICAL OXIDATIONS IN CELL MEMBRANES

PubMed Central

Schafer, Freya Q.; Yue Qian, Steven; Buettner, Garry R.

2013-01-01

Brain tissue being rich in polyunsaturated fatty acids, is very susceptible to lipid peroxidation. Iron is well known to be an important initiator of free radical oxidations. We propose that the principal route to iron-mediated lipid peroxidations is via iron-oxygen complexes rather than the reaction of iron with hydrogen peroxide, the Fenton reaction. To test this hypothesis, we enriched leukemia cells (K-562 and L1210 cells) with docosahexaenoic acid (DHA) as a model for brain tissue, increasing the amount of DHA from approximately 3 mole % to 32 mole %. These cells were then subjected to ferrous iron and dioxygen to initiate lipid peroxidation in the presence or absence of hydrogen peroxide. Lipid-derived radicals were detected using EPR spin trapping with α-(4-pyridyl-1-oxide)-N-t-butylnitrone (POBN). As expected, lipid-derived radical formation increases with increasing cellular lipid unsaturation. Experiments with Desferal demonstrate that iron is required for the formation of lipid radicals from these cells. Addition of iron to DHA-enriched L1210 cells resulted in significant amounts of radical formation; radical formation increased with increasing amount of iron. However, the exposure of cells to hydrogen peroxide before the addition of ferrous iron did not increase cellular radical formation, but actually decreased spin adduct formation. These data suggest that iron-oxygen complexes are the primary route to the initiation of biological free radical oxidations. This model proposes a mechanism to explain how catalytic iron in brain tissue can be so destructive. PMID:10872752

The significance of ACTH for the process of formation of complex heparin compounds in the blood during immobilization stress

NASA Technical Reports Server (NTRS)

Kudryashov, B. A.; Shapiro, F. B.; Lomovskaya, F. B.; Lyapina, L. A.

1979-01-01

Adrenocorticotropin (ACTH) was administered to rats at different times following adrenalectomy. Adrenocorticotropin caused a significant increase in the formation of heparin complexes even in the absence of stress factor. When ACTH secretion is blocked, immobilization stress is not accompanied by an increase in the process of complex formation. The effect of ACTH on the formation of heparin complexes was mediated through its stimulation of the adrenal cortex.

C-C bond forming radical SAM enzymes involved in the construction of carbon skeletons of cofactors and natural products.

PubMed

Yokoyama, Kenichi; Lilla, Edward A

2018-04-10

Covering: up to the end of 2017C-C bond formations are frequently the key steps in cofactor and natural product biosynthesis. Historically, C-C bond formations were thought to proceed by two electron mechanisms, represented by Claisen condensation in fatty acids and polyketide biosynthesis. These types of mechanisms require activated substrates to create a nucleophile and an electrophile. More recently, increasing number of C-C bond formations catalyzed by radical SAM enzymes are being identified. These free radical mediated reactions can proceed between almost any sp3 and sp2 carbon centers, allowing introduction of C-C bonds at unconventional positions in metabolites. Therefore, free radical mediated C-C bond formations are frequently found in the construction of structurally unique and complex metabolites. This review discusses our current understanding of the functions and mechanisms of C-C bond forming radical SAM enzymes and highlights their important roles in the biosynthesis of structurally complex, naturally occurring organic molecules. Mechanistic consideration of C-C bond formation by radical SAM enzymes identifies the significance of three key mechanistic factors: radical initiation, acceptor substrate activation and radical quenching. Understanding the functions and mechanisms of these characteristic enzymes will be important not only in promoting our understanding of radical SAM enzymes, but also for understanding natural product and cofactor biosynthesis.

Drosophila Chitinase 2 is expressed in chitin producing organs for cuticle formation.

PubMed

Pesch, Yanina-Yasmin; Riedel, Dietmar; Behr, Matthias

2017-01-01

The architecture of the outer body wall cuticle is fundamental to protect arthropods against invading pathogens and numerous other harmful stresses. Such robust cuticles are formed by parallel running chitin microfibrils. Molting and also local wounding leads to dynamic assembly and disassembly of the chitin-matrix throughout development. However, the underlying molecular mechanisms that organize proper chitin-matrix formation are poorly known. Recently we identified a key region for cuticle thickening at the apical cell surface, the cuticle assembly zone, where Obstructor-A (Obst-A) coordinates the formation of the chitin-matrix. Obst-A binds chitin and the deacetylase Serpentine (Serp) in a core complex, which is required for chitin-matrix maturation and preservation. Here we present evidence that Chitinase 2 (Cht2) could be essential for this molecular machinery. We show that Cht2 is expressed in the chitin-matrix of epidermis, trachea, and the digestive system. There, Cht2 is enriched at the apical cell surface and the dense chitin-matrix. We further show that in Cht2 knockdown larvae the assembly zone is rudimentary, preventing normal cuticle formation and pore canal organization. As sequence similarities of Cht2 and the core complex proteins indicate evolutionarily conserved molecular mechanisms, our findings suggest that Cht2 is involved in chitin formation also in other insects. Copyright © 2016 Elsevier Ltd. All rights reserved.

Type 4 pili are dispensable for biofilm development in the cyanobacterium Synechococcus elongatus.

PubMed

Nagar, Elad; Zilberman, Shaul; Sendersky, Eleonora; Simkovsky, Ryan; Shimoni, Eyal; Gershtein, Diana; Herzberg, Moshe; Golden, Susan S; Schwarz, Rakefet

2017-07-01

The hair-like cell appendages denoted as type IV pili are crucial for biofilm formation in diverse eubacteria. The protein complex responsible for type IV pilus assembly is homologous with the type II protein secretion complex. In the cyanobacterium Synechococcus elongatus PCC 7942, the gene Synpcc7942_2071 encodes an ATPase homologue of type II/type IV systems. Here, we report that inactivation of Synpcc7942_2071 strongly affected the suite of proteins present in the extracellular milieu (exo-proteome) and eliminated pili observable by electron microscopy. These results support a role for this gene product in protein secretion as well as in pili formation. As we previously reported, inactivation of Synpcc7942_2071 enables biofilm formation and suppresses the planktonic growth of S. elongatus. Thus, pili are dispensable for biofilm development in this cyanobacterium, in contrast to their biofilm-promoting function in type IV pili-producing heterotrophic bacteria. Nevertheless, pili removal is not required for biofilm formation as evident by a piliated mutant of S. elongatus that develops biofilms. We show that adhesion and timing of biofilm development differ between the piliated and non-piliated strains. The study demonstrates key differences in the process of biofilm formation between cyanobacteria and well-studied type IV pili-producing heterotrophic bacteria. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Keeping a Step Ahead: formative phase of a workplace intervention trial to prevent obesity.

PubMed

Zapka, Jane; Lemon, Stephenie C; Estabrook, Barbara B; Jolicoeur, Denise G

2007-11-01

Ecological interventions hold promise for promoting overweight and obesity prevention in worksites. Given the paucity of evaluative research in the hospital worksite setting, considerable formative work is required for successful implementation and evaluation. This paper describes the formative phases of Step Ahead, a site-randomized controlled trial of a multilevel intervention that promotes physical activity and healthy eating in six hospitals in central Massachusetts. The purpose of the formative research phase was to increase the feasibility, effectiveness, and likelihood of sustainability of the intervention. The Step Ahead ecological intervention approach targets change at the organization, interpersonal work environment, and individual levels. The intervention was developed using fundamental steps of intervention mapping and important tenets of participatory research. Formative research methods were used to engage leadership support and assistance and to develop an intervention plan that is both theoretically and practically grounded. This report uses observational data, program minutes and reports, and process tracking data. Leadership involvement (key informant interviews and advisory boards), employee focus groups and advisory boards, and quantitative environmental assessments cultivated participation and support. Determining multiple foci of change and designing measurable objectives and generic assessment tools to document progress are complex challenges encountered in planning phases. Multilevel trials in diverse organizations require flexibility and balance of theory application and practice-based perspectives to affect impact and outcome objectives. Formative research is an essential component.

OIL—Output input language for data connectivity between geoscientific software applications

NASA Astrophysics Data System (ADS)

Amin Khan, Khalid; Akhter, Gulraiz; Ahmad, Zulfiqar

2010-05-01

Geoscientific computing has become so complex that no single software application can perform all the processing steps required to get the desired results. Thus for a given set of analyses, several specialized software applications are required, which must be interconnected for electronic flow of data. In this network of applications the outputs of one application become inputs of other applications. Each of these applications usually involve more than one data type and may have their own data formats, making them incompatible with other applications in terms of data connectivity. Consequently several data format conversion utilities are developed in-house to provide data connectivity between applications. Practically there is no end to this problem as each time a new application is added to the system, a set of new data conversion utilities need to be developed. This paper presents a flexible data format engine, programmable through a platform independent, interpreted language named; Output Input Language (OIL). Its unique architecture allows input and output formats to be defined independent of each other by two separate programs. Thus read and write for each format is coded only once and data connectivity link between two formats is established by a combination of their read and write programs. This results in fewer programs with no redundancy and maximum reuse, enabling rapid application development and easy maintenance of data connectivity links.

Molecular cell biology and physiology of solute transport

PubMed Central

Caplan, Michael J.; Seo-Mayer, Patricia; Zhang, Li

2010-01-01

Purpose of review An enormous body of research has been focused on exploring the mechanisms through which epithelial cells establish their characteristic polarity. It is clear that under normal circumstances cell–cell contacts mediated by the calcium-dependent adhesion proteins of the intercellular adhesion junctions are required to initiate complete polarization. Furthermore, formation of the tight, or occluding, junctions that limit paracellular permeability has long been thought to help to establish polarity by preventing the diffusion of membrane proteins between the two plasmalemmal domains. This review will discuss several selected kinases and protein complexes and highlight their relevance to transporting epithelial cell polarization. Recent findings Recent work has shed new light on the roles of junctional complexes in establishing and maintaining epithelial cell polarity. In addition, work from several laboratories, suggests that the formation of these junctions is tied to processes that regulate cellular energy metabolism. Summary Junctional complexes and energy sensing kinases constitute a novel class of machinery whose capacity to generate and modulate epithelial cell polarity is likely to have wide ranging and important physiological ramifications. PMID:18695392

A Design Principle for an Autonomous Post-translational Pattern Formation.

PubMed

Sugai, Shuhei S; Ode, Koji L; Ueda, Hiroki R

2017-04-25

Previous autonomous pattern-formation models often assumed complex molecular and cellular networks. This theoretical study, however, shows that a system composed of one substrate with multisite phosphorylation and a pair of kinase and phosphatase can generate autonomous spatial information, including complex stripe patterns. All (de-)phosphorylation reactions are described with a generic Michaelis-Menten scheme, and all species freely diffuse without pre-existing gradients. Computational simulation upon >23,000,000 randomly generated parameter sets revealed the design motifs of cyclic reaction and enzyme sequestration by slow-diffusing substrates. These motifs constitute short-range positive and long-range negative feedback loops to induce Turing instability. The width and height of spatial patterns can be controlled independently by distinct reaction-diffusion processes. Therefore, multisite reversible post-translational modification can be a ubiquitous source for various patterns without requiring other complex regulations such as autocatalytic regulation of enzymes and is applicable to molecular mechanisms for inducing subcellular localization of proteins driven by post-translational modifications. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Decision analysis in clinical cardiology: When is coronary angiography required in aortic stenosis

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

Georgeson, S.; Meyer, K.B.; Pauker, S.G.

1990-03-15

Decision analysis offers a reproducible, explicit approach to complex clinical decisions. It consists of developing a model, typically a decision tree, that separates choices from chances and that specifies and assigns relative values to outcomes. Sensitivity analysis allows exploration of alternative assumptions. Cost-effectiveness analysis shows the relation between dollars spent and improved health outcomes achieved. In a tutorial format, this approach is applied to the decision whether to perform coronary angiography in a patient who requires aortic valve replacement for critical aortic stenosis.

Exercise habit formation in new gym members: a longitudinal study.

PubMed

Kaushal, Navin; Rhodes, Ryan E

2015-08-01

Reasoned action approaches have primarily been applied to understand exercise behaviour for the past three decades, yet emerging findings in unconscious and Dual Process research show that behavior may also be predicted by automatic processes such as habit. The purpose of this study was to: (1) investigate the behavioral requirements for exercise habit formation, (2) how Dual Process approach predicts behaviour, and (3) what predicts habit by testing a model (Lally and Gardner in Health Psychol Rev 7:S137-S158, 2013). Participants (n = 111) were new gym members who completed surveys across 12 weeks. It was found that exercising for at least four bouts per week for 6 weeks was the minimum requirement to establish an exercise habit. Dual Process analysis using Linear Mixed Models (LMM) revealed habit and intention to be parallel predictors of exercise behavior in the trajectory analysis. Finally, the habit antecedent model in LLM showed that consistency (β = .21), low behavioral complexity (β = .19), environment (β = .17) and affective judgments (β = .13) all significantly (p < .05) predicted changes in habit formation over time. Trainers should keep exercises fun and simple for new clients and focus on consistency which could lead to habit formation in nearly 6 weeks.

Morphogenic Protein RodZ Interacts with Sporulation Specific SpoIIE in Bacillus subtilis.

PubMed

Muchová, Katarína; Chromiková, Zuzana; Bradshaw, Niels; Wilkinson, Anthony J; Barák, Imrich

2016-01-01

The first landmark in sporulation of Bacillus subtilis is the formation of an asymmetric septum followed by selective activation of the transcription factor σF in the resulting smaller cell. How the morphological transformations that occur during sporulation are coupled to cell-specific activation of transcription is largely unknown. The membrane protein SpoIIE is a constituent of the asymmetric sporulation septum and is a crucial determinant of σF activation. Here we report that the morphogenic protein, RodZ, which is essential for cell shape determination, is additionally required for asymmetric septum formation and sporulation. In cells depleted of RodZ, formation of asymmetric septa is disturbed and σF activation is perturbed. During sporulation, we found that SpoIIE recruits RodZ to the asymmetric septum. Moreover, we detected a direct interaction between SpoIIE and RodZ in vitro and in vivo, indicating that SpoIIE-RodZ may form a complex to coordinate asymmetric septum formation and σF activation. We propose that RodZ could provide a link between the cell shape machinery and the coordinated morphological and developmental transitions required to form a resistant spore.

An important role for Myb-MuvB and its target gene KIF23 in a mouse model of lung adenocarcinoma.

PubMed

Iltzsche, F; Simon, K; Stopp, S; Pattschull, G; Francke, S; Wolter, P; Hauser, S; Murphy, D J; Garcia, P; Rosenwald, A; Gaubatz, S

2017-01-05

The conserved Myb-MuvB (MMB) multiprotein complex has an important role in transcriptional activation of mitotic genes. MMB target genes are overexpressed in several different cancer types and their elevated expression is associated with an advanced tumor state and a poor prognosis. This suggests that MMB could contribute to tumorigenesis by mediating overexpression of mitotic genes. However, although MMB has been extensively characterized biochemically, the requirement for MMB in tumorigenesis in vivo has not been investigated. Here we demonstrate that MMB is required for tumor formation in a mouse model of lung cancer driven by oncogenic K-RAS. We also identify a requirement for the mitotic kinesin KIF23, a key target gene of MMB, in tumorigenesis. RNA interference-mediated depletion of KIF23 inhibited lung tumor formation in vivo and induced apoptosis in lung cancer cell lines. Our results suggest that inhibition of KIF23 could be a strategy for treatment of lung cancer.

Genetic Interactions Between the Meiosis-Specific Cohesin Components, STAG3, REC8, and RAD21L.

PubMed

Ward, Ayobami; Hopkins, Jessica; Mckay, Matthew; Murray, Steve; Jordan, Philip W

2016-06-01

Cohesin is an essential structural component of chromosomes that ensures accurate chromosome segregation during mitosis and meiosis. Previous studies have shown that there are cohesin complexes specific to meiosis, required to mediate homologous chromosome pairing, synapsis, recombination, and segregation. Meiosis-specific cohesin complexes consist of two structural maintenance of chromosomes proteins (SMC1α/SMC1β and SMC3), an α-kleisin protein (RAD21, RAD21L, or REC8), and a stromal antigen protein (STAG1, 2, or 3). STAG3 is exclusively expressed during meiosis, and is the predominant STAG protein component of cohesin complexes in primary spermatocytes from mouse, interacting directly with each α-kleisin subunit. REC8 and RAD21L are also meiosis-specific cohesin components. Stag3 mutant spermatocytes arrest in early prophase ("zygotene-like" stage), displaying failed homolog synapsis and persistent DNA damage, as a result of unstable loading of cohesin onto the chromosome axes. Interestingly, Rec8, Rad21L double mutants resulted in an earlier "leptotene-like" arrest, accompanied by complete absence of STAG3 loading. To assess genetic interactions between STAG3 and α-kleisin subunits RAD21L and REC8, our lab generated Stag3, Rad21L, and Stag3, Rec8 double knockout mice, and compared them to the Rec8, Rad21L double mutant. These double mutants are phenotypically distinct from one another, and more severe than each single knockout mutant with regards to chromosome axis formation, cohesin loading, and sister chromatid cohesion. The Stag3, Rad21L, and Stag3, Rec8 double mutants both progress further into prophase I than the Rec8, Rad21L double mutant. Our genetic analysis demonstrates that cohesins containing STAG3 and REC8 are the main complex required for centromeric cohesion, and RAD21L cohesins are required for normal clustering of pericentromeric heterochromatin. Furthermore, the STAG3/REC8 and STAG3/RAD21L cohesins are the primary cohesins required for axis formation. Copyright © 2016 Ward et al.

FANCG promotes formation of a newly identified protein complex containing BRCA2, FANCD2 and XRCC3.

PubMed

Wilson, J B; Yamamoto, K; Marriott, A S; Hussain, S; Sung, P; Hoatlin, M E; Mathew, C G; Takata, M; Thompson, L H; Kupfer, G M; Jones, N J

2008-06-12

Fanconi anemia (FA) is a human disorder characterized by cancer susceptibility and cellular sensitivity to DNA crosslinks and other damages. Thirteen complementation groups and genes are identified, including BRCA2, which is defective in the FA-D1 group. Eight of the FA proteins, including FANCG, participate in a nuclear core complex that is required for the monoubiquitylation of FANCD2 and FANCI. FANCD2, like FANCD1/BRCA2, is not part of the core complex, and we previously showed direct BRCA2-FANCD2 interaction using yeast two-hybrid analysis. We now show in human and hamster cells that expression of FANCG protein, but not the other core complex proteins, is required for co-precipitation of BRCA2 and FANCD2. We also show that phosphorylation of FANCG serine 7 is required for its co-precipitation with BRCA2, XRCC3 and FANCD2, as well as the direct interaction of BRCA2-FANCD2. These results argue that FANCG has a role independent of the FA core complex, and we propose that phosphorylation of serine 7 is the signalling event required for forming a discrete complex comprising FANCD1/BRCA2-FANCD2-FANCG-XRCC3 (D1-D2-G-X3). Cells that fail to express either phospho-Ser7-FANCG, or full length BRCA2 protein, lack the interactions amongst the four component proteins. A role for D1-D2-G-X3 in homologous recombination repair (HRR) is supported by our finding that FANCG and the RAD51-paralog XRCC3 are epistatic for sensitivity to DNA crosslinking compounds in DT40 chicken cells. Our findings further define the intricate interface between FANC and HRR proteins in maintaining chromosome stability.

Osmotically Induced Reversible Transitions in Lipid-DNA Mesophases

PubMed Central

Danino, Dganit; Kesselman, Ellina; Saper, Gadiel; Petrache, Horia I.; Harries, Daniel

2009-01-01

We follow the effect of osmotic pressure on isoelectric complexes that self-assemble from mixtures of DNA and mixed neutral and cationic lipids. Using small angle x-ray diffraction and freeze-fracture cryo-electron microscopy, we find that lamellar complexes known to form in aqueous solutions can reversibly transition to hexagonal mesophases under high enough osmotic stress exerted by adding a neutral polymer. Using molecular spacings derived from x-ray diffraction, we estimate the reversible osmotic pressure-volume (Π-V) work needed to induce this transition. We find that the transition free energy is comparable to the work required to elastically bend lipid layers around DNA. Consistent with this, the required work is significantly lowered by an addition of hexanol, which is known to soften lipid bilayers. Our findings not only help to resolve the free-energy contributions associated with lipid-DNA complex formation, but they also demonstrate the importance that osmotic stress can have to the macromolecular phase geometry in realistic biological environments. PMID:19348739

The Fanconi anemia pathway requires FAA phosphorylation and FAA/FAC nuclear accumulation

PubMed Central

Yamashita, Takayuki; Kupfer, Gary M.; Naf, Dieter; Suliman, Ahmed; Joenje, Hans; Asano, Shigetaka; D’Andrea, Alan D.

1998-01-01

Fanconi anemia (FA) is an autosomal recessive cancer susceptibility syndrome with at least eight complementation groups (A–H). Two FA genes, corresponding to complementation groups A and C, have been cloned, but the function of the FAA and FAC proteins remains unknown. We have recently shown that the FAA and FAC proteins bind and form a nuclear complex. In the current study, we analyzed the FAA and FAC proteins in normal lymphoblasts and lymphoblasts from multiple FA complementation groups. In contrast to normal controls, FA cells derived from groups A, B, C, E, F, G, and H were defective in the formation of the FAA/FAC protein complex, the phosphorylation of the FAA protein, and the accumulation of the FAA/FAC protein complex in the nucleus. These biochemical events seem to define a signaling pathway required for the maintenance of genomic stability and normal hematopoiesis. Our results support the idea that multiple gene products cooperate in the FA Pathway. PMID:9789045

Formation, Migration, and Reactivity of Au CO Complexes on Gold Surfaces

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

Wang, Jun; McEntee, Monica; Tang, Wenjie

2016-01-12

Here, we report experimental as well as theoretical evidence that suggests Au CO complex formation upon the exposure of CO to active sites (step edges and threading dislocations) on a Au(111) surface. Room-temperature scanning tunneling microscopy (STM), X-ray photoelectron spectroscopy, transmission infrared spectroscopy, and density functional theory calculations point to Au CO complex formation and migration. Room-temperature STM of the Au(111) surface at CO pressures in the range from 10^ 8 to 10^ 4 Torr (dosage up to 10^6 langmuir) indicates Au atom extraction from dislocation sites of the herringbone reconstruction, mobile Au CO complex formation and diffusion, and Aumore » adatom cluster formation on both elbows and step edges on the Au surface. The formation and mobility of the Au CO complex result from the reduced Au Au bonding at elbows and step edges leading to stronger Au CO bonding and to the formation of a more positively charged CO (CO +) on Au. These studies indicate that the mobile Au CO complex is involved in the Au nanoparticle formation and reactivity, and that the positive charge on CO increases due to the stronger adsorption of CO at Au sites with lower coordination numbers.« less

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

Vital Roles of the Second DNA-binding Site of Rad52 Protein in Yeast Homologous Recombination*

PubMed Central

Arai, Naoto; Kagawa, Wataru; Saito, Kengo; Shingu, Yoshinori; Mikawa, Tsutomu; Kurumizaka, Hitoshi; Shibata, Takehiko

2011-01-01

RecA/Rad51 proteins are essential in homologous DNA recombination and catalyze the ATP-dependent formation of D-loops from a single-stranded DNA and an internal homologous sequence in a double-stranded DNA. RecA and Rad51 require a “recombination mediator” to overcome the interference imposed by the prior binding of single-stranded binding protein/replication protein A to the single-stranded DNA. Rad52 is the prototype of recombination mediators, and the human Rad52 protein has two distinct DNA-binding sites: the first site binds to single-stranded DNA, and the second site binds to either double- or single-stranded DNA. We previously showed that yeast Rad52 extensively stimulates Rad51-catalyzed D-loop formation even in the absence of replication protein A, by forming a 2:1 stoichiometric complex with Rad51. However, the precise roles of Rad52 and Rad51 within the complex are unknown. In the present study, we constructed yeast Rad52 mutants in which the amino acid residues corresponding to the second DNA-binding site of the human Rad52 protein were replaced with either alanine or aspartic acid. We found that the second DNA-binding site is important for the yeast Rad52 function in vivo. Rad51-Rad52 complexes consisting of these Rad52 mutants were defective in promoting the formation of D-loops, and the ability of the complex to associate with double-stranded DNA was specifically impaired. Our studies suggest that Rad52 within the complex associates with double-stranded DNA to assist Rad51-mediated homologous pairing. PMID:21454474

Concerted formation of macromolecular Suppressor–mutator transposition complexes

PubMed Central

Raina, Ramesh; Schläppi, Michael; Karunanandaa, Balasulojini; Elhofy, Adam; Fedoroff, Nina

1998-01-01

Transposition of the maize Suppressor–mutator (Spm) transposon requires two element-encoded proteins, TnpA and TnpD. Although there are multiple TnpA binding sites near each element end, binding of TnpA to DNA is not cooperative, and the binding affinity is not markedly affected by the number of binding sites per DNA fragment. However, intermolecular complexes form cooperatively between DNA fragments with three or more TnpA binding sites. TnpD, itself not a sequence-specific DNA-binding protein, binds to TnpA and stabilizes the TnpA–DNA complex. The high redundancy of TnpA binding sites at both element ends and the protein–protein interactions between DNA-bound TnpA complexes and between these and TnpD imply a concerted transition of the element from a linear to a protein crosslinked transposition complex within a very narrow protein concentration range. PMID:9671711

Nucleic acids, proteins, and chirality

NASA Technical Reports Server (NTRS)

Usher, D. A.; Profy, A. T.; Walstrum, S. A.; Needels, M. C.; Bulack, S. C.; Lo, K. M.

1984-01-01

The present investigation is concerned with experimental results related, in one case, to the chirality of nucleotides, and, in another case, to the possibility of a link between the chirality of nucleic acids, and that of peptides. It has been found that aminoacylation of the 'internal' hydroxyl group of a dinucleoside monophosphate can occur stereoselectively. However, this reaction has not yet been made a part of a working peptide synthesis scheme. The formation and cleavage of oligonucleotides is considered. In the event of the formation of a helical complex between the oligonucleotide and the polymer, 1-prime,5-prime-bonds in the oligomer are found to become more resistant towards cleavage. The conditions required for peptide bond formation are examined, taking into account the known structures of RNA and possible mechanisms for prebiotic peptide bond formation. The possibility is considered that the 2-prime,5-prime-internucleotide linkage could have played an important part in the early days of biological peptide synthesis.

New and old ways to control meiotic recombination.

PubMed

Phadnis, Naina; Hyppa, Randy W; Smith, Gerald R

2011-10-01

The unique segregation of homologs, rather than sister chromatids, at the first meiotic division requires the formation of crossovers (COs) between homologs by meiotic recombination in most species. Crossovers do not form at random along chromosomes. Rather, their formation is carefully controlled, both at the stage of formation of DNA double-strand breaks (DSBs) that can initiate COs and during the repair of these DSBs. Here, we review control of DSB formation and two recently recognized controls of DSB repair: CO homeostasis and CO invariance. Crossover homeostasis maintains a constant number of COs per cell when the total number of DSBs in a cell is experimentally or stochastically reduced. Crossover invariance maintains a constant CO density (COs per kb of DNA) across much of the genome despite strong DSB hotspots in some intervals. These recently uncovered phenomena show that CO control is even more complex than previously suspected. Copyright © 2011 Elsevier Ltd. All rights reserved.

Switch-Hitting Immune Cells: From Tumor Protection to Metastasis Promotion | Center for Cancer Research

Cancer.gov

The leading cause of death from cancer is not a primary tumor but is the metastases, or invasion of tumor cells into other locations in the body, that result from it. A complex and incompletely understood process, metastatic tumor formation is thought to require several steps in which tumor cells invade the tissue surrounding the primary tumor, enter local blood vessels,

The Formation and Thermochemical Properties of Multiligand Complexes

DTIC Science & Technology

1987-08-25

SUBJECT TERMS (Continue on revers, if necessary and identify by block numoer) FIELD GROUP SUB-GROUP Ion-molecule reactions, clusters, multiligand...mercaptans, and phosphonates for which the results may be useful in the development of detection techniques that employ ion mobility analyzers or... field involve the use of ion mobility and mass spectrometers. Detection of a species by such instruments in an atmospheric environment requires that the

The Relationship between State High School Exit Exams and Mathematical Proficiency: Analyses of the Complexity, Content, and Format of Items and Assessment Protocols

ERIC Educational Resources Information Center

Regan, Blake B.

2012-01-01

This study examined the relationship between high school exit exams and mathematical proficiency. With the No Child Left Behind (NCLB) Act requiring all students to be proficient in mathematics by 2014, it is imperative that high-stakes assessments accurately evaluate all aspects of student achievement, appropriately set the yardstick by which…

High-Speed Systolic Array Testbed.

DTIC Science & Technology

1987-10-01

applications since the concept was introduced by H.T. Kung In 1978. This highly parallel architecture of nearet neighbor data communciation and...must be addressed. For instance, should bit-serial or bit parallei computation be utilized. Does the dynamic range of the candidate applications or...numericai stability of the algorithms used require computations In fixed point and Integer format or the architecturally more complex and slower floating

Intrahippocampal Glutamine Administration Inhibits mTORC1 Signaling and Impairs Long-Term Memory

ERIC Educational Resources Information Center

Rozas, Natalia S.; Redell, John B.; Pita-Almenar, Juan D.; McKenna, James, III.; Moore, Anthony N.; Gambello, Michael J.; Dash, Pramod K.

2015-01-01

The mechanistic Target of Rapamycin Complex 1 (mTORC1), a key regulator of protein synthesis and cellular growth, is also required for long-term memory formation. Stimulation of mTORC1 signaling is known to be dependent on the availability of energy and growth factors, as well as the presence of amino acids. In vitro studies using serum- and amino…

Segmental expression of Hoxb2 in r4 requires two separate sites that integrate cooperative interactions between Prep1, Pbx and Hox proteins.

PubMed

Ferretti, E; Marshall, H; Pöpperl, H; Maconochie, M; Krumlauf, R; Blasi, F

2000-01-01

Direct auto- and cross-regulatory interactions between Hox genes serve to establish and maintain segmentally restricted patterns in the developing hindbrain. Rhombomere r4-specific expression of both Hoxb1 and Hoxb2 depends upon bipartite cis Hox response elements for the group 1 paralogous proteins, Hoxal and Hoxbl. The DNA-binding ability and selectivity of these proteins depend upon the formation of specific heterodimeric complexes with members of the PBC homeodomain protein family (Pbx genes). The r4 enhancers from Hoxb1 and Hoxb2 have the same activity, but differ with respect to the number and organisation of bipartite Pbx/Hox (PH) sites required, suggesting the intervention of other components/sequences. We report here that another family of homeodomain proteins (TALE, Three-Amino acids-Loop-Extension: Prep1, Meis, HTH), capable of dimerizing with Pbx/EXD, is involved in the mechanisms of r4-restricted expression. We show that: (1) the r4-specific Hoxb1 and Hoxb2 enhancers are complex elements containing separate PH and Prep/Meis (PM) sites; (2) the PM site of the Hoxb2, but not Hoxb1, enhancer is essential in vivo for r4 expression and also influences other sites of expression; (3) both PM and PH sites are required for in vitro binding of Prepl-Pbx and formation and binding of a ternary Hoxbl-Pbxla (or 1b)-Prepl complex. (4) A similar ternary association forms in nuclear extracts from embryonal P19 cells, but only upon retinoic acid induction. This requires synthesis of Hoxbl and also contains Pbx with either Prepl or Meisl. Together these findings highlight the fact that PM sites are found in close proximity to bipartite PH motifs in several Hox responsive elements shown to be important in vivo and that such sites play an essential role in potentiating regulatory activity in combination with the PH motifs.

NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidase-DNA and neutrophil elastase-DNA complexes.

PubMed

Yoo, Dae-goon; Floyd, Madison; Winn, Matthew; Moskowitz, Samuel M; Rada, Balázs

2014-08-01

Cystic fibrosis (CF) airway disease is characterized by Pseudomonas aeruginosa infection and recruitment of neutrophil granulocytes. Neutrophil granule components (myeloperoxidase (MPO), human neutrophil elastase (HNE)), extracellular DNA and P. aeruginosa can all be found in the CF respiratory tract and have all been associated with worsening CF lung function. Pseudomonas-induced formation of neutrophil extracellular traps (NETs) offers a likely mechanism for release of MPO, HNE and DNA from neutrophils. NETs are composed of a DNA backbone decorated with granule proteins like MPO and HNE. Here we sought to examine whether CF clinical isolates of Pseudomonas are capable of inducing NET release from human neutrophil granulocytes. We used two methods to quantify NETs. We modified a previously employed ELISA that detects MPO-DNA complexes and established a new HNE-DNA ELISA. We show that these methods reliably quantify MPO-DNA and HNE-DNA complexes, measures of NET formation. We have found that CF isolates of P. aeruginosa stimulate robust respiratory burst and NET release in human neutrophils. By comparing paired "early" and "late" bacterial isolates obtained from the same CF patient we have found that early isolates induced significantly more NET release than late isolates. Our data support that Pseudomonas-induced NET release represents an important mechanism for release of neutrophil-derived CF inflammatory mediators, and confirm that decreased induction of NET formation is required for long-term adaptation of P. aeruginosa to CF airways. Copyright © 2014 Elsevier B.V. All rights reserved.

Principles for the formation of an effective concept of multifunctional high-rise construction investment projects

NASA Astrophysics Data System (ADS)

Beliakov, Sergei

2018-03-01

Investment projects of high-rise construction have a number of features that determine specific risks and additional opportunities that require analysis and accounting in the formation of an effective project concept. The most significant features of high-rise construction include long construction time, complexity of technical and technological solutions, complexity of decisions on the organization of construction and operation, high cost of construction and operation, complexity in determining the ratio of areas designed to accommodate different functional areas, when organizing and coordinating the operation of the facility, with internal zoning. Taking into account the specificity of high-rise construction, among the factors determining the effectiveness of projects, it is advisable to consider as key factors: organizational, technological and investment factors. Within the framework of the article, the author singled out key particular functions for each group of factors under consideration, and also developed a system of principles for the formation of an effective concept of multifunctional high-rise construction investment projects, including the principle of logistic efficiency, the principle of optimal functional zoning, the principle of efficiency of equipment use, the principle of optimizing technological processes, the principle maximization of income, the principle of fund management, the principle of risk management . The model of formation of an effective concept of investment projects of multifunctional high-rise construction developed by the author can contribute to the development of methodological tools in the field of managing the implementation of high-rise construction projects, taking into account their specificity in the current economic conditions.

Papillae formation on trichome cell walls requires the function of the mediator complex subunit Med25.

PubMed

Fornero, Christy; Suo, Bangxia; Zahde, Mais; Juveland, Katelyn; Kirik, Viktor

2017-11-01

Glassy Hair 1 (GLH1) gene that promotes papillae formation on trichome cell walls was identified as a subunit of the transcriptional mediator complex MED25. The MED25 gene is shown to be expressed in trichomes. The expression of the trichome development marker genes GLABRA2 (GL2) and Ethylene Receptor2 (ETR2) is not affected in the glh1 mutant. Presented data suggest that Arabidopsis MED25 mediator component is likely involved in the transcription of genes promoting papillae deposition in trichomes. The plant cell wall plays an important role in communication, defense, organization and support. The importance of each of these functions varies by cell type. Specialized cells, such as Arabidopsis trichomes, exhibit distinct cell wall characteristics including papillae. To better understand the molecular processes important for papillae deposition on the cell wall surface, we identified the GLASSY HAIR 1 (GLH1) gene, which is necessary for papillae formation. We found that a splice-site mutation in the component of the transcriptional mediator complex MED25 gene is responsible for the near papillae-less phenotype of the glh1 mutant. The MED25 gene is expressed in trichomes. Reporters for trichome developmental marker genes GLABRA2 (GL2) and Ethylene Receptor2 (ETR2) were not affected in the glh1 mutant. Collectively, the presented results show that MED25 is necessary for papillae formation on the cell wall surface of leaf trichomes and suggest that the Arabidopsis MED25 mediator component is likely involved in the transcription of a subset of genes that promote papillae deposition in trichomes.

Inner/Outer nuclear membrane fusion in nuclear pore assembly: biochemical demonstration and molecular analysis.

PubMed

Fichtman, Boris; Ramos, Corinne; Rasala, Beth; Harel, Amnon; Forbes, Douglass J

2010-12-01

Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membranes, which carry out nucleocytoplasmic exchange. The mechanism of nuclear pore assembly involves a unique challenge, as it requires creation of a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel has little evolutionary precedent. Here we mapped inner/outer nuclear membrane fusion in NPC assembly biochemically by using novel assembly intermediates and membrane fusion inhibitors. Incubation of a Xenopus in vitro nuclear assembly system at 14°C revealed an early pore intermediate where nucleoporin subunits POM121 and the Nup107-160 complex were organized in a punctate pattern on the inner nuclear membrane. With time, this intermediate progressed to diffusion channel formation and finally to complete nuclear pore assembly. Correct channel formation was blocked by the hemifusion inhibitor lysophosphatidylcholine (LPC), but not if a complementary-shaped lipid, oleic acid (OA), was simultaneously added, as determined with a novel fluorescent dextran-quenching assay. Importantly, recruitment of the bulk of FG nucleoporins, characteristic of mature nuclear pores, was not observed before diffusion channel formation and was prevented by LPC or OA, but not by LPC+OA. These results map the crucial inner/outer nuclear membrane fusion event of NPC assembly downstream of POM121/Nup107-160 complex interaction and upstream or at the time of FG nucleoporin recruitment.

Central region component1, a novel synaptonemal complex component, is essential for meiotic recombination initiation in rice.

PubMed

Miao, Chunbo; Tang, Ding; Zhang, Honggen; Wang, Mo; Li, Yafei; Tang, Shuzhu; Yu, Hengxiu; Gu, Minghong; Cheng, Zhukuan

2013-08-01

In meiosis, homologous recombination entails programmed DNA double-strand break (DSB) formation and synaptonemal complex (SC) assembly coupled with the DSB repair. Although SCs display extensive structural conservation among species, their components identified are poorly conserved at the sequence level. Here, we identified a novel SC component, designated central region component1 (CRC1), in rice (Oryza sativa). CRC1 colocalizes with ZEP1, the rice SC transverse filament protein, to the central region of SCs in a mutually dependent fashion. Consistent with this colocalization, CRC1 interacts with ZEP1 in yeast two-hybrid assays. CRC1 is orthologous to Saccharomyces cerevisiae pachytene checkpoint2 (Pch2) and Mus musculus THYROID receptor-interacting protein13 (TRIP13) and may be a conserved SC component. Additionally, we provide evidence that CRC1 is essential for meiotic DSB formation. CRC1 interacts with homologous pairing aberration in rice meiosis1 (PAIR1) in vitro, suggesting that these proteins act as a complex to promote DSB formation. PAIR2, the rice ortholog of budding yeast homolog pairing1, is required for homologous chromosome pairing. We found that CRC1 is also essential for the recruitment of PAIR2 onto meiotic chromosomes. The roles of CRC1 identified here have not been reported for Pch2 or TRIP13.

A multiscale modelling approach to understand atherosclerosis formation: A patient-specific case study in the aortic bifurcation

PubMed Central

Alimohammadi, Mona; Pichardo-Almarza, Cesar; Agu, Obiekezie; Díaz-Zuccarini, Vanessa

2017-01-01

Atherogenesis, the formation of plaques in the wall of blood vessels, starts as a result of lipid accumulation (low-density lipoprotein cholesterol) in the vessel wall. Such accumulation is related to the site of endothelial mechanotransduction, the endothelial response to mechanical stimuli and haemodynamics, which determines biochemical processes regulating the vessel wall permeability. This interaction between biomechanical and biochemical phenomena is complex, spanning different biological scales and is patient-specific, requiring tools able to capture such mathematical and biological complexity in a unified framework. Mathematical models offer an elegant and efficient way of doing this, by taking into account multifactorial and multiscale processes and mechanisms, in order to capture the fundamentals of plaque formation in individual patients. In this study, a mathematical model to understand plaque and calcification locations is presented: this model provides a strong interpretability and physical meaning through a multiscale, complex index or metric (the penetration site of low-density lipoprotein cholesterol, expressed as volumetric flux). Computed tomography scans of the aortic bifurcation and iliac arteries are analysed and compared with the results of the multifactorial model. The results indicate that the model shows potential to predict the majority of the plaque locations, also not predicting regions where plaques are absent. The promising results from this case study provide a proof of concept that can be applied to a larger patient population. PMID:28427316

CENTRAL REGION COMPONENT1, a Novel Synaptonemal Complex Component, Is Essential for Meiotic Recombination Initiation in Rice[C][W

PubMed Central

Miao, Chunbo; Tang, Ding; Zhang, Honggen; Wang, Mo; Li, Yafei; Tang, Shuzhu; Yu, Hengxiu; Gu, Minghong; Cheng, Zhukuan

2013-01-01

In meiosis, homologous recombination entails programmed DNA double-strand break (DSB) formation and synaptonemal complex (SC) assembly coupled with the DSB repair. Although SCs display extensive structural conservation among species, their components identified are poorly conserved at the sequence level. Here, we identified a novel SC component, designated CENTRAL REGION COMPONENT1 (CRC1), in rice (Oryza sativa). CRC1 colocalizes with ZEP1, the rice SC transverse filament protein, to the central region of SCs in a mutually dependent fashion. Consistent with this colocalization, CRC1 interacts with ZEP1 in yeast two-hybrid assays. CRC1 is orthologous to Saccharomyces cerevisiae pachytene checkpoint2 (Pch2) and Mus musculus THYROID RECEPTOR-INTERACTING PROTEIN13 (TRIP13) and may be a conserved SC component. Additionally, we provide evidence that CRC1 is essential for meiotic DSB formation. CRC1 interacts with HOMOLOGOUS PAIRING ABERRATION IN RICE MEIOSIS1 (PAIR1) in vitro, suggesting that these proteins act as a complex to promote DSB formation. PAIR2, the rice ortholog of budding yeast homolog pairing1, is required for homologous chromosome pairing. We found that CRC1 is also essential for the recruitment of PAIR2 onto meiotic chromosomes. The roles of CRC1 identified here have not been reported for Pch2 or TRIP13. PMID:23943860

Improved Efficacy of Synthesizing *MIII-Labeled DOTA Complexes in Binary Mixtures of Water and Organic Solvents. A Combined Radio- and Physicochemical Study.

PubMed

Pérez-Malo, Marylaine; Szabó, Gergely; Eppard, Elisabeth; Vagner, Adrienn; Brücher, Ernő; Tóth, Imre; Maiocchi, Alessandro; Suh, Eul Hyun; Kovács, Zoltán; Baranyai, Zsolt; Rösch, Frank

2018-05-21

Typically, the synthesis of radiometal-based radiopharmaceuticals is performed in buffered aqueous solutions. We found that the presence of organic solvents like ethanol increased the radiolabeling yields of [ 68 Ga]Ga-DOTA (DOTA = 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacatic acid). In the present study, the effect of organic cosolvents [ethanol (EtOH), isopropyl alcohol, and acetonitrile] on the radiolabeling yields of the macrocyclic chelator DOTA with several trivalent radiometals (gallium-68, scandium-44, and lutetium-177) was systematically investigated. Various binary water (H 2 O)/organic solvent mixtures allowed the radiolabeling of DOTA at a significantly lower temperature than 95 °C, which is relevant for the labeling of sensitive biological molecules. Simultaneously, much lower amounts of the chelators were required. This strategy may have a fundamental impact on the formulation of trivalent radiometal-based radiopharmaceuticals. The equilibrium properties and formation kinetics of [M(DOTA)] - (M III = Ga III , Ce III , Eu III , Y III , and Lu III ) complexes were investigated in H 2 O/EtOH mixtures (up to 70 vol % EtOH). The protonation constants of DOTA were determined by pH potentiometry in H 2 O/EtOH mixtures (0-70 vol % EtOH, 0.15 M NaCl, 25 °C). The log K 1 H and log K 2 H values associated with protonation of the ring N atoms decreased with an increase of the EtOH content. The formation rates of [M(DOTA)] - complexes increase with an increase of the pH and [EtOH]. Complexation occurs through rapid formation of the diprotonated [M(H 2 DOTA)] + intermediates, which are in equilibrium with the kinetically active monoprotonated [M(HDOTA)] intermediates. The rate-controlling step is deprotonation (and rearrangement) of the monoprotonated intermediate, which occurs through H 2 O ( *M(HL) k H 2 O ) and OH - ( *M(HL) k OH ) assisted reaction pathways. The rate constants are essentially independent of the EtOH concentration, but the M(HL) k H2O values increase from Ce III to Lu III . However, the log K M(HL) H protonation constants, analogous to the log K H 2 value, decrease with increasing [EtOH], which increases the concentration of the monoprotonated M(HDOTA) intermediate and accelerates formation of the final complexes. The overall rates of complex formation calculated by the obtained rate constants at different EtOH concentrations show a trend similar to that of the complexation rates determined with the use of radioactive isotopes.

Observability and Estimation of Distributed Space Systems via Local Information-Exchange Networks

NASA Technical Reports Server (NTRS)

Fathpour, Nanaz; Hadaegh, Fred Y.; Mesbahi, Mehran; Rahmani, Amirreza

2011-01-01

Spacecraft formation flying involves the coordination of states among multiple spacecraft through relative sensing, inter-spacecraft communication, and control. Most existing formation-flying estimation algorithms can only be supported via highly centralized, all-to-all, static relative sensing. New algorithms are proposed that are scalable, modular, and robust to variations in the topology and link characteristics of the formation exchange network. These distributed algorithms rely on a local information exchange network, relaxing the assumptions on existing algorithms. Distributed space systems rely on a signal transmission network among multiple spacecraft for their operation. Control and coordination among multiple spacecraft in a formation is facilitated via a network of relative sensing and interspacecraft communications. Guidance, navigation, and control rely on the sensing network. This network becomes more complex the more spacecraft are added, or as mission requirements become more complex. The observability of a formation state was observed by a set of local observations from a particular node in the formation. Formation observability can be parameterized in terms of the matrices appearing in the formation dynamics and observation matrices. An agreement protocol was used as a mechanism for observing formation states from local measurements. An agreement protocol is essentially an unforced dynamic system whose trajectory is governed by the interconnection geometry and initial condition of each node, with a goal of reaching a common value of interest. The observability of the interconnected system depends on the geometry of the network, as well as the position of the observer relative to the topology. For the first time, critical GN&C (guidance, navigation, and control estimation) subsystems are synthesized by bringing the contribution of the spacecraft information-exchange network to the forefront of algorithmic analysis and design. The result is a formation estimation algorithm that is modular and robust to variations in the topology and link properties of the underlying formation network.

The Mediator complex: a master coordinator of transcription and cell lineage development.

PubMed

Yin, Jing-wen; Wang, Gang

2014-03-01

Mediator is a multiprotein complex that is required for gene transcription by RNA polymerase II. Multiple subunits of the complex show specificity in relaying information from signals and transcription factors to the RNA polymerase II machinery, thus enabling control of the expression of specific genes. Recent studies have also provided novel mechanistic insights into the roles of Mediator in epigenetic regulation, transcriptional elongation, termination, mRNA processing, noncoding RNA activation and super enhancer formation. Based on these specific roles in gene regulation, Mediator has emerged as a master coordinator of development and cell lineage determination. Here, we describe the most recent advances in understanding the mechanisms of Mediator function, with an emphasis on its role during development and disease.

Neuropeptides: Developmental Signals in Placode Progenitor Formation

PubMed Central

Lleras-Forero, Laura; Tambalo, Monica; Christophorou, Nicolas; Chambers, David; Houart, Corinne; Streit, Andrea

2013-01-01

Summary Few families of signaling factors have been implicated in the control of development. Here, we identify the neuropeptides nociceptin and somatostatin, a neurotransmitter and neuroendocrine hormone, as a class of developmental signals in both chick and zebrafish. We show that signals from the anterior mesendoderm are required for the formation of anterior placode progenitors, with one of the signals being somatostatin. Somatostatin controls ectodermal expression of nociceptin, and both peptides regulate Pax6 in lens and olfactory progenitors. Consequently, loss of somatostatin and nociceptin signaling leads to severe reduction of lens formation. Our findings not only uncover these neuropeptides as developmental signals but also identify a long-sought-after mechanism that initiates Pax6 in placode progenitors and may explain the ancient evolutionary origin of neuropeptides, predating a complex nervous system. PMID:23906067

Reference Proteome Extracts for Mass Spec Instrument Performance Validation and Method Development

PubMed Central

Rosenblatt, Mike; Urh, Marjeta; Saveliev, Sergei

2014-01-01

Biological samples of high complexity are required to test protein mass spec sample preparation procedures and validate mass spec instrument performance. Total cell protein extracts provide the needed sample complexity. However, to be compatible with mass spec applications, such extracts should meet a number of design requirements: compatibility with LC/MS (free of detergents, etc.)high protein integrity (minimal level of protein degradation and non-biological PTMs)compatibility with common sample preparation methods such as proteolysis, PTM enrichment and mass-tag labelingLot-to-lot reproducibility Here we describe total protein extracts from yeast and human cells that meet the above criteria. Two extract formats have been developed: Intact protein extracts with primary use for sample preparation method development and optimizationPre-digested extracts (peptides) with primary use for instrument validation and performance monitoring

Spindle formation in the mouse embryo requires Plk4 in the absence of centrioles.

PubMed

Coelho, Paula A; Bury, Leah; Sharif, Bedra; Riparbelli, Maria G; Fu, Jingyan; Callaini, Giuliano; Glover, David M; Zernicka-Goetz, Magdalena

2013-12-09

During the first five rounds of cell division in the mouse embryo, spindles assemble in the absence of centrioles. Spindle formation initiates around chromosomes, but the microtubule nucleating process remains unclear. Here we demonstrate that Plk4, a protein kinase known as a master regulator of centriole formation, is also essential for spindle assembly in the absence of centrioles. Depletion of maternal Plk4 prevents nucleation and growth of microtubules and results in monopolar spindle formation. This leads to cytokinesis failure and, consequently, developmental arrest. We show that Plk4 function depends on its kinase activity and its partner protein, Cep152. Moreover, tethering Cep152 to cellular membranes sequesters Plk4 and is sufficient to trigger spindle assembly from ectopic membranous sites. Thus, the Plk4-Cep152 complex has an unexpected role in promoting microtubule nucleation in the vicinity of chromosomes to mediate bipolar spindle formation in the absence of centrioles. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Centromere Pairing in Early Meiotic Prophase Requires Active Centromeres and Precedes Installation of the Synaptonemal Complex in Maize[W

PubMed Central

Zhang, Jing; Pawlowski, Wojciech P.; Han, Fangpu

2013-01-01

Pairing of homologous chromosomes in meiosis is critical for their segregation to daughter cells. In most eukaryotes, clustering of telomeres precedes and facilitates chromosome pairing. In several species, centromeres also form pairwise associations, known as coupling, before the onset of pairing. We found that, in maize (Zea mays), centromere association begins at the leptotene stage and occurs earlier than the formation of the telomere bouquet. We established that centromere pairing requires centromere activity and the sole presence of centromeric repeats is not sufficient for pairing. In several species, homologs of the ZIP1 protein, which forms the central element of the synaptonemal complex in budding yeast (Saccharomyces cerevisiae), play essential roles in centromere coupling. However, we found that the maize ZIP1 homolog ZYP1 installs in the centromeric regions of chromosomes after centromeres form associations. Instead, we found that maize STRUCTURAL MAINTENANCE OF CHROMOSOMES6 homolog forms a central element of the synaptonemal complex, which is required for centromere associations. These data shed light on the poorly understood mechanism of centromere interactions and suggest that this mechanism may vary somewhat in different species. PMID:24143803

Centromere pairing in early meiotic prophase requires active centromeres and precedes installation of the synaptonemal complex in maize.

PubMed

Zhang, Jing; Pawlowski, Wojciech P; Han, Fangpu

2013-10-01

Pairing of homologous chromosomes in meiosis is critical for their segregation to daughter cells. In most eukaryotes, clustering of telomeres precedes and facilitates chromosome pairing. In several species, centromeres also form pairwise associations, known as coupling, before the onset of pairing. We found that, in maize (Zea mays), centromere association begins at the leptotene stage and occurs earlier than the formation of the telomere bouquet. We established that centromere pairing requires centromere activity and the sole presence of centromeric repeats is not sufficient for pairing. In several species, homologs of the ZIP1 protein, which forms the central element of the synaptonemal complex in budding yeast (Saccharomyces cerevisiae), play essential roles in centromere coupling. However, we found that the maize ZIP1 homolog ZYP1 installs in the centromeric regions of chromosomes after centromeres form associations. Instead, we found that maize structural maintenance of chromosomes6 homolog forms a central element of the synaptonemal complex, which is required for centromere associations. These data shed light on the poorly understood mechanism of centromere interactions and suggest that this mechanism may vary somewhat in different species.

A novel medical image data-based multi-physics simulation platform for computational life sciences.

PubMed

Neufeld, Esra; Szczerba, Dominik; Chavannes, Nicolas; Kuster, Niels

2013-04-06

Simulating and modelling complex biological systems in computational life sciences requires specialized software tools that can perform medical image data-based modelling, jointly visualize the data and computational results, and handle large, complex, realistic and often noisy anatomical models. The required novel solvers must provide the power to model the physics, biology and physiology of living tissue within the full complexity of the human anatomy (e.g. neuronal activity, perfusion and ultrasound propagation). A multi-physics simulation platform satisfying these requirements has been developed for applications including device development and optimization, safety assessment, basic research, and treatment planning. This simulation platform consists of detailed, parametrized anatomical models, a segmentation and meshing tool, a wide range of solvers and optimizers, a framework for the rapid development of specialized and parallelized finite element method solvers, a visualization toolkit-based visualization engine, a Python scripting interface for customized applications, a coupling framework, and more. Core components are cross-platform compatible and use open formats. Several examples of applications are presented: hyperthermia cancer treatment planning, tumour growth modelling, evaluating the magneto-haemodynamic effect as a biomarker and physics-based morphing of anatomical models.

Exosites in the substrate specificity of blood coagulation reactions.

PubMed

Bock, P E; Panizzi, P; Verhamme, I M A

2007-07-01

The specificity of blood coagulation proteinases for substrate, inhibitor, and effector recognition is mediated by exosites on the surfaces of the catalytic domains, physically separated from the catalytic site. Some thrombin ligands bind specifically to either exosite I or II, while others engage both exosites. The involvement of different, overlapping constellations of exosite residues enables binding of structurally diverse ligands. The flexibility of the thrombin structure is central to the mechanism of complex formation and the specificity of exosite interactions. Encounter complex formation is driven by electrostatic ligand-exosite interactions, followed by conformational rearrangement to a stable complex. Exosites on some zymogens are in low affinity proexosite states and are expressed concomitant with catalytic site activation. The requirement for exosite expression controls the specificity of assembly of catalytic complexes on the coagulation pathway, such as the membrane-bound factor Xa*factor Va (prothrombinase) complex, and prevents premature assembly. Substrate recognition by prothrombinase involves a two-step mechanism with initial docking of prothrombin to exosites, followed by a conformational change to engage the FXa catalytic site. Prothrombin and its activation intermediates bind prothrombinase in two alternative conformations determined by the zymogen to proteinase transition that are hypothesized to involve prothrombin (pro)exosite I interactions with FVa, which underpin the sequential activation pathway. The role of exosites as the major source of substrate specificity has stimulated development of exosite-targeted anticoagulants for treatment of thrombosis.

Dynamical and topological aspects of consensus formation in complex networks

NASA Astrophysics Data System (ADS)

Chacoma, A.; Mato, G.; Kuperman, M. N.

2018-04-01

The present work analyzes a particular scenario of consensus formation, where the individuals navigate across an underlying network defining the topology of the walks. The consensus, associated to a given opinion coded as a simple message, is generated by interactions during the agent's walk and manifest itself in the collapse of the various opinions into a single one. We analyze how the topology of the underlying networks and the rules of interaction between the agents promote or inhibit the emergence of this consensus. We find that non-linear interaction rules are required to form consensus and that consensus is more easily achieved in networks whose degree distribution is narrower.

The effects of task difficulty and resource requirements on attention strategies

NASA Technical Reports Server (NTRS)

King, Teresa

1991-01-01

The patterns of attention strategies for task difficulty/resource tasks for which experimental results are presented and analyzed support the hypothesis that subjects may adopt an alternating (rather than concurrent one) when compelled to do so by either the size or the complexity of a visual display. According to the multiple resource model, if subjects had been performing the two tasks concurrently, the cost of this strategy would have been shown by a decrement in the spatial format, rather than the verbal format, due to competition for the same resource. Subjects may apply different strategies as a function of task difficulty and/or resource demand.

A cooperative game theory approach to transmission planning in power systems

NASA Astrophysics Data System (ADS)

Contreras, Javier

The rapid restructuring of the electric power industry from a vertically integrated entity into a decentralized industry has given rise to complex problems. In particular, the transmission component of the electric power system requires new methodologies to fully capture this emerging competitive industry. Game theory models are used to model strategic interactions in a competitive environment. This thesis presents a new decentralized framework to study the transmission network expansion problem using cooperative game theory. First, the players and the rules of the game are defined. Second, a coalition formation scheme is developed. Finally, the optimized cost of expansion is allocated based on the history of the coalition formation.

C-Cbl reverses HER2-mediated tamoxifen resistance in human breast cancer cells.

PubMed

Li, Wei; Xu, Ling; Che, Xiaofang; Li, Haizhou; Zhang, Ye; Song, Na; Wen, Ti; Hou, Kezuo; Yang, Yi; Zhou, Lu; Xin, Xing; Xu, Lu; Zeng, Xue; Shi, Sha; Liu, Yunpeng; Qu, Xiujuan; Teng, Yuee

2018-05-02

Tamoxifen is a frontline therapy for estrogen receptor (ER)-positive breast cancer in premenopausal women. However, many patients develop resistance to tamoxifen, and the mechanism underlying tamoxifen resistance is not well understood. Here we examined whether ER-c-Src-HER2 complex formation is involved in tamoxifen resistance. MTT and colony formation assays were used to measure cell viability and proliferation. Western blot was used to detect protein expression and protein complex formations were detected by immunoprecipitation and immunofluorescence. SiRNA was used to examine the function of HER2 in of BT474 cells. An in vivo xenograft animal model was established to examine the role of c-Cbl in tumor growth. MTT and colony formation assay showed that BT474 cells are resistant to tamoxifen and T47D cells are sensitive to tamoxifen. Immunoprecipitation experiments revealed ER-c-Src-HER2 complex formation in BT474 cells but not in T47D cells. However, ER-c-Src-HER2 complex formation was detected after overexpressing HER2 in T47D cells and these cells were more resistant to tamoxifen. HER2 knockdown by siRNA in BT474 cells reduced ER-c-Src-HER2 complex formation and reversed tamoxifen resistance. ER-c-Src-HER2 complex formation was also disrupted and tamoxifen resistance was reversed in BT474 cells by the c-Src inhibitor PP2 and HER2 antibody trastuzumab. Nystatin, a lipid raft inhibitor, reduced ER-c-Src-HER2 complex formation and partially reversed tamoxifen resistance. ER-c-Src-HER2 complex formation was disrupted by overexpression of c-Cbl but not by the c-Cbl ubiquitin ligase mutant. In addition, c-Cbl could reverse tamoxifen resistance in BT474 cells, but the ubiquitin ligase mutant had no effect. The effect of c-Cbl was validated in BT474 tumor-bearing nude mice in vivo. Immunofluorescence also revealed ER-c-Src-HER2 complex formation was reduced in tumor tissues of nude mice with c-Cbl overexpression. Our results suggested that c-Cbl can reverse tamoxifen resistance in HER2-overexpressing breast cancer cells by inhibiting the formation of the ER-c-Src-HER2 complex.

The MARVEL domain protein, Singles Bar, is required for progression past the pre-fusion complex stage of myoblast fusion.

PubMed

Estrada, Beatriz; Maeland, Anne D; Gisselbrecht, Stephen S; Bloor, James W; Brown, Nicholas H; Michelson, Alan M

2007-07-15

Multinucleated myotubes develop by the sequential fusion of individual myoblasts. Using a convergence of genomic and classical genetic approaches, we have discovered a novel gene, singles bar (sing), that is essential for myoblast fusion. sing encodes a small multipass transmembrane protein containing a MARVEL domain, which is found in vertebrate proteins involved in processes such as tight junction formation and vesicle trafficking where--as in myoblast fusion--membrane apposition occurs. sing is expressed in both founder cells and fusion competent myoblasts preceding and during myoblast fusion. Examination of embryos injected with double-stranded sing RNA or embryos homozygous for ethane methyl sulfonate-induced sing alleles revealed an identical phenotype: replacement of multinucleated myofibers by groups of single, myosin-expressing myoblasts at a stage when formation of the mature muscle pattern is complete in wild-type embryos. Unfused sing mutant myoblasts form clusters, suggesting that early recognition and adhesion of these cells are unimpaired. To further investigate this phenotype, we undertook electron microscopic ultrastructural studies of fusing myoblasts in both sing and wild-type embryos. These experiments revealed that more sing mutant myoblasts than wild-type contain pre-fusion complexes, which are characterized by electron-dense vesicles paired on either side of the fusing plasma membranes. In contrast, embryos mutant for another muscle fusion gene, blown fuse (blow), have a normal number of such complexes. Together, these results lead to the hypothesis that sing acts at a step distinct from that of blow, and that sing is required on both founder cell and fusion-competent myoblast membranes to allow progression past the pre-fusion complex stage of myoblast fusion, possibly by mediating fusion of the electron-dense vesicles to the plasma membrane.

DnaA protein DNA-binding domain binds to Hda protein to promote inter-AAA+ domain interaction involved in regulatory inactivation of DnaA.

PubMed

Keyamura, Kenji; Katayama, Tsutomu

2011-08-19

Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis.

DnaA Protein DNA-binding Domain Binds to Hda Protein to Promote Inter-AAA+ Domain Interaction Involved in Regulatory Inactivation of DnaA*

PubMed Central

Keyamura, Kenji; Katayama, Tsutomu

2011-01-01

Chromosomal replication is initiated from the replication origin oriC in Escherichia coli by the active ATP-bound form of DnaA protein. The regulatory inactivation of DnaA (RIDA) system, a complex of the ADP-bound Hda and the DNA-loaded replicase clamp, represses extra initiations by facilitating DnaA-bound ATP hydrolysis, yielding the inactive ADP-bound form of DnaA. However, the mechanisms involved in promoting the DnaA-Hda interaction have not been determined except for the involvement of an interaction between the AAA+ domains of the two. This study revealed that DnaA Leu-422 and Pro-423 residues within DnaA domain IV, including a typical DNA-binding HTH motif, are specifically required for RIDA-dependent ATP hydrolysis in vitro and that these residues support efficient interaction with the DNA-loaded clamp·Hda complex and with Hda in vitro. Consistently, substitutions of these residues caused accumulation of ATP-bound DnaA in vivo and oriC-dependent inhibition of cell growth. Leu-422 plays a more important role in these activities than Pro-423. By contrast, neither of these residues is crucial for DNA replication from oriC, although they are highly conserved in DnaA orthologues. Structural analysis of a DnaA·Hda complex model suggested that these residues make contact with residues in the vicinity of the Hda AAA+ sensor I that participates in formation of a nucleotide-interacting surface. Together, the results show that functional DnaA-Hda interactions require a second interaction site within DnaA domain IV in addition to the AAA+ domain and suggest that these interactions are crucial for the formation of RIDA complexes that are active for DnaA-ATP hydrolysis. PMID:21708944

Autonomous Performance Monitoring System: Monitoring and Self-Tuning (MAST)

NASA Technical Reports Server (NTRS)

Peterson, Chariya; Ziyad, Nigel A.

2000-01-01

Maintaining the long-term performance of software onboard a spacecraft can be a major factor in the cost of operations. In particular, the task of controlling and maintaining a future mission of distributed spacecraft will undoubtedly pose a great challenge, since the complexity of multiple spacecraft flying in formation grows rapidly as the number of spacecraft in the formation increases. Eventually, new approaches will be required in developing viable control systems that can handle the complexity of the data and that are flexible, reliable and efficient. In this paper we propose a methodology that aims to maintain the accuracy of flight software, while reducing the computational complexity of software tuning tasks. The proposed Monitoring and Self-Tuning (MAST) method consists of two parts: a flight software monitoring algorithm and a tuning algorithm. The dependency on the software being monitored is mostly contained in the monitoring process, while the tuning process is a generic algorithm independent of the detailed knowledge on the software. This architecture will enable MAST to be applicable to different onboard software controlling various dynamics of the spacecraft, such as attitude self-calibration, and formation control. An advantage of MAST over conventional techniques such as filter or batch least square is that the tuning algorithm uses machine learning approach to handle uncertainty in the problem domain, resulting in reducing over all computational complexity. The underlying concept of this technique is a reinforcement learning scheme based on cumulative probability generated by the historical performance of the system. The success of MAST will depend heavily on the reinforcement scheme used in the tuning algorithm, which guarantees the tuning solutions exist.

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

The Gβγ-Src signaling pathway regulates TNF-induced necroptosis via control of necrosome translocation

PubMed Central

Li, Lisheng; Chen, Wanze; Liang, Yaoji; Ma, Huabin; Li, Wenjuan; Zhou, Zhenru; Li, Jie; Ding, Yan; Ren, Junming; Lin, Juan; Han, Felicia; Wu, Jianfeng; Han, Jiahuai

2014-01-01

Formation of multi-component signaling complex necrosomes is essential for tumor necrosis factor α (TNF)-induced programmed necrosis (also called necroptosis). However, the mechanisms of necroptosis are still largely unknown. We isolated a TNF-resistant L929 mutant cell line generated by retrovirus insertion and identified that disruption of the guanine nucleotide-binding protein γ 10 (Gγ10) gene is responsible for this phenotype. We further show that Gγ10 is involved in TNF-induced necroptosis and Gβ2 is the partner of Gγ10. Src is the downstream effector of Gβ2γ10 in TNF-induced necroptosis because TNF-induced Src activation was impaired upon Gγ10 knockdown. Gγ10 does not affect TNF-induced activation of NF-κB and MAPKs and the formation of necrosomes, but is required for trafficking of necrosomes to their potential functioning site, an unidentified subcellular organelle that can be fractionated into heterotypic membrane fractions. The TNF-induced Gβγ-Src signaling pathway is independent of RIP1/RIP3 kinase activity and necrosome formation, but is required for the necrosome to function. PMID:24513853

Association of herpes simplex virus regulatory protein ICP22 with transcriptional complexes containing EAP, ICP4, RNA polymerase II, and viral DNA requires posttranslational modification by the U(L)13 proteinkinase.

PubMed Central

Leopardi, R; Ward, P L; Ogle, W O; Roizman, B

1997-01-01

The expression of herpes simplex virus 1 gamma (late) genes requires functional alpha proteins (gamma1 genes) and the onset of viral DNA synthesis (gamma2 genes). We report that late in infection after the onset of viral DNA synthesis, cell nuclei exhibit defined structures which contain two viral regulatory proteins (infected cell proteins 4 and 22) required for gamma gene expression, RNA polymerase II, a host nucleolar protein (EAP or L22) known to be associated with ribosomes and to bind small RNAs, including the Epstein-Barr virus small nuclear RNAs, and newly synthesized progeny DNA. The formation of these complexes required the onset of viral DNA synthesis. The association of infected cell protein 22, a highly posttranslationally processed protein, with these structures did not occur in cells infected with a viral mutant deleted in the genes U(L)13 and U(S)3, each of which specifies a protein kinase known to phosphorylate the protein. PMID:8995634

Formation of a PKCζ/β-catenin complex in endothelial cells promotes angiopoietin-1–induced collective directional migration and angiogenic sprouting

PubMed Central

Oubaha, Malika; Lin, Michelle I.; Margaron, Yoran; Filion, Dominic; Price, Emily N.; Zon, Leonard I.; Côté, Jean-François

2012-01-01

Angiogenic sprouting requires that cell-cell contacts be maintained during migration of endothelial cells. Angiopoietin-1 (Ang-1) and vascular endothelial growth factor act oppositely on endothelial cell junctions. We found that Ang-1 promotes collective and directional migration and, in contrast to VEGF, induces the formation of a complex formed of atypical protein kinase C (PKC)-ζ and β-catenin at cell-cell junctions and at the leading edge of migrating endothelial cells. This complex brings Par3, Par6, and adherens junction proteins at the front of migrating cells to locally activate Rac1 in response to Ang-1. The colocalization of PKCζ and β-catenin at leading edge along with PKCζ-dependent stabilization of cell-cell contacts promotes directed and collective endothelial cell migration. Consistent with these results, down-regulation of PKCζ in endothelial cells alters Ang-1–induced sprouting in vitro and knockdown in developing zebrafish results in intersegmental vessel defects caused by a perturbed directionality of tip cells and by loss of cell contacts between tip and stalk cells. These results reveal that PKCζ and β-catenin function in a complex at adherens junctions and at the leading edge of migrating endothelial cells to modulate collective and directional migration during angiogenesis. PMID:22936663

WAVE2 signaling mediates invasion of polarized epithelial cells by Salmonella typhimurium.

PubMed

Shi, Jing; Scita, Giorgio; Casanova, James E

2005-08-19

The bacterial pathogen Salmonella penetrates the intestinal epithelium by inducing its own phagocytosis into epithelial cells. The dramatic reorganization of the actin cytoskeleton required for internalization is driven by bacterial manipulation of host signaling pathways, including activation of the Rho family GTPase Rac1 and subsequent activation of the Arp2/3 complex. However, the mechanisms linking these two events remain poorly understood. Rac1 is thought to promote activation of the Arp2/3 complex through its interaction with suppressor of cAMP receptor/WASP family verprolin-homologous (SCAR/WAVE) family proteins, but this interaction is apparently indirect. Two different Rac1 effectors have been shown to bind WAVE2: IRSp53, the SH3 domain of which binds the WAVE2 proline-rich domain, and PIR121/Sra-1, which forms a pentameric complex containing WAVE, Abi1, Nap1, and HSPC300. However, the extent to which each of these complexes contributes to Arp2/3 complex activation in the context of Salmonella infection is unclear. Here, we show that WAVE2 is necessary for efficient invasion of epithelial cells by Salmonella typhimurium. We found that although Salmonella infection strongly promotes the formation of an IRSp53/WAVE2 complex, IRSp53 is not necessary for bacterial internalization. In contrast, disruption of the PIR121/Nap1/Abi1/WAVE2/HSPC300 complex potently inhibits bacterial uptake. These results indicate that WAVE2 is an important component in signaling pathways leading to Salmonella invasion. Although infection leads to the formation of an IRSp53/WAVE2 complex, it is the association of WAVE2 with the Abi1/Nap1/PIR121/HSPC300 complex that regulates bacterial internalization.

A Conserved Coatomer-related Complex Containing Sec13 and Seh1 Dynamically Associates With the Vacuole in Saccharomyces cerevisiae*

PubMed Central

Dokudovskaya, Svetlana; Waharte, Francois; Schlessinger, Avner; Pieper, Ursula; Devos, Damien P.; Cristea, Ileana M.; Williams, Rosemary; Salamero, Jean; Chait, Brian T.; Sali, Andrej; Field, Mark C.; Rout, Michael P.; Dargemont, Catherine

2011-01-01

The presence of multiple membrane-bound intracellular compartments is a major feature of eukaryotic cells. Many of the proteins required for formation and maintenance of these compartments share an evolutionary history. Here, we identify the SEA (Seh1-associated) protein complex in yeast that contains the nucleoporin Seh1 and Sec13, the latter subunit of both the nuclear pore complex and the COPII coating complex. The SEA complex also contains Npr2 and Npr3 proteins (upstream regulators of TORC1 kinase) and four previously uncharacterized proteins (Sea1–Sea4). Combined computational and biochemical approaches indicate that the SEA complex proteins possess structural characteristics similar to the membrane coating complexes COPI, COPII, the nuclear pore complex, and, in particular, the related Vps class C vesicle tethering complexes HOPS and CORVET. The SEA complex dynamically associates with the vacuole in vivo. Genetic assays indicate a role for the SEA complex in intracellular trafficking, amino acid biogenesis, and response to nitrogen starvation. These data demonstrate that the SEA complex is an additional member of a family of membrane coating and vesicle tethering assemblies, extending the repertoire of protocoatomer-related complexes. PMID:21454883

Highly polygenic variation in environmental perception determines dauer larvae formation in growing populations of Caenorhabditis elegans.

PubMed

Green, James W M; Stastna, Jana J; Orbidans, Helen E; Harvey, Simon C

2014-01-01

Determining how complex traits are genetically controlled is a requirement if we are to predict how they evolve and how they might respond to selection. This requires understanding how distinct, and often more simple, life history traits interact and change in response to environmental conditions. In order to begin addressing such issues, we have been analyzing the formation of the developmentally arrested dauer larvae of Caenorhabditis elegans under different conditions. We find that 18 of 22 previously identified quantitative trait loci (QTLs) affecting dauer larvae formation in growing populations, assayed by determining the number of dauer larvae present at food patch exhaustion, can be recovered under various environmental conditions. We also show that food patch size affects both the ability to detect QTLs and estimates of effect size, and demonstrate that an allele of nath-10 affects dauer larvae formation in growing populations. To investigate the component traits that affect dauer larvae formation in growing populations we map, using the same introgression lines, QTLs that affect dauer larvae formation in response to defined amounts of pheromone. This identifies 36 QTLs, again demonstrating the highly polygenic nature of the genetic variation underlying dauer larvae formation. These data indicate that QTLs affecting the number of dauer larvae at food exhaustion in growing populations of C. elegans are highly reproducible, and that nearly all can be explained by variation affecting dauer larvae formation in response to defined amounts of pheromone. This suggests that most variation in dauer larvae formation in growing populations is a consequence of variation in the perception of the food and pheromone environment (i.e. chemosensory variation) and in the integration of these cues.

Centriole maturation requires regulated Plk1 activity during two consecutive cell cycles.

PubMed

Kong, Dong; Farmer, Veronica; Shukla, Anil; James, Jana; Gruskin, Richard; Kiriyama, Shigeo; Loncarek, Jadranka

2014-09-29

Newly formed centrioles in cycling cells undergo a maturation process that is almost two cell cycles long before they become competent to function as microtubule-organizing centers and basal bodies. As a result, each cell contains three generations of centrioles, only one of which is able to form cilia. It is not known how this long and complex process is regulated. We show that controlled Plk1 activity is required for gradual biochemical and structural maturation of the centrioles and timely appendage assembly. Inhibition of Plk1 impeded accumulation of appendage proteins and appendage formation. Unscheduled Plk1 activity, either in cycling or interphase-arrested cells, accelerated centriole maturation and appendage and cilia formation on the nascent centrioles, erasing the age difference between centrioles in one cell. These findings provide a new understanding of how the centriole cycle is regulated and how proper cilia and centrosome numbers are maintained in the cells.

Centriole maturation requires regulated Plk1 activity during two consecutive cell cycles

PubMed Central

Kong, Dong; Farmer, Veronica; Shukla, Anil; James, Jana; Gruskin, Richard; Kiriyama, Shigeo

2014-01-01

Newly formed centrioles in cycling cells undergo a maturation process that is almost two cell cycles long before they become competent to function as microtubule-organizing centers and basal bodies. As a result, each cell contains three generations of centrioles, only one of which is able to form cilia. It is not known how this long and complex process is regulated. We show that controlled Plk1 activity is required for gradual biochemical and structural maturation of the centrioles and timely appendage assembly. Inhibition of Plk1 impeded accumulation of appendage proteins and appendage formation. Unscheduled Plk1 activity, either in cycling or interphase-arrested cells, accelerated centriole maturation and appendage and cilia formation on the nascent centrioles, erasing the age difference between centrioles in one cell. These findings provide a new understanding of how the centriole cycle is regulated and how proper cilia and centrosome numbers are maintained in the cells. PMID:25246616

Coordination properties of tridentate (N,O,O) heterocyclic alcohol (PDC) with Cu(II). Mixed ligand complex formation reactions of Cu(II) with PDC and some bio-relevant ligands.

PubMed

El-Sherif, Ahmed A; Shoukry, Mohamed M

2007-03-01

The formation equilibria of copper(II) complexes and the ternary complexes Cu(PDC)L (PDC=2,6-bis-(hydroxymethyl)-pyridine, HL=amino acid, amides or DNA constituents) have been investigated. Ternary complexes are formed by a simultaneous mechanism. The results showed the formation of Cu(PDC)L, Cu(PDC, H(-1))(L) and Cu(PDC, H(-2))(L) complexes. The concentration distribution of the complexes in solution is evaluated as a function of pH. The effect of dioxane as a solvent on the protonation constant of PDC and the formation constants of Cu(II) complexes are discussed. The thermodynamic parameters DeltaH degrees and DeltaS degrees calculated from the temperature dependence of the equilibrium constants are investigated.

Positronium formation studies in solid molecular complexes: Triphenylphosphine oxide-triphenylmethanol

NASA Astrophysics Data System (ADS)

Oliveira, F. C.; Denadai, A. M. L.; Fulgêncio, F. H.; Magalhães, W. F.; Alcântara, A. F. C.; Windmöller, D.; Machado, J. C.

2012-06-01

Positronium formation in triphenylphosphine oxide (TPPO), triphenylmethanol (TPM), and systems [TPPO(1-X)ṡTPMX] has been studied. The low probability of positronium formation in complex [TPPO0.5ṡTPM0.5] was attributed to strong hydrogen bond and sixfold phenyl embrace interactions. These strong interactions in complex reduce the possibility of the n- and π-electrons to interact with positrons on the spur and consequently, the probability of positronium formation is lower. The τ3 parameter and free volume (correlated to τ3) were also sensitive to the formation of hydrogen bonds and sixfold phenyl embrace interactions within the complex. For physical mixture the positron annihilation parameters remained unchanged throughout the composition range.

Amide-Directed Photoredox Catalyzed C-C Bond Formation at Unactivated sp3 C-H Bonds

PubMed Central

Chu, John C. K.; Rovis, Tomislav

2017-01-01

Carbon-carbon (C-C) bond formation is paramount in the synthesis of biologically relevant molecules, modern synthetic materials and commodity chemicals such as fuels and lubricants. Traditionally, the presence of a functional group is required at the site of C-C bond formation. Strategies that allow C-C bond formation at inert carbon-hydrogen (C-H) bonds allow scientists to access molecules which would otherwise be inaccessible and to develop more efficient syntheses of complex molecules.1,2 Herein we report a method for the formation of C-C bonds by directed cleavage of traditionally non-reactive C-H bonds and their subsequent coupling with readily available alkenes. Our methodology allows for the selective C-C bond formation at single C-H bonds in molecules that contain a multitude of seemingly indifferentiable such bonds. Selectivity arises through a relayed photoredox catalyzed oxidation of an N-H bond. We anticipate our findings to serve as a starting point for functionalization at inert C-H bonds through a hydrogen atom transfer strategy. PMID:27732580

KEEPING A STEP AHEAD - FORMATIVE PHASE OF A WORKPLACE INTERVENTION TRIAL TO PREVENT OBESITY

PubMed Central

Zapka, Jane; Lemon, Stephenie C.; Estabrook, Barbara B.; Jolicoeur, Denise G.

2008-01-01

Background Ecological interventions hold promise for promoting overweight and obesity prevention in worksites. Given the paucity of evaluative research in the hospital worksite setting, considerable formative work is required for successful implementation and evaluation. Purpose This paper describes the formative phases of Step Ahead, a site-randomized controlled trial of a multi-level intervention that promotes physical activity and healthy eating in 6 hospitals in central Massachusetts. The purpose of the formative research phase was to increase the feasibility, effectiveness and likelihood of sustainability of the intervention. Design and Procedures The Step Ahead ecological intervention approach targets change at the organization, the interpersonal work environment and the individual levels. The intervention was developed using fundamental steps of intervention mapping and important tenets of participatory research. Formative research methods were used to engage leadership support and assistance and to develop an intervention plan that is both theoretically and practically grounded. This report uses observational data, program minutes and reports, and process tracking data. Developmental Strategies and Observations Leadership involvement (key informant interviews and advisory boards), employee focus groups and advisory boards, and quantitative environmental assessments cultivated participation and support. Determining multiple foci of change and designing measurable objectives and generic assessment tools to document progress are complex challenges encountered in planning phases. Lessons Learned Multi-level trials in diverse organizations require flexibility and balance of theory application and practice-based perspectives to affect impact and outcome objectives. Formative research is an essential component. PMID:18073339

Examination of the superoxide/hydrogen peroxide forming and quenching potential of mouse liver mitochondria.

PubMed

Slade, Liam; Chalker, Julia; Kuksal, Nidhi; Young, Adrian; Gardiner, Danielle; Mailloux, Ryan J

2017-08-01

Pyruvate dehydrogenase (PDHC) and α-ketoglutarate dehydrogenase complex (KGDHC) are important sources of reactive oxygen species (ROS). In addition, it has been found that mitochondria can also serve as sinks for cellular hydrogen peroxide (H 2 O 2 ). However, the ROS forming and quenching capacity of liver mitochondria has never been thoroughly examined. Here, we show that mouse liver mitochondria use catalase, glutathione (GSH), and peroxiredoxin (PRX) systems to quench ROS. Incubation of mitochondria with catalase inhibitor 3-amino-1,2,4-triazole (triazole) induced a significant increase in pyruvate or α-ketoglutarate driven O 2 - /H 2 O 2 formation. 1-Choro-2,4-dinitrobenzene (CDNB), which depletes glutathione (GSH), elicited a similar effect. Auranofin (AF), a thioredoxin reductase-2 (TR2) inhibitor which disables the PRX system, did not significantly change O 2 - /H 2 O 2 formation. By contrast catalase, GSH, and PRX were all required to scavenging extramitochondrial H 2 O 2 . In this study, the ROS forming potential of PDHC, KGDHC, Complex I, and Complex III was also profiled. Titration of mitochondria with 3-methyl-2-oxovaleric acid (KMV), a specific inhibitor for O 2 - /H 2 O 2 production by KGDHC, induced a ~86% and ~84% decrease in ROS production during α-ketoglutarate and pyruvate oxidation. Titration of myxothiazol, a Complex III inhibitor, decreased O 2 - /H 2 O 2 formation by ~45%. Rotenone also lowered ROS production in mitochondria metabolizing pyruvate or α-ketoglutarate indicating that Complex I does not contribute to ROS production during forward electron transfer from NADH. Taken together, our results indicate that KGDHC and Complex III are high capacity sites for O 2 - /H 2 O 2 production in mouse liver mitochondria. We also confirm that catalase plays a role in quenching either exogenous or intramitochondrial H 2 O 2 . Copyright © 2017 Elsevier B.V. All rights reserved.

Autolysosomal β-catenin degradation regulates Wnt-autophagy-p62 crosstalk

PubMed Central

Petherick, Katy J; Williams, Ann C; Lane, Jon D; Ordóñez-Morán, Paloma; Huelsken, Joerg; Collard, Tracey J; Smartt, Helena JM; Batson, Jennifer; Malik, Karim; Paraskeva, Chris; Greenhough, Alexander

2013-01-01

The Wnt/β-catenin signalling and autophagy pathways each play important roles during development, adult tissue homeostasis and tumorigenesis. Here we identify the Wnt/β-catenin signalling pathway as a negative regulator of both basal and stress-induced autophagy. Manipulation of β-catenin expression levels in vitro and in vivo revealed that β-catenin suppresses autophagosome formation and directly represses p62/SQSTM1 (encoding the autophagy adaptor p62) via TCF4. Furthermore, we show that during nutrient deprivation β-catenin is selectively degraded via the formation of a β-catenin–LC3 complex, attenuating β-catenin/TCF-driven transcription and proliferation to favour adaptation during metabolic stress. Formation of the β-catenin–LC3 complex is mediated by a W/YXXI/L motif and LC3-interacting region (LIR) in β-catenin, which is required for interaction with LC3 and non-proteasomal degradation of β-catenin. Thus, Wnt/β-catenin represses autophagy and p62 expression, while β-catenin is itself targeted for autophagic clearance in autolysosomes upon autophagy induction. These findings reveal a regulatory feedback mechanism that place β-catenin at a key cellular integration point coordinating proliferation with autophagy, with implications for targeting these pathways for cancer therapy. PMID:23736261

Crystal structure of the Haemophilus influenzae Hap adhesin reveals an intercellular oligomerization mechanism for bacterial aggregation

PubMed Central

Meng, Guoyu; Spahich, Nicole; Kenjale, Roma; Waksman, Gabriel; St Geme, Joseph W

2011-01-01

Bacterial biofilms are complex microbial communities that are common in nature and are being recognized increasingly as an important determinant of bacterial virulence. However, the structural determinants of bacterial aggregation and eventual biofilm formation have been poorly defined. In Gram-negative bacteria, a major subgroup of extracellular proteins called self-associating autotransporters (SAATs) can mediate cell–cell adhesion and facilitate biofilm formation. In this study, we used the Haemophilus influenzae Hap autotransporter as a prototype SAAT to understand how bacteria associate with each other. The crystal structure of the H. influenzae HapS passenger domain (harbouring the SAAT domain) was determined to 2.2 Å by X-ray crystallography, revealing an unprecedented intercellular oligomerization mechanism for cell–cell interaction. The C-terminal SAAT domain folds into a triangular-prism-like structure that can mediate Hap–Hap dimerization and higher degrees of multimerization through its F1–F2 edge and F2 face. The intercellular multimerization can give rise to massive buried surfaces that are required for overcoming the repulsive force between cells, leading to bacterial cell–cell interaction and formation of complex microcolonies. PMID:21841773

The requirement for bivalent cations in formation of nicotinamide–adenine dinucleotide by nicotinamide mononucleotide adenylyltransferase of pig-liver nuclei

PubMed Central

Jackson, J. F.; Atkinson, M. R.

1966-01-01

1. The requirement for bivalent cations in catalysis of NAD formation from ATP and NMN in the presence of NMN adenylyltransferase of pig-liver nuclei was studied. Rates of NAD formation in the presence of the activating cations Cd2+, Mn2+, Mg2+, Zn2+, Co2+ and Ni2+ were approximately a linear function of heats of hydration of the corresponding ions. Ba2+, Sr2+, Ca2+, Cu2+ and Be2+ did not activate the enzyme; Be2+ inhibited the reaction in the presence of Mg2+ and, to a greater extent, in the presence of Ni2+. 2. Michaelis constants for NAD formation, measured in a coupled assay with NMN adenylyltransferase and alcohol dehydrogenase at pH8·0 and 25°, in the presence of 3mm concentrations of the unvaried reactants, were 88±7μm-ATP, 42±4μm-NMN and 85±4μm-Mg2+. The results at this pH and at pH7·5 were consistent with mechanisms in which Mg2+–ATP complex is a reactant and free ATP a competitive inhibitor. 3. Formation of nicotinamide–hypoxanthine dinucleotide from NMN and ITP in the presence of the transferase was also more rapid with Ni2+ and Co2+ than with Mg2+. PMID:4291356

Formation of a parallel-stranded DNA homoduplex by d(GGA) repeat oligonucleotides.

PubMed Central

Suda, T; Mishima, Y; Asakura, H; Kominami, R

1995-01-01

The GGA9-H molecules consisting of a double helical stretch followed by a single-stranded 3'-terminal overhang of nine GGA sequence repeats exhibited a gel mobility-shifted band in a concentration-dependent manner, suggestive of the intermolecular complex formation. The position of the shifted band in a gel was almost identical to that of the Y-shaped dimer marker of the same molecular weight that had the two double-helices at one side. This suggests that GGA9-H dimerizes in a parallel orientation without the formation of four-stranded hairpin structure. Since the GGA9-H homoduplex was stably formed at pH 4, 7 and 9, the formation does not require protonation or deprotonation of the N1 position of adenines. Neither does it require the N7 group of guanines responsible for Hoogsteen base pairing from the methylation interference and modification studies. Modification of the N7 group of guanines with dimethyl sulfate (DMS) did not inhibit the association and also the N7 group in the homoduplex was not protected from DMS. On the other hand, the GAA9-H having the G to A base substitution did not show such an association with either GGA9-H or GAA9-H. These results suggest that the homoduplex formation may be due to G.G base pairing through non-Hoogsteen hydrogen bonds. Images PMID:7479009

Implications of latest Pennsylvanian to Middle Permian paleontological and U-Pb SHRIMP data from the Tecomate Formation to re-dating tectonothermal events in the Acatlán Complex, Southern Mexico

USGS Publications Warehouse

Keppie, J. Duncan; Sandberg, Charles A.; Miller, B.V.; Sanchez-Zavala, J. L.; Nance, R.D.; Poole, Forrest G.

2004-01-01

Limestones in the highly deformed Tecomate Formation, uppermost unit of the Acatla??n Complex, are latest Pennsylvanian-earliest Middle Permian in age rather than Devonian, the latter based on less diagnostic fossils. Conodont collections from two marble horizons now constrain its age to range from latest Pennsylvanian to latest Early Permian or early Middle Permian. The older collection contains Gondolella sp., Neostreptognathodus sp., and Streptognathodus sp., suggesting an oldest age limit close to the Pennsylvanian-Permian time boundary. The other collection contains Sweet-ognathus subsymmetricus, a short-lived species ranging only from Kungurian (latest Leonardian) to Wordian (earliest Guadelupian: 272 ?? 4 to 264 ?? 2 Ma). A fusilinid, Parafusulina c.f. P. antimonioensis Dunbar, in a third Tecomate marble horizon is probably Wordian (early Guadelupian, early Middle Permian). Furthermore, granite pebbles in a Tecomate conglomerate have yielded ???320-264 Ma U-Pb SHRIMP ages probably derived from the ???288 Ma, arc-related Totoltepec pluton. Collectively, these data suggest a correlation with two nearby units: (1) the Missourian-Leonardian carbonate horizons separated by a Wolfcampian(?) conglomerate in the upper part of the less deformed San Salvador Patlanoaya Formation; and (2) the clastic, Westphalian-Leonardian Matzitzi Formation. This requires that deformation in the Tecomate Formation be of Early-Middle Permian age rather than Devonian. These three formations are re-interpreted as periarc deposits with deformation related to oblique subduction. The revised dating of the Tecomate Formation is consistent with new data, which indicates that the unconformity between the Tecomate and the Piaxtla Group is mid-Carboniferous and corresponds to a tectonothermal event. ?? 2004 by V. H. Winston and Son, Inc. All rights reserved.

Training & Personnel Systems Technology. R&D Program Description FY 84-85.

DTIC Science & Technology

1984-04-01

performance requirements in terms of rapid response times, high rates of information processing, and complex decision making that tax the capabilities...makers to make linguistic and format changes to texts to enhance general literacy rates , (d) begin integrating human and animal data on stress ;ffects...systems are being Integrated Into the force at unprecedented rates , arrival of this sophisticated, high-technology equipment will coincide with increased

Joint Vision for the Korean Peninsula -- Can We Get There?

DTIC Science & Technology

2012-03-11

complex problem that requires a multifaceted approach. Trilateral cooperation with China coupled with all the elements of the Alliance’s elements of...national power can set the conditions for the Joint Vision Statement to become a reality in this century. 15. SUBJECT TERMS Northeast Asia, China ...We Get There? FORMAT: Strategy Research Project DATE: 11 March 2012 WORD COUNT: 5,917 PAGES: 30 KEY TERMS: Northeast Asia, China

A semi-automated workflow for biodiversity data retrieval, cleaning, and quality control

PubMed Central

Mathew, Cherian; Obst, Matthias; Vicario, Saverio; Haines, Robert; Williams, Alan R.; de Jong, Yde; Goble, Carole

2014-01-01

Abstract The compilation and cleaning of data needed for analyses and prediction of species distributions is a time consuming process requiring a solid understanding of data formats and service APIs provided by biodiversity informatics infrastructures. We designed and implemented a Taverna-based Data Refinement Workflow which integrates taxonomic data retrieval, data cleaning, and data selection into a consistent, standards-based, and effective system hiding the complexity of underlying service infrastructures. The workflow can be freely used both locally and through a web-portal which does not require additional software installations by users. PMID:25535486

The structure of a ring-opened proliferating cell nuclear antigen-replication factor C complex revealed by fluorescence energy transfer.

PubMed

Zhuang, Zhihao; Yoder, Bonita L; Burgers, Peter M J; Benkovic, Stephen J

2006-02-21

Numerous proteins that function in DNA metabolic pathways are known to interact with the proliferating cell nuclear antigen (PCNA). The important function of PCNA in stimulating various cellular activities requires its topological linkage with DNA. Loading of the circular PCNA onto duplex DNA requires the activity of a clamp-loader [replication factor C (RFC)] complex and the energy derived from ATP hydrolysis. The mechanistic and structural details regarding PCNA loading by the RFC complex are still developing. In particular, the positive identification of a long-hypothesized structure of an open clamp-RFC complex as an intermediate in loading has remained elusive. In this study, we capture an open yeast PCNA clamp in a complex with RFC through fluorescence energy transfer experiments. We also follow the topological transitions of PCNA in the various steps of the clamp-loading pathway through both steady-state and stopped-flow fluorescence studies. We find that ATP effectively drives the clamp-loading process to completion with the formation of the closed PCNA bound to DNA, whereas ATPgammaS cannot. The information derived from this work complements that obtained from previous structural and mechanistic studies and provides a more complete picture of a eukaryotic clamp-loading pathway using yeast as a paradigm.

Cyclodextrin inclusion complex formation and solid-state characterization of the natural antioxidants alpha-tocopherol and quercetin.

PubMed

Koontz, John L; Marcy, Joseph E; O'Keefe, Sean F; Duncan, Susan E

2009-02-25

Cyclodextrin (CD) complexation procedures are relatively simple processes, but these techniques often require very specific conditions for each individual guest molecule. Variations of the coprecipitation from aqueous solution technique were optimized for the CD complexation of the natural antioxidants alpha-tocopherol and quercetin. Solid inclusion complex products of alpha-tocopherol/beta-CD and quercetin/gamma-CD had molar ratios of 1.7:1, which were equivalent to 18.1% (w/w) alpha-tocopherol and 13.0% (w/w) quercetin. The molar reactant ratios of CD/antioxidant were optimized at 8:1 to improve the yield of complexation. The product yields of alpha-tocopherol/beta-CD and quercetin/gamma-CD complexes from their individual reactants were calculated as 24 and 21% (w/w), respectively. ATR/FT-IR, 13C CP/MAS NMR, TGA, and DSC provided evidence of antioxidant interaction with CD at the molecular level, which indicated true CD inclusion complexation in the solid state. Natural antioxidant/CD inclusion complexes may serve as novel additives in controlled-release active packaging to extend the oxidative stability of foods.

Formation kinetics and mechanism of metastable vacancy-dioxygen complex in neutron irradiated Czochralski silicon

NASA Astrophysics Data System (ADS)

Dong, Peng; Wang, Rong; Yu, Xuegong; Chen, Lin; Ma, Xiangyang; Yang, Deren

2017-07-01

We have quantitatively investigated the formation kinetics of metastable vacancy-dioxygen (VO2) complex in a structure of [VO + Oi], where a VO complex is trapped in a next-neighbor position to an interstitial oxygen atom (Oi). It is found that the VO annihilation is accompanied by the generation of metastable [VO + Oi] complex during annealing in the temperature range of 220-250 °C. The activation energy for [VO + Oi] generation appears at around 0.48 eV, which is much lower than the counterpart of stable VO2 complex. This indicates that the formation of [VO + Oi] complex originates from the reaction between VO and Oi. The ab initio calculations show that the formation energy of [VO + Oi] complex is larger than that of VO2 complex, which means that [VO + Oi] complex is thermodynamically unfavorable as compared to VO2 complex. However, the binding energy of [VO + Oi] complex is positive, indicating that [VO + Oi] complex is stable against decomposition of VO and Oi in silicon. It is believed that [VO + Oi] complex serves as the intermediate for VO to VO2 conversion.

The CCR4-NOT Complex Is Implicated in the Viability of Aneuploid Yeasts

PubMed Central

Tange, Yoshie; Kurabayashi, Atsushi; Goto, Bunshiro; Hoe, Kwang-Lae; Kim, Dong-Uk; Park, Han-Oh; Hayles, Jacqueline; Chikashige, Yuji; Tsutumi, Chihiro; Hiraoka, Yasushi; Yamao, Fumiaki; Nurse, Paul; Niwa, Osami

2012-01-01

To identify the genes required to sustain aneuploid viability, we screened a deletion library of non-essential genes in the fission yeast Schizosaccharomyces pombe, in which most types of aneuploidy are eventually lethal to the cell. Aneuploids remain viable for a period of time and can form colonies by reducing the extent of the aneuploidy. We hypothesized that a reduction in colony formation efficiency could be used to screen for gene deletions that compromise aneuploid viability. Deletion mutants were used to measure the effects on the viability of spores derived from triploid meiosis and from a chromosome instability mutant. We found that the CCR4-NOT complex, an evolutionarily conserved general regulator of mRNA turnover, and other related factors, including poly(A)-specific nuclease for mRNA decay, are involved in aneuploid viability. Defective mutations in CCR4-NOT complex components in the distantly related yeast Saccharomyces cerevisiae also affected the viability of spores produced from triploid cells, suggesting that this complex has a conserved role in aneuploids. In addition, our findings suggest that the genes required for homologous recombination repair are important for aneuploid viability. PMID:22737087

The mammalian INO80 chromatin remodeling complex is required for replication stress recovery

PubMed Central

Vassileva, Ivelina; Yanakieva, Iskra; Peycheva, Michaela; Gospodinov, Anastas; Anachkova, Boyka

2014-01-01

A number of studies have implicated the yeast INO80 chromatin remodeling complex in DNA replication, but the function of the human INO80 complex during S phase remains poorly understood. Here, we have systematically investigated the involvement of the catalytic subunit of the human INO80 complex during unchallenged replication and under replication stress by following the effects of its depletion on cell survival, S-phase checkpoint activation, the fate of individual replication forks, and the consequences of fork collapse. We report that INO80 was specifically needed for efficient replication elongation, while it was not required for initiation of replication. In the absence of the Ino80 protein, cells became hypersensitive to hydroxyurea and displayed hyperactive ATR-Chk1 signaling. Using bulk and fiber labeling of DNA, we found that cells deficient for Ino80 and Arp8 had impaired replication restart after treatment with replication inhibitors and accumulated double-strand breaks as evidenced by the formation of γ-H2AX and Rad51 foci. These data indicate that under conditions of replication stress mammalian INO80 protects stalled forks from collapsing and allows their subsequent restart. PMID:25016522

Carbon Nanotubes: Molecular Electronic Components

NASA Technical Reports Server (NTRS)

Srivastava, Deepak; Saini, Subhash; Menon, Madhu

1997-01-01

The carbon Nanotube junctions have recently emerged as excellent candidates for use as the building blocks in the formation of nanoscale molecular electronic networks. While the simple joint of two dissimilar tubes can be generated by the introduction of a pair of heptagon-pentagon defects in an otherwise perfect hexagonal graphene sheet, more complex joints require other mechanisms. In this work we explore structural characteristics of complex 3-point junctions of carbon nanotubes using a generalized tight-binding molecular-dynamics scheme. The study of pi-electron local densities of states (LDOS) of these junctions reveal many interesting features, most prominent among them being the defect-induced states in the gap.

Assembly of the Herpes Simplex Virus Capsid: Preformed Triplexes Bind to the Nascent Capsid

PubMed Central

Spencer, Juliet V.; Newcomb, William W.; Thomsen, Darrell R.; Homa, Fred L.; Brown, Jay C.

1998-01-01

The herpes simplex virus type 1 (HSV-1) capsid is a T=16 icosahedral shell that forms in the nuclei of infected cells. Capsid assembly also occurs in vitro in reaction mixtures created from insect cell extracts containing recombinant baculovirus-expressed HSV-1 capsid proteins. During capsid formation, the major capsid protein, VP5, and the scaffolding protein, pre-VP22a, condense to form structures that are extended into procapsids by addition of the triplex proteins, VP19C and VP23. We investigated whether triplex proteins bind to the major capsid-scaffold protein complexes as separate polypeptides or as preformed triplexes. Assembly products from reactions lacking one triplex protein were immunoprecipitated and examined for the presence of the other. The results showed that neither triplex protein bound unless both were present, suggesting that interaction between VP19C and VP23 is required before either protein can participate in the assembly process. Sucrose density gradient analysis was employed to determine the sedimentation coefficients of VP19C, VP23, and VP19C-VP23 complexes. The results showed that the two proteins formed a complex with a sedimentation coefficient of 7.2S, a value that is consistent with formation of a VP19C-VP232 heterotrimer. Furthermore, VP23 was observed to have a sedimentation coefficient of 4.9S, suggesting that this protein exists as a dimer in solution. Deletion analysis of VP19C revealed two domains that may be required for attachment of the triplex to major capsid-scaffold protein complexes; none of the deletions disrupted interaction of VP19C with VP23. We propose that preformed triplexes (VP19C-VP232 heterotrimers) interact with major capsid-scaffold protein complexes during assembly of the HSV-1 capsid. PMID:9557680

Stabilization and activation of alpha-chymotrypsin in water-organic solvent systems by complex formation with oligoamines.

PubMed

Kudryashova, Elena V; Artemova, Tatiana M; Vinogradov, Alexei A; Gladilin, Alexander K; Mozhaev, Vadim V; Levashov, Andrey V

2003-04-01

Formation of enzyme-oligoamine complexes was suggested as an approach to obtain biocatalysts with enhanced resistance towards inactivation in water-organic media. Complex formation results in broadening (by 20-40% v/v ethanol) of the range of cosolvent concentrations where the enzyme retains its catalytic activity (stabilization effect). At moderate cosolvent concentrations (20-40% v/v) complex formation activates the enzyme (by 3-6 times). The magnitude of activation and stabilization effects increases with the number of possible electrostatic contacts between the protein surface and the molecules of oligoamines (OA). Circular dichroism spectra in the far-UV region show that complex formation stabilizes protein conformation and prevents aggregation in water-organic solvent mixtures. Two populations of the complexes with different thermodynamic stabilities were found in alpha-chymotrypsin (CT)-OA systems depending on the CT/OA ratio. The average dissociation constants and stoichiometries of both low- and high-affinity populations of the complexes were estimated. It appears that it is the low-affinity sites on the CT surface that are responsible for the activation effect.

Multi-stage origin of the Coast Range ophiolite, California: Implications for the life cycle of supra-subduction zone ophiolites

USGS Publications Warehouse

Shervais, J.W.; Kimbrough, D.L.; Renne, P.; Hanan, B.B.; Murchey, B.; Snow, C.A.; Zoglman, Schuman M.M.; Beaman, J.

2004-01-01

The Coast Range ophiolite of California is one of the most extensive ophiolite terranes in North America, extending over 700 km from the northernmost Sacramento Valley to the southern Transverse Ranges in central California. This ophiolite, and other ophiolite remnants with similar mid-Jurassic ages, represent a major but short-lived episode of oceanic crust formation that affected much of western North America. The history of this ophiolite is important for models of the tectonic evolution of western North America during the Mesozoic, and a range of conflicting interpretations have arisen. Current petrologic, geochemical, stratigraphic, and radiometric age data all favor the interpretation that the Coast Range ophiolite formed to a large extent by rapid extension in the forearc region of a nascent subduction zone. Closer inspection of these data, however, along with detailed studies of field relationships at several locales, show that formation of the ophiolite was more complex, and requires several stages of formation. Our work shows that exposures of the Coast Range ophiolite preserve evidence for four stages of magmatic development. The first three stages represent formation of the ophiolite above a nascent subduction zone. Rocks associated with the first stage include ophiolite layered gabbros, a sheeted complex, and volcanic rocks vith arc tholeiitic or (roore rarely) low-K calc-alkaline affinities. The second stage is characterized by intrusive wehrlite-clinopyroxenite complexes, intrusive gabbros, Cr-rich diorites, and volcanic rocks with high-Ca boninitic or tholeiitic ankaramite affinities. The third stage includes diorite and quartz diorite plutons, felsic dike and sill complexes, and calc-alkaline volcanic rocks. The first three stages of ophiolite formation were terminated by the intrusion of mid-ocean ridge basalt dikes, and the eruption of mid-ocean ridge basalt or ocean-island basalt volcanic suites. We interpret this final magmatic event (MORB dikes) to represent the collision of an active spreading ridge. Subsequent reorganization of relative plate motions led to sinistral transpression, along with renewed subduction and accretion of the Franciscan Complex. The latter event resulted in uplift and exhumation of the ophiolite by the process of accretionary uplift. ?? 2004 by V. H. Winston and Son, Inc. All rights reserved.

An automated graphics tool for comparative genomics: the Coulson plot generator

PubMed Central

2013-01-01

Background Comparative analysis is an essential component to biology. When applied to genomics for example, analysis may require comparisons between the predicted presence and absence of genes in a group of genomes under consideration. Frequently, genes can be grouped into small categories based on functional criteria, for example membership of a multimeric complex, participation in a metabolic or signaling pathway or shared sequence features and/or paralogy. These patterns of retention and loss are highly informative for the prediction of function, and hence possible biological context, and can provide great insights into the evolutionary history of cellular functions. However, representation of such information in a standard spreadsheet is a poor visual means from which to extract patterns within a dataset. Results We devised the Coulson Plot, a new graphical representation that exploits a matrix of pie charts to display comparative genomics data. Each pie is used to describe a complex or process from a separate taxon, and is divided into sectors corresponding to the number of proteins (subunits) in a complex/process. The predicted presence or absence of proteins in each complex are delineated by occupancy of a given sector; this format is visually highly accessible and makes pattern recognition rapid and reliable. A key to the identity of each subunit, plus hierarchical naming of taxa and coloring are included. A java-based application, the Coulson plot generator (CPG) automates graphic production, with a tab or comma-delineated text file as input and generating an editable portable document format or svg file. Conclusions CPG software may be used to rapidly convert spreadsheet data to a graphical matrix pie chart format. The representation essentially retains all of the information from the spreadsheet but presents a graphically rich format making comparisons and identification of patterns significantly clearer. While the Coulson plot format is highly useful in comparative genomics, its original purpose, the software can be used to visualize any dataset where entity occupancy is compared between different classes. Availability CPG software is available at sourceforge http://sourceforge.net/projects/coulson and http://dl.dropbox.com/u/6701906/Web/Sites/Labsite/CPG.html PMID:23621955

Structure of UreG/UreF/UreH Complex Reveals How Urease Accessory Proteins Facilitate Maturation of Helicobacter pylori Urease

PubMed Central

Fong, Yu Hang; Wong, Ho Chun; Yuen, Man Hon; Lau, Pak Ho; Chen, Yu Wai; Wong, Kam-Bo

2013-01-01

Urease is a metalloenzyme essential for the survival of Helicobacter pylori in acidic gastric environment. Maturation of urease involves carbamylation of Lys219 and insertion of two nickel ions at its active site. This process requires GTP hydrolysis and the formation of a preactivation complex consisting of apo-urease and urease accessory proteins UreF, UreH, and UreG. UreF and UreH form a complex to recruit UreG, which is a SIMIBI class GTPase, to the preactivation complex. We report here the crystal structure of the UreG/UreF/UreH complex, which illustrates how UreF and UreH facilitate dimerization of UreG, and assembles its metal binding site by juxtaposing two invariant Cys66-Pro67-His68 metal binding motif at the interface to form the (UreG/UreF/UreH)2 complex. Interaction studies revealed that addition of nickel and GTP to the UreG/UreF/UreH complex releases a UreG dimer that binds a nickel ion at the dimeric interface. Substitution of Cys66 and His68 with alanine abolishes the formation of the nickel-charged UreG dimer. This nickel-charged UreG dimer can activate urease in vitro in the presence of the UreF/UreH complex. Static light scattering and atomic absorption spectroscopy measurements demonstrated that the nickel-charged UreG dimer, upon GTP hydrolysis, reverts to its monomeric form and releases nickel to urease. Based on our results, we propose a mechanism on how urease accessory proteins facilitate maturation of urease. PMID:24115911

Loss of laminin alpha 1 results in multiple structural defects and divergent effects on adhesion during vertebrate optic cup morphogenesis

PubMed Central

Bryan, Chase D.; Chien, Chi-Bin; Kwan, Kristen M.

2016-01-01

The vertebrate eye forms via a complex set of morphogenetic events. The optic vesicle evaginates and undergoes transformative shape changes to form the optic cup, in which neural retina and retinal pigmented epithelium enwrap the lens. It has long been known that a complex, glycoprotein-rich extracellular matrix layer surrounds the developing optic cup throughout the process, yet the functions of the matrix and its specific molecular components have remained unclear. Previous work established a role for laminin extracellular matrix in particular steps of eye development, including optic vesicle evagination, lens differentiation, and retinal ganglion cell polarization, yet it is unknown what role laminin might play in the early process of optic cup formation subsequent to the initial step of optic vesicle evagination. Here, we use the zebrafish lama1 mutant (lama1UW1) to determine the function of laminin during optic cup morphogenesis. Using live imaging, we find, surprisingly, that loss of laminin leads to divergent effects on focal adhesion assembly in a spatiotemporally-specific manner, and that laminin is required for multiple steps of optic cup morphogenesis, including optic stalk constriction, invagination, and formation of a spherical lens. Laminin is not required for single cell behaviors and changes in cell shape. Rather, in lama1UW1 mutants, loss of epithelial polarity and altered adhesion lead to defective tissue architecture and formation of a disorganized retina. These results demonstrate that the laminin extracellular matrix plays multiple critical roles regulating adhesion and polarity to establish and maintain tissue structure during optic cup morphogenesis. PMID:27339294

Actomyosin tension is required for correct recruitment of adherens junction components and zonula occludens formation.

PubMed

Miyake, Yuka; Inoue, Naoko; Nishimura, Koji; Kinoshita, Nagatoki; Hosoya, Hiroshi; Yonemura, Shigenobu

2006-05-15

The adherens junction (AJ) densely associated with actin filaments is a major cell-cell adhesion structure. To understand the importance of actin filament association in AJ formation, we first analyzed punctate AJs in NRK fibroblasts where one actin cable binds to one AJ structure unit. The accumulation of AJ components such as the cadherin/catenin complex and vinculin, as well as the formation of AJ-associated actin cables depended on Rho activity. Inhibitors for the Rho target, ROCK, which regulates myosin II activity, and for myosin II ATPase prevented the accumulation of AJ components, indicating that myosin II activity is more directly involved than Rho activity. Depletion of myosin II by RNAi showed similar results. The inhibition of myosin II activity in polarized epithelial MTD-1A cells affected the accumulation of vinculin to circumferential AJ (zonula adherens). Furthermore, correct zonula occludens (tight junction) formation along the apicobasal axis that requires cadherin activity was also impaired. Although MDCK cells which are often used as typical epithelial cells do not have a typical zonula adherens, punctate AJs formed dependently on myosin II activity by inducing wound closure in a MDCK cell sheet. These findings suggest that tension generated by actomyosin is essential for correct AJ assembly.

Membrane microdomains and the cytoskeleton constrain AtHIR1 dynamics and facilitate the formation of an AtHIR1-associated immune complex.

PubMed

Lv, Xueqin; Jing, Yanping; Xiao, Jianwei; Zhang, Yongdeng; Zhu, Yingfang; Julian, Russell; Lin, Jinxing

2017-04-01

Arabidopsis hypersensitive-induced reaction (AtHIR) proteins function in plant innate immunity. However, the underlying mechanisms by which AtHIRs participate in plant immunity remain elusive. Here, using VA-TIRFM and FLIM-FRET, we revealed that AtHIR1 is present in membrane microdomains and co-localizes with the membrane microdomain marker REM1.3. Single-particle tracking analysis revealed that membrane microdomains and the cytoskeleton, especially microtubules, restrict the lateral mobility of AtHIR1 at the plasma membrane and facilitate its oligomerization. Furthermore, protein proximity index measurements, fluorescence cross-correlation spectroscopy, and biochemical experiments demonstrated that the formation of the AtHIR1 complex upon pathogen perception requires intact microdomains and cytoskeleton. Taken together, these findings suggest that microdomains and the cytoskeleton constrain AtHIR1 dynamics, promote AtHIR1 oligomerization, and increase the efficiency of the interactions of AtHIR1 with components of the AtHIR1 complex in response to pathogens, thus providing valuable insight into the mechanisms of defense-related responses in plants. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.

Emerging role of ILK and ELMO2 in the integration of adhesion and migration pathways

PubMed Central

Ho, Ernest; Dagnino, Lina

2012-01-01

Integrins and their associated proteins are essential components of the cellular machinery that modulates adhesion and migration. In particular, integrin-linked kinase (ILK), which binds to the cytoplasmic tail of β1 integrins, is required for migration in a variety of cell types. We previously identified engulfment and motility 2 (ELMO2) as an ILK-binding protein in epidermal keratinocytes. Recently, we investigated the biological role of the ILK/ELMO2 complexes, and found that they exist in the cytoplasm. ILK/ELMO2 species are recruited by active RhoG to the plasma membrane, where they induce Rac1 activation and formation of lamellipodia at the leading edge of migrating cells. A large number of growth factors and cytokines induce keratinocyte migration. However, we found that formation of RhoG/ELMO2/ILK complexes occurs selectively upon stimulation by epidermal growth factor, but not by transforming growth factor-β1 or keratinocyte growth factor. Herein we discuss the relevance of these complexes to our understanding of the molecular mechanisms involved in cell migration, as well as their potential functions in morphogenesis and tissue regeneration following injury. PMID:22568984

Emerging role of ILK and ELMO2 in the integration of adhesion and migration pathways.

PubMed

Ho, Ernest; Dagnino, Lina

2012-01-01

Integrins and their associated proteins are essential components of the cellular machinery that modulates adhesion and migration. In particular, integrin-linked kinase (ILK), which binds to the cytoplasmic tail of β1 integrins, is required for migration in a variety of cell types. We previously identified engulfment and motility 2 (ELMO2) as an ILK-binding protein in epidermal keratinocytes. Recently, we investigated the biological role of the ILK/ELMO2 complexes, and found that they exist in the cytoplasm. ILK/ELMO2 species are recruited by active RhoG to the plasma membrane, where they induce Rac1 activation and formation of lamellipodia at the leading edge of migrating cells. A large number of growth factors and cytokines induce keratinocyte migration. However, we found that formation of RhoG/ELMO2/ILK complexes occurs selectively upon stimulation by epidermal growth factor, but not by transforming growth factor-β1 or keratinocyte growth factor. Herein we discuss the relevance of these complexes to our understanding of the molecular mechanisms involved in cell migration, as well as their potential functions in morphogenesis and tissue regeneration following injury.

The nucleoporins Nup170p and Nup157p are essential for nuclear pore complex assembly

PubMed Central

Makio, Tadashi; Stanton, Leslie H.; Lin, Cheng-Chao; Goldfarb, David S.; Weis, Karsten

2009-01-01

We have established that two homologous nucleoporins, Nup170p and Nup157p, play an essential role in the formation of nuclear pore complexes (NPCs) in Saccharomyces cerevisiae. By regulating their synthesis, we showed that the loss of these nucleoporins triggers a decrease in NPCs caused by a halt in new NPC assembly. Preexisting NPCs are ultimately lost by dilution as cells grow, causing the inhibition of nuclear transport and the loss of viability. Significantly, the loss of Nup170p/Nup157p had distinct effects on the assembly of different architectural components of the NPC. Nucleoporins (nups) positioned on the cytoplasmic face of the NPC rapidly accumulated in cytoplasmic foci. These nup complexes could be recruited into new NPCs after reinitiation of Nup170p synthesis, and may represent a physiological intermediate. Loss of Nup170p/Nup157p also caused core and nucleoplasmically positioned nups to accumulate in NPC-like structures adjacent to the inner nuclear membrane, which suggests that these nucleoporins are required for formation of the pore membrane and the incorporation of cytoplasmic nups into forming NPCs. PMID:19414608

A synthetic lethal screen identifies FAT1 as an antagonist of caspase-8 in extrinsic apoptosis

PubMed Central

Kranz, Dominique; Boutros, Michael

2014-01-01

The extrinsic apoptosis pathway is initiated by binding of death ligands to death receptors resulting in the formation of the death-inducing signaling complex (DISC). Activation of procaspase-8 within the DISC and its release from the signaling complex is required for processing executor caspases and commiting cell death. Here, we report that the atypical cadherin FAT1 interacts with caspase-8 preventing the association of caspase-8 with the DISC. We identified FAT1 in a genome-wide siRNA screen for synthetic lethal interactions with death receptor-mediated apoptosis. Knockdown of FAT1 sensitized established and patient-derived glioblastoma cell lines for apoptosis transduced by cell death ligands. Depletion of FAT1 resulted in enhanced procaspase-8 recruitment to the DISC and increased formation of caspase-8 containing secondary signaling complexes. In addition, FAT1 knockout cell lines generated by CRISPR/Cas9-mediated genome engineering were more susceptible for death receptor-mediated apoptosis. Our findings provide evidence for a mechanism to control caspase-8-dependent cell death by the atypical cadherin FAT1. These results contribute towards the understanding of effector caspase regulation in physiological conditions. PMID:24442637

A synthetic lethal screen identifies FAT1 as an antagonist of caspase-8 in extrinsic apoptosis.

PubMed

Kranz, Dominique; Boutros, Michael

2014-02-03

The extrinsic apoptosis pathway is initiated by binding of death ligands to death receptors resulting in the formation of the death-inducing signaling complex (DISC). Activation of procaspase-8 within the DISC and its release from the signaling complex is required for processing executor caspases and commiting cell death. Here, we report that the atypical cadherin FAT1 interacts with caspase-8 preventing the association of caspase-8 with the DISC. We identified FAT1 in a genome-wide siRNA screen for synthetic lethal interactions with death receptor-mediated apoptosis. Knockdown of FAT1 sensitized established and patient-derived glioblastoma cell lines for apoptosis transduced by cell death ligands. Depletion of FAT1 resulted in enhanced procaspase-8 recruitment to the DISC and increased formation of caspase-8 containing secondary signaling complexes. In addition, FAT1 knockout cell lines generated by CRISPR/Cas9-mediated genome engineering were more susceptible for death receptor-mediated apoptosis. Our findings provide evidence for a mechanism to control caspase-8-dependent cell death by the atypical cadherin FAT1. These results contribute towards the understanding of effector caspase regulation in physiological conditions.

Protein Kinase Cδ and Calmodulin Regulate Epidermal Growth Factor Receptor Recycling from Early Endosomes through Arp2/3 Complex and Cortactin

PubMed Central

Lladó, Anna; Timpson, Paul; Vilà de Muga, Sandra; Moretó, Jemina; Pol, Albert; Grewal, Thomas; Daly, Roger J.

2008-01-01

The intracellular trafficking of the epidermal growth factor receptor (EGFR) is regulated by a cross-talk between calmodulin (CaM) and protein kinase Cδ (PKCδ). On inhibition of CaM, PKCδ promotes the formation of enlarged early endosomes and blocks EGFR recycling and degradation. Here, we show that PKCδ impairs EGFR trafficking due to the formation of an F-actin coat surrounding early endosomes. The PKCδ-induced polymerization of actin is orchestrated by the Arp2/3 complex and requires the interaction of cortactin with PKCδ. Accordingly, inhibition of actin polymerization by using cytochalasin D or by overexpression of active cofilin, restored the normal morphology of the organelle and the recycling of EGFR. Similar results were obtained after down-regulation of cortactin and the sequestration of the Arp2/3 complex. Furthermore we demonstrate an interaction of cortactin with CaM and PKCδ, the latter being dependent on CaM inhibition. In summary, this study provides the first evidence that CaM and PKCδ organize actin dynamics in the early endosomal compartment, thereby regulating the intracellular trafficking of EGFR. PMID:17959830

Prebiotic selection and assembly of proteinogenic amino acids and natural nucleotides from complex mixtures

NASA Astrophysics Data System (ADS)

Islam, Saidul; Bučar, Dejan-Krešimir; Powner, Matthew W.

2017-06-01

A central problem for the prebiotic synthesis of biological amino acids and nucleotides is to avoid the concomitant synthesis of undesired or irrelevant by-products. Additionally, multistep pathways require mechanisms that enable the sequential addition of reactants and purification of intermediates that are consistent with reasonable geochemical scenarios. Here, we show that 2-aminothiazole reacts selectively with two- and three-carbon sugars (glycolaldehyde and glyceraldehyde, respectively), which results in their accumulation and purification as stable crystalline aminals. This permits ribonucleotide synthesis, even from complex sugar mixtures. Remarkably, aminal formation also overcomes the thermodynamically favoured isomerization of glyceraldehyde into dihydroxyacetone because only the aminal of glyceraldehyde separates from the equilibrating mixture. Finally, we show that aminal formation provides a novel pathway to amino acids that avoids the synthesis of the non-proteinogenic α,α-disubstituted analogues. The common physicochemical mechanism that controls the proteinogenic amino acid and ribonucleotide assembly from prebiotic mixtures suggests that these essential classes of metabolite had a unified chemical origin.

An evidence based hypothesis on the existence of two pathways of mitochondrial crista formation

PubMed Central

Harner, Max E; Unger, Ann-Katrin; Geerts, Willie JC; Mari, Muriel; Izawa, Toshiaki; Stenger, Maria; Geimer, Stefan; Reggiori, Fulvio; Westermann, Benedikt; Neupert, Walter

2016-01-01

Metabolic function and architecture of mitochondria are intimately linked. More than 60 years ago, cristae were discovered as characteristic elements of mitochondria that harbor the protein complexes of oxidative phosphorylation, but how cristae are formed, remained an open question. Here we present experimental results obtained with yeast that support a novel hypothesis on the existence of two molecular pathways that lead to the generation of lamellar and tubular cristae. Formation of lamellar cristae depends on the mitochondrial fusion machinery through a pathway that is required also for homeostasis of mitochondria and mitochondrial DNA. Tubular cristae are formed via invaginations of the inner boundary membrane by a pathway independent of the fusion machinery. Dimerization of the F1FO-ATP synthase and the presence of the MICOS complex are necessary for both pathways. The proposed hypothesis is suggested to apply also to higher eukaryotes, since the key components are conserved in structure and function throughout evolution. DOI: http://dx.doi.org/10.7554/eLife.18853.001 PMID:27849155

Modification of the SERS spectrum of cyanide traces due to complex formation between cyanide and silver

NASA Astrophysics Data System (ADS)

Cao Dao, Tran; Kieu, Ngoc Minh; Quynh Ngan Luong, Truc; Cao, Tuan Anh; Hai Nguyen, Ngoc; Le, Van Vu

2018-06-01

It is well known that cyanide is an extremely toxic lethal poison with human death within minutes after exposure to only 300 ppm cyanide. On the other hand, cyanide is released into the environment (mainly through waste water) every day from various human activities. Therefore, rapid, sensitive and cost-effective cyanide trace detection is an urgent need. Surface-enhanced Raman scattering (SERS) is a method that meets these requirements. It should be noted, however, that in this technique SERS substrates, which are usually made of gold or silver, will be leached with aqueous cyanide by the formation of complexes between gold or silver with cyanide. This will cause the SERS spectrum of cyanide to be modified. When determining cyanide concentrations by SERS analysis, this spectral modification should be taken into account. This report presents the SERS spectral modification of aqueous cyanide traces (in ppm and lower concentration range) when the SERS substrates used are flower-like silver micro-structures.

The Transcription Factor Rbf1 Is the Master Regulator for b-Mating Type Controlled Pathogenic Development in Ustilago maydis

PubMed Central

Vranes, Miroslav; Wahl, Ramon; Pothiratana, Chetsada; Schuler, David; Vincon, Volker; Finkernagel, Florian; Flor-Parra, Ignacio; Kämper, Jörg

2010-01-01

In the phytopathogenic basidiomycete Ustilago maydis, sexual and pathogenic development are tightly connected and controlled by the heterodimeric bE/bW transcription factor complex encoded by the b-mating type locus. The formation of the active bE/bW heterodimer leads to the formation of filaments, induces a G2 cell cycle arrest, and triggers pathogenicity. Here, we identify a set of 345 bE/bW responsive genes which show altered expression during these developmental changes; several of these genes are associated with cell cycle coordination, morphogenesis and pathogenicity. 90% of the genes that show altered expression upon bE/bW-activation require the zinc finger transcription factor Rbf1, one of the few factors directly regulated by the bE/bW heterodimer. Rbf1 is a novel master regulator in a multilayered network of transcription factors that facilitates the complex regulatory traits of sexual and pathogenic development. PMID:20700446

Modulation of Correlated Segment Fluctuations in IDPs upon Complex Formation as an Allosteric Regulatory Mechanism.

PubMed

Beier, Andreas; Schwarz, Thomas C; Kurzbach, Dennis; Platzer, Gerald; Tribuzio, Francesca; Konrat, Robert

2018-05-05

Molecular recognition of and by intrinsically disordered proteins (IDPs) is an intriguing and still largely elusive phenomenon. Typically, protein recognition involving IDPs requires either folding upon binding or, alternatively, the formation of "fuzzy complexes." Here we show via correlation analyses of paramagnetic relaxation enhancement data unprecedented and striking alterations of the concerted fluctuations within the conformational ensemble of IDPs upon ligand binding. We study the binding of α-synuclein to calmodulin, a ubiquitous calcium-binding protein, and the binding of the extracellular matrix IDP osteopontin to heparin, a mimic of the extracellular matrix ligand hyaluronic acid. In both cases, binding leads to reduction of correlated long-range motions in these two IDPs and thus indicates a loosening of structural compaction upon binding. Most importantly, however, the simultaneous presence of correlated and anti-correlated fluctuations in IDPs suggests the prevalence of "energetic frustration" and provides an explanation for the puzzling observation of disordered allostery in IDPs. Copyright © 2018. Published by Elsevier Ltd.

Disease-enhancing antibodies improve the efficacy of bacterial toxin-neutralizing antibodies

PubMed Central

Chow, Siu-Kei; Smith, Cameron; MacCarthy, Thomas; Pohl, Mary Ann; Bergman, Aviv; Casadevall, Arturo

2013-01-01

SUMMARY During infection, humoral immunity produces a polyclonal response with various immunoglobulins recognizing different epitopes within the microbe or toxin. Despite this diverse response, the biological activity of an antibody (Ab) is usually assessed by the action of a monoclonal population. We demonstrate that a combination of monoclonal antibodies (mAbs) that are individually disease-enhancing or neutralizing to Bacillus anthracis protective antigen (PA), a component of anthrax toxin, results in significantly augmented protection against the toxin. This boosted protection is Fc gamma receptor (FcγR)-dependent and involves the formation of stoichiometrically defined mAb-PA complexes that requires immunoglobulin bivalence and simultaneous interaction between PA and the two mAbs. The formation of these mAb-PA complexes inhibits PA oligomerization, resulting in protection. These data suggest that functional assessments of single Abs may inaccurately predict how the same Abs will operate in polyclonal preparations and imply that potentially therapeutic mAbs may be overlooked in single Ab screens. PMID:23601104

The subcortical maternal complex controls symmetric division of mouse zygotes by regulating F-actin dynamics.

PubMed

Yu, Xing-Jiang; Yi, Zhaohong; Gao, Zheng; Qin, Dandan; Zhai, Yanhua; Chen, Xue; Ou-Yang, Yingchun; Wang, Zhen-Bo; Zheng, Ping; Zhu, Min-Sheng; Wang, Haibin; Sun, Qing-Yuan; Dean, Jurrien; Li, Lei

2014-09-11

Maternal effect genes play critical roles in early embryogenesis of model organisms where they have been intensively investigated. However, their molecular function in mammals remains largely unknown. Recently, we identified a subcortical maternal complex (SCMC) that contains four proteins encoded by maternal effect genes (Mater, Filia, Floped and Tle6). Here we report that TLE6, similar to FLOPED and MATER, stabilizes the SCMC and is necessary for cleavage beyond the two-cell stage of development. We document that the SCMC is required for formation of the cytoplasmic F-actin meshwork that controls the central position of the spindle and ensures symmetric division of mouse zygotes. We further demonstrate that the SCMC controls formation of the actin cytoskeleton specifically via Cofilin, a key regulator of F-actin assembly. Our results provide molecular insight into the physiological function of TLE6, its interaction with the SCMC and their roles in the symmetric division of the zygote in early mouse development.

FOXO1 expression in keratinocytes promotes connective tissue healing

PubMed Central

Zhang, Chenying; Lim, Jason; Liu, Jian; Ponugoti, Bhaskar; Alsadun, Sarah; Tian, Chen; Vafa, Rameen; Graves, Dana T.

2017-01-01

Wound healing is complex and highly orchestrated. It is well appreciated that leukocytes, particularly macrophages, are essential for inducing the formation of new connective tissue, which requires the generation of signals that stimulate mesenchymal stem cells (MSC), myofibroblasts and fibroblasts. A key role for keratinocytes in this complex process has yet to be established. To this end, we investigated possible involvement of keratinocytes in connective tissue healing. By lineage-specific deletion of the forkhead box-O 1 (FOXO1) transcription factor, we demonstrate for the first time that keratinocytes regulate proliferation of fibroblasts and MSCs, formation of myofibroblasts and production of collagen matrix in wound healing. This stimulation is mediated by a FOXO1 induced TGFβ1/CTGF axis. The results provide direct evidence that epithelial cells play a key role in stimulating connective tissue healing through a FOXO1-dependent mechanism. Thus, FOXO1 and keratinocytes may be an important therapeutic target where healing is deficient or compromised by a fibrotic outcome. PMID:28220813

Room temperature deintercalation of alkali metal atoms from epitaxial graphene by formation of charge-transfer complexes

NASA Astrophysics Data System (ADS)

Shin, H.-C.; Ahn, S. J.; Kim, H. W.; Moon, Y.; Rai, K. B.; Woo, S. H.; Ahn, J. R.

2016-08-01

Atom (or molecule) intercalations and deintercalations have been used to control the electronic properties of graphene. In general, finite energies above room temperature (RT) thermal energy are required for the intercalations and deintercalations. Here, we demonstrate that alkali metal atoms can be deintercalated from epitaxial graphene on a SiC substrate at RT, resulting in the reduction in density of states at the Fermi level. The change in density of states at the Fermi level at RT can be applied to a highly sensitive graphene sensor operating at RT. Na atoms, which were intercalated at a temperature of 80 °C, were deintercalated at a high temperature above 1000 °C when only a thermal treatment was used. In contrast to the thermal treatment, the intercalated Na atoms were deintercalated at RT when tetrafluorotetracyanoquinodimethane (F4-TCNQ) molecules were adsorbed on the surface. The RT deintercalation occurred via the formation of charge-transfer complexes between Na atoms and F4-TCNQ molecules.

On the mechanical theory for biological pattern formation

NASA Astrophysics Data System (ADS)

Bentil, D. E.; Murray, J. D.

1993-02-01

We investigate the pattern-forming potential of mechanical models in embryology proposed by Oster, Murray and their coworkers. We show that the presence of source terms in the tissue extracellular matrix and cell density equations give rise to spatio-temporal oscillations. An extension of one such model to include ‘biologically realistic long range effects induces the formation of stationary spatial patterns. Previous attempts to solve the full system were in one dimension only. We obtain solutions in one dimension and extend our simulations to two dimensions. We show that a single mechanical model alone is capable of generating complex but regular spatial patterns rather than the requirement of model interaction as suggested by Nagorcka et al. and Shaw and Murray. We discuss some biological applications of the models among which are would healing and formation of dermatoglyphic (fingerprint) patterns.

Integrated geometry and grid generation system for complex configurations

NASA Technical Reports Server (NTRS)

Akdag, Vedat; Wulf, Armin

1992-01-01

A grid generation system was developed that enables grid generation for complex configurations. The system called ICEM/CFD is described and its role in computational fluid dynamics (CFD) applications is presented. The capabilities of the system include full computer aided design (CAD), grid generation on the actual CAD geometry definition using robust surface projection algorithms, interfacing easily with known CAD packages through common file formats for geometry transfer, grid quality evaluation of the volume grid, coupling boundary condition set-up for block faces with grid topology generation, multi-block grid generation with or without point continuity and block to block interface requirement, and generating grid files directly compatible with known flow solvers. The interactive and integrated approach to the problem of computational grid generation not only substantially reduces manpower time but also increases the flexibility of later grid modifications and enhancements which is required in an environment where CFD is integrated into a product design cycle.

Analytic network process model for sustainable lean and green manufacturing performance indicator

NASA Astrophysics Data System (ADS)

Aminuddin, Adam Shariff Adli; Nawawi, Mohd Kamal Mohd; Mohamed, Nik Mohd Zuki Nik

2014-09-01

Sustainable manufacturing is regarded as the most complex manufacturing paradigm to date as it holds the widest scope of requirements. In addition, its three major pillars of economic, environment and society though distinct, have some overlapping among each of its elements. Even though the concept of sustainability is not new, the development of the performance indicator still needs a lot of improvement due to its multifaceted nature, which requires integrated approach to solve the problem. This paper proposed the best combination of criteria en route a robust sustainable manufacturing performance indicator formation via Analytic Network Process (ANP). The integrated lean, green and sustainable ANP model can be used to comprehend the complex decision system of the sustainability assessment. The finding shows that green manufacturing is more sustainable than lean manufacturing. It also illustrates that procurement practice is the most important criteria in the sustainable manufacturing performance indicator.

Application of ion exchange resin in floating drug delivery system.

PubMed

Upadhye, Abhijeet A; Ambike, Anshuman A; Mahadik, Kakasaheb R; Paradkar, Anant

2008-10-01

The purpose of this study was to explore the application of low-density ion exchange resin (IER) Tulsion(R) 344, for floating drug delivery system (FDDS), and study the effect of its particle size on rate of complexation, water uptake, drug release, and in situ complex formation. Batch method was used for the preparation of complexes, which were characterized by physical methods. Tablet containing resin with high degree of crosslinking showed buoyancy lag time (BLT) of 5-8 min. Decreasing the particle size of resin showed decrease in water uptake and drug release, with no significant effect on the rate of complexation and in situ complex formation for both preformed complexes (PCs) and physical mixtures (PMs). Thus, low-density and high degree of crosslinking of resin and water uptake may be the governing factor for controlling the initial release of tablet containing PMs but not in situ complex formation. However, further sustained release may be due to in situ complex formation.

Hydrogen Cyanide In Protoplanetary Disks

NASA Astrophysics Data System (ADS)

Walker, Ashley L.; Oberg, Karin; Cleeves, L. Ilsedore

2018-01-01

The chemistry behind star and planet formation is extremely complex and important in the formation of habitable planets. Life requires molecules containing carbon, oxygen, and importantly, nitrogen. Hydrogen cyanide, or HCN, one of the main interstellar nitrogen carriers, is extremely dangerous here on Earth. However, it could be used as a vital tool for tracking the chemistry of potentially habitable planets. As we get closer to identifying other habitable planets, we must understand the beginnings of how those planets are formed in the early protoplanetary disk. This project investigates HCN chemistry in different locations in the disk, and what this might mean for forming planets at different distances from the star. HCN is a chemically diverse molecule. It is connected to the formation for other more complex molecules and is commonly used as a nitrogen tracer. Using computational chemical models we look at how the HCN abundance changes at different locations. We use realistic and physically motivated conditions for the gas in the protoplanetary disk: temperature, density, and radiation (UV flux). We analyze the reaction network, formation, and destruction of HCN molecules in the disk environment. The disk environment informs us about stability of habitable planets that are created based on HCN molecules. We reviewed and compared the difference in the molecules with a variety of locations in the disk and ultimately giving us a better understanding on how we view protoplanetary disks.

Expression of DNAJB12 or DNAJB14 Causes Coordinate Invasion of the Nucleus by Membranes Associated with a Novel Nuclear Pore Structure

PubMed Central

Goodwin, Edward C.; Motamedi, Nasim; Lipovsky, Alex; Fernández-Busnadiego, Rubén; DiMaio, Daniel

2014-01-01

DNAJB12 and DNAJB14 are transmembrane proteins in the endoplasmic reticulum (ER) that serve as co-chaperones for Hsc70/Hsp70 heat shock proteins. We demonstrate that over-expression of DNAJB12 or DNAJB14 causes the formation of elaborate membranous structures within cell nuclei, which we designate DJANGOS for DNAJ-associated nuclear globular structures. DJANGOS contain DNAJB12, DNAJB14, Hsc70 and markers of the ER lumen and ER and nuclear membranes. Strikingly, they are evenly distributed underneath the nuclear envelope and are of uniform size in any one nucleus. DJANGOS are composed primarily of single-walled membrane tubes and sheets that connect to the nuclear envelope via a unique configuration of membranes, in which the nuclear pore complex appears anchored exclusively to the outer nuclear membrane, allowing both the inner and outer nuclear membranes to flow past the circumference of the nuclear pore complex into the nucleus. DJANGOS break down rapidly during cell division and reform synchronously in the daughter cell nuclei, demonstrating that they are dynamic structures that undergo coordinate formation and dissolution. Genetic studies showed that the chaperone activity of DNAJ/Hsc70 is required for the formation of DJANGOS. Further analysis of these structures will provide insight into nuclear pore formation and function, activities of molecular chaperones, and mechanisms that maintain membrane identity. PMID:24732912

A G protein alpha null mutation confers prolificacy potential in maize

DOE PAGES

Urano, Daisuke; Jackson, David; Jones, Alan M.

2015-05-06

Plasticity in plant development is controlled by environmental signals through largely unknown signalling networks. Signalling coupled by the heterotrimeric G protein complex underlies various developmental pathways in plants. The morphology of two plastic developmental pathways, root system architecture and female inflorescence formation, was quantitatively assessed in a mutant compact plant 2 (ct2) lacking the alpha subunit of the heterotrimeric G protein complex in maize. The ct2 mutant partially compensated for a reduced shoot height by increased total leaf number, and had far more ears, even in the presence of pollination signals. Lastly, the maize heterotrimeric G protein complex is importantmore » in some plastic developmental traits in maize. In particular, the maize Gα subunit is required to dampen the overproduction of female inflorescences.« less

Absorption spectrometric study of charge transfer complex formation between 4-acetamidophenol (paracetamol) and a series of quinones including Vitamin K 3

NASA Astrophysics Data System (ADS)

Saha, Avijit; Mukherjee, Asok K.

2004-07-01

The formation of charge transfer (CT) complexes of 4-acetamidophenol (commonly called 'paracetamol') and a series of quinones (including Vitamin K 3) has been studied spectrophotometrically in ethanol medium. The vertical ionisation potential of paracetamol and the degrees of charge transfer of the complexes in their ground state has been estimated from the trends in the charge transfer bands. The oscillator and transition dipole strengths of the complexes have been determined from the CT absorption spectra at 298 K. The complexes have been found by Job's method of continuous variation to have the uncommon 2:1 (paracetamol:quinone) stoichiometry in each case. The enthalpies and entropies of formation of the complexes have been obtained by determining their formation constants at five different temperatures.

Bacterial formate hydrogenlyase complex.

PubMed

McDowall, Jennifer S; Murphy, Bonnie J; Haumann, Michael; Palmer, Tracy; Armstrong, Fraser A; Sargent, Frank

2014-09-23

Under anaerobic conditions, Escherichia coli can carry out a mixed-acid fermentation that ultimately produces molecular hydrogen. The enzyme directly responsible for hydrogen production is the membrane-bound formate hydrogenlyase (FHL) complex, which links formate oxidation to proton reduction and has evolutionary links to Complex I, the NADH:quinone oxidoreductase. Although the genetics, maturation, and some biochemistry of FHL are understood, the protein complex has never been isolated in an intact form to allow biochemical analysis. In this work, genetic tools are reported that allow the facile isolation of FHL in a single chromatographic step. The core complex is shown to comprise HycE (a [NiFe] hydrogenase component termed Hyd-3), FdhF (the molybdenum-dependent formate dehydrogenase-H), and three iron-sulfur proteins: HycB, HycF, and HycG. A proportion of this core complex remains associated with HycC and HycD, which are polytopic integral membrane proteins believed to anchor the core complex to the cytoplasmic side of the membrane. As isolated, the FHL complex retains formate hydrogenlyase activity in vitro. Protein film electrochemistry experiments on Hyd-3 demonstrate that it has a unique ability among [NiFe] hydrogenases to catalyze production of H2 even at high partial pressures of H2. Understanding and harnessing the activity of the FHL complex is critical to advancing future biohydrogen research efforts.

Suitable configurations for triangular formation flying about collinear libration points under the circular and elliptic restricted three-body problems

NASA Astrophysics Data System (ADS)

Ferrari, Fabio; Lavagna, Michèle

2018-06-01

The design of formations of spacecraft in a three-body environment represents one of the most promising challenges for future space missions. Two or more cooperating spacecraft can greatly answer some very complex mission goals, not achievable by a single spacecraft. The dynamical properties of a low acceleration environment such as the vicinity of libration points associated to a three-body system, can be effectively exploited to design spacecraft configurations able of satisfying tight relative position and velocity requirements. This work studies the evolution of an uncontrolled formation orbiting in the proximity of periodic orbits about collinear libration points under the Circular and Elliptic Restricted Three-Body Problems. A three spacecraft triangularly-shaped formation is assumed as a representative geometry to be investigated. The study identifies initial configurations that provide good performance in terms of formation keeping, and investigates key parameters that control the relative dynamics between the spacecraft within the three-body system. Formation keeping performance is quantified by monitoring shape and size changes of the triangular formation. The analysis has been performed under five degrees of freedom to define the geometry, the orientation and the location of the triangle in the synodic rotating frame.

Non-Linear Pattern Formation in Bone Growth and Architecture

PubMed Central

Salmon, Phil

2014-01-01

The three-dimensional morphology of bone arises through adaptation to its required engineering performance. Genetically and adaptively bone travels along a complex spatiotemporal trajectory to acquire optimal architecture. On a cellular, micro-anatomical scale, what mechanisms coordinate the activity of osteoblasts and osteoclasts to produce complex and efficient bone architectures? One mechanism is examined here – chaotic non-linear pattern formation (NPF) – which underlies in a unifying way natural structures as disparate as trabecular bone, swarms of birds flying, island formation, fluid turbulence, and others. At the heart of NPF is the fact that simple rules operating between interacting elements, and Turing-like interaction between global and local signals, lead to complex and structured patterns. The study of “group intelligence” exhibited by swarming birds or shoaling fish has led to an embodiment of NPF called “particle swarm optimization” (PSO). This theoretical model could be applicable to the behavior of osteoblasts, osteoclasts, and osteocytes, seeing them operating “socially” in response simultaneously to both global and local signals (endocrine, cytokine, mechanical), resulting in their clustered activity at formation and resorption sites. This represents problem-solving by social intelligence, and could potentially add further realism to in silico computer simulation of bone modeling. What insights has NPF provided to bone biology? One example concerns the genetic disorder juvenile Pagets disease or idiopathic hyperphosphatasia, where the anomalous parallel trabecular architecture characteristic of this pathology is consistent with an NPF paradigm by analogy with known experimental NPF systems. Here, coupling or “feedback” between osteoblasts and osteoclasts is the critical element. This NPF paradigm implies a profound link between bone regulation and its architecture: in bone the architecture is the regulation. The former is the emergent consequence of the latter. PMID:25653638

Non-linear pattern formation in bone growth and architecture.

PubMed

Salmon, Phil

2014-01-01

The three-dimensional morphology of bone arises through adaptation to its required engineering performance. Genetically and adaptively bone travels along a complex spatiotemporal trajectory to acquire optimal architecture. On a cellular, micro-anatomical scale, what mechanisms coordinate the activity of osteoblasts and osteoclasts to produce complex and efficient bone architectures? One mechanism is examined here - chaotic non-linear pattern formation (NPF) - which underlies in a unifying way natural structures as disparate as trabecular bone, swarms of birds flying, island formation, fluid turbulence, and others. At the heart of NPF is the fact that simple rules operating between interacting elements, and Turing-like interaction between global and local signals, lead to complex and structured patterns. The study of "group intelligence" exhibited by swarming birds or shoaling fish has led to an embodiment of NPF called "particle swarm optimization" (PSO). This theoretical model could be applicable to the behavior of osteoblasts, osteoclasts, and osteocytes, seeing them operating "socially" in response simultaneously to both global and local signals (endocrine, cytokine, mechanical), resulting in their clustered activity at formation and resorption sites. This represents problem-solving by social intelligence, and could potentially add further realism to in silico computer simulation of bone modeling. What insights has NPF provided to bone biology? One example concerns the genetic disorder juvenile Pagets disease or idiopathic hyperphosphatasia, where the anomalous parallel trabecular architecture characteristic of this pathology is consistent with an NPF paradigm by analogy with known experimental NPF systems. Here, coupling or "feedback" between osteoblasts and osteoclasts is the critical element. This NPF paradigm implies a profound link between bone regulation and its architecture: in bone the architecture is the regulation. The former is the emergent consequence of the latter.

Required, tissue-specific roles for Fgf8 in outflow tract formation and remodeling.

PubMed

Park, Eon Joo; Ogden, Lisa A; Talbot, Amy; Evans, Sylvia; Cai, Chen-Leng; Black, Brian L; Frank, Deborah U; Moon, Anne M

2006-06-01

Fibroblast growth factor 8 (Fgf8) is a secreted signaling protein expressed in numerous temporospatial domains that are potentially relevant to cardiovascular development. However, the pathogenesis of complex cardiac and outflow tract defects observed in Fgf8-deficient mice, and the specific source(s) of Fgf8 required for outflow tract formation and subsequent remodeling are unknown. A detailed examination of the timing and location of Fgf8 production revealed previously unappreciated expression in a subset of primary heart field cells; Fgf8 is also expressed throughout the anterior heart field (AHF) mesoderm and in pharyngeal endoderm at the crescent and early somite stages. We used conditional mutagenesis to examine the requirements for Fgf8 function in these different expression domains during heart and outflow tract morphogenesis. Formation of the primary heart tube and the addition of right ventricular and outflow tract myocardium depend on autocrine Fgf8 signaling in cardiac crescent mesoderm. Loss of Fgf8 in this domain resulted in decreased expression of the Fgf8 target gene Erm, and aberrant production of Isl1 and its target Mef2c in the anterior heart field, thus linking Fgf8 signaling with transcription factor networks that regulate survival and proliferation of the anterior heart field. We further found that mesodermal- and endodermal-derived Fgf8 perform specific functions during outflow tract remodeling: mesodermal Fgf8 is required for correct alignment of the outflow tract and ventricles, whereas activity of Fgf8 emanating from pharyngeal endoderm regulates outflow tract septation. These findings provide a novel insight into how the formation and remodeling of primary and anterior heart field-derived structures rely on Fgf8 signals from discrete temporospatial domains.

Diversity in Requirement of Genetic and Epigenetic Factors for Centromere Function in Fungi ▿

PubMed Central

Roy, Babhrubahan; Sanyal, Kaustuv

2011-01-01

A centromere is a chromosomal region on which several proteins assemble to form the kinetochore. The centromere-kinetochore complex helps in the attachment of chromosomes to spindle microtubules to mediate segregation of chromosomes to daughter cells during mitosis and meiosis. In several budding yeast species, the centromere forms in a DNA sequence-dependent manner, whereas in most other fungi, factors other than the DNA sequence also determine the centromere location, as centromeres were able to form on nonnative sequences (neocentromeres) when native centromeres were deleted in engineered strains. Thus, in the absence of a common DNA sequence, the cues that have facilitated centromere formation on a specific DNA sequence for millions of years remain a mystery. Kinetochore formation is facilitated by binding of a centromere-specific histone protein member of the centromeric protein A (CENP-A) family that replaces a canonical histone H3 to form a specialized centromeric chromatin structure. However, the process of kinetochore formation on the rapidly evolving and seemingly diverse centromere DNAs in different fungal species is largely unknown. More interestingly, studies in various yeasts suggest that the factors required for de novo centromere formation (establishment) may be different from those required for maintenance (propagation) of an already established centromere. Apart from the DNA sequence and CENP-A, many other factors, such as posttranslational modification (PTM) of histones at centric and pericentric chromatin, RNA interference, and DNA methylation, are also involved in centromere formation, albeit in a species-specific manner. In this review, we discuss how several genetic and epigenetic factors influence the evolution of structure and function of centromeres in fungal species. PMID:21908596

Alternative process for thin layer etching: Application to nitride spacer etching stopping on silicon germanium

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

Posseme, N., E-mail: nicolas.posseme@cea.fr; Pollet, O.; Barnola, S.

2014-08-04

Silicon nitride spacer etching realization is considered today as one of the most challenging of the etch process for the new devices realization. For this step, the atomic etch precision to stop on silicon or silicon germanium with a perfect anisotropy (no foot formation) is required. The situation is that none of the current plasma technologies can meet all these requirements. To overcome these issues and meet the highly complex requirements imposed by device fabrication processes, we recently proposed an alternative etching process to the current plasma etch chemistries. This process is based on thin film modification by light ionsmore » implantation followed by a selective removal of the modified layer with respect to the non-modified material. In this Letter, we demonstrate the benefit of this alternative etch method in term of film damage control (silicon germanium recess obtained is less than 6 A), anisotropy (no foot formation), and its compatibility with other integration steps like epitaxial. The etch mechanisms of this approach are also addressed.« less

Neuromechanical factors involved in the formation and propulsion of fecal pellets in the guinea-pig colon.

PubMed

Costa, M; Wiklendt, L; Simpson, P; Spencer, N J; Brookes, S J; Dinning, P G

2015-10-01

The neuromechanical processes involved in the formation and propulsion of fecal pellets remain incompletely understood. We analyzed motor patterns in isolated segments of the guinea-pig proximal and distal colon, using video imaging, during oral infusion of liquid, viscous material, or solid pellets. Colonic migrating motor complexes (CMMCs) in the proximal colon divided liquid or natural semisolid contents into elongated shallow boluses. At the colonic flexure these boluses were formed into shorter, pellet-shaped boluses. In the non-distended distal colon, spontaneous CMMCs produced small dilations. Both high- and low-viscosity infusions evoked a distinct motor pattern that produced pellet-shaped boluses. These were propelled at speeds proportional to their surface area. Solid pellets were propelled at a speed that increased with diameter, to a maximum that matched the diameter of natural pellets. Pellet speed was reduced by increasing resistive load. Tetrodotoxin blocked all propulsion. Hexamethonium blocked normal motor patterns, leaving irregular propagating contractions, indicating the existence of neural pathways that did not require nicotinic transmission. Colonic migrating motor complexes are responsible for the slow propulsion of the soft fecal content in the proximal colon, while the formation of pellets at the colonic flexure involves a content-dependent mechanism in combination with content-independent spontaneous CMMCs. Bolus size and consistency affects propulsion speed suggesting that propulsion is not a simple reflex but rather a more complex process involving an adaptable neuromechanical loop. © 2015 John Wiley & Sons Ltd.

Dorsal medial prefrontal cortex contributes to conditioned taste aversion memory consolidation and retrieval.

PubMed

Gonzalez, Maria Carolina; Villar, Maria Eugenia; Igaz, Lionel M; Viola, Haydée; Medina, Jorge H

2015-12-01

The medial prefrontal cortex (mPFC) is known for its role in decision making and memory processing, including the participation in the formation of extinction memories. However, little is known regarding its contribution to aversive memory consolidation. Here we demonstrate that neural activity and protein synthesis are required in the dorsal mPFC for memory formation of a conditioned taste aversion (CTA) task and that this region is involved in the retrieval of recent and remote long-term CTA memory. In addition, both NMDA receptor and CaMKII activity in dorsal mPFC are needed for CTA memory consolidation, highlighting the complexity of mPFC functions. Copyright © 2015 Elsevier Inc. All rights reserved.

Nup53 Is Required for Nuclear Envelope and Nuclear Pore Complex Assembly

PubMed Central

Hawryluk-Gara, Lisa A.; Platani, Melpomeni; Santarella, Rachel

2008-01-01

Transport across the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs). These structures are composed of various subcomplexes of proteins that are each present in multiple copies and together establish the eightfold symmetry of the NPC. One evolutionarily conserved subcomplex of the NPC contains the nucleoporins Nup53 and Nup155. Using truncation analysis, we have defined regions of Nup53 that bind to neighboring nucleoporins as well as those domains that target Nup53 to the NPC in vivo. Using this information, we investigated the role of Nup53 in NE and NPC assembly using Xenopus egg extracts. We show that both events require Nup53. Importantly, the analysis of Nup53 fragments revealed that the assembly activity of Nup53 depleted extracts could be reconstituted using a region of Nup53 that binds specifically to its interacting partner Nup155. On the basis of these results, we propose that the formation of a Nup53–Nup155 complex plays a critical role in the processes of NPC and NE assembly. PMID:18256286

Exploring protein-DNA interactions in 3D using in situ construction, manipulation, and visualization of individual DNA-dumbbells with optical traps, microfluidics, and fluorescence microscopy

PubMed Central

Forget, Anthony L.; Dombrowski, Christopher C.; Amitani, Ichiro; Kowalczykowski, Stephen C.

2015-01-01

In this Protocol, we describe a procedure to generate ‘DNA-dumbbells’ — single molecules of DNA with a microscopic bead attached at each end — and techniques for manipulating individual DNA-dumbbells. We also detail the design and fabrication of a microfluidic device (flow cell) used in conjunction with dual optical trapping to manipulate DNA-dumbbells and to visualize individual protein–DNA complexes by single-molecule epifluorescence microscopy. Our design of the flow cell enables the rapid movement of trapped molecules between laminar flow channels and a flow-free ‘reservoir’. The reservoir provides the means to examine formation of DNA–protein complexes in solution in the absence of external flow forces, while still maintaining a predetermined end-to-end extension of the DNA. These features facilitate examination of the role of three-dimensional DNA conformation and dynamics in protein–DNA interactions. Preparation of flow cells and reagents requires two days each; in situ DNA-dumbbell assembly and imaging of single protein–DNA complexes requires another day. PMID:23411634

Designed Proteins Induce the Formation of Nanocage-containing Extracellular Vesicles

PubMed Central

Votteler, Jörg; Ogohara, Cassandra; Yi, Sue; Hsia, Yang; Nattermann, Una; Belnap, David M.; King, Neil P.; Sundquist, Wesley I.

2017-01-01

Complex biological processes are often performed by self-organizing nanostructures comprising multiple classes of macromolecules, such as ribosomes (proteins and RNA) or enveloped viruses (proteins, nucleic acids, and lipids). Approaches have been developed for designing self-assembling structures consisting of either nucleic acids1,2 or proteins3–5, but strategies for engineering hybrid biological materials are only beginning to emerge6,7. Here, we describe the design of self-assembling protein nanocages that direct their own release from human cells inside small vesicles in a manner that resembles some viruses. We refer to these hybrid biomaterials as Enveloped Protein Nanocages (EPNs). Robust EPN biogenesis required protein sequence elements that encode three distinct functions: membrane binding, self-assembly, and recruitment of the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery8. A variety of synthetic proteins with these functional elements induced EPN biogenesis, highlighting the modularity and generality of the design strategy. Biochemical and electron cryomicroscopic (cryo-EM) analyses revealed that one design, EPN-01, comprised small (~100 nm) vesicles containing multiple protein nanocages that closely matched the structure of the designed 60-subunit self-assembling scaffold9. EPNs that incorporated the vesicular stomatitis viral glycoprotein (VSV-G) could fuse with target cells and deliver their contents, thereby transferring cargoes from one cell to another. These studies show how proteins can be programmed to direct the formation of hybrid biological materials that perform complex tasks, and establish EPNs as a novel class of designed, modular, genetically-encoded nanomaterials that can transfer molecules between cells. PMID:27919066

Dimerization site 2 of the bacterial DNA-binding protein H-NS is required for gene silencing and stiffened nucleoprotein filament formation.

PubMed

Yamanaka, Yuki; Winardhi, Ricksen S; Yamauchi, Erika; Nishiyama, So-Ichiro; Sowa, Yoshiyuki; Yan, Jie; Kawagishi, Ikuro; Ishihama, Akira; Yamamoto, Kaneyoshi

2018-06-15

The bacterial nucleoid-associated protein H-NS is a DNA-binding protein, playing a major role in gene regulation. To regulate transcription, H-NS silences genes, including horizontally acquired foreign genes. Escherichia coli H-NS is 137 residues long and consists of two discrete and independent structural domains: an N-terminal oligomerization domain and a C-terminal DNA-binding domain, joined by a flexible linker. The N-terminal oligomerization domain is composed of two dimerization sites, dimerization sites 1 and 2, which are both required for H-NS oligomerization, but the exact role of dimerization site 2 in gene silencing is unclear. To this end, we constructed a whole set of single amino acid substitution variants spanning residues 2 to 137. Using a well-characterized H-NS target, the slp promoter of the glutamic acid-dependent acid resistance (GAD) cluster promoters, we screened for any variants defective in gene silencing. Focusing on the function of dimerization site 2, we analyzed four variants, I70C/I70A and L75C/L75A, which all could actively bind DNA but are defective in gene silencing. Atomic force microscopy analysis of DNA-H-NS complexes revealed that all of these four variants formed condensed complexes on DNA, whereas WT H-NS formed rigid and extended nucleoprotein filaments, a conformation required for gene silencing. Single-molecule stretching experiments confirmed that the four variants had lost the ability to form stiffened filaments. We conclude that dimerization site 2 of H-NS plays a key role in the formation of rigid H-NS nucleoprotein filament structures required for gene silencing. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanistic investigation of the formation of H2 from HCOOH with a dinuclear Ru model complex for formate hydrogen lyase.

PubMed

Tokunaga, Taisuke; Yatabe, Takeshi; Matsumoto, Takahiro; Ando, Tatsuya; Yoon, Ki-Seok; Ogo, Seiji

2017-01-01

We report the mechanistic investigation of catalytic H 2 evolution from formic acid in water using a formate-bridged dinuclear Ru complex as a formate hydrogen lyase model. The mechanistic study is based on isotope-labeling experiments involving hydrogen isotope exchange reaction.

Substrate-Triggered Addition of Dioxygen to the Diferrous Cofactor of Aldehyde-Deformylating Oxygenase to form a Diferric-Peroxide Intermediate†

PubMed Central

Nørgaard, Hanne; Warui, Douglas M.; Rajakovich, Lauren J.; Chang, Wei-chen; Booker, Squire J.; Krebs, Carsten; Bollinger, J. Martin

2013-01-01

Cyanobacterial aldehyde-deformylating oxygenases (ADOs) belong to the ferritin-like diiron-carboxylate superfamily of dioxygen-activating proteins. They catalyze conversion of saturated or mono-unsaturated Cn fatty aldehydes to formate and the corresponding Cn-1 alkanes or alkenes, respectively. This unusual, apparently redox-neutral transformation actually requires four electrons per turnover to reduce the O2 co-substrate to the oxidation state of water and incorporates one O-atom from O2 into the formate co-product. We show here that the complex of the diiron(II/II) form of ADO from Nostoc punctiforme (Np) with an aldehyde substrate reacts with O2 to form a colored intermediate with spectroscopic properties suggestive of a Fe2III/III complex with a bound peroxide. Its Mössbauer spectra reveal that the intermediate possesses an antiferromagnetically (AF) coupled Fe2III/III center with resolved sub-sites. The intermediate is long-lived in the absence of a reducing system, decaying slowly (t1/2 ~ 400 s at 5 °C) to produce a very modest yield of formate (< 0.15 enzyme equivalents), but reacts rapidly with the fully reduced form of 1-methoxy-5-methylphenazine (MeOPMS) to yield product, albeit at only ~ 50% of the maximum theoretical yield (owing to competition from one or more unproductive pathway). The results represent the most definitive evidence to date that ADO can use a diiron cofactor (rather than a homo- or hetero-dinuclear cluster involving another transition metal) and provide support for a mechanism involving attack on the carbonyl of the bound substrate by the reduced O2 moiety to form a Fe2III/III-peroxyhemiacetal complex, which undergoes reductive O-O-bond cleavage, leading to C1–C2 radical fragmentation and formation of the alk(a/e)ne and formate products. PMID:23987523

Substrate-triggered addition of dioxygen to the diferrous cofactor of aldehyde-deformylating oxygenase to form a diferric-peroxide intermediate.

PubMed

Pandelia, Maria E; Li, Ning; Nørgaard, Hanne; Warui, Douglas M; Rajakovich, Lauren J; Chang, Wei-Chen; Booker, Squire J; Krebs, Carsten; Bollinger, J Martin

2013-10-23

Cyanobacterial aldehyde-deformylating oxygenases (ADOs) belong to the ferritin-like diiron-carboxylate superfamily of dioxygen-activating proteins. They catalyze conversion of saturated or monounsaturated C(n) fatty aldehydes to formate and the corresponding C(n-1) alkanes or alkenes, respectively. This unusual, apparently redox-neutral transformation actually requires four electrons per turnover to reduce the O2 cosubstrate to the oxidation state of water and incorporates one O-atom from O2 into the formate coproduct. We show here that the complex of the diiron(II/II) form of ADO from Nostoc punctiforme (Np) with an aldehyde substrate reacts with O2 to form a colored intermediate with spectroscopic properties suggestive of a Fe2(III/III) complex with a bound peroxide. Its Mössbauer spectra reveal that the intermediate possesses an antiferromagnetically (AF) coupled Fe2(III/III) center with resolved subsites. The intermediate is long-lived in the absence of a reducing system, decaying slowly (t(1/2) ~ 400 s at 5 °C) to produce a very modest yield of formate (<0.15 enzyme equivalents), but reacts rapidly with the fully reduced form of 1-methoxy-5-methylphenazinium methylsulfate ((MeO)PMS) to yield product, albeit at only ~50% of the maximum theoretical yield (owing to competition from one or more unproductive pathway). The results represent the most definitive evidence to date that ADO can use a diiron cofactor (rather than a homo- or heterodinuclear cluster involving another transition metal) and provide support for a mechanism involving attack on the carbonyl of the bound substrate by the reduced O2 moiety to form a Fe2(III/III)-peroxyhemiacetal complex, which undergoes reductive O-O-bond cleavage, leading to C1-C2 radical fragmentation and formation of the alk(a/e)ne and formate products.

The First Prokaryotic Trehalose Synthase Complex Identified in the Hyperthermophilic Crenarchaeon Thermoproteus tenax

PubMed Central

Bräsen, Christopher; Hensel, Reinhard; Lupas, Andrei N.; Brinkmann, Henner; Siebers, Bettina

2013-01-01

The role of the disaccharide trehalose, its biosynthesis pathways and their regulation in Archaea are still ambiguous. In Thermoproteus tenax a fused trehalose-6-phosphate synthase/phosphatase (TPSP), consisting of an N-terminal trehalose-6-phosphate synthase (TPS) and a C-terminal trehalose-6-phosphate phosphatase (TPP) domain, was identified. The tpsp gene is organized in an operon with a putative glycosyltransferase (GT) and a putative mechanosensitive channel (MSC). The T. tenax TPSP exhibits high phosphatase activity, but requires activation by the co-expressed GT for bifunctional synthase-phosphatase activity. The GT mediated activation of TPS activity relies on the fusion of both, TPS and TPP domain, in the TPSP enzyme. Activation is mediated by complex-formation in vivo as indicated by yeast two-hybrid and crude extract analysis. In combination with first evidence for MSC activity the results suggest a sophisticated stress response involving TPSP, GT and MSC in T. tenax and probably in other Thermoproteales species. The monophyletic prokaryotic TPSP proteins likely originated via a single fusion event in the Bacteroidetes with subsequent horizontal gene transfers to other Bacteria and Archaea. Furthermore, evidence for the origin of eukaryotic TPSP fusions via HGT from prokaryotes and therefore a monophyletic origin of eukaryotic and prokaryotic fused TPSPs is presented. This is the first report of a prokaryotic, archaeal trehalose synthase complex exhibiting a much more simple composition than the eukaryotic complex described in yeast. Thus, complex formation and a complex-associated regulatory potential might represent a more general feature of trehalose synthesizing proteins. PMID:23626675

Ribosome binding induces repositioning of the signal recognition particle receptor on the translocon

PubMed Central

Kuhn, Patrick; Draycheva, Albena; Vogt, Andreas; Petriman, Narcis-Adrian; Sturm, Lukas; Drepper, Friedel; Warscheid, Bettina; Wintermeyer, Wolfgang

2015-01-01

Cotranslational protein targeting delivers proteins to the bacterial cytoplasmic membrane or to the eukaryotic endoplasmic reticulum membrane. The signal recognition particle (SRP) binds to signal sequences emerging from the ribosomal tunnel and targets the ribosome-nascent-chain complex (RNC) to the SRP receptor, termed FtsY in bacteria. FtsY interacts with the fifth cytosolic loop of SecY in the SecYEG translocon, but the functional role of the interaction is unclear. By using photo-cross-linking and fluorescence resonance energy transfer measurements, we show that FtsY–SecY complex formation is guanosine triphosphate independent but requires a phospholipid environment. Binding of an SRP–RNC complex exposing a hydrophobic transmembrane segment induces a rearrangement of the SecY–FtsY complex, which allows the subsequent contact between SecY and ribosomal protein uL23. These results suggest that direct RNC transfer to the translocon is guided by the interaction between SRP and translocon-bound FtsY in a quaternary targeting complex. PMID:26459600

Structure and function of the C-terminal domain of MrpA in the Bacillus subtilis Mrp-antiporter complex--the evolutionary progenitor of the long horizontal helix in complex I.

PubMed

Virzintiene, Egle; Moparthi, Vamsi K; Al-Eryani, Yusra; Shumbe, Leonard; Górecki, Kamil; Hägerhäll, Cecilia

2013-10-11

MrpA and MrpD are homologous to NuoL, NuoM and NuoN in complex I over the first 14 transmembrane helices. In this work, the C-terminal domain of MrpA, outside this conserved area, was investigated. The transmembrane orientation was found to correspond to that of NuoJ in complex I. We have previously demonstrated that the subunit NuoK is homologous to MrpC. The function of the MrpA C-terminus was tested by expression in a previously used Bacillus subtilis model system. At neutral pH, the truncated MrpA still worked, but at pH 8.4, where Mrp-complex formation is needed for function, the C-terminal domain of MrpA was absolutely required. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Kinetic competence of the cADP-ribose-CD38 complex as an intermediate in the CD38/NAD+ glycohydrolase-catalysed reactions: implication for CD38 signalling.

PubMed Central

Cakir-Kiefer, C; Muller-Steffner, H; Oppenheimer, N; Schuber, F

2001-01-01

CD38/NAD(+) glycohydrolase is a type II transmembrane glycoprotein widely used to study T- and B-cell activation and differentiation. CD38 is endowed with two different activities: it is a signal transduction molecule and an ectoenzyme that converts NAD(+) into ADP-ribose (NAD(+) glycohydrolase activity) and small proportions of cADP-ribose (cADPR; ADP-ribosyl cyclase activity), a calcium-mobilizing metabolite, which, ultimately, can also be hydrolysed (cADPR hydrolase activity). The relationship between these two properties, and strikingly the requirement for signalling in the formation of free or enzyme-complexed cADPR, is still ill-defined. In the present study we wanted to test whether the CD38-cADPR complex is kinetically competent in the conversion of NAD(+) into the reaction product ADP-ribose. In principle, such a complex could be invoked for cross-talk, via conformational changes, with neighbouring partner(s) of CD38 thus triggering the signalling phenomena. Analysis of the kinetic parameters measured for the CD38/NAD(+) glycohydrolase-catalysed hydrolysis of 2'-deoxy-2'-aminoribo-NAD(+) and ADP-cyclo[N1,C1']-2'-deoxy-2'-aminoribose (slowly hydrolysable analogues of NAD(+) and cADPR respectively) ruled out that the CD38-cADPR complex can accumulate under steady-state conditions. This was borne out by simulation of the prevalent kinetic mechanism of CD38, which involve the partitioning of a common E.ADP-ribosyl intermediate in the formation of the enzyme-catalysed reaction products. Using this mechanism, microscopic rate conditions were found which transform a NAD(+) glycohydrolase into an ADP-ribosyl cyclase. Altogether, the present work shows that if the cross-talk with a partner depends on a conformational change of CD38, this is most probably not attributable to the formation of the CD38-cADPR complex. In line with recent results on the conformational change triggered by CD38 ligands [Berthelier, Laboureau, Boulla, Schuber and Deterre (2000) Eur. J. Biochem. 267, 3056-3064], we believe that the Michaelis CD38-NAD(+) complex could play such a role instead. PMID:11513738

GTSF-1 is required for formation of a functional RNA-dependent RNA Polymerase complex in Caenorhabditis elegans.

PubMed

Almeida, Miguel Vasconcelos; Dietz, Sabrina; Redl, Stefan; Karaulanov, Emil; Hildebrandt, Andrea; Renz, Christian; Ulrich, Helle D; König, Julian; Butter, Falk; Ketting, René F

2018-05-16

Argonaute proteins and their associated small RNAs (sRNAs) are evolutionarily conserved regulators of gene expression. Gametocyte-specific factor 1 (Gtsf1) proteins, characterized by two tandem CHHC zinc fingers and an unstructured C-terminal tail, are conserved in animals and have been shown to interact with Piwi clade Argonautes, thereby assisting their activity. We identified the Caenorhabditis elegans Gtsf1 homolog, named it gtsf-1 and characterized it in the context of the sRNA pathways of C. elegans We report that GTSF-1 is not required for Piwi-mediated gene silencing. Instead, gtsf-1 mutants show a striking depletion of 26G-RNAs, a class of endogenous sRNAs, fully phenocopying rrf-3 mutants. We show, both in vivo and in vitro , that GTSF-1 interacts with RRF-3 via its CHHC zinc fingers. Furthermore, we demonstrate that GTSF-1 is required for the assembly of a larger RRF-3 and DCR-1-containing complex (ERIC), thereby allowing for 26G-RNA generation. We propose that GTSF-1 homologs may act to drive the assembly of larger complexes that act in sRNA production and/or in imposing sRNA-mediated silencing activities. © 2018 The Authors.

Dlg3 Trafficking and Apical Tight Junction Formation Is Regulated by Nedd4 and Nedd4-2 E3 Ubiquitin Ligases

PubMed Central

Van Campenhout, Claude A.; Eitelhuber, Andrea; Gloeckner, Christian J.; Giallonardo, Patrizia; Gegg, Moritz; Oller, Heide; Grant, Seth G.N.; Krappmann, Daniel; Ueffing, Marius; Lickert, Heiko

2011-01-01

Summary The Drosophila Discs large (Dlg) scaffolding protein acts as a tumor suppressor regulating basolateral epithelial polarity and proliferation. In mammals, four Dlg homologs have been identified; however, their functions in cell polarity remain poorly understood. Here, we demonstrate that the X-linked mental retardation gene product Dlg3 contributes to apical-basal polarity and epithelial junction formation in mouse organizer tissues, as well as to planar cell polarity in the inner ear. We purified complexes associated with Dlg3 in polarized epithelial cells, including proteins regulating directed trafficking and tight junction formation. Remarkably, of the four Dlg family members, Dlg3 exerts a distinct function by recruiting the ubiquitin ligases Nedd4 and Nedd4-2 through its PPxY motifs. We found that these interactions are required for Dlg3 monoubiquitination, apical membrane recruitment, and tight junction consolidation. Our findings reveal an unexpected evolutionary diversification of the vertebrate Dlg family in basolateral epithelium formation. PMID:21920314

E3 ligase Rad18 promotes monoubiquitination rather than ubiquitin chain formation by E2 enzyme Rad6

PubMed Central

Hibbert, Richard G.; Huang, Anding; Boelens, Rolf; Sixma, Titia K.

2011-01-01

In ubiquitin conjugation, different combinations of E2 and E3 enzymes catalyse either monoubiquitination or ubiquitin chain formation. The E2/E3 complex Rad6/Rad18 exclusively monoubiquitinates the proliferating cell nuclear antigen (PCNA) to signal for “error prone” DNA damage tolerance, whereas a different set of conjugation enzymes is required for ubiquitin chain formation on PCNA. Here we show that human E2 enzyme Rad6b is intrinsically capable of catalyzing ubiquitin chain formation. This activity is prevented during PCNA ubiquitination by the interaction of Rad6 with E3 enzyme Rad18. Using NMR and X-ray crystallography we show that the R6BD of Rad18 inhibits this activity by competing with ubiquitin for a noncovalent “backside” binding site on Rad6. Our findings provide mechanistic insights into how E3 enzymes can regulate the ubiquitin conjugation process. PMID:21422291

Analysis of the Enhanced Stability of R(+)-Alpha Lipoic Acid by the Complex Formation with Cyclodextrins

PubMed Central

Ikuta, Naoko; Sugiyama, Hironori; Shimosegawa, Hiroshi; Nakane, Rie; Ishida, Yoshiyuki; Uekaji, Yukiko; Nakata, Daisuke; Pallauf, Kathrin; Rimbach, Gerald; Terao, Keiji; Matsugo, Seiichi

2013-01-01

R(+)-alpha lipoic acid (RALA) is one of the cofactors for mitochondrial enzymes and, therefore, plays a central role in energy metabolism. RALA is unstable when exposed to low pH or heat, and therefore, it is difficult to use enantiopure RALA as a pharma- and nutra-ceutical. In this study, we have aimed to stabilize RALA through complex formation with cyclodextrins (CDs). α-CD, β-CD and γ-CD were used for the formation of these RALA-CD complexes. We confirmed the complex formation using differential scanning calorimetry and showed by using HPLC analysis that complexed RALA is more stable than free RALA when subjected to humidity and high temperature or acidic pH conditions. Scanning electron microscopy studies showed that the particle size and shape differed depending on the cyclodextrin used for complexation. Further, the complexes of CD and RALA showed a different particle size distribution pattern compared with that of CD itself or that of the physical mixture of RALA and CD. PMID:23434662

Cl-doping of Te-rich CdTe: Complex formation, self-compensation and self-purification from first principles

NASA Astrophysics Data System (ADS)

Lindström, A.; Klintenberg, M.; Sanyal, B.; Mirbt, S.

2015-08-01

The coexistence in Te-rich CdTe of substitutional Cl-dopants, ClTe, which act as donors, and Cd vacancies, VC d - 1 , which act as electron traps, was studied from first principles utilising the HSE06 hybrid functional. We find ClTe to preferably bind to VC d - 1 and to form an acceptor complex, (ClTe-VCd)-1. The complex has a (0,-1) charge transfer level close to the valence band and shows no trap state (deep level) in the band gap. During the complex formation, the defect state of VCd-1 is annihilated and leaves the Cl-doped CdTe bandgap without any trap states (self-purification). We calculate Cl-doped CdTe to be semi-insulating with a Fermi energy close to midgap. We calculate the formation energy of the complex to be sufficiently low to allow for spontanous defect formation upon Cl-doping (self-compensation). In addition, we quantitatively analyse the geometries, DOS, binding energies and formation energies of the (ClTe-VCd) complexes.

Selective Co-Encapsulation Inside an M6 L4 Cage.

PubMed

Leenders, Stefan H A M; Becker, René; Kumpulainen, Tatu; de Bruin, Bas; Sawada, Tomohisa; Kato, Taito; Fujita, Makoto; Reek, Joost N H

2016-10-17

There is broad interest in molecular encapsulation as such systems can be utilized to stabilize guests, facilitate reactions inside a cavity, or give rise to energy-transfer processes in a confined space. Detailed understanding of encapsulation events is required to facilitate functional molecular encapsulation. In this contribution, it is demonstrated that Ir and Rh-Cp-type metal complexes can be encapsulated inside a self-assembled M 6 L 4 metallocage only in the presence of an aromatic compound as a second guest. The individual guests are not encapsulated, suggesting that only the pair of guests can fill the void of the cage. Hence, selective co-encapsulation is observed. This principle is demonstrated by co-encapsulation of a variety of combinations of metal complexes and aromatic guests, leading to several ternary complexes. These experiments demonstrate that the efficiency of formation of the ternary complexes depends on the individual components. Moreover, selective exchange of the components is possible, leading to formation of the most favorable complex. Besides the obvious size effect, a charge-transfer interaction may also contribute to this effect. Charge-transfer bands are clearly observed by UV/Vis spectrophotometry. A change in the oxidation potential of the encapsulated electron donor also leads to a shift in the charge-transfer energy bands. As expected, metal complexes with a higher oxidation potential give rise to a higher charge-transfer energy and a larger hypsochromic shift in the UV/Vis spectrum. These subtle energy differences may potentially be used to control the binding and reactivity of the complexes bound in a confined space. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Cancer-associated fibroblast-derived annexin A6+ extracellular vesicles support pancreatic cancer aggressiveness

PubMed Central

Leca, Julie; Martinez, Sébastien; Lac, Sophie; Nigri, Jérémy; Secq, Véronique; Rubis, Marion; Bressy, Christian; Lavaut, Marie-Noelle; Dusetti, Nelson; Loncle, Céline; Roques, Julie; Pietrasz, Daniel; Bousquet, Corinne; Garcia, Stéphane; Granjeaud, Samuel; Ouaissi, Mehdi; Bachet, Jean Baptiste; Iovanna, Juan L.; Zimmermann, Pascale; Vasseur, Sophie

2016-01-01

The intratumoral microenvironment, or stroma, is of major importance in the pathobiology of pancreatic ductal adenocarcinoma (PDA), and specific conditions in the stroma may promote increased cancer aggressiveness. We hypothesized that this heterogeneous and evolving compartment drastically influences tumor cell abilities, which in turn influences PDA aggressiveness through crosstalk that is mediated by extracellular vesicles (EVs). Here, we have analyzed the PDA proteomic stromal signature and identified a contribution of the annexin A6/LDL receptor-related protein 1/thrombospondin 1 (ANXA6/LRP1/TSP1) complex in tumor cell crosstalk. Formation of the ANXA6/LRP1/TSP1 complex was restricted to cancer-associated fibroblasts (CAFs) and required physiopathologic culture conditions that improved tumor cell survival and migration. Increased PDA aggressiveness was dependent on tumor cell–mediated uptake of CAF-derived ANXA6+ EVs carrying the ANXA6/LRP1/TSP1 complex. Depletion of ANXA6 in CAFs impaired complex formation and subsequently impaired PDA and metastasis occurrence, while injection of CAF-derived ANXA6+ EVs enhanced tumorigenesis. We found that the presence of ANXA6+ EVs in serum was restricted to PDA patients and represents a potential biomarker for PDA grade. These findings suggest that CAF–tumor cell crosstalk supported by ANXA6+ EVs is predictive of PDA aggressiveness, highlighting a therapeutic target and potential biomarker for PDA. PMID:27701147

A New Paradigm for New Oceans

NASA Astrophysics Data System (ADS)

Foulger, G. R.; Doré, A. G.; Franke, D.; Geoffroy, L.; Gernigon, L.; Hole, M.; Hoskuldsson, A.; Julian, B. R.; Kusznir, N.; Martinez, F.; Natland, J. H.; Peace, A.; Petersen, K. D.; Schiffer, C.; Stephenson, R.; Stoker, M. S.

2017-12-01

The original simple theory of plate tectonics had to be refined to accommodate second-order geological features such as back-arc basins and continental deformation zones. We propose an additional refinement that is required by complexities that form and persist in new oceans when inhomogeneous continental lithosphere/tectosphere disintegrates. Such complexities include continual plate-boundary reorganizations and migrations, distributed continental material in the ocean, propagating and dying ridges, and sagging, flexing and tilting in the oceans and at continent-ocean boundary zones. Reorganizations of stress and motion persist, resulting in variable orientations over short distances, tectonic reactivations, complex plate boundary configurations including multiple triple junctions, and the formation and abandonment of oceanic microplates. Resulting local compressions and extensions are manifest as bathymetric anomalies, vertical motions, and distributed volcanism at various times and places as the new ocean grows. Examples of regions that exhibit some or all of these features include the North Atlantic, the Rio Grande Rise/Walvis Ridge region of the South Atlantic, and the Seychelles-Mauritius region in the Indian Ocean. We suggest that these complexities arise as a result of the formation of new spreading plate boundaries by rifts propagating through continental lithosphere/tectosphere that is anisotropic as a result of inherited structure/composition and/or a sub-lithospheric mantle destabilized by lithospheric-controlled processes. Such scenarios result in complicated disintegration of continents and local persistent dynamic instability in the new ocean.

IAA RAS Radio Telescope Monitoring System

NASA Astrophysics Data System (ADS)

Mikhailov, A.; Lavrov, A.

2007-07-01

Institute of Applied Astronomy of the Russian Academy of Sciences (IAA RAS) has three identical radio telescopes, the receiving complex of which consists of five two-channel receivers of different bands, six cryogen systems, and additional devices: four local oscillators, phase calibration generators and IF commutator. The design, hardware and data communication protocol are described. The most convenient way to join the devices of the receiving complex into the common monitoring system is to use the interface which allows to connect numerous devices to the data bus. For the purpose of data communication regulation and to exclude conflicts, a data communication protocol has been designed, which operates with complex formatted data sequences. Formation of such sequences requires considerable data processing capability. That is provided by a microcontroller chip in each slave device. The test version of the software for the central computer has been developed in IAA RAS. We are developing the Mark IV FS software extension modules, which will allow us to control the receiving complex of the radio telescope by special SNAP commands from both operator input and schedule files. We are also developing procedures of automatic measurements of SEFD, system noise temperature and other parameters, available both in VLBI and single-dish modes of operation. The system described has been installed on all IAA RAS radio telescopes at "Svetloe", "Zelenchukskaya" and "Badary" observatories. It has proved to be working quite reliably and to show the perfonmance expected.

Exploring inclusion complexes of ionic liquids with α- and β- cyclodextrin by NMR, IR, mass, density, viscosity, surface tension and conductance study

NASA Astrophysics Data System (ADS)

Barman, Biraj Kumar; Rajbanshi, Biplab; Yasmin, Ananya; Roy, Mahendra Nath

2018-05-01

The formation of the host-guest inclusion complexes of ionic liquids namely [BMIm]Cl and [HMIm]Cl with α-CD and β-CD were studied by means of physicochemical and spectroscopic methods. Conductivity and surface tension study were in good agreement with the 1H NMR and FT-IR studies which confirm the formation of the inclusion complexes. The Density and viscosity study also supported the formation of the ICs. Further the stoichiometry was determined 1:1 for each case and the association constants and thermodynamic parameters derived supported the most feasible formation of the [BMIm]Cl- β-CD inclusion complex.

Transforming Growth Factor-β-Activated Kinase 1 Is Required for Human FcγRIIIb-Induced Neutrophil Extracellular Trap Formation.

PubMed

Alemán, Omar Rafael; Mora, Nancy; Cortes-Vieyra, Ricarda; Uribe-Querol, Eileen; Rosales, Carlos

2016-01-01

Neutrophils (PMNs) are the most abundant leukocytes in the blood. PMN migrates from the circulation to sites of infection where they are responsible for antimicrobial functions. PMN uses phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. Several stimuli, including bacteria, fungi, and parasites, and some pharmacological compounds, such as Phorbol 12-myristate 13-acetate (PMA), are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. Recently, it was reported that FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. Direct cross-linking of FcγRIIA or integrins did not promote NET formation. FcγRIIIb-induced NET formation presented different kinetics from PMA-induced NET formation, suggesting differences in signaling. Because FcγRIIIb also induces a strong activation of extracellular signal-regulated kinase (ERK) and nuclear factor Elk-1, and the transforming growth factor-β-activated kinase 1 (TAK1) has recently been implicated in ERK signaling, in the present report, we explored the role of TAK1 in the signaling pathway activated by FcγRIIIb leading to NET formation. FcγRIIIb was stimulated by specific monoclonal antibodies, and NET formation was evaluated in the presence or absence of pharmacological inhibitors. The antibiotic LL Z1640-2, a selective inhibitor of TAK1 prevented FcγRIIIb-induced, but not PMA-induced NET formation. Both PMA and FcγRIIIb cross-linking induced phosphorylation of ERK. But, LL Z1640-2 only inhibited the FcγRIIIb-mediated activation of ERK. Also, only FcγRIIIb, similarly to transforming growth factor-β-induced TAK1 phosphorylation. A MEK (ERK kinase)-specific inhibitor was able to prevent ERK phosphorylation induced by both PMA and FcγRIIIb. These data show for the first time that FcγRIIIb cross-linking activates TAK1, and that this kinase is required for triggering the MEK/ERK signaling pathway to NETosis.

Rapamycin-induced oligomer formation system of FRB-FKBP fusion proteins.

PubMed

Inobe, Tomonao; Nukina, Nobuyuki

2016-07-01

Most proteins form larger protein complexes and perform multiple functions in the cell. Thus, artificial regulation of protein complex formation controls the cellular functions that involve protein complexes. Although several artificial dimerization systems have already been used for numerous applications in biomedical research, cellular protein complexes form not only simple dimers but also larger oligomers. In this study, we showed that fusion proteins comprising the induced heterodimer formation proteins FRB and FKBP formed various oligomers upon addition of rapamycin. By adjusting the configuration of fusion proteins, we succeeded in generating an inducible tetramer formation system. Proteins of interest also formed tetramers by fusing to the inducible tetramer formation system, which exhibits its utility in a broad range of biological applications. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Molecular-scale characterization of uranium sorption by bone apatite materials for a permeable reactive barrier demonstration

USGS Publications Warehouse

Fuller, C.C.; Bargar, J.R.; Davis, J.A.

2003-01-01

Uranium binding to bone charcoal and bone meal apatite materials was investigated using U LIII-edge EXAFS spectroscopy and synchrotron source XRD measurements of laboratory batch preparations in the absence and presence of dissolved carbonate. Pelletized bone char apatite recovered from a permeable reactive barrier (PRB) at Fry Canyon, UT, was also studied. EXAFS analyses indicate that U(VI) sorption in the absence of dissolved carbonate occurred by surface complexation of U(VI) for sorbed concentrations ??? 5500 ??g U(VI)/g for all materials with the exception of crushed bone char pellets. Either a split or a disordered equatorial oxygen shell was observed, consistent with complexation of uranyl by the apatite surface. A second shell of atoms at a distance of 2.9 A?? was required to fit the spectra of samples prepared in the presence of dissolved carbonate (4.8 mM total) and is interpreted as formation of ternary carbonate complexes with sorbed U(VI). A U-P distance at 3.5-3.6 A?? was found for most samples under conditions where uranyl phosphate phases did not form, which is consistent with monodentate coordination of uranyl by phosphate groups in the apatite surface. At sorbed concentrations ??? 5500 ??g U(VI)/g in the absence of dissolved carbonate, formation of the uranyl phosphate solid phase, chernikovite, was observed. The presence of dissolved carbonate (4.8 mM total) suppressed the formation of chernikovite, which was not detected even with sorbed U(VI) up to 12 300 ??g U(VI)/g in batch samples of bone meal, bone charcoal, and reagent-grade hydroxyapatite. EXAFS spectra of bone char samples recovered from the Fry Canyon PRB were comparable to laboratory samples in the presence of dissolved carbonate where U(VI) sorption occurred by surface complexation. Our findings demonstrate that uranium uptake by bone apatite will probably occur by surface complexation instead of precipitation of uranyl phosphate phases under the groundwater conditions found at many U-contaminated sites.

Determining Serpin Conformational Distributions with Single Molecule Fluorescence

PubMed Central

Mushero, Nicole; Gershenson, Anne

2012-01-01

Conformational plasticity is key to inhibitory serpin function, and this plasticity gives serpins relatively easy access to alternative, dysfunctional conformations. Thus, a given serpin population may contain both functional and dysfunctional proteins. Single molecule fluorescence (SMF), with its ability to interrogate one fluorescently labeled protein at a time, is a powerful method for elucidating conformational distributions and monitoring how these distributions change over time. SMF and related methods have been particularly valuable for characterizing serpin polymerization. Fluorescence correlation spectroscopy experiments have revealed a second lag phase during in vitro α1-antitrypsin polymerization associated with the formation of smaller oligomers that then condense to form longer polymers [Purkayastha, P., Klemke, J. W., Lavender, S., Oyola, R., Cooperman, B. S., and Gai, F. (2005). Alpha 1-antitrypsin polymerization: A fluorescence correlation spectroscopic study. Biochemistry 44, 2642–2649.]. SMF studies of in vitro neuroserpin polymerization have confirmed that a monomeric intermediate is required for polymer formation while providing a test of proposed polymerization mechanisms [Chiou, A., Hägglöf, P., Orte, A., Chen, A. Y., Dunne, P. D., Belorgey, D., Karlsson-Li, S., Lomas, D., and Klenerman, D. (2009)]. Probing neuroserpin polymerization and interaction with amyloid-beta peptides using single molecule fluorescence. Biophys. J. 97, 2306–2315.]. SMF has also been used to monitor protease–serpin interactions. Single pair Förster resonance energy transfer studies of covalent protease–serpin complexes suggest that the extent of protease structural disruption in the complex is protease dependent [Liu, L., Mushero, N., Hedstrom, L., and Gershenson, A. (2006). Conformational distributions of protease-serpin complexes: A partially translocated complex. Biochemistry 45, 10865–10872.]. SMF techniques are still evolving and the combination of SMF with encapsulation methods has the potential to provide more detailed information on the conformational changes associated with serpin polymerization, protease–serpin complex formation, and serpin folding. PMID:22078542

Mangifera indica L. extract (Vimang) inhibits Fe2+-citrate-induced lipoperoxidation in isolated rat liver mitochondria.

PubMed

Pardo Andreu, Gilberto; Delgado, René; Velho, Jesus; Inada, Natalia M; Curti, Carlos; Vercesi, Anibal E

2005-05-01

The extract of Mangifera indica L. (Vimang) is able to prevent iron mediated mitochondrial damage by means of oxidation of reduced transition metals required for the production of superoxide and hydroxyl radicals and direct free radical scavenging activity. In this study we report for the first time the iron-complexing ability of Vimang as a primary mechanism for protection of rat liver mitochondria against Fe2+ -citrate-induced lipoperoxidation. Thiobarbituric acid reactive substances (TBARS) and antimycin A-insensitive oxygen consumption were used as quantitative measures of lipoperoxidation. Vimang at 10 microM mangiferin concentration equivalent induced near-full protection against 50 microM Fe2+ -citrate-induced mitochondrial swelling and loss of mitochondrial transmembrane potential (DeltaPsi). The IC50 value for Vimang protection against Fe2+ -citrate-induced mitochondrial TBARS formation (7.89+/-1.19 microM) was around 10 times lower than that for tert-butylhydroperoxide mitochondrial induction of TBARS formation. The extract also inhibited the iron citrate induction of mitochondrial antimycin A-insensitive oxygen consumption, stimulated oxygen consumption due to Fe2+ autoxidation and prevented Fe3+ ascorbate reduction. The extracted polyphenolic compound, mainly mangiferin, could form a complex with Fe2+, accelerating Fe2+ oxidation and the formation of more stable Fe3+ -polyphenol complexes, unable to participate in Fenton-type reactions and lipoperoxidation propagation phase. The strong DPPH radical scavenging activity with an apparent IC50 of 2.45+/-0.08 microM suggests that besides its iron-complexing capacity, Vimang could also protect mitochondria from Fe2+ -citrate lipoperoxidation through direct free radical scavenging ability, mainly lipoperoxyl and alcoxyl radicals, acting as both a chain-breaking and iron-complexing antioxidant. These results are of pharmacological relevance since Vimang could be a potential candidate for antioxidant therapy in diseases related to abnormal intracellular iron distribution or iron overload.

Spectroscopic Case-Based Studies in a Flipped Quantum Mechanics Course

NASA Astrophysics Data System (ADS)

Shipman, Steven

2015-06-01

Students in a flipped Quantum Mechanics course were expected to apply their knowledge of spectroscopy to a variety of case studies involving complex mixtures of chemicals. They used simulated data, prepared in advance by the instructor, to determine the major chemical constituents of complex mixtures. Students were required to request the appropriate data in order to ultimately make plausible guesses about the composition of the mixtures, allowing them ownership over the discovery process. This talk will describe how these activities worked in practice, give caveats for instructors who wish to adopt them in the future, and discuss how the results of these exercises can be used for both formative and summative assessment.

Implementation of Super-Encryption with Trithemius Algorithm and Double Transposition Cipher in Securing PDF Files on Android Platform

NASA Astrophysics Data System (ADS)

Budiman, M. A.; Rachmawati, D.; Jessica

2018-03-01

This study aims to combine the trithemus algorithm and double transposition cipher in file security that will be implemented to be an Android-based application. The parameters being examined are the real running time, and the complexity value. The type of file to be used is a file in PDF format. The overall result shows that the complexity of the two algorithms with duper encryption method is reported as Θ (n 2). However, the processing time required in the encryption process uses the Trithemius algorithm much faster than using the Double Transposition Cipher. With the length of plaintext and password linearly proportional to the processing time.

[Development method of healthcare information system integration based on business collaboration model].

PubMed

Li, Shasha; Nie, Hongchao; Lu, Xudong; Duan, Huilong

2015-02-01

Integration of heterogeneous systems is the key to hospital information construction due to complexity of the healthcare environment. Currently, during the process of healthcare information system integration, people participating in integration project usually communicate by free-format document, which impairs the efficiency and adaptability of integration. A method utilizing business process model and notation (BPMN) to model integration requirement and automatically transforming it to executable integration configuration was proposed in this paper. Based on the method, a tool was developed to model integration requirement and transform it to integration configuration. In addition, an integration case in radiology scenario was used to verify the method.

Study of the formation of soluble complexes of sodium caseinate and xanthan in solution.

PubMed

Bouhannache, Bouchra; HadjSadok, Abdelkader; Touabet, Abdelkrim

2017-09-01

The main objective of this work was to determinate the optimum conditions for the formation of soluble complexes between sodium caseinate and xanthan in solution at neutral pH, in the presence of the NaCl. The study of the influence of the concentrations of these three substances showed that salt was the most influent factor. It worsens the thermodynamic incompatibility of the two biopolymers in solution, when they are present at large amounts. However, it contributes to soluble complexes formation, when sodium caseinate concentration is below 5.5%. In this case, gels with enhanced rheological properties were obtained. Infrared spectroscopy confirmed that the complexes formation within these gels involves hydrophobic interactions. On the other hand, dynamic light scattering revealed that dilution cause their dissociation. These soluble complexes are promising ingredients to ensure new texturing properties.

ArrayInitiative - a tool that simplifies creating custom Affymetrix CDFs

PubMed Central

2011-01-01

Background Probes on a microarray represent a frozen view of a genome and are quickly outdated when new sequencing studies extend our knowledge, resulting in significant measurement error when analyzing any microarray experiment. There are several bioinformatics approaches to improve probe assignments, but without in-house programming expertise, standardizing these custom array specifications as a usable file (e.g. as Affymetrix CDFs) is difficult, owing mostly to the complexity of the specification file format. However, without correctly standardized files there is a significant barrier for testing competing analysis approaches since this file is one of the required inputs for many commonly used algorithms. The need to test combinations of probe assignments and analysis algorithms led us to develop ArrayInitiative, a tool for creating and managing custom array specifications. Results ArrayInitiative is a standalone, cross-platform, rich client desktop application for creating correctly formatted, custom versions of manufacturer-provided (default) array specifications, requiring only minimal knowledge of the array specification rules and file formats. Users can import default array specifications, import probe sequences for a default array specification, design and import a custom array specification, export any array specification to multiple output formats, export the probe sequences for any array specification and browse high-level information about the microarray, such as version and number of probes. The initial release of ArrayInitiative supports the Affymetrix 3' IVT expression arrays we currently analyze, but as an open source application, we hope that others will contribute modules for other platforms. Conclusions ArrayInitiative allows researchers to create new array specifications, in a standard format, based upon their own requirements. This makes it easier to test competing design and analysis strategies that depend on probe definitions. Since the custom array specifications are easily exported to the manufacturer's standard format, researchers can analyze these customized microarray experiments using established software tools, such as those available in Bioconductor. PMID:21548938

Dynamic epigenetic states of maize centromeres

PubMed Central

Liu, Yalin; Su, Handong; Zhang, Jing; Liu, Yang; Han, Fangpu; Birchler, James A.

2015-01-01

The centromere is a specialized chromosomal region identified as the major constriction, upon which the kinetochore complex is formed, ensuring accurate chromosome orientation and segregation during cell division. The rapid evolution of centromere DNA sequence and the conserved centromere function are two contradictory aspects of centromere biology. Indeed, the sole presence of genetic sequence is not sufficient for centromere formation. Various dicentric chromosomes with one inactive centromere have been recognized. It has also been found that de novo centromere formation is common on fragments in which centromeric DNA sequences are lost. Epigenetic factors play important roles in centromeric chromatin assembly and maintenance. Non-disjunction of the supernumerary B chromosome centromere is independent of centromere function, but centromere pairing during early prophase of meiosis I requires an active centromere. This review discusses recent studies in maize about genetic and epigenetic elements regulating formation and maintenance of centromere chromatin, as well as centromere behavior in meiosis. PMID:26579154

Lattice-Matched Epitaxial Graphene Grown on Boron Nitride.

PubMed

Davies, Andrew; Albar, Juan D; Summerfield, Alex; Thomas, James C; Cheng, Tin S; Korolkov, Vladimir V; Stapleton, Emily; Wrigley, James; Goodey, Nathan L; Mellor, Christopher J; Khlobystov, Andrei N; Watanabe, Kenji; Taniguchi, Takashi; Foxon, C Thomas; Eaves, Laurence; Novikov, Sergei V; Beton, Peter H

2018-01-10

Lattice-matched graphene on hexagonal boron nitride is expected to lead to the formation of a band gap but requires the formation of highly strained material and has not hitherto been realized. We demonstrate that aligned, lattice-matched graphene can be grown by molecular beam epitaxy using substrate temperatures in the range 1600-1710 °C and coexists with a topologically modified moiré pattern with regions of strained graphene which have giant moiré periods up to ∼80 nm. Raman spectra reveal narrow red-shifted peaks due to isotropic strain, while the giant moiré patterns result in complex splitting of Raman peaks due to strain variations across the moiré unit cell. The lattice-matched graphene has a lower conductance than both the Frenkel-Kontorova-type domain walls and also the topological defects where they terminate. We relate these results to theoretical models of band gap formation in graphene/boron nitride heterostructures.

Dynamic epigenetic states of maize centromeres.

PubMed

Liu, Yalin; Su, Handong; Zhang, Jing; Liu, Yang; Han, Fangpu; Birchler, James A

2015-01-01

The centromere is a specialized chromosomal region identified as the major constriction, upon which the kinetochore complex is formed, ensuring accurate chromosome orientation and segregation during cell division. The rapid evolution of centromere DNA sequence and the conserved centromere function are two contradictory aspects of centromere biology. Indeed, the sole presence of genetic sequence is not sufficient for centromere formation. Various dicentric chromosomes with one inactive centromere have been recognized. It has also been found that de novo centromere formation is common on fragments in which centromeric DNA sequences are lost. Epigenetic factors play important roles in centromeric chromatin assembly and maintenance. Non-disjunction of the supernumerary B chromosome centromere is independent of centromere function, but centromere pairing during early prophase of meiosis I requires an active centromere. This review discusses recent studies in maize about genetic and epigenetic elements regulating formation and maintenance of centromere chromatin, as well as centromere behavior in meiosis.

Learning and memory: Steroids and epigenetics.

PubMed

Colciago, Alessandra; Casati, Lavinia; Negri-Cesi, Paola; Celotti, Fabio

2015-06-01

Memory formation and utilization is a complex process involving several brain structures in conjunction as the hippocampus, the amygdala and the adjacent cortical areas, usually defined as medial temporal lobe structures (MTL). The memory processes depend on the formation and modulation of synaptic connectivity affecting synaptic strength, synaptic plasticity and synaptic consolidation. The basic neurocognitive mechanisms of learning and memory are shortly recalled in the initial section of this paper. The effect of sex hormones (estrogens, androgens and progesterone) and of adrenocortical steroids on several aspects of memory processes are then analyzed on the basis of animal and human studies. A specific attention has been devoted to the different types of steroid receptors (membrane or nuclear) involved and on local metabolic transformations when required. The review is concluded by a short excursus on the steroid activated epigenetic mechanisms involved in memory formation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Advances in synapse formation: forging connections in the worm.

PubMed

Cherra, Salvatore J; Jin, Yishi

2015-01-01

Synapse formation is the quintessential process by which neurons form specific connections with their targets to enable the development of functional circuits. Over the past few decades, intense research efforts have identified thousands of proteins that localize to the pre- and postsynaptic compartments. Genetic dissection has provided important insights into the nexus of the molecular and cellular network, and has greatly advanced our knowledge about how synapses form and function physiologically. Moreover, recent studies have highlighted the complex regulation of synapse formation with the identification of novel mechanisms involving cell interactions from non-neuronal sources. In this review, we cover the conserved pathways required for synaptogenesis and place specific focus on new themes of synapse modulation arising from studies in Caenorhabditis elegans. For further resources related to this article, please visit the WIREs website. The authors have declared no conflicts of interest for this article. © 2014 Wiley Periodicals, Inc.

Positive Disintegration as a Process of Symmetry Breaking.

PubMed

Laycraft, Krystyna

2017-04-01

This article presents an analysis of the positive disintegration as a process of symmetry breaking. Symmetry breaking plays a major role in self-organized patterns formation and correlates directly to increasing complexity and function specialization. According to Dabrowski, a creator of the Theory of Positive Disintegration, the change from lower to higher levels of human development requires a major restructuring of an individual's psychological makeup. Each level of human development is a relatively stable and coherent configuration of emotional-cognitive patterns called developmental dynamisms. Their main function is to restructure a mental structure by breaking the symmetry of a low level and bringing differentiation and then integration to higher levels. The positive disintegration is then the process of transitions from a lower level of high symmetry and low complexity to higher levels of low symmetry and high complexity of mental structure.

Nuclear magnetic resonance studies of formyltetrahydrofolate synthetase interactions with formate and methylammonium ion.

PubMed

Wendland, M F; Stevens, T H; Buttlaire, D H; Everett, G W; Himes, R H

1983-02-15

Using nuclear magnetic resonance techniques, we have measured the internuclear distances separating the nucleotide-bound metal from the carbon and hydrogen nuclei of formate as well as the carbon of methylammonium cation when bound to formyltetrahydrofolate synthetase. Measurements were made of the paramagnetic effect on the spin-lattice relaxation rates (1/T1) of 13C and 1H nuclei arising from the replacement of Mg2+ with Mn2+, which binds to the enzyme in the form of a metal-nucleotide complex. Distances from Mn2+ to the formate carbon and proton were found to be 6.3 and 7.4 A, respectively, in the E . ATP . Mn2+ . formate complex and 6.0 and 7.1 A, respectively, in the E . ADP . Mn2+ . formate complex. When tetrahydrofolate was added to the latter complex, the exchange of formate was greatly reduced and became rate limiting for relaxation. These results are consistent with substantial conformational effects produced by the binding of the cofactor. The distance from Mn2+ to the methylammonium carbon in the E . ADP . Mn2+ . CH3NH+3, E . ADP . Mn2+ . formate . CH3NH3+, and E . ADP . Mn2+ . tetrahydrofolate . CH3NH3+ complexes was estimated to be in the range of 7.4-12 A. However, in the E . ADP . Mn2+ formate . tetrahydrofolate . CH3NH3+ complex, the data suggest that exchange of cation contributes significantly to relaxation. These results, combined with other known features of the enzyme, suggest that there may be a monovalent cation site within the active site of the enzyme.

SEPALLATA3: the 'glue' for MADS box transcription factor complex formation

PubMed Central

Immink, Richard GH; Tonaco, Isabella AN; de Folter, Stefan; Shchennikova, Anna; van Dijk, Aalt DJ; Busscher-Lange, Jacqueline; Borst, Jan W; Angenent, Gerco C

2009-01-01

Background Plant MADS box proteins play important roles in a plethora of developmental processes. In order to regulate specific sets of target genes, MADS box proteins dimerize and are thought to assemble into multimeric complexes. In this study a large-scale yeast three-hybrid screen is utilized to provide insight into the higher-order complex formation capacity of the Arabidopsis MADS box family. SEPALLATA3 (SEP3) has been shown to mediate complex formation and, therefore, special attention is paid to this factor in this study. Results In total, 106 multimeric complexes were identified; in more than half of these at least one SEP protein was present. Besides the known complexes involved in determining floral organ identity, various complexes consisting of combinations of proteins known to play a role in floral organ identity specification, and flowering time determination were discovered. The capacity to form this latter type of complex suggests that homeotic factors play essential roles in down-regulation of the MADS box genes involved in floral timing in the flower via negative auto-regulatory loops. Furthermore, various novel complexes were identified that may be important for the direct regulation of the floral transition process. A subsequent detailed analysis of the APETALA3, PISTILLATA, and SEP3 proteins in living plant cells suggests the formation of a multimeric complex in vivo. Conclusions Overall, these results provide strong indications that higher-order complex formation is a general and essential molecular mechanism for plant MADS box protein functioning and attribute a pivotal role to the SEP3 'glue' protein in mediating multimerization. PMID:19243611

Distinct functions for IFT140 and IFT20 in opsin transport.

PubMed Central

Crouse, Jacquelin A.; Lopes, Vanda S.; SanAgustin, Jovenal T.; Keady, Brian T.; Williams, David S.; Pazour, Gregory J.

2014-01-01

In the vertebrate retina, light is detected by the outer segments of photoreceptor rods and cones, which are highly modified cilia. Like other cilia, outer segments have no protein synthetic capacity and depend on proteins made in the cell body for their formation and maintenance. The mechanism of transport into the outer segment is not fully understood but intraflagellar transport (IFT) is thought to be a major mechanism for moving protein from the cell body into the cilium. In the case of photoreceptor cells, the high density of receptors and the disk turnover that occurs daily necessitates much higher rates of transport than would be required in other cilia. In this work, we show that the IFT complex A protein IFT140 is required for development and maintenance of outer segments. In earlier work we found that acute deletion of Ift20 caused opsin to accumulate at the Golgi complex. In this work we find that acute deletion of Ift140 does not cause opsin to accumulate at the Golgi complex but rather it accumulates in the plasma membrane of the inner segments. This work is strong support of a model of opsin transport where IFT20 is involved in the movement from the Golgi complex to the base of the cilium. Then, once at the base, the opsin is carried through the connecting cilium by an IFT complex that includes IFT140. PMID:24619649

Loss of the Mammalian DREAM Complex Deregulates Chondrocyte Proliferation

PubMed Central

Forristal, Chantal; Henley, Shauna A.; MacDonald, James I.; Bush, Jason R.; Ort, Carley; Passos, Daniel T.; Talluri, Srikanth; Ishak, Charles A.; Thwaites, Michael J.; Norley, Chris J.; Litovchick, Larisa; DeCaprio, James A.; DiMattia, Gabriel; Holdsworth, David W.; Beier, Frank

2014-01-01

Mammalian DREAM is a conserved protein complex that functions in cellular quiescence. DREAM contains an E2F, a retinoblastoma (RB)-family protein, and the MuvB core (LIN9, LIN37, LIN52, LIN54, and RBBP4). In mammals, MuvB can alternatively bind to BMYB to form a complex that promotes mitotic gene expression. Because BMYB-MuvB is essential for proliferation, loss-of-function approaches to study MuvB have generated limited insight into DREAM function. Here, we report a gene-targeted mouse model that is uniquely deficient for DREAM complex assembly. We have targeted p107 (Rbl1) to prevent MuvB binding and combined it with deficiency for p130 (Rbl2). Our data demonstrate that cells from these mice preferentially assemble BMYB-MuvB complexes and fail to repress transcription. DREAM-deficient mice show defects in endochondral bone formation and die shortly after birth. Micro-computed tomography and histology demonstrate that in the absence of DREAM, chondrocytes fail to arrest proliferation. Since DREAM requires DYRK1A (dual-specificity tyrosine phosphorylation-regulated protein kinase 1A) phosphorylation of LIN52 for assembly, we utilized an embryonic bone culture system and pharmacologic inhibition of (DYRK) kinase to demonstrate a similar defect in endochondral bone growth. This reveals that assembly of mammalian DREAM is required to induce cell cycle exit in chondrocytes. PMID:24710275

ASF1 is required to load histones on the HIRA complex in preparation of paternal chromatin assembly at fertilization.

PubMed

Horard, Béatrice; Sapey-Triomphe, Laure; Bonnefoy, Emilie; Loppin, Benjamin

2018-05-11

Anti-Silencing Factor 1 (ASF1) is a conserved H3-H4 histone chaperone involved in both Replication-Coupled and Replication-Independent (RI) nucleosome assembly pathways. At DNA replication forks, ASF1 plays an important role in regulating the supply of H3.1/2 and H4 to the CAF-1 chromatin assembly complex. ASF1 also provides H3.3-H4 dimers to HIRA and DAXX chaperones for RI nucleosome assembly. The early Drosophila embryo is an attractive system to study chromatin assembly in a developmental context. The formation of a diploid zygote begins with the unique, genome-wide RI assembly of paternal chromatin following sperm protamine eviction. Then, within the same cytoplasm, syncytial embryonic nuclei undergo a series of rapid, synchronous S and M phases to form the blastoderm embryo. Here, we have investigated the implication of ASF1 in these two distinct assembly processes. We show that depletion of the maternal pool of ASF1 with a specific shRNA induces a fully penetrant, maternal effect embryo lethal phenotype. Unexpectedly, despite the depletion of ASF1 protein to undetectable levels, we show that asf1 knocked-down (KD) embryos can develop to various stages, thus demonstrating that ASF1 is not absolutely required for the amplification of cleavage nuclei. Remarkably, we found that ASF1 is required for the formation of the male pronucleus, although ASF1 protein does not reside in the decondensing sperm nucleus. In asf1 KD embryos, HIRA localizes to the male nucleus but is only capable of limited and insufficient chromatin assembly. Finally, we show that the conserved HIRA B domain, which is involved in ASF1-HIRA interaction, is dispensable for female fertility. We conclude that ASF1 is critically required to load H3.3-H4 dimers on the HIRA complex prior to histone deposition on paternal DNA. This separation of tasks could optimize the rapid assembly of paternal chromatin within the gigantic volume of the egg cell. In contrast, ASF1 is surprisingly dispensable for the amplification of cleavage nuclei, although chromatin integrity is likely compromised in KD embryos.

Advanced glycoxidation and lipoxidation end products (AGEs and ALEs): an overview of their mechanisms of formation.

PubMed

Vistoli, G; De Maddis, D; Cipak, A; Zarkovic, N; Carini, M; Aldini, G

2013-08-01

Advanced lipoxidation end products (ALEs) and advanced glycation end products (AGEs) have a pathogenetic role in the development and progression of different oxidative-based diseases including diabetes, atherosclerosis, and neurological disorders. AGEs and ALEs represent a quite complex class of compounds that are formed by different mechanisms, by heterogeneous precursors and that can be formed either exogenously or endogenously. There is a wide interest in AGEs and ALEs involving different aspects of research which are essentially focused on set-up and application of analytical strategies (1) to identify, characterize, and quantify AGEs and ALEs in different pathophysiological conditions; (2) to elucidate the molecular basis of their biological effects; and (3) to discover compounds able to inhibit AGEs/ALEs damaging effects not only as biological tools aimed at validating AGEs/ALEs as drug target, but also as promising drugs. All the above-mentioned research stages require a clear picture of the chemical formation of AGEs/ALEs but this is not simple, due to the complex and heterogeneous pathways, involving different precursors and mechanisms. In view of this intricate scenario, the aim of the present review is to group the main AGEs and ALEs and to describe, for each of them, the precursors and mechanisms of formation.

Computational study of C(sp3)-O bond formation at a PdIV centre.

PubMed

Canty, Allan J; Ariafard, Alireza; Camasso, Nicole M; Higgs, Andrew T; Yates, Brian F; Sanford, Melanie S

2017-03-14

This report describes a computational study of C(sp 3 )-OR bond formation from Pd IV complexes of general structure Pd IV (CH 2 CMe 2 -o-C 6 H 4 -C,C')(F)(OR)(bpy-N,N') (bpy = 2,2'-bipyridine). Dissociation of - OR from the different octahedral Pd IV starting materials results in a common square-pyramidal Pd IV cation. An S N 2-type attack by - OR ( - OR = phenoxide, acetate, difluoroacetate, and nitrate) then leads to C(sp 3 )-OR bond formation. In contrast, when - OR = triflate, concerted C(sp 3 )-C(sp 2 ) bond-forming reductive elimination takes place, and the calculations indicate this outcome is the result of thermodynamic rather than kinetic control. The energy requirements for the dissociation and S N 2 steps with different - OR follow opposing trends. The S N 2 transition states exhibit "PdCO" angles in a tight range of 151.5 to 153.0°, resulting from steric interactions between the oxygen atom and the gem-dimethyl group of the ligand. Conformational effects for various OR ligands and isomerisation of the complexes were also examined as components of the solution dynamics in these systems. In all cases, the trends observed computationally agree with those observed experimentally.

A Simple Explanation of Complexation

ERIC Educational Resources Information Center

Elliott, J. Richard

2010-01-01

The topics of solution thermodynamics, activity coefficients, and complex formation are introduced through computational exercises and sample applications. The presentation is designed to be accessible to freshmen in a chemical engineering computations course. The MOSCED model is simplified to explain complex formation in terms of hydrogen…

Curcumin complexation with cyclodextrins by the autoclave process: Method development and characterization of complex formation.

PubMed

Hagbani, Turki Al; Nazzal, Sami

2017-03-30

One approach to enhance curcumin (CUR) aqueous solubility is to use cyclodextrins (CDs) to form inclusion complexes where CUR is encapsulated as a guest molecule within the internal cavity of the water-soluble CD. Several methods have been reported for the complexation of CUR with CDs. Limited information, however, is available on the use of the autoclave process (AU) in complex formation. The aims of this work were therefore to (1) investigate and evaluate the AU cycle as a complex formation method to enhance CUR solubility; (2) compare the efficacy of the AU process with the freeze-drying (FD) and evaporation (EV) processes in complex formation; and (3) confirm CUR stability by characterizing CUR:CD complexes by NMR, Raman spectroscopy, DSC, and XRD. Significant differences were found in the saturation solubility of CUR from its complexes with CD when prepared by the three complexation methods. The AU yielded a complex with expected chemical and physical fingerprints for a CUR:CD inclusion complex that maintained the chemical integrity and stability of CUR and provided the highest solubility of CUR in water. Physical and chemical characterizations of the AU complexes confirmed the encapsulated of CUR inside the CD cavity and the transformation of the crystalline CUR:CD inclusion complex to an amorphous form. It was concluded that the autoclave process with its short processing time could be used as an alternate and efficient methods for drug:CD complexation. Copyright © 2017 Elsevier B.V. All rights reserved.

Electrocatalytic Oxidation of Formate by [Ni(P R 2N R' 2) 2(CH 3CN)] 2+ Complexes

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

Galan, Brandon R.; Schöffel, Julia; Linehan, John C.

2011-08-17

[Ni(P R 2N R' 2) 2(CH 3CN)] 2+ complexes with R = Ph, R' = 4-MeOPh or R = Cy, R' = Ph , and a mixed-ligand [Ni(P R 2N R' 2)(P R" 2N R' 2)(CH 3CN)] 2+ with R = Cy, R' = Ph, R" = Ph, have been synthesized and characterized by single-crystal X-ray crystallography. These and previously reported complexes are shown to be electrocatalysts for the oxidation of formate in solution to produce CO 2, protons, and electrons, with rates that are first-order in catalyst and formate at formate concentrations below ~0.04 M (34 equiv). At concentrationsmore » above ~0.06 M formate (52 equiv), catalytic rates become nearly independent of formate concentration. For the catalysts studied, maximum observed turnover frequencies vary from <1.1 to 15.8 s –1 at room temperature, which are the highest rates yet reported for formate oxidation by homogeneous catalysts. These catalysts are the only base-metal electrocatalysts as well as the only homogeneous electrocatalysts reported to date for the oxidation of formate. An acetate complex demonstrating an η 1-OC(O)CH 3 binding mode to nickel has also been synthesized and characterized by single-crystal X-ray crystallography. Based on this structure and the electrochemical and spectroscopic data, a mechanistic scheme for electrocatalytic formate oxidation is proposed which involves formate binding followed by a rate-limiting proton and two-electron transfer step accompanied by CO 2 liberation. Finally, the pendant amines have been demonstrated to be essential for electrocatalysis, as no activity toward formate oxidation was observed for the similar [Ni(depe) 2] 2+ (depe = 1,2-bis(diethylphosphino)ethane) complex.« less

Electrocatalytic oxidation of formate by [Ni(P(R)2N(R')2)2(CH3CN)]2+ complexes.

PubMed

Galan, Brandon R; Schöffel, Julia; Linehan, John C; Seu, Candace; Appel, Aaron M; Roberts, John A S; Helm, Monte L; Kilgore, Uriah J; Yang, Jenny Y; DuBois, Daniel L; Kubiak, Clifford P

2011-08-17

[Ni(P(R)(2)N(R')(2))(2)(CH(3)CN)](2+) complexes with R = Ph, R' = 4-MeOPh or R = Cy, R' = Ph , and a mixed-ligand [Ni(P(R)(2)N(R')(2))(P(R''(2))N(R'(2)))(CH(3)CN)](2+) with R = Cy, R' = Ph, R'' = Ph, have been synthesized and characterized by single-crystal X-ray crystallography. These and previously reported complexes are shown to be electrocatalysts for the oxidation of formate in solution to produce CO(2), protons, and electrons, with rates that are first-order in catalyst and formate at formate concentrations below ∼0.04 M (34 equiv). At concentrations above ∼0.06 M formate (52 equiv), catalytic rates become nearly independent of formate concentration. For the catalysts studied, maximum observed turnover frequencies vary from <1.1 to 15.8 s(-1) at room temperature, which are the highest rates yet reported for formate oxidation by homogeneous catalysts. These catalysts are the only base-metal electrocatalysts as well as the only homogeneous electrocatalysts reported to date for the oxidation of formate. An acetate complex demonstrating an η(1)-OC(O)CH(3) binding mode to nickel has also been synthesized and characterized by single-crystal X-ray crystallography. Based on this structure and the electrochemical and spectroscopic data, a mechanistic scheme for electrocatalytic formate oxidation is proposed which involves formate binding followed by a rate-limiting proton and two-electron transfer step accompanied by CO(2) liberation. The pendant amines have been demonstrated to be essential for electrocatalysis, as no activity toward formate oxidation was observed for the similar [Ni(depe)(2)](2+) (depe = 1,2-bis(diethylphosphino)ethane) complex.

Decarboxylative aldol reactions of allyl beta-keto esters via heterobimetallic catalysis.

PubMed

Lou, Sha; Westbrook, John A; Schaus, Scott E

2004-09-22

Mild and selective heterobimetallic-catalyzed decarboxylative aldol reactions involving allyl beta-keto esters have been developed. The reaction is promoted by Pd(0)- and Yb(III)-DIOP complexes at room temperature and involves the in situ formation of a ketone enolate from allyl beta-keto esters followed by addition of the enolate to aldehydes. The reaction is a new example of heterobimetallic catalysis in which the optimized reaction conditions require the addition of both metals.

Thermodynamics of complexation in an aqueous solution of Tb(III) nitrate at 298 K

NASA Astrophysics Data System (ADS)

Lobacheva, O. L.; Berlinskii, I. V.; Dzhevaga, N. V.

2017-01-01

The pH of the formation of hydroxo complexes and hydrates in an aqueous solution of terbium Tb(III) is determined using combined means of potentiometric and conductometric titration. The stability constants of the hydroxo complexes, the products of hydroxide solubility, and the Gibbs energy of terbium hydroxo complex formation are calculated.

The Effect of Complex Formation upon the Redox Potentials of Metallic Ions. Cyclic Voltammetry Experiments.

ERIC Educational Resources Information Center

Ibanez, Jorge G.; And Others

1988-01-01

Describes experiments in which students prepare in situ soluble complexes of metal ions with different ligands and observe and estimate the change in formal potential that the ion undergoes upon complexation. Discusses student formation and analysis of soluble complexes of two different metal ions with the same ligand. (CW)

Noncanonical Gβ Gib2 is a scaffolding protein promoting cAMP signaling through functions of Ras1 and Cac1 proteins in Cryptococcus neoformans.

PubMed

Wang, Yanli; Shen, Gui; Gong, Jinjun; Shen, Danyu; Whittington, Amy; Qing, Jiang; Treloar, Joshua; Boisvert, Scott; Zhang, Zhengguang; Yang, Cai; Wang, Ping

2014-05-02

Gβ-like/RACK1 functions as a key mediator of various pathways and contributes to numerous cellular functions in eukaryotic organisms. In the pathogenic fungus Cryptococcus neoformans, noncanonical Gβ Gib2 promotes cAMP signaling in cells lacking normal Gpa1 function while displaying versatility in interactions with Gα Gpa1, protein kinase Pkc1, and endocytic intersectin Cin1. To elucidate the Gib2 functional mechanism(s), we demonstrate that Gib2 is required for normal growth and virulence. We show that Gib2 directly binds to Gpa1 and Gγ Gpg1/Gpg2 and that it interacts with phosphodiesterase Pde2 and monomeric GTPase Ras1. Pde2 remains functionally dispensable, but Ras1 is found to associate with adenylyl cyclase Cac1 through the conserved Ras association domain. In addition, the ras1 mutant exhibits normal capsule formation, whereas the ras1 gpa1 mutant displays enhanced capsule formation, and the ras1 gpa1 cac1 mutant is acapsular. Collectively, these findings suggest that Gib2 promotes cAMP levels by relieving an inhibitory function of Ras1 on Cac1 in the absence of Gpa1. In addition, using GST affinity purification combined with mass spectrometry, we identified 47 additional proteins that interact with Gib2. These proteins have putative functions ranging from signal transduction, energy generation, metabolism, and stress response to ribosomal function. After establishing and validating a protein-protein interactive network, we believe Gib2 to be a key adaptor/scaffolding protein that drives the formation of various protein complexes required for growth and virulence. Our study reveals Gib2 as an essential component in deciphering the complexity of regulatory networks that control growth and virulence in C. neoformans.

The HUSH complex cooperates with TRIM28 to repress young retrotransposons and new genes.

PubMed

Robbez-Masson, Luisa; Tie, Christopher H C; Conde, Lucia; Tunbak, Hale; Husovsky, Connor; Tchasovnikarova, Iva A; Timms, Richard T; Herrero, Javier; Lehner, Paul J; Rowe, Helen M

2018-05-04

Retrotransposons encompass half of the human genome and contribute to the formation of heterochromatin, which provides nuclear structure and regulates gene expression. Here, we asked if the human silencing hub (HUSH) complex is necessary to silence retrotransposons and whether it collaborates with TRIM28 and the chromatin remodeler ATRX at specific genomic loci. We show that the HUSH complex contributes to de novo repression and DNA methylation of a SVA retrotransposon reporter. By using naïve vs. primed mouse pluripotent stem cells, we reveal a critical role for the HUSH complex in naïve cells, implicating it in programming epigenetic marks in development. While the HUSH component FAM208A binds to endogenous retroviruses (ERVs) and long interspersed element-1s (LINE-1s or L1s), it is mainly required to repress evolutionarily young L1s (mouse-specific lineages less than 5 million years old). TRIM28, in contrast, is necessary to repress both ERVs and young L1s. Genes co-repressed by TRIM28 and FAM208A are evolutionarily young, or exhibit tissue-specific expression, are enriched in young L1s and display evidence for regulation through LTR promoters. Finally, we demonstrate that the HUSH complex is also required to repress L1 elements in human cells. Overall, these data indicate that the HUSH complex and TRIM28 co-repress young retrotransposons and new genes rewired by retrotransposon non-coding DNA. Published by Cold Spring Harbor Laboratory Press.

Controlled assembly of artificial protein-protein complexes via DNA duplex formation.

PubMed

Płoskoń, Eliza; Wagner, Sara C; Ellington, Andrew D; Jewett, Michael C; O'Reilly, Rachel; Booth, Paula J

2015-03-18

DNA-protein conjugates have found a wide range of applications. This study demonstrates the formation of defined, non-native protein-protein complexes via the site specific labeling of two proteins of interest with complementary strands of single-stranded DNA in vitro. This study demonstrates that the affinity of two DNA-protein conjugates for one another may be tuned by the use of variable lengths of DNA allowing reversible control of complex formation.

Statistical analysis of dimer formation in supersaturated metal vapor based on molecular dynamics simulation

NASA Astrophysics Data System (ADS)

Korenchenko, Anna E.; Vorontsov, Alexander G.; Gelchinski, Boris R.; Sannikov, Grigorii P.

2018-04-01

We discuss the problem of dimer formation during the homogeneous nucleation of atomic metal vapor in an inert gas environment. We simulated nucleation with molecular dynamics and carried out the statistical analysis of double- and triple-atomic collisions as the two ways of long-lived diatomic complex formation. Close pair of atoms with lifetime greater than the mean time interval between atom-atom collisions is called a long-lived diatomic complex. We found that double- and triple-atomic collisions gave approximately the same probabilities of long-lived diatomic complex formation, but internal energy of the resulted state was essentially lower in the second case. Some diatomic complexes formed in three-particle collisions are stable enough to be a critical nucleus.

User-friendly tools on handheld devices for observer performance study

NASA Astrophysics Data System (ADS)

Matsumoto, Takuya; Hara, Takeshi; Shiraishi, Junji; Fukuoka, Daisuke; Abe, Hiroyuki; Matsusako, Masaki; Yamada, Akira; Zhou, Xiangrong; Fujita, Hiroshi

2012-02-01

ROC studies require complex procedures to select cases from many data samples, and to set confidence levels in each selected case to generate ROC curves. In some observer performance studies, researchers have to develop software with specific graphical user interface (GUI) to obtain confidence levels from readers. Because ROC studies could be designed for various clinical situations, it is difficult task for preparing software corresponding to every ROC studies. In this work, we have developed software for recording confidence levels during observer studies on tiny personal handheld devices such as iPhone, iPod touch, and iPad. To confirm the functions of our software, three radiologists performed observer studies to detect lung nodules by using public database of chest radiograms published by Japan Society of Radiological Technology. The output in text format conformed to the format for the famous ROC kit from the University of Chicago. Times required for the reading each case was recorded very precisely.

Frog virus 3 ORF 53R, a putative myristoylated membrane protein, is essential for virus replication in vitro

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

Whitley, Dexter S.; Yu, Kwang; Sample, Robert C.

2010-09-30

Although previous work identified 12 complementation groups with possible roles in virus assembly, currently only one frog virus 3 protein, the major capsid protein (MCP), has been linked with virion formation. To identify other proteins required for assembly, we used an antisense morpholino oligonucleotide to target 53R, a putative myristoylated membrane protein, and showed that treatment resulted in marked reductions in 53R levels and a 60% drop in virus titers. Immunofluorescence assays confirmed knock down and showed that 53R was found primarily within viral assembly sites, whereas transmission electron microscopy detected fewer mature virions and, in some cells, dense granularmore » bodies that may represent unencapsidated DNA-protein complexes. Treatment with a myristoylation inhibitor (2-hydroxymyristic acid) resulted in an 80% reduction in viral titers. Collectively, these data indicate that 53R is an essential viral protein that is required for replication in vitro and suggest it plays a critical role in virion formation.« less

Artificial photosynthesis: understanding water splitting in nature

PubMed Central

Cox, Nicholas; Pantazis, Dimitrios A.; Neese, Frank; Lubitz, Wolfgang

2015-01-01

In the context of a global artificial photosynthesis (GAP) project, we review our current work on nature's water splitting catalyst. In a recent report (Cox et al. 2014 Science 345, 804–808 (doi:10.1126/science.1254910)), we showed that the catalyst—a Mn4O5Ca cofactor—converts into an ‘activated’ form immediately prior to the O–O bond formation step. This activated state, which represents an all MnIV complex, is similar to the structure observed by X-ray crystallography but requires the coordination of an additional water molecule. Such a structure locates two oxygens, both derived from water, in close proximity, which probably come together to form the product O2 molecule. We speculate that formation of the activated catalyst state requires inherent structural flexibility. These features represent new design criteria for the development of biomimetic and bioinspired model systems for water splitting catalysts using first-row transition metals with the aim of delivering globally deployable artificial photosynthesis technologies. PMID:26052426

Dynamic resource allocation engine for cloud-based real-time video transcoding in mobile cloud computing environments

NASA Astrophysics Data System (ADS)

Adedayo, Bada; Wang, Qi; Alcaraz Calero, Jose M.; Grecos, Christos

2015-02-01

The recent explosion in video-related Internet traffic has been driven by the widespread use of smart mobile devices, particularly smartphones with advanced cameras that are able to record high-quality videos. Although many of these devices offer the facility to record videos at different spatial and temporal resolutions, primarily with local storage considerations in mind, most users only ever use the highest quality settings. The vast majority of these devices are optimised for compressing the acquired video using a single built-in codec and have neither the computational resources nor battery reserves to transcode the video to alternative formats. This paper proposes a new low-complexity dynamic resource allocation engine for cloud-based video transcoding services that are both scalable and capable of being delivered in real-time. Firstly, through extensive experimentation, we establish resource requirement benchmarks for a wide range of transcoding tasks. The set of tasks investigated covers the most widely used input formats (encoder type, resolution, amount of motion and frame rate) associated with mobile devices and the most popular output formats derived from a comprehensive set of use cases, e.g. a mobile news reporter directly transmitting videos to the TV audience of various video format requirements, with minimal usage of resources both at the reporter's end and at the cloud infrastructure end for transcoding services.

Catastrophic ice lake collapse in Aram Chaos, Mars

NASA Astrophysics Data System (ADS)

Roda, Manuel; Kleinhans, Maarten G.; Zegers, Tanja E.; Oosthoek, Jelmer H. P.

2014-07-01

Hesperian chaotic terrains have been recognized as the source of outflow channels formed by catastrophic outflows. Four main scenarios have been proposed for the formation of chaotic terrains that involve different amounts of water and single or multiple outflow events. Here, we test these scenarios with morphological and structural analyses of imagery and elevation data for Aram Chaos in conjunction with numerical modeling of the morphological evolution of the catastrophic carving of the outflow valley. The morphological and geological analyses of Aram Chaos suggest large-scale collapse and subsidence (1500 m) of the entire area, which is consistent with a massive expulsion of liquid water from the subsurface in one single event. The combined observations suggest a complex process starting with the outflow of water from two small channels, followed by continuous groundwater sapping and headward erosion and ending with a catastrophic lake rim collapse and carving of the Aram Valley, which is synchronous with the 2.5 Ga stage of the Ares Vallis formation. The water volume and formative time scale required to carve the Aram channels indicate that a single, rapid (maximum tens of days) and catastrophic (flood volume of 9.3 × 104 km3) event carved the outflow channel. We conclude that a sub-ice lake collapse model can best explain the features of the Aram Chaos Valley system as well as the time scale required for its formation.

Deprotonated Dicarboxylic Acid Homodimers: Hydrogen Bonds and Atmospheric Implications

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

Hou, Gao-Lei; Valiev, Marat; Wang, Xue-Bin

Dicarboxylic acids represent an important class of water-soluble organic compounds found in the atmosphere. In this work we are studying properties of dicarboxylic acid homodimer complexes (HO 2(CH 2) nCO 2 -[HO 2(CH 2) nCO 2H], n = 0-12), as potentially important intermediates in aerosol formation processes. Our approach is based on experimental data from negative ion photoelectron spectra of the dimer complexes combined with updated measurements of the corresponding monomer species. These results are analyzed with quantum-mechanical calculations, which provide further information about equilibrium structures, thermochemical parameters associated with the complex formation, and evaporation rates. We find that uponmore » formation of the dimer complexes the electron binding energies increase by 1.3–1.7 eV (30.0–39.2 kcal/mol), indicating increased stability of the dimerized complexes. Calculations indicate that these dimer complexes are characterized by the presence of strong intermolecular hydrogen bonds with high binding energies and are thermodynamically favorable to form with low evaporation rates. Comparison with previously studied HSO 4 -[HO 2(CH 2) 2CO 2H] complex (J. Phys. Chem. Lett. 2013, 4, 779-785) shows that HO 2(CH 2) 2CO 2 -[HO 2(CH 2) 2CO 2H] has very similar thermochemical properties. These results imply that dicarboxylic acids not only can contribute to the heterogeneous complexes formation involving sulfuric acid and dicarboxylic acids, but also can promote the formation of homogenous complexes by involving dicarboxylic acids themselves.« less

Dynamics of nanoparticle-protein corona complex formation: analytical results from population balance equations.

PubMed

Darabi Sahneh, Faryad; Scoglio, Caterina; Riviere, Jim

2013-01-01

Nanoparticle-protein corona complex formation involves absorption of protein molecules onto nanoparticle surfaces in a physiological environment. Understanding the corona formation process is crucial in predicting nanoparticle behavior in biological systems, including applications of nanotoxicology and development of nano drug delivery platforms. This paper extends the modeling work in to derive a mathematical model describing the dynamics of nanoparticle corona complex formation from population balance equations. We apply nonlinear dynamics techniques to derive analytical results for the composition of nanoparticle-protein corona complex, and validate our results through numerical simulations. The model presented in this paper exhibits two phases of corona complex dynamics. In the first phase, proteins rapidly bind to the free surface of nanoparticles, leading to a metastable composition. During the second phase, continuous association and dissociation of protein molecules with nanoparticles slowly changes the composition of the corona complex. Given sufficient time, composition of the corona complex reaches an equilibrium state of stable composition. We find analytical approximate formulae for metastable and stable compositions of corona complex. Our formulae are very well-structured to clearly identify important parameters determining corona composition. The dynamics of biocorona formation constitute vital aspect of interactions between nanoparticles and living organisms. Our results further understanding of these dynamics through quantitation of experimental conditions, modeling results for in vitro systems to better predict behavior for in vivo systems. One potential application would involve a single cell culture medium related to a complex protein medium, such as blood or tissue fluid.

Phosphodiester Cleavage in Ribonuclease H Occurs via an Associative Two-Metal-Aided Catalytic Mechanism

PubMed Central

De Vivo, Marco; Dal Peraro, Matteo; Klein, Michael L.

2009-01-01

Ribonuclease H (RNase H) belongs to the nucleotidyl-transferase (NT) superfamily and hydrolyzes the phosphodiester linkages that form the backbone of the RNA strand in RNA·DNA hybrids. This enzyme is implicated in replication initiation and DNA topology restoration and represents a very promising target for anti-HIV drug design. Structural information has been provided by high-resolution crystal structures of the complex RNase H/RNA·DNA from Bacillus halodurans (Bh), which reveals that two metal ions are required for formation of a catalytic active complex. Here, we use classical force field-based and quantum mechanics/molecular mechanics calculations for modeling the nucleotidyl transfer reaction in RNase H, clarifying the role of the metal ions and the nature of the nucleophile (water versus hydroxide ion). During the catalysis, the two metal ions act cooperatively, facilitating nucleophile formation and stabilizing both transition state and leaving group. Importantly, the two Mg2+ metals also support the formation of a meta-stable phosphorane intermediate along the reaction, which resembles the phosphorane intermediate structure obtained only in the debated β-phosphoglucomutase crystal. The nucleophile formation (i.e., water deprotonation) can be achieved in situ, after migration of one proton from the water to the scissile phosphate in the transition state. This proton transfer is actually mediated by solvation water molecules. Due to the highly conserved nature of the enzymatic bimetal motif, these results might also be relevant for structurally similar enzymes belonging to the NT superfamily. PMID:18662000

Intervention description is not enough: evidence from an in-depth multiple case study on the untold role and impact of context in randomised controlled trials of seven complex interventions

PubMed Central

2012-01-01

Background A number of single case reports have suggested that the context within which intervention studies take place may challenge the assumptions that underpin randomised controlled trials (RCTs). However, the diverse ways in which context may challenge the central tenets of the RCT, and the degree to which this information is known to researchers or subsequently reported, has received much less attention. In this paper, we explore these issues by focusing on seven RCTs of interventions varying in type and degree of complexity, and across diverse contexts. Methods This in-depth multiple case study using interviews, focus groups and documentary analysis was conducted in two phases. In phase one, a RCT of a nurse-led intervention provided a single exploratory case and informed the design, sampling and data collection within the main study. Phase two consisted of a multiple explanatory case study covering a spectrum of trials of different types of complex intervention. A total of eighty-four data sources across the seven trials were accessed. Results We present consistent empirical evidence across all trials to indicate that four key elements of context (personal, organisational, trial and problem context) are crucial to understanding how a complex intervention works and to enable both assessments of internal validity and likely generalisability to other settings. The ways in which context challenged trial operation was often complex, idiosyncratic, and subtle; often falling outside of current trial reporting formats. However, information on such issues appeared to be available via first hand ‘insider accounts’ of each trial suggesting that improved reporting on the role of context is possible. Conclusions Sufficient detail about context needs to be understood and reported in RCTs of complex interventions, in order for the transferability of complex interventions to be assessed. Improved reporting formats that require and encourage the clarification of both general and project-specific threats to the likely internal and external validity need to be developed. In addition, a cultural change is required in which the open and honest reporting of such issues is seen as an indicator of study strength and researcher integrity, rather than a symbol of a poor quality study or investigator ability. PMID:22742939

Synchronization for Optical PPM with Inter-Symbol Guard Times

NASA Astrophysics Data System (ADS)

Rogalin, R.; Srinivasan, M.

2017-05-01

Deep space optical communications promises orders of magnitude growth in communication capacity, supporting high data rate applications such as video streaming and high-bandwidth science instruments. Pulse position modulation is the modulation format of choice for deep space applications, and by inserting inter-symbol guard times between the symbols, the signal carries the timing information needed by the demodulator. Accurately extracting this timing information is crucial to demodulating and decoding this signal. In this article, we propose a number of timing and frequency estimation schemes for this modulation format, and in particular highlight a low complexity maximum likelihood timing estimator that significantly outperforms the prior art in this domain. This method does not require an explicit synchronization sequence, freeing up channel resources for data transmission.

Mission and science activity scheduling language

NASA Technical Reports Server (NTRS)

Hull, Larry G.

1993-01-01

To support the distributed and complex operational scheduling required for future National Aeronautics and Space Administration (NASA) missions, a formal, textual language, the Scheduling Applications Interface Language (SAIL), has been developed. Increased geographic dispersion of investigators is leading to distributed mission and science activity planning, scheduling, and operations. SAIL is an innovation which supports the effective and efficient communication of scheduling information among physically dispersed applications in distributed scheduling environments. SAIL offers a clear, concise, unambiguous expression of scheduling information in a readable, hardware independent format. The language concept, syntax, and semantics incorporate language features found useful during five years of research and prototyping with scheduling languages in physically distributed environments. SAIL allows concise specification of mission and science activity plans in a format which promotes repetition and reuse.

Thermodynamic properties of minerals

USGS Publications Warehouse

Robie, Richard A.

1962-01-01

In the ten years since the publication of the national Bureau of Standards comprehensive tables of thermochemical properties, by Rossini and other (1952), a very large body of modern calorimetric and equilibrium data has become available. Because of the complex interrelations among many thermochemical data and the necessity for internal consistency among these values, a complete revision of this standard reference is required. This is also true of the summaries of thermochemical data for the sulfides (Richardson and Jeffes 1952) and for the oxides (Coughlin 1954). The following tables present critically selected values for the heat and free energy of formation, the logarithm of the equilibrium constant of formation Log Kf, the entropy and the molar volume, at 298.15°K (25.0°C) and one atmosphere for minerals.

Reaction of iminopropadienones with amines: mechanistic explanations of zwitterionic intermediate, ketene and ketenimine formation.

PubMed

Koch, Rainer; Finnerty, Justin J; Bruhn, Torsten; Borget, Fabien; Wentrup, Curt

2008-09-25

The complex reaction of thermally generated iminopropadienones with amines in the gas phase and upon matrix deposition and its varying product composition is investigated using density functional theory. In the high energy gas phase addition a single amine molecule reacts readily with iminopropadienone with the decisive step being a 1,3-hydrogen shift and activation barriers of at least 100 kJ/mol. In accordance with the experiment, the formation of ketenes is favored. In the condensed phase of an amine matrix, the utilization of amine dimers both as reagents and as explicit solvents lowers the activation energy required to a feasible 20-30 kJ/mol and predicts ketenimines as the main products, as observed experimentally.

Wrestling with Chromosomes: The Roles of SUMO During Meiosis.

PubMed

Nottke, Amanda C; Kim, Hyun-Min; Colaiácovo, Monica P

2017-01-01

Meiosis is a specialized form of cell division required for the formation of haploid gametes and therefore is essential for successful sexual reproduction. Various steps are exquisitely coordinated to ensure accurate chromosome segregation during meiosis, thereby promoting the formation of haploid gametes from diploid cells. Recent studies are demonstrating that an important form of regulation during meiosis is exerted by the post-translational protein modification known as sumoylation. Here, we review and discuss the various critical steps of meiosis in which SUMO-mediated regulation has been implicated thus far. These include the maintenance of meiotic centromeric heterochromatin , meiotic DNA double-strand break repair and homologous recombination, centromeric coupling, and the assembly of a proteinaceous scaffold between homologous chromosomes known as the synaptonemal complex.

Visible-Light-Induced Nickel-Catalyzed Negishi Cross-Couplings by Exogenous-Photosensitizer-Free Photocatalysis.

PubMed

Abdiaj, Irini; Fontana, Alberto; Gomez, M Victoria; de la Hoz, Antonio; Alcázar, Jesús

2018-03-22

The merging of photoredox and transition-metal catalysis has become one of the most attractive approaches for carbon-carbon bond formation. Such reactions require the use of two organo-transition-metal species, one of which acts as a photosensitizer and the other one as a cross-coupling catalyst. We report herein an exogenous-photosensitizer-free photocatalytic process for the formation of carbon-carbon bonds by direct acceleration of the well-known nickel-catalyzed Negishi cross-coupling that is based on the use of two naturally abundant metals. This finding will open new avenues in cross-coupling chemistry that involve the direct visible-light absorption of organometallic catalytic complexes. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A study of swing-curve physics in diffraction-based overlay

NASA Astrophysics Data System (ADS)

Bhattacharyya, Kaustuve; den Boef, Arie; Storms, Greet; van Heijst, Joost; Noot, Marc; An, Kevin; Park, Noh-Kyoung; Jeon, Se-Ra; Oh, Nang-Lyeom; McNamara, Elliott; van de Mast, Frank; Oh, SeungHwa; Lee, Seung Yoon; Hwang, Chan; Lee, Kuntack

2016-03-01

With the increase of process complexity in advanced nodes, the requirements of process robustness in overlay metrology continues to tighten. Especially with the introduction of newer materials in the film-stack along with typical stack variations (thickness, optical properties, profile asymmetry etc.), the signal formation physics in diffraction-based overlay (DBO) becomes an important aspect to apply in overlay metrology target and recipe selection. In order to address the signal formation physics, an effort is made towards studying the swing-curve phenomena through wavelength and polarizations on production stacks using simulations as well as experimental technique using DBO. The results provide a wealth of information on target and recipe selection for robustness. Details from simulation and measurements will be reported in this technical publication.

Synthesis, crystal growth, structural and physicochemical studies of novel binary organic complex: 4-chloroaniline-3-hydroxy-4-methoxybenzaldehyde

NASA Astrophysics Data System (ADS)

Sharma, K. P.; Reddi, R. S. B.; Bhattacharya, S.; Rai, R. N.

2012-06-01

The solid-state reaction, which is solvent free and green synthesis, has been adopted to explore the novel compound. The phase diagram of 4-chloroaniline (CA) and 3-hydroxy-4-methoxybenzaldehyde (HMB) system shows the formation of a novel 1:1 molecular complex, and two eutectics on either sides of complex. Thermochemical studies of complex and eutectics have been carried out for various properties such as heat of fusion, entropy of fusion, Jackson's parameters, interfacial energy and excess thermodynamic functions. The formation of molecular complex was also studied by IR, NMR, elemental analysis and UV-Vis absorption spectra. The single crystal of molecular complex was grown and its XRD study confirms the formation of complex and identifies the crystal structure and atomic packing of crystal of complex. Transmission spectra of grown crystal of the complex show 70% transmittance efficiency with cut off wavelength 412 nm. The band gap and refractive index of the crystal of complex have also been studied.

Quantitative Analysis of HIV-1 Preintegration Complexes

PubMed Central

Engelman, Alan; Oztop, Ilker; Vandegraaff, Nick; Raghavendra, Nidhanapati K.

2009-01-01

Retroviral replication proceeds through the formation of a provirus, an integrated DNA copy of the viral RNA genome. The linear cDNA product of reverse transcription is the integration substrate and two different integrase activities, 3′ processing and DNA strand transfer, are required for provirus formation. Integrase nicks the cDNA ends adjacent to phylogenetically-conserved CA dinucleotides during 3′ processing. After nuclear entry and locating a suitable chromatin acceptor site, integrase joins the recessed 3′-OHs to the 5′-phosphates of a double-stranded staggered cut in the DNA target. Integrase functions in the context of a large nucleoprotein complex, called the preintegration complex (PIC), and PICs are analyzed to determine levels of integrase 3′ processing and DNA strand transfer activities that occur during acute virus infection. Denatured cDNA end regions are monitored by indirect end-labeling to measure the extent of 3′ processing. Native PICs can efficiently integrate their viral cDNA into exogenously added target DNA in vitro, and Southern blotting or nested PCR assays are used to quantify the resultant DNA strand transfer activity. This study details HIV-1 infection, PIC extraction, partial purification, and quantitative analyses of integrase 3′ processing and DNA strand transfer activities. PMID:19233280

Ca2+-dependent localization of integrin-linked kinase to cell junctions in differentiating keratinocytes.

PubMed

Vespa, Alisa; Darmon, Alison J; Turner, Christopher E; D'Souza, Sudhir J A; Dagnino, Lina

2003-03-28

Integrin complexes are necessary for proper proliferation and differentiation of epidermal keratinocytes. Differentiation of these cells is accompanied by down-regulation of integrins and focal adhesions as well as formation of intercellular adherens junctions through E-cadherin homodimerization. A central component of integrin adhesion complexes is integrin-linked kinase (ILK), which can induce loss of E-cadherin expression and epithelial-mesenchymal transformation when ectopically expressed in intestinal and mammary epithelia. In cultured primary mouse keratinocytes, we find that ILK protein levels are independent of integrin expression and signaling, since they remain constant during Ca(2+)-induced differentiation. In contrast, keratinocyte differentiation is accompanied by marked reduction in kinase activity in ILK immunoprecipitates and altered ILK subcellular distribution. Specifically, ILK distributes in close apposition to actin fibers along intercellular junctions in differentiated but not in undifferentiated keratinocytes. ILK localization to cell-cell borders occurs independently of integrin signaling and requires Ca(2+) as well as an intact actin cytoskeleton. Further, and in contrast to what is observed in other epithelial cells, ILK overexpression in differentiated keratinocytes does not promote E-cadherin down-regulation and epithelial-mesenchymal transition. Thus, novel tissue-specific mechanisms control the formation of ILK complexes associated with cell-cell junctions in differentiating murine epidermal keratinocytes.

Analyzing a suitable elastic geomechanical model for Vaca Muerta Formation

NASA Astrophysics Data System (ADS)

Sosa Massaro, Agustin; Espinoza, D. Nicolas; Frydman, Marcelo; Barredo, Silvia; Cuervo, Sergio

2017-11-01

Accurate geomechanical evaluation of oil and gas reservoir rocks is important to provide design parameters for drilling, completion and predict production rates. In particular, shale reservoir rocks are geologically complex and heterogeneous. Wells need to be hydraulically fractured for stimulation and, in complex tectonic environments, it is to consider that rock fabric and in situ stress, strongly influence fracture propagation geometry. This article presents a combined wellbore-laboratory characterization of the geomechanical properties of a well in El Trapial/Curamched Field, over the Vaca Muerta Formation, located in the Neuquén Basin in Argentina. The study shows the results of triaxial tests with acoustic measurements in rock plugs from outcrops and field cores, and corresponding dynamic to static correlations considering various elastic models. The models, with increasing complexity, include the Isotropic Elastic Model (IEM), the Anisotropic Elastic Model (AEM) and the Detailed Anisotropic Elastic Model (DAEM). Each model shows advantages over the others. An IEM offers a quick overview, being easy to run without much detailed data for heterogeneous and anisotropic rocks. The DAEM requires significant amounts of data, time and a multidisciplinary team to arrive to a detailed model. Finally, an AEM suits well to an anisotropic and realistic rock without the need of massive amounts of data.

Dual chromatin recognition by the histone deacetylase complex HCHC is required for proper DNA methylation in Neurospora crassa

PubMed Central

Honda, Shinji; Bicocca, Vincent T.; Gessaman, Jordan D.; Rountree, Michael R.; Yokoyama, Ayumi; Yu, Eun Y.; Selker, Jeanne M. L.; Selker, Eric U.

2016-01-01

DNA methylation, heterochromatin protein 1 (HP1), histone H3 lysine 9 (H3K9) methylation, histone deacetylation, and highly repeated sequences are prototypical heterochromatic features, but their interrelationships are not fully understood. Prior work showed that H3K9 methylation directs DNA methylation and histone deacetylation via HP1 in Neurospora crassa and that the histone deacetylase complex HCHC is required for proper DNA methylation. The complex consists of the chromodomain proteins HP1 and chromodomain protein 2 (CDP-2), the histone deacetylase HDA-1, and the AT-hook motif protein CDP-2/HDA-1–associated protein (CHAP). We show that the complex is required for proper chromosome segregation, dissect its function, and characterize interactions among its components. Our analyses revealed the existence of an HP1-based DNA methylation pathway independent of its chromodomain. The pathway partially depends on CHAP but not on the CDP-2 chromodomain. CDP-2 serves as a bridge between the recognition of H3K9 trimethylation (H3K9me3) by HP1 and the histone deacetylase activity of HDA-1. CHAP is also critical for HDA-1 localization to heterochromatin. Specifically, the CHAP zinc finger interacts directly with the HDA-1 argonaute-binding protein 2 (Arb2) domain, and the CHAP AT-hook motifs recognize heterochromatic regions by binding to AT-rich DNA. Our data shed light on the interrelationships among the prototypical heterochromatic features and support a model in which dual recognition by the HP1 chromodomain and the CHAP AT-hooks are required for proper heterochromatin formation. PMID:27681634

Levels of control exerted by the Isc iron-sulfur cluster system on biosynthesis of the formate hydrogenlyase complex.

PubMed

Pinske, Constanze; Jaroschinsky, Monique; Sawers, R Gary

2013-06-01

The membrane-associated formate hydrogenlyase (FHL) complex of bacteria like Escherichia coli is responsible for the disproportionation of formic acid into the gaseous products carbon dioxide and dihydrogen. It comprises minimally seven proteins including FdhF and HycE, the catalytic subunits of formate dehydrogenase H and hydrogenase 3, respectively. Four proteins of the FHL complex have iron-sulphur cluster ([Fe-S]) cofactors. Biosynthesis of [Fe-S] is principally catalysed by the Isc or Suf systems and each comprises proteins for assembly and for delivery of [Fe-S]. This study demonstrates that the Isc system is essential for biosynthesis of an active FHL complex. In the absence of the IscU assembly protein no hydrogen production or activity of FHL subcomponents was detected. A deletion of the iscU gene also resulted in reduced intracellular formate levels partially due to impaired synthesis of pyruvate formate-lyase, which is dependent on the [Fe-S]-containing regulator FNR. This caused reduced expression of the formate-inducible fdhF gene. The A-type carrier (ATC) proteins IscA and ErpA probably deliver [Fe-S] to specific apoprotein components of the FHL complex because mutants lacking either protein exhibited strongly reduced hydrogen production. Neither ATC protein could compensate for the lack of the other, suggesting that they had independent roles in [Fe-S] delivery to complex components. Together, the data indicate that the Isc system modulates FHL complex biosynthesis directly by provision of [Fe-S] as well as indirectly by influencing gene expression through the delivery of [Fe-S] to key regulators and enzymes that ultimately control the generation and oxidation of formate.

Elongator complex influences telomeric gene silencing and DNA damage response by its role in wobble uridine tRNA modification.

PubMed

Chen, Changchun; Huang, Bo; Eliasson, Mattias; Rydén, Patrik; Byström, Anders S

2011-09-01

Elongator complex is required for formation of the side chains at position 5 of modified nucleosides 5-carbamoylmethyluridine (ncm⁵U₃₄), 5-methoxycarbonylmethyluridine (mcm⁵U₃₄), and 5-methoxycarbonylmethyl-2-thiouridine (mcm⁵s²U₃₄) at wobble position in tRNA. These modified nucleosides are important for efficient decoding during translation. In a recent publication, Elongator complex was implicated to participate in telomeric gene silencing and DNA damage response by interacting with proliferating cell nuclear antigen (PCNA). Here we show that elevated levels of tRNA(Lys)(s²UUU), tRNA(Gln)(s²UUG), and tRNA(Glu)(s²UUC), which in a wild-type background contain the mcm⁵s²U nucleoside at position 34, suppress the defects in telomeric gene silencing and DNA damage response observed in the Elongator mutants. We also found that the reported differences in telomeric gene silencing and DNA damage response of various elp3 alleles correlated with the levels of modified nucleosides at U₃₄. Defects in telomeric gene silencing and DNA damage response are also observed in strains with the tuc2Δ mutation, which abolish the formation of the 2-thio group of the mcm⁵s²U nucleoside in tRNA(Lys)(mcm⁵s²UUU), tRNA(Gln)(mcm⁵s²UUG), and tRNA(Glu)(mcm⁵s²UUC). These observations show that Elongator complex does not directly participate in telomeric gene silencing and DNA damage response, but rather that modified nucleosides at U₃₄ are important for efficient expression of gene products involved in these processes. Consistent with this notion, we found that expression of Sir4, a silent information regulator required for assembly of silent chromatin at telomeres, was decreased in the elp3Δ mutants.

Elongator Complex Influences Telomeric Gene Silencing and DNA Damage Response by Its Role in Wobble Uridine tRNA Modification

PubMed Central

Chen, Changchun; Huang, Bo; Eliasson, Mattias; Rydén, Patrik; Byström, Anders S.

2011-01-01

Elongator complex is required for formation of the side chains at position 5 of modified nucleosides 5-carbamoylmethyluridine (ncm5U34), 5-methoxycarbonylmethyluridine (mcm5U34), and 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U34) at wobble position in tRNA. These modified nucleosides are important for efficient decoding during translation. In a recent publication, Elongator complex was implicated to participate in telomeric gene silencing and DNA damage response by interacting with proliferating cell nuclear antigen (PCNA). Here we show that elevated levels of tRNALys s2 UUU, tRNAGln s2 UUG, and tRNAGlu s2 UUC, which in a wild-type background contain the mcm5s2U nucleoside at position 34, suppress the defects in telomeric gene silencing and DNA damage response observed in the Elongator mutants. We also found that the reported differences in telomeric gene silencing and DNA damage response of various elp3 alleles correlated with the levels of modified nucleosides at U34. Defects in telomeric gene silencing and DNA damage response are also observed in strains with the tuc2Δ mutation, which abolish the formation of the 2-thio group of the mcm5s2U nucleoside in tRNALys mcm5s2UUU, tRNAGln mcm5s2UUG, and tRNAGlu mcm5s2UUC. These observations show that Elongator complex does not directly participate in telomeric gene silencing and DNA damage response, but rather that modified nucleosides at U34 are important for efficient expression of gene products involved in these processes. Consistent with this notion, we found that expression of Sir4, a silent information regulator required for assembly of silent chromatin at telomeres, was decreased in the elp3Δ mutants. PMID:21912530

Regulation of substrate adhesion dynamics during cell motility.

PubMed

Kaverina, Irina; Krylyshkina, Olga; Small, J Victor

2002-07-01

The movement of a metazoan cell entails the regulated creation and turnover of adhesions with the surface on which it moves. Adhesion sites form as a result of signaling between the extracellular matrix on the outside and the actin cytoskeleton on the inside, and they are associated with specific assembles of actin filaments. Two broad categories of adhesion sites can be distinguished: (1) "focal complexes" associated with lamellipodia and filopodia that support protrusion and traction at the cell front; and (2) "focal adhesions" at the termini of stress fibre bundles that serve in longer term anchorage. Focal complexes are signaled via Rac1 or Cdc42 and can either turnover on a minute scale or differentiate, via intervention of the RhoA pathway, into longer-lived focal adhesions. All classes of adhesion sites depend on the stress in the actin cytoskeleton for their formation and maintenance. Different cell types use different adhesion strategies to move, in terms of the relative engagement of filopodia and lamellipodia in focal complex formation and protrusion and the extent of focal adhesion formation. These differences can be attributed to variations in the relative activities of Rho family members. However, the Rho GTPases alone are unable to signal asymmetry in the actin cytoskeleton, necessary for polarisation and movement. Polarisation requires the collaboration of the microtubule cytoskeleton. Changes in the polymerisation state of microtubules influences the activities of both Rac1 and RhoA and microtubules interact directly with adhesion foci and promote their turnover. Possible mechanisms of cross-talk between the microtubule and actin cytoskeletons in determining polarity are discussed.

Pattern Formation in Complex Fluids

NASA Astrophysics Data System (ADS)

Shelley, Michael

2000-03-01

Classical fluid instabilities -- such as the Saffman-Taylor instability in a Hele-Shaw cell -- are dramatically modified by using complex fluids. For example, polymeric liquids driven in a Hele-Shaw cell yield "dendritic" patterns with an apparent directional anisotropy. The dynamics of complex liquids can also lead to new instabilities and patterns, such as space-filling patterns formed by successive bucklings of growing "elastica" seen in the phase transition of a liquid crystalline material. Understanding such problems requires an interplay between physical modeling, mathematical analysis, and sophisticated nonlinear simulation. For the first problem, I will discuss a non-Newtonian version of Darcy's law for Hele-Shaw flow. This yields a free-boundary problem for the pattern formation, and requires the solution of a nonlinear elliptic equation in a time-dependent domain. This is pushing the development of adaptive grid methods that represent the geometry accurately and efficiently. Our simulations yield insight into how shear-thinning, as is evinced by polymeric liquids, can produce patterns reminiscent of experiment, with "dendritic fingers", side-branching, and reduced tip-splitting. In the second problem, a long filament in a smectic-A phase grows within an isotropic fluid. The splay deformation of the material gives this filament an elastic response. The macroscopic model describes the dynamics of a growing, elastic filament immersed in a Stokesian fluid. The model marries filament elasticity and tensile forces with a numerically tractable nonlocal slender-body theory. Analysis shows that growth of the filament, despite fluid drag, produces a buckling instability. When coupled to a nonlocal hydrodynamic self-interaction, our fully nonlinear simulations show that such instabilities iterate along the filament, and give "space-filling" patterns.

A novel germ cell protein, SPIF (sperm PKA interacting factor), is essential for the formation of a PKA/TCP11 complex that undergoes conformational and phosphorylation changes upon capacitation.

PubMed

Stanger, Simone J; Law, Estelle A; Jamsai, Duangporn; O'Bryan, Moira K; Nixon, Brett; McLaughlin, Eileen A; Aitken, R John; Roman, Shaun D

2016-08-01

Spermatozoa require the process of capacitation to enable them to fertilize an egg. PKA is crucial to capacitation and the development of hyperactivated motility. Sperm PKA is activated by cAMP generated by the germ cell-enriched adenylyl cyclase encoded by Adcy10 Male mice lacking Adcy10 are sterile, because their spermatozoa are immotile. The current study was designed to identify binding partners of the sperm-specific (Cα2) catalytic subunit of PKA (PRKACA) by using it as the "bait" in a yeast 2-hybrid system. This approach was used to identify a novel germ cell-enriched protein, sperm PKA interacting factor (SPIF), in 25% of the positive clones. Homozygous Spif-null mice were embryonically lethal. SPIF was coexpressed and coregulated with PRKACA and with t-complex protein (TCP)-11, a protein associated with PKA signaling. We established that these 3 proteins form part of a novel complex in mouse spermatozoa. Upon capacitation, the SPIF protein becomes tyrosine phosphorylated in >95% of sperm. An apparent molecular rearrangement in the complex occurs, bringing PRKACA and TCP11 into proximity. Taken together, these results suggest a role for the novel complex of SPIF, PRKACA, and TCP11 during sperm capacitation, fertilization, and embryogenesis.-Stanger, S. J., Law, E. A., Jamsai, D., O'Bryan, M. K., Nixon, B., McLaughlin, E. A., Aitken, R. J., Roman, S. D. A novel germ cell protein, SPIF (sperm PKA interacting factor), is essential for the formation of a PKA/TCP11 complex that undergoes conformational and phosphorylation changes upon capacitation. © FASEB.

G-protein-coupled receptor-2-interacting protein-1 is required for endothelial cell directional migration and tumor angiogenesis via cortactin-dependent lamellipodia formation.

PubMed

Majumder, Syamantak; Sowden, Mark P; Gerber, Scott A; Thomas, Tamlyn; Christie, Christine K; Mohan, Amy; Yin, Guoyong; Lord, Edith M; Berk, Bradford C; Pang, Jinjiang

2014-02-01

Recent evidence suggests G-protein-coupled receptor-2-interacting protein-1 (GIT1) overexpression in several human metastatic tumors, including breast, lung, and prostate. Tumor metastasis is associated with an increase in angiogenesis. We have showed previously that GIT1 is required for postnatal angiogenesis during lung development. However, the functional role of GIT1 in pathological angiogenesis during tumor growth is unknown. In the present study, we show inhibition of angiogenesis in matrigel implants as well as reduced tumor angiogenesis and melanoma tumor growth in GIT1-knockout mice. We demonstrate that this is a result of impaired directional migration of GIT1-depleted endothelial cells toward a vascular endothelial growth factor gradient. Cortactin-mediated lamellipodia formation in the leading edge is critical for directional migration. We observed a significant reduction in cortactin localization and lamellipodia formation in the leading edge of GIT1-depleted endothelial cells. We specifically identified that the Spa homology domain (aa 250-420) of GIT1 is required for GIT1-cortactin complex localization to the leading edge. The mechanisms involved extracellular signal-regulated kinases 1 and 2-mediated Cortactin-S405 phosphorylation and activation of Rac1/Cdc42. Finally, using gain of function studies, we show that a constitutively active mutant of cortactin restored directional migration of GIT1-depleted cells. Our data demonstrated that a GIT1-cortactin association through GIT1-Spa homology domain is required for cortactin localization to the leading edge and is essential for endothelial cell directional migration and tumor angiogenesis.

Complex organic molecules and star formation

NASA Astrophysics Data System (ADS)

Bacmann, A.; Faure, A.

2014-12-01

Star forming regions are characterised by the presence of a wealth of chemical species. For the past two to three decades, ever more complex organic species have been detected in the hot cores of protostars. The evolution of these molecules in the course of the star forming process is still uncertain, but it is likely that they are partially incorporated into protoplanetary disks and then into planetesimals and the small bodies of planetary systems. The complex organic molecules seen in star forming regions are particularly interesting since they probably make up building blocks for prebiotic chemistry. Recently we showed that these species were also present in the cold gas in prestellar cores, which represent the very first stages of star formation. These detections question the models which were until now accepted to account for the presence of complex organic molecules in star forming regions. In this article, we shortly review our current understanding of complex organic molecule formation in the early stages of star formation, in hot and cold cores alike and present new results on the formation of their likely precursor radicals.

Mammalian polo-like kinase 1-dependent regulation of the PBIP1-CENP-Q complex at kinetochores.

PubMed

Kang, Young H; Park, Chi Hoon; Kim, Tae-Sung; Soung, Nak-Kyun; Bang, Jeong K; Kim, Bo Y; Park, Jung-Eun; Lee, Kyung S

2011-06-03

Mammalian polo-like kinase 1 (Plk1) plays a pivotal role during M-phase progression. Plk1 localizes to specific subcellular structures through the targeting activity of the C-terminal polo-box domain (PBD). Disruption of the PBD function results in improper bipolar spindle formation, chromosome missegregation, and cytokinesis defect that ultimately lead to the generation of aneuploidy. It has been shown that Plk1 recruits itself to centromeres by phosphorylating and binding to a centromere scaffold, PBIP1 (also called MLF1IP and CENP-U[50]) through its PBD. However, how PBIP1 itself is targeted to centromeres and what roles it plays in the regulation of Plk1-dependent mitotic events remain unknown. Here, we demonstrated that PBIP1 directly interacts with CENP-Q, and this interaction was mutually required not only for their stability but also for their centromere localization. Plk1 did not appear to interact with CENP-Q directly. However, Plk1 formed a ternary complex with PBIP1 and CENP-Q through a self-generated p-T78 motif on PBIP1. This complex formation was central for Plk1-dependent phosphorylation of PBIP1-bound CENP-Q and delocalization of the PBIP1-CENP-Q complex from mitotic centromeres. This study reveals a unique mechanism of how PBIP1 mediates Plk1-dependent phosphorylation event onto a third protein, and provides new insights into the mechanism of how Plk1 and its recruitment scaffold, PBIP1-CENP-Q complex, are localized to and delocalized from centromeres.

Mammalian Polo-like Kinase 1-dependent Regulation of the PBIP1-CENP-Q Complex at Kinetochores*

PubMed Central

Kang, Young H.; Park, Chi Hoon; Kim, Tae-Sung; Soung, Nak-Kyun; Bang, Jeong K.; Kim, Bo Y.; Park, Jung-Eun; Lee, Kyung S.

2011-01-01

Mammalian polo-like kinase 1 (Plk1) plays a pivotal role during M-phase progression. Plk1 localizes to specific subcellular structures through the targeting activity of the C-terminal polo-box domain (PBD). Disruption of the PBD function results in improper bipolar spindle formation, chromosome missegregation, and cytokinesis defect that ultimately lead to the generation of aneuploidy. It has been shown that Plk1 recruits itself to centromeres by phosphorylating and binding to a centromere scaffold, PBIP1 (also called MLF1IP and CENP-U[50]) through its PBD. However, how PBIP1 itself is targeted to centromeres and what roles it plays in the regulation of Plk1-dependent mitotic events remain unknown. Here, we demonstrated that PBIP1 directly interacts with CENP-Q, and this interaction was mutually required not only for their stability but also for their centromere localization. Plk1 did not appear to interact with CENP-Q directly. However, Plk1 formed a ternary complex with PBIP1 and CENP-Q through a self-generated p-T78 motif on PBIP1. This complex formation was central for Plk1-dependent phosphorylation of PBIP1-bound CENP-Q and delocalization of the PBIP1-CENP-Q complex from mitotic centromeres. This study reveals a unique mechanism of how PBIP1 mediates Plk1-dependent phosphorylation event onto a third protein, and provides new insights into the mechanism of how Plk1 and its recruitment scaffold, PBIP1-CENP-Q complex, are localized to and delocalized from centromeres. PMID:21454580

Coenzyme Q supplementation or over-expression of the yeast Coq8 putative kinase stabilizes multi-subunit Coq polypeptide complexes in yeast coq null mutants.

PubMed

He, Cuiwen H; Xie, Letian X; Allan, Christopher M; Tran, Uyenphuong C; Clarke, Catherine F

2014-04-04

Coenzyme Q biosynthesis in yeast requires a multi-subunit Coq polypeptide complex. Deletion of any one of the COQ genes leads to respiratory deficiency and decreased levels of the Coq4, Coq6, Coq7, and Coq9 polypeptides, suggesting that their association in a high molecular mass complex is required for stability. Over-expression of the putative Coq8 kinase in certain coq null mutants restores steady-state levels of the sensitive Coq polypeptides and promotes the synthesis of late-stage Q-intermediates. Here we show that over-expression of Coq8 in yeast coq null mutants profoundly affects the association of several of the Coq polypeptides in high molecular mass complexes, as assayed by separation of digitonin extracts of mitochondria by two-dimensional blue-native/SDS PAGE. The Coq4 polypeptide persists at high molecular mass with over-expression of Coq8 in coq3, coq5, coq6, coq7, coq9, and coq10 mutants, indicating that Coq4 is a central organizer of the Coq complex. Supplementation with exogenous Q6 increased the steady-state levels of Coq4, Coq7, and Coq9, and several other mitochondrial polypeptides in select coq null mutants, and also promoted the formation of late-stage Q-intermediates. Q supplementation may stabilize this complex by interacting with one or more of the Coq polypeptides. The stabilizing effects of exogenously added Q6 or over-expression of Coq8 depend on Coq1 and Coq2 production of a polyisoprenyl intermediate. Based on the observed interdependence of the Coq polypeptides, the effect of exogenous Q6, and the requirement for an endogenously produced polyisoprenyl intermediate, we propose a new model for the Q-biosynthetic complex, termed the CoQ-synthome. Copyright © 2014 Elsevier B.V. All rights reserved.

Coenzyme Q supplementation or over-expression of the yeast Coq8 putative kinase stabilizes multi-subunit Coq polypeptide complexes in yeast coq null mutants*

PubMed Central

He, Cuiwen H.; Xie, Letian X.; Allan, Christopher M.; Tran, UyenPhuong C.; Clarke, Catherine F.

2014-01-01

Coenzyme Q biosynthesis in yeast requires a multi-subunit Coq polypeptide complex. Deletion of any one of the COQ genes leads to respiratory deficiency and decreased levels of the Coq4, Coq6, Coq7, and Coq9 polypeptides, suggesting that their association in a high molecular mass complex is required for stability. Over-expression of the putative Coq8 kinase in certain coq null mutants restores steady-state levels of the sensitive Coq polypeptides and promotes the synthesis of late-stage Q-intermediates. Here we show that over-expression of Coq8 in yeast coq null mutants profoundly affects the association of several of the Coq polypeptides in high molecular mass complexes, as assayed by separation of digitonin extracts of mitochondria by two-dimensional blue-native/SDS PAGE. The Coq4 polypeptide persists at high molecular mass with over-expression of Coq8 in coq3, coq5, coq6, coq7, coq9, and coq10 mutants, indicating that Coq4 is a central organizer of the Coq complex. Supplementation with exogenous Q6 increased the steady-state levels of Coq4, Coq7, Coq9, and several other mitochondrial polypeptides in select coq null mutants, and also promoted the formation of late-stage Q-intermediates. Q supplementation may stabilize this complex by interacting with one or more of the Coq polypeptides. The stabilizing effects of exogenously added Q6 or over-expression of Coq8 depend on Coq1 and Coq2 production of a polyisoprenyl intermediate. Based on the observed interdependence of the Coq polypeptides, the effect of exogenous Q6, and the requirement for an endogenously produced polyisoprenyl intermediate, we propose a new model for the Q-biosynthetic complex, termed the CoQ-synthome. PMID:24406904

The influence of water-ethanol mixture on the thermodynamics of complex formation between 18-crown-6 ether and L-phenylalanine

NASA Astrophysics Data System (ADS)

Usacheva, T. R.; Sharnin, V. A.; Chernov, I. V.; Matteoli, E.; Terekhova, I. V.; Kumeev, R. S.

2012-08-01

The influence of water-ethanol mixture composition on the complex formation between 18-crown-6 ether and L-phenylalanine was studied by titration calorimetry at Т = 298.15 K. The standard thermodynamic parameters (ΔrGо, ΔrHо, ТΔrSо) of formation of [Phe18C6] molecular complex were calculated from data obtained by means of the microcalorimetric system TAM III (TA Instruments, USA) at X(EtOH) = 0.0/0.6 mol fraction. The stability of [Phe18C6] and the mechanism of complexation in water were investigated using the 1Н and 13С NMR spectroscopy. The increase of EtOH concentration results in an increase of the complex stability and of the exothermicity of complexation.

Structure of soybean serine acetyltransferase and formation of the cysteine regulatory complex as a molecular chaperone

USDA-ARS?s Scientific Manuscript database

Serine acetyltransferase (SAT) catalyzes the limiting reaction in plant and microbial biosynthesis of cysteine. In addition to its enzymatic function, SAT forms a macromolecular complex with O-acetylserine sulfhydrylase (OASS). Formation of the cysteine regulatory complex (CRC) is a critical biochem...

Room temperature deintercalation of alkali metal atoms from epitaxial graphene by formation of charge-transfer complexes

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

Shin, H.-C.; Ahn, S. J.; Kim, H. W.

2016-08-22

Atom (or molecule) intercalations and deintercalations have been used to control the electronic properties of graphene. In general, finite energies above room temperature (RT) thermal energy are required for the intercalations and deintercalations. Here, we demonstrate that alkali metal atoms can be deintercalated from epitaxial graphene on a SiC substrate at RT, resulting in the reduction in density of states at the Fermi level. The change in density of states at the Fermi level at RT can be applied to a highly sensitive graphene sensor operating at RT. Na atoms, which were intercalated at a temperature of 80 °C, were deintercalatedmore » at a high temperature above 1000 °C when only a thermal treatment was used. In contrast to the thermal treatment, the intercalated Na atoms were deintercalated at RT when tetrafluorotetracyanoquinodimethane (F4-TCNQ) molecules were adsorbed on the surface. The RT deintercalation occurred via the formation of charge-transfer complexes between Na atoms and F4-TCNQ molecules.« less

Physical Conditions of Eta Car Complex Environment Revealed From Photoionization Modeling

NASA Technical Reports Server (NTRS)

Verner, E. M.; Bruhweiler, F.; Nielsen, K. E.; Gull, T.; Kober, G. Vieira; Corcoran, M.

2006-01-01

The very massive star, Eta Carinae, is enshrouded in an unusual complex environment of nebulosities and structures. The circumstellar gas gives rise to distinct absorption and emission components at different velocities and distances from the central source(s). Through photoionization modeling, we find that the radiation field from the more massive B-star companion supports the low ionization structure throughout the 5.54 year period. The radiation field of an evolved O-star is required to produce the higher ionization . emission seen across the broad maximum. Our studies utilize the HST/STIS data and model calculations of various regimes from doubly ionized species (T= 10,000K) to the low temperature (T = 760 K) conditions conductive to molecule formation (CH and OH). Overall analysis suggests the high depletion in C and O and the enrichment in He and N. The sharp molecular and ionic absorptions in this extensively CNO - processed material offers a unique environment for studying the chemistry, dust formation processes, and nucleosynthesis in the ejected layers of a highly evolved massive star.

A biochemical network can control formation of a synthetic material by sensing numerous specific stimuli

NASA Astrophysics Data System (ADS)

Hun Yeon, Ju; Chan, Karen Y. T.; Wong, Ting-Chia; Chan, Kelvin; Sutherland, Michael R.; Ismagilov, Rustem F.; Pryzdial, Edward L. G.; Kastrup, Christian J.

2015-05-01

Developing bio-compatible smart materials that assemble in response to environmental cues requires strategies that can discriminate multiple specific stimuli in a complex milieu. Synthetic materials have yet to achieve this level of sensitivity, which would emulate the highly evolved and tailored reaction networks of complex biological systems. Here we show that the output of a naturally occurring network can be replaced with a synthetic material. Exploiting the blood coagulation system as an exquisite biological sensor, the fibrin clot end-product was replaced with a synthetic material under the biological control of a precisely regulated cross-linking enzyme. The functions of the coagulation network remained intact when the material was incorporated. Clot-like polymerization was induced in indirect response to distinct small molecules, phospholipids, enzymes, cells, viruses, an inorganic solid, a polyphenol, a polysaccharide, and a membrane protein. This strategy demonstrates for the first time that an existing stimulus-responsive biological network can be used to control the formation of a synthetic material by diverse classes of physiological triggers.

NADP-Specific Electron-Bifurcating [FeFe]-Hydrogenase in a Functional Complex with Formate Dehydrogenase in Clostridium autoethanogenum Grown on CO

PubMed Central

Wang, Shuning; Huang, Haiyan; Kahnt, Jörg; Mueller, Alexander P.; Köpke, Michael

2013-01-01

Flavin-based electron bifurcation is a recently discovered mechanism of coupling endergonic to exergonic redox reactions in the cytoplasm of anaerobic bacteria and archaea. Among the five electron-bifurcating enzyme complexes characterized to date, one is a heteromeric ferredoxin- and NAD-dependent [FeFe]-hydrogenase. We report here a novel electron-bifurcating [FeFe]-hydrogenase that is NADP rather than NAD specific and forms a complex with a formate dehydrogenase. The complex was found in high concentrations (6% of the cytoplasmic proteins) in the acetogenic Clostridium autoethanogenum autotrophically grown on CO, which was fermented to acetate, ethanol, and 2,3-butanediol. The purified complex was composed of seven different subunits. As predicted from the sequence of the encoding clustered genes (fdhA/hytA-E) and from chemical analyses, the 78.8-kDa subunit (FdhA) is a selenocysteine- and tungsten-containing formate dehydrogenase, the 65.5-kDa subunit (HytB) is an iron-sulfur flavin mononucleotide protein harboring the NADP binding site, the 51.4-kDa subunit (HytA) is the [FeFe]-hydrogenase proper, and the 18.1-kDa (HytC), 28.6-kDa (HytD), 19.9-kDa (HytE1), and 20.1-kDa (HytE2) subunits are iron-sulfur proteins. The complex catalyzed both the reversible coupled reduction of ferredoxin and NADP+ with H2 or formate and the reversible formation of H2 and CO2 from formate. We propose the complex to have two functions in vivo, namely, to normally catalyze CO2 reduction to formate with NADPH and reduced ferredoxin in the Wood-Ljungdahl pathway and to catalyze H2 formation from NADPH and reduced ferredoxin when these redox mediators get too reduced during unbalanced growth of C. autoethanogenum on CO (E0′ = −520 mV). PMID:23893107

NADP-specific electron-bifurcating [FeFe]-hydrogenase in a functional complex with formate dehydrogenase in Clostridium autoethanogenum grown on CO.

PubMed

Wang, Shuning; Huang, Haiyan; Kahnt, Jörg; Mueller, Alexander P; Köpke, Michael; Thauer, Rudolf K

2013-10-01

Flavin-based electron bifurcation is a recently discovered mechanism of coupling endergonic to exergonic redox reactions in the cytoplasm of anaerobic bacteria and archaea. Among the five electron-bifurcating enzyme complexes characterized to date, one is a heteromeric ferredoxin- and NAD-dependent [FeFe]-hydrogenase. We report here a novel electron-bifurcating [FeFe]-hydrogenase that is NADP rather than NAD specific and forms a complex with a formate dehydrogenase. The complex was found in high concentrations (6% of the cytoplasmic proteins) in the acetogenic Clostridium autoethanogenum autotrophically grown on CO, which was fermented to acetate, ethanol, and 2,3-butanediol. The purified complex was composed of seven different subunits. As predicted from the sequence of the encoding clustered genes (fdhA/hytA-E) and from chemical analyses, the 78.8-kDa subunit (FdhA) is a selenocysteine- and tungsten-containing formate dehydrogenase, the 65.5-kDa subunit (HytB) is an iron-sulfur flavin mononucleotide protein harboring the NADP binding site, the 51.4-kDa subunit (HytA) is the [FeFe]-hydrogenase proper, and the 18.1-kDa (HytC), 28.6-kDa (HytD), 19.9-kDa (HytE1), and 20.1-kDa (HytE2) subunits are iron-sulfur proteins. The complex catalyzed both the reversible coupled reduction of ferredoxin and NADP(+) with H2 or formate and the reversible formation of H2 and CO2 from formate. We propose the complex to have two functions in vivo, namely, to normally catalyze CO2 reduction to formate with NADPH and reduced ferredoxin in the Wood-Ljungdahl pathway and to catalyze H2 formation from NADPH and reduced ferredoxin when these redox mediators get too reduced during unbalanced growth of C. autoethanogenum on CO (E0' = -520 mV).

Crystal structure and solution species of Ce(III) and Ce(IV) formates: from mononuclear to hexanuclear complexes.

PubMed

Hennig, Christoph; Ikeda-Ohno, Atsushi; Kraus, Werner; Weiss, Stephan; Pattison, Philip; Emerich, Hermann; Abdala, Paula M; Scheinost, Andreas C

2013-10-21

Cerium(III) and cerium(IV) both form formate complexes. However, their species in aqueous solution and the solid-state structures are surprisingly different. The species in aqueous solutions were investigated with Ce K-edge EXAFS spectroscopy. Ce(III) formate shows only mononuclear complexes, which is in agreement with the predicted mononuclear species of Ce(HCOO)(2+) and Ce(HCOO)2(+). In contrast, Ce(IV) formate forms in aqueous solution a stable hexanuclear complex of [Ce6(μ3-O)4(μ3-OH)4(HCOO)x(NO3)y](12-x-y). The structural differences reflect the different influence of hydrolysis, which is weak for Ce(III) and strong for Ce(IV). Hydrolysis of Ce(IV) ions causes initial polymerization while complexation through HCOO(-) results in 12 chelate rings stabilizing the hexanuclear Ce(IV) complex. Crystals were grown from the above-mentioned solutions. Two crystal structures of Ce(IV) formate were determined. Both form a hexanuclear complex with a [Ce6(μ3-O)4(μ3-OH)4](12+) core in aqueous HNO3/HCOOH solution. The pH titration with NaOH resulted in a structure with the composition [Ce6(μ3-O)4(μ3-OH)4(HCOO)10(NO3)2(H2O)3]·(H2O)9.5, while the pH adjustment with NH3 resulted in [Ce6(μ3-O)4(μ3-OH)4(HCOO)10(NO3)4]·(NO3)3(NH4)5(H2O)5. Furthermore, the crystal structure of Ce(III) formate, Ce(HCOO)3, was determined. The coordination polyhedron is a tricapped trigonal prism which is formed exclusively by nine HCOO(-) ligands. The hexanuclear Ce(IV) formate species from aqueous solution is widely preserved in the crystal structure, whereas the mononuclear solution species of Ce(III) formate undergoes a polymerization during the crystallization process.

Resolving protein interactions and organization downstream the T cell antigen receptor using single-molecule localization microscopy: a review

NASA Astrophysics Data System (ADS)

Sherman, Eilon

2016-06-01

Signal transduction is mediated by heterogeneous and dynamic protein complexes. Such complexes play a critical role in diverse cell functions, with the important example of T cell activation. Biochemical studies of signalling complexes and their imaging by diffraction limited microscopy have resulted in an intricate network of interactions downstream the T cell antigen receptor (TCR). However, in spite of their crucial roles in T cell activation, much remains to be learned about these signalling complexes, including their heterogeneous contents and size distribution, their complex arrangements in the PM, and the molecular requirements for their formation. Here, we review how recent advancements in single molecule localization microscopy have helped to shed new light on the organization of signalling complexes in single molecule detail in intact T cells. From these studies emerges a picture where cells extensively employ hierarchical and dynamic patterns of nano-scale organization to control the local concentration of interacting molecular species. These patterns are suggested to play a critical role in cell decision making. The combination of SMLM with more traditional techniques is expected to continue and critically contribute to our understanding of multimolecular protein complexes and their significance to cell function.

Interactions of the Human LIP5 Regulatory Protein with Endosomal Sorting Complexes Required for Transport*♦

PubMed Central

Skalicky, Jack J.; Arii, Jun; Wenzel, Dawn M.; Stubblefield, William-May B.; Katsuyama, Angela; Uter, Nathan T.; Bajorek, Monika; Myszka, David G.; Sundquist, Wesley I.

2012-01-01

The endosomal sorting complex required for transport (ESCRT) pathway remodels membranes during multivesicular body biogenesis, the abscission stage of cytokinesis, and enveloped virus budding. The ESCRT-III and VPS4 ATPase complexes catalyze the membrane fission events associated with these processes, and the LIP5 protein helps regulate their interactions by binding directly to a subset of ESCRT-III proteins and to VPS4. We have investigated the biochemical and structural basis for different LIP5-ligand interactions and show that the first microtubule-interacting and trafficking (MIT) module of the tandem LIP5 MIT domain binds CHMP1B (and other ESCRT-III proteins) through canonical type 1 MIT-interacting motif (MIM1) interactions. In contrast, the second LIP5 MIT module binds with unusually high affinity to a novel MIM element within the ESCRT-III protein CHMP5. A solution structure of the relevant LIP5-CHMP5 complex reveals that CHMP5 helices 5 and 6 and adjacent linkers form an amphipathic “leucine collar” that wraps almost completely around the second LIP5 MIT module but makes only limited contacts with the first MIT module. LIP5 binds MIM1-containing ESCRT-III proteins and CHMP5 and VPS4 ligands independently in vitro, but these interactions are coupled within cells because formation of stable VPS4 complexes with both LIP5 and CHMP5 requires LIP5 to bind both a MIM1-containing ESCRT-III protein and CHMP5. Our studies thus reveal how the tandem MIT domain of LIP5 binds different types of ESCRT-III proteins, promoting assembly of active VPS4 enzymes on the polymeric ESCRT-III substrate. PMID:23105106