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Sample records for acidic tail domain

  1. Mutations in type 3 reovirus that determine binding to sialic acid are contained in the fibrous tail domain of viral attachment protein sigma1.

    PubMed

    Chappell, J D; Gunn, V L; Wetzel, J D; Baer, G S; Dermody, T S

    1997-03-01

    The reovirus attachment protein, sigma1, determines numerous aspects of reovirus-induced disease, including viral virulence, pathways of spread, and tropism for certain types of cells in the central nervous system. The sigma1 protein projects from the virion surface and consists of two distinct morphologic domains, a virion-distal globular domain known as the head and an elongated fibrous domain, termed the tail, which is anchored into the virion capsid. To better understand structure-function relationships of sigma1 protein, we conducted experiments to identify sequences in sigma1 important for viral binding to sialic acid, a component of the receptor for type 3 reovirus. Three serotype 3 reovirus strains incapable of binding sialylated receptors were adapted to growth in murine erythroleukemia (MEL) cells, in which sialic acid is essential for reovirus infectivity. MEL-adapted (MA) mutant viruses isolated by serial passage in MEL cells acquired the capacity to bind sialic acid-containing receptors and demonstrated a dependence on sialic acid for infection of MEL cells. Analysis of reassortant viruses isolated from crosses of an MA mutant virus and a reovirus strain that does not bind sialic acid indicated that the sigma1 protein is solely responsible for efficient growth of MA mutant viruses in MEL cells. The deduced sigma1 amino acid sequences of the MA mutant viruses revealed that each strain contains a substitution within a short region of sequence in the sigma1 tail predicted to form beta-sheet. These studies identify specific sequences that determine the capacity of reovirus to bind sialylated receptors and suggest a location for a sialic acid-binding domain. Furthermore, the results support a model in which type 3 sigma1 protein contains discrete receptor binding domains, one in the head and another in the tail that binds sialic acid.

  2. Binding of chara Myosin globular tail domain to phospholipid vesicles.

    PubMed

    Nunokawa, Shun-Ya; Anan, Hiromi; Shimada, Kiyo; Hachikubo, You; Kashiyama, Taku; Ito, Kohji; Yamamoto, Keiichi

    2007-11-01

    Binding of Chara myosin globular tail domain to phospholipid vesicles was investigated quantitatively. It was found that the globular tail domain binds to vesicles made from acidic phospholipids but not to those made from neutral phospholipids. This binding was weakened at high KCl concentration, suggesting that the binding is electrostatic by nature. The dissociation constant for the binding of the globular tail domain to 20% phosphatidylserine vesicles (similar to endoplasmic reticulum in acidic phospholipid contents) at 150 mM KCl was 273 nM. The free energy change due to this binding calculated from the dissociation constant was -37.3 kJ mol(-1). Thus the bond between the globular tail domain and membrane phospholipids would not be broken when the motor domain of Chara myosin moves along the actin filament using the energy of ATP hydrolysis (DeltaG degrees ' = -30.5 kJ mol(-1)). Our results suggested that direct binding of Chara myosin to the endoplasmic reticulum membrane through the globular tail domain could work satisfactorily in Chara cytoplasmic streaming. We also suggest a possible regulatory mechanism of cytoplasmic streaming including phosphorylation-dependent dissociation of the globular tail domain from the endoplasmic reticulum membrane.

  3. The terminal six amino-acids of the carboxy cytoplasmic tail of CD36 contain a functional domain implicated in the binding and capture of oxidized low-density lipoprotein.

    PubMed Central

    Malaud, Eric; Hourton, Delphine; Giroux, Louise Marie; Ninio, Ewa; Buckland, Robin; McGregor, John L

    2002-01-01

    CD36, a major adhesion molecule expressed by monocytes/macrophages, plays a key role in the binding and internalization of oxidized low-density lipoprotein (OxLDL). This adhesion molecule, a member of an important scavenger receptor family, contains a very short C-terminal cytoplasmic tail that is known to induce intracellular signalling events. However, the domains on the cytoplasmic tail involved in such signal transduction are unknown. In this study, we have investigated the functional components of the cytoplasmic tail by site-directed mutagenesis coupled with functional OxLDL and monoclonal antibody (mAb) binding studies. Seven truncated or punctual CD36 constructs, localized in the cytoplasmic tail, were produced by site-directed mutagenesis. Each construct was stably expressed in HEK293 cells. We used a quantitative and a qualitative method, labelling OxLDL with either iodine or rhodamine, to determine the functional importance of the cytoplasmic domains in OxLDL internalization. Results indicate that: (1) a deletion of the last amino-acid (construct K472STOP) significantly reduces, compared with wild-type, the binding, internalization and degradation of OxLDL; (2) truncation of the last six amino-acids (construct R467STOP) significantly reduces OxLDL binding; (3) the above two constructs (K472STOP and R467STOP) showed a reduced rate of OxLDL internalization compared with wild-type; (4) the binding and rate of internalization of an anti-CD36 monoclonal antibody (10/5) was not affected by the above mentioned mutants (K472STOP and R467STOP), compared with wild-type. This study shows, for the first time, a specific site on the CD36 cytoplasmic tail that is critical for the binding, endocytosis and targeting of OxLDL. PMID:12023894

  4. Functional analysis of a phosphatidic acid binding domain in human Raf-1 kinase: mutations in the phosphatidate binding domain lead to tail and trunk abnormalities in developing zebrafish embryos.

    PubMed

    Ghosh, Sujoy; Moore, Sean; Bell, Robert M; Dush, Michael

    2003-11-14

    Previously, we and others identified a 35-amino acid segment within human Raf-1 kinase that preferentially binds phosphatidic acid. The presence of phosphatidic acid was found to be necessary for the translocation of Raf-1 to the plasma membrane. We have now employed a combination of alanine-scanning and deletion mutagenesis to identify the critical amino acid residues in Raf-1 necessary for interaction with phosphatidic acid. Progressive mutations within a tetrapeptide motif (residues 398-401 of human Raf-1) reduced and finally eliminated binding of Raf-1 to phosphatidic acid. We then injected zebrafish embryos with RNA encoding wild-type Raf-1 kinase or a mutant version with triple alanine mutations in the tetrapeptide motif and followed the morphological fate of embryonic development. Embryos with mutant but not wild-type Raf-1 exhibited defects in posterior axis formation exemplified by bent trunk and tail structures. Molecular evidence for lack of signaling through mutated Raf-1 was obtained by aberrant in situ hybridization of the ntl (no tail) gene, which functions downstream of Raf-1. Our results demonstrate that a functional phosphatidate binding site is necessary for Raf-1 function in embryonic development.

  5. Structure and stability of the lamin A tail domain and HGPS mutant.

    PubMed

    Qin, Zhao; Kalinowski, Agnieszka; Dahl, Kris Noel; Buehler, Markus J

    2011-09-01

    Hutchinson-Gilford progeria syndrome (HGPS) is a premature aging syndrome caused by the expression and accumulation of a mutant form of lamin A, Δ50 lamin A. As a component of the cell's nucleoskeleton, lamin A plays an important role in the mechanical stabilization of the nuclear envelope and in other nuclear functions. It is largely unknown how the characteristic 50 amino acid deletion affects the conformation of the mostly intrinsically disordered tail domain of lamin A. Here we perform replica exchange molecular dynamics simulations of the tail domain and determine an ensemble of semi-stable structures. Based on these structures we show that the ZMPSTE 24 cleavage site on the precursor form of the lamin A tail domain orients itself in such a way as to facilitate cleavage during the maturation process. We confirm our simulated structures by comparing the thermodynamic properties of the ensemble structures to in vitro stability measurements. Using this combination of experimental and computational techniques, we compare the size, heterogeneity of size, thermodynamic stability of the Ig-fold, as well as the mechanisms of force-induced denaturation. Our data shows that the Δ50 lamin A tail domain is more compact and displays less heterogeneity than the mature lamin A tail domain. Altogether these results suggest that the altered structure and stability of the tail domain can explain changed protein-protein and protein-DNA interactions and may represent an etiology of the disease. Also, this study provides the first molecular structure(s) of the lamin A tail domain, which is confirmed by thermodynamic tests in experiment. PMID:21635954

  6. The extended arms of DNA-binding domains: a tale of tails.

    PubMed

    Crane-Robinson, Colyn; Dragan, Anatoly I; Privalov, Peter L

    2006-10-01

    DNA-binding domains (DBDs) frequently have N- or C-terminal tails, rich in lysine and/or arginine and disordered in free solution, that bind the DNA separately from and in the opposite groove to the folded domain. Is their role simply to increase affinity for DNA or do they have a role in specificity, that is, sequence recognition? One approach to answering this question is to analyze the contribution of such tails to the overall energetics of binding. It turns out that, despite similarities of amino acid sequence, three distinct categories of DBD extension exist: (i) those that are purely electrostatic and lack specificity, (ii) those that are largely non-electrostatic with a high contribution to specificity and (iii) those of mixed character that show sequence preference. Because in all cases the tails also increase the affinity for target DNA, they represent a crucial component of the machinery for selective gene activation or repression. PMID:16920361

  7. Calcium-triggered membrane interaction of the alpha-synuclein acidic tail.

    PubMed

    Tamamizu-Kato, Shiori; Kosaraju, Malathi G; Kato, Hiroyuki; Raussens, Vincent; Ruysschaert, Jean-Marie; Narayanaswami, Vasanthy

    2006-09-12

    Alpha-synuclein (alpha-syn) is a 140-residue protein that aggregates in intraneuronal inclusions called Lewy bodies in Parkinson's disease (PD). It is composed of an N-terminal domain with a propensity to bind lipids and a C-terminal domain rich in acidic residues (the acidic tail). The objective of this study was to examine the effect of Ca(2+) on the acidic tail conformation in lipid-bound alpha-syn. We exploit the extreme sensitivity of the band III fluorescence emission peak of the pyrene fluorophore to the polarity of its microenvironment to monitor subtle conformational response of the alpha-syn acidic tail to Ca(2+). Using recombinant human alpha-syn bearing a pyrene to probe either the N-terminal domain or the acidic tail, we noted that lipid binding resulted in an increase in band III emission intensity in the pyrene probe tagging the N-terminal domain but not that in the acidic tail. This suggests that the protein is anchored to the lipid surface via the N-terminal domain. However, addition of Ca(2+) caused an increase in band III emission intensity in the pyrene tagging the acidic tail, with a corresponding increased susceptibility to quenching by quenchers located in the lipid milieu, indicative of lipid interaction of this domain. Taken together with the increased beta-sheet content of membrane-associated alpha-syn in the presence of Ca(2+), we propose a model wherein initial lipid interaction occurs via the N-terminal domain, followed by a Ca(2+)-triggered membrane association of the acidic tail as a potential mechanism leading to alpha-syn aggregation. These observations have direct implications in the role of age-related oxidative stress and the attendant cellular Ca(2+) dysregulation as critical factors in alpha-syn aggregation in PD.

  8. Fungal mediator tail subunits contain classical transcriptional activation domains.

    PubMed

    Liu, Zhongle; Myers, Lawrence C

    2015-04-01

    Classical activation domains within DNA-bound eukaryotic transcription factors make weak interactions with coactivator complexes, such as Mediator, to stimulate transcription. How these interactions stimulate transcription, however, is unknown. The activation of reporter genes by artificial fusion of Mediator subunits to DNA binding domains that bind to their promoters has been cited as evidence that the primary role of activators is simply to recruit Mediator. We have identified potent classical transcriptional activation domains in the C termini of several tail module subunits of Saccharomyces cerevisiae, Candida albicans, and Candida dubliniensis Mediator, while their N-terminal domains are necessary and sufficient for their incorporation into Mediator but do not possess the ability to activate transcription when fused to a DNA binding domain. This suggests that Mediator fusion proteins actually are functioning in a manner similar to that of a classical DNA-bound activator rather than just recruiting Mediator. Our finding that deletion of the activation domains of S. cerevisiae Med2 and Med3, as well as C. dubliniensis Tlo1 (a Med2 ortholog), impairs the induction of certain genes shows these domains function at native promoters. Activation domains within coactivators are likely an important feature of these complexes and one that may have been uniquely leveraged by a common fungal pathogen.

  9. Mutational Analysis Defines a C-Terminal Tail Domain of Rap1 Essential for Telomeric Silencing in Saccharomyces Cerevisiae

    PubMed Central

    Liu, C.; Mao, X.; Lustig, A. J.

    1994-01-01

    Alleles specifically defective in telomeric silencing were generated by in vitro mutagenesis of the yeast RAP1 gene. The most severe phenotypes occur with three mutations in the C-terminal 28 amino acids. Two of the alleles are nonsense mutations resulting in truncated repressor/activator protein 1 (RAP1) species lacking the C-terminal 25-28 amino acids; the third allele is a missense mutation within this region. These alleles define a novel 28-amino acid region, termed the C-terminal tail domain, that is essential for telomeric and HML silencing. Using site-directed mutagenesis, an 8-amino acid region (amino acids 818-825) that is essential for telomeric silencing has been localized within this domain. Further characterization of these alleles has indicated that the C-terminal tail domain also plays a role in telomere size control. The function of the C-terminal tail in telomere maintenance is not mediated through the RAP1 interacting factor RIF1: rap1 alleles defective in both the C-terminal tail and RIF1 interaction domains have additive effects on telomere length. Overproduction of SIR3, a dose-dependent enhancer of telomeric silencing, suppresses the telomeric silencing, but not length, phenotypes of a subset of C-terminal tail alleles. In contrast, an allele that truncates the terminal 28 amino acids of RAP1 is refractory to SIR3 overproduction. These results indicate that the C-terminal tail domain is required for SIR3-dependent enhancement of telomeric silencing. These data also suggest a distinct set of C-terminal requirements for telomere size control and telomeric silencing. PMID:7896088

  10. The Tail Domain Is Essential but the Head Domain Dispensable for C. elegans Intermediate Filament IFA-2 Function

    PubMed Central

    Williams, Kyle; Williams, Kristen; Baucher, Hallie M.; Plenefisch, John

    2015-01-01

    The intermediate filament protein IFA-2 is essential for the structural integrity of the Caenorhabditis elegans epidermis. It is one of the major components of the fibrous organelle, an epidermal structure comprised of apical and basal hemidesmosomes linked by cytoplasmic intermediate filaments that serve to transmit force from the muscle to the cuticle. Mutations of IFA-2 result in epidermal fragility and separation of the apical and basal epidermal surfaces during postembryonic growth. An IFA-2 lacking the head domain fully rescues the IFA-2 null mutant, whereas an IFA-2 lacking the tail domain cannot. Conversely, an isolated IFA-2 head was able to localize to fibrous organelles whereas the tail was not. Taken together these results suggest that the head domain contains redundant signals for IF localization, whereas non-redundant essential functions map to the IFA-2, tail, although the tail is unlikely to be directly involved in fibrous organelle localization. PMID:25742641

  11. The tail domain of tomosyn controls membrane fusion through tomosyn displacement by VAMP2

    SciTech Connect

    Yamamoto, Yasunori; Fujikura, Kohei; Sakaue, Mio; Okimura, Kenjiro; Kobayashi, Yuta; Nakamura, Toshihiro; Sakisaka, Toshiaki

    2010-08-13

    Research highlights: {yields} The tail domain of tomosyn has no effect on the tomosyn-SNARE complex formation. {yields} The tail domain binding to the VAMP-like domain allows VAMP2 to displace tomosyn. {yields} Tomosyn displacement by VAMP2 leads to SNARE complex formation. {yields} The SNARE complex formation drives membrane fusion. -- Abstract: Neurotransmitter release is regulated by SNARE complex-mediated synaptic vesicle fusion. Tomosyn sequesters target SNAREs (t-SNAREs) through its C-terminal VAMP-like domain (VLD). Cumulative biochemical results suggest that the tomosyn-SNARE complex is so tight that VAMP2 cannot displace tomosyn. Based on these results, the tomosyn-SNARE complex has been believed to be a dead-end complex to inhibit neurotransmitter release. On the other hand, some studies using siRNA depletion of tomosyn suggest that tomosyn positively regulates exocytosis. Therefore, it is still controversial whether tomosyn is a simple inhibitor for neurotransmitter release. We recently reported that the inhibitory activity of tomosyn is regulated by the tail domain binding to the VLD. In this study, we employed the liposome fusion assay in order to further understand modes of action of tomosyn in detail. The tail domain unexpectedly had no effect on binding of the VLD to t-SNARE-bearing liposomes. Nonetheless, the tail domain decreased the inhibitory activity of the VLD on the SNARE complex-mediated liposome fusion. These results indicate that the tail domain controls membrane fusion through tomosyn displacement by VAMP2. Deletion of the tail domain-binding region in the VLD retained the binding to t-SNAREs and promoted the liposome fusion. Together, we propose here a novel mechanism of tomosyn that controls synaptic vesicle fusion positively by serving as a placeholder for VAMP2.

  12. Divalent cations crosslink vimentin intermediate filament tail domains to regulate network mechanics.

    PubMed

    Lin, Yi-Chia; Broedersz, Chase P; Rowat, Amy C; Wedig, Tatjana; Herrmann, Harald; Mackintosh, Frederick C; Weitz, David A

    2010-06-18

    Intermediate filament networks in the cytoplasm and nucleus are critical for the mechanical integrity of metazoan cells. However, the mechanism of crosslinking in these networks and the origins of their mechanical properties are not understood. Here, we study the elastic behavior of in vitro networks of the intermediate filament protein vimentin. Rheological experiments reveal that vimentin networks stiffen with increasing concentrations of Ca(2+) and Mg(2+), showing that divalent cations act as crosslinkers. We quantitatively describe the elastic response of vimentin networks over five decades of applied stress using a theory that treats the divalent cations as crosslinkers: at low stress, the behavior is entropic in origin, and increasing stress pulls out thermal fluctuations from single filaments, giving rise to a nonlinear response; at high stress, enthalpic stretching of individual filaments significantly modifies the nonlinearity. We investigate the elastic properties of networks formed by a series of protein variants with stepwise tail truncations and find that the last 11 amino acids of the C-terminal tail domain mediate crosslinking by divalent ions. We determined the single-filament persistence length, l(P) approximately 0.5 mum, and Young's modulus, Y approximately 9 MPa; both are consistent with literature values. Our results provide insight into a crosslinking mechanism for vimentin networks and suggest that divalent ions may help regulate the cytoskeletal structure and mechanical properties of cells.

  13. The tail-elicited tail withdrawal reflex of Aplysia is mediated centrally at tail sensory-motor synapses and exhibits sensitization across multiple temporal domains

    PubMed Central

    Philips, Gary T.; Sherff, Carolyn M.; Menges, Steven A.; Carew, Thomas J.

    2011-01-01

    The defensive withdrawal reflexes of Aplysia californica have provided powerful behavioral systems for studying the cellular and molecular basis of memory formation. Among these reflexes the tail-elicited tail withdrawal reflex (T-TWR) has been especially useful. In vitro studies examining the monosynaptic circuit for the T-TWR, the tail sensory-motor (SN-MN) synapses, have identified the induction requirements and molecular basis of different temporal phases of synaptic facilitation that underlie sensitization in this system. They have also permitted more recent studies elucidating the role of synaptic and nuclear signaling during synaptic facilitation. Here we report the development of a novel, compartmentalized semi-intact T-TWR preparation that allows examination of the unique contributions of processing in the SN somatic compartment (the pleural ganglion) and the SN-MN synaptic compartment (the pedal ganglion) during the induction of sensitization. Using this preparation we find that the T-TWR is mediated entirely by central connections in the synaptic compartment. Moreover, the reflex is stably expressed for at least 24 h, and can be modified by tail shocks that induce sensitization across multiple temporal domains, as well as direct application of the modulatory neurotransmitter serotonin. This preparation now provides an experimentally powerful system in which to directly examine the unique and combined roles of synaptic and nuclear signaling in different temporal domains of memory formation. PMID:21450911

  14. Assessment of Phytostabilization Success in Metalliferous Acid Mine Tailings

    NASA Astrophysics Data System (ADS)

    Wang, Y.; Root, R. A.; Hammond, C.; Amistadi, M. K.; Maier, R. M.; Chorover, J.

    2014-12-01

    Legacy mine tailings are a significant source of metal(loid)s due to wind and water erosion, especially in the arid southwest, and exposure to fugative dusts presents a health risk to surrounding populations. Compost assisted phytostabilization has been implemented to reduce off site emissions at the Iron King Mine U.S. Superfund Site in central Arizona, concurrent with a greenhouse mesocosm study for detailed study of subsurface mechanisms. Quantification of plant available toxic metal(loid)s in the amended tailings was accessed with a targeted single extraction of diethylenetriaminepentaactic acid (DTPA). Greenhouse mesocosms (1m dia, 0.4 m deep), run in triplicate, mimicked field treatments with: i) tailings only control (TO), ii) tailings plus 15 wt% compost (TC), iii) TC + quailbush seeds (TCA), and iv) TC + buffalo grass seeds (TCB). Core samples collected at 3-month intervals for 1 year were dissected by depth (10 cm each) for analysis. DTPA results indicated that compost treated samples decreased plant availability of Al, As, Cd, Cu, Fe, and Pb but increased Mn and Zn compared with TO. TCB decreased plant available metal(loid)s at all depths, whereas TCA plant available Al, As, Cd, Cu, Fe, Mn and Zn increased in the deeper 20-30cm and 30-40 cm relative to TCB. Samples from the greenhouse were compared to tailings from both the field site and tailings impacted soils used to grow vegetables. Mineral transformations and metal complexation, in the pre- and post-extracted tailings were analyzed by synchrotron transmission XRD and FTIR spectroscopy. The temporal change in plant available metal(loid)s in response to phytostabilization indicates mineralogical alteration that improves soil quality by reducing plant available metal(loid)s. These results will aid in the understanding and efficacy of phytostabilization as a means of remediating and reducing toxicity on mine tailings as well as providing information on health risk management in the region.

  15. Interfacial binding and aggregation of lamin A tail domains associated with Hutchinson-Gilford progeria syndrome.

    PubMed

    Kalinowski, Agnieszka; Yaron, Peter N; Qin, Zhao; Shenoy, Siddharth; Buehler, Markus J; Lösche, Mathias; Dahl, Kris Noel

    2014-12-01

    Hutchinson-Gilford progeria syndrome is a premature aging disorder associated with the expression of ∆50 lamin A (∆50LA), a mutant form of the nuclear structural protein lamin A (LA). ∆50LA is missing 50 amino acids from the tail domain and retains a C-terminal farnesyl group that is cleaved from the wild-type LA. Many of the cellular pathologies of HGPS are thought to be a consequence of protein-membrane association mediated by the retained farnesyl group. To better characterize the protein-membrane interface, we quantified binding of purified recombinant ∆50LA tail domain (∆50LA-TD) to tethered bilayer membranes composed of phosphatidylserine and phosphocholine using surface plasmon resonance. Farnesylated ∆50LA-TD binds to the membrane interface only in the presence of Ca(2+) or Mg(2+) at physiological ionic strength. At extremely low ionic strength, both the farnesylated and non-farnesylated forms of ∆50LA-TD bind to the membrane surface in amounts that exceed those expected for a densely packed protein monolayer. Interestingly, the wild-type LA-TD with no farnesylation also associates with membranes at low ionic strength but forms only a single layer. We suggest that electrostatic interactions are mediated by charge clusters with a net positive charge that we calculate on the surface of the LA-TDs. These studies suggest that the accumulation of ∆50LA at the inner nuclear membrane observed in cells is due to a combination of aggregation and membrane association rather than simple membrane binding; electrostatics plays an important role in mediating this association.

  16. Interfacial binding and aggregation of lamin A tail domains associated with Hutchinson-Gilford progeria syndrome

    PubMed Central

    Kalinowski, Agnieszka; Yaron, Peter N.; Qin, Zhao; Shenoy, Siddharth; Buehler, Markus J.; Lösche, Mathias; Dahl, Kris Noel

    2014-01-01

    Hutchinson-Gilford progeria syndrome is a premature aging disorder associated with the expression of Δ50 lamin A (Δ50LA), a mutant form of the nuclear structural protein lamin A (LA). Δ50LA is missing 50 amino acids from the tail domain and retains a C-terminal farnesyl group that is cleaved from the wild-type LA. Many of the cellular pathologies of HGPS are thought to be a consequence of protein-membrane association mediated by the retained farnesyl group. To better characterize the protein-membrane interface, we quantified binding of purified recombinant Δ50LA tail domain (Δ50LA-TD) to tethered bilayer membranes composed of phosphatidylserine and phosphocholine using surface plasmon resonance. Farnesylated Δ50LATD binds to the membrane interface only in the presence of Ca2+ or Mg2+ at physiological ionic strength. At extremely low ionic strength, both the farnesylated and non-farnesylated forms of Δ50LA-TD bind to the membrane surface in amounts that exceed those expected for a densely packed protein monolayer. Interestingly, the wild-type LA-TD with no farnesylation also associates with membranes at low ionic strength but forms only a single layer. We suggest that electrostatic interactions are mediated by charge clusters with a net positive charge that we calculate on the surface of the LA-TDs. These studies suggest that the accumulation of Δ50LA at the inner nuclear membrane observed in cells is due to a combination of aggregation and membrane association rather than simple membrane binding; electrostatics plays an important role in mediating this association. PMID:25194277

  17. Intramolecular interaction in the tail of Acanthamoeba myosin IC between the SH3 domain and a putative pleckstrin homology domain

    PubMed Central

    Hwang, Kae-Jung; Mahmoodian, Fatemeh; Ferretti, James A.; Korn, Edward D.; Gruschus, James M.

    2007-01-01

    The 466-aa tail of the heavy chain of Acanthamoeba myosin IC (AMIC) comprises an N-terminal 220-residue basic region (BR) followed by a 56-residue Gly/Pro/Ala-rich region (GPA1), a 55-residue Src homology 3 (SH3) domain, and a C-terminal 135-residue Gly/Pro/Ala-rich region (GPA2). Cryo-electron microscopy of AMIC had shown previously that the AMIC tail is folded back on itself, suggesting the possibility of interactions between its N- and C-terminal regions. We now show specific differences between the NMR spectrum of bacterially expressed full-length tail and the sum of the spectra of individually expressed BR and GPA1-SH3-GPA2 (GSG) regions. These results are indicative of interactions between the two subdomains in the full-length tail. From the NMR data, we could assign many of the residues in BR and GSG that are involved in these interactions. By combining homology modeling with the NMR data, we identify a putative pleckstrin homology (PH) domain within BR, and show that the PH domain interacts with the SH3 domain. PMID:17215368

  18. Charge compensation of head-to-head and tail-to-tail domain walls in barium titanate and its influence on conductivity

    SciTech Connect

    Zuo, Yinan; Genenko, Yuri A.; Xu, Bai-Xiang

    2014-07-28

    The effect of the polarization charge compensation by ionic and electronic space charges on domain properties in ferroelectrics with semiconducting features is considered, in particular, the conductivity of head-to-head and tail-to-tail domain walls is studied. It is shown that the domain wall conductivity that is enhanced by electrons or holes depends on the configuration and the types of domains as well as on the energy levels and concentrations of the defects involved. Phase field simulation results are used to explain recent equivocal experimental results on conductivity of charged domain walls in different ferroelectrics.

  19. Naturally occurring hybrids derived from γ-amino acids and sugars with potential tail to tail ether-bonds

    PubMed Central

    Feng, Zi-ming; Zhan, Zhi-lai; Yang, Ya-nan; Jiang, Jian-shuang; Zhang, Pei-cheng

    2016-01-01

    The basic substances of life include various amino acids and sugars. To search such molecules is the precondition to understand the essential nature. Here we reported four unprecedented hybrids of γ-amino acids and sugars from the roots of Ranunculus ternatus, which possess potential tail to tail ether-connected (6,6-ether-connected) modes in the sugar moiety. The structures of these hybrids were elucidated by extensive analyses of spectra and calculated electronic circular dichroism (ECD) method. PMID:27166276

  20. Naturally occurring hybrids derived from γ-amino acids and sugars with potential tail to tail ether-bonds

    NASA Astrophysics Data System (ADS)

    Feng, Zi-Ming; Zhan, Zhi-Lai; Yang, Ya-Nan; Jiang, Jian-Shuang; Zhang, Pei-Cheng

    2016-05-01

    The basic substances of life include various amino acids and sugars. To search such molecules is the precondition to understand the essential nature. Here we reported four unprecedented hybrids of γ-amino acids and sugars from the roots of Ranunculus ternatus, which possess potential tail to tail ether-connected (6,6-ether-connected) modes in the sugar moiety. The structures of these hybrids were elucidated by extensive analyses of spectra and calculated electronic circular dichroism (ECD) method.

  1. Sulfur Oxidation, Microbes And Acidity In A Mine Tailings Lake

    NASA Astrophysics Data System (ADS)

    Warren, L. A.; Bernier, L.

    2003-12-01

    Disposal of tailings (waste rock) aqueously is a common approach at mine sites to minimize oxidation of the associated sulfur minerals (pyrrhotite, pyrite) and the associated generation of acidity that accompanies this process. The study site, Moose Lake, receives tailings runoff at a nickel mine in Northern Ontario, which has rendered the lake highly acidic (surface pH values less than 3.5) with high metal loads and on-going acid export to off-site, downstream systems. To investigate the potential influence of microbial processes for acid generation, as well as characterizing any attendant influences for metal behaviour, the biogeochemistry of Moose Lake was characterized on a seasonal and a diel basis during the summer of 2002. Physico-chemical profiles were used to identify the area of strong redox gradient across the thermocline (typically 1 to 2 metres across this zone) on each sampling day. Samples at five depths within this redox gradient, were then collected for Fe3+/Fe2+, SO42-/H2S, metal and microbial samples, in addition to more highly resolved Hydrolab profiling. Samples were collected both during the lighted portion of the day (10am-12pm) and at dusk (6pm-8pm) to evaluate any contributions to S and Fe cycling attributed to photosynthetic activity. Results indicate a clear seasonal increase in acidity in the upper waters of the lake: pH values dropped from 3.19 in May to 2.90 in September. Further, a strong diel trend of increasing acidity (lower pH) from mid morning to dusk was also observed for each sampling period. Biotic control on S processes appears to be important associated with the thermocline region of the lake, whilst surficial processes occurring in the upper one to three meters are more consistent with a dominant abiotic control. Both pathways contribute to acidity generation, however the controls and rates differ. These results and implications for mitigation strategies will be presented.

  2. Formation of salt bridges mediates internal dimerization of myosin VI medial tail domain.

    PubMed

    Kim, Hyeongjun; Hsin, Jen; Liu, Yanxin; Selvin, Paul R; Schulten, Klaus

    2010-11-10

    The unconventional motor protein, myosin VI, is known to dimerize upon cargo binding to its C-terminal end. It has been shown that one of its tail domains, called the medial tail domain, is a dimerization region. The domain contains an unusual pattern of alternating charged residues and a few hydrophobic residues. To reveal the unknown dimerization mechanism of the medial tail domain, we employed molecular dynamics and single-molecule experimental techniques. Both techniques suggest that the formation of electrostatic-based interhelical salt bridges between oppositely charged residues is a key dimerization factor. For the dimerization to occur, the two identical helices within the dimer do not bind in a symmetric fashion, but rather with an offset of about one helical repeat. Calculations of the dimer-dissociation energy find the contribution of hydrophobic residues to the dimerization process to be minor; they also find that the asymmetric homodimer state is energetically favorable over a state of separate helices. PMID:21070943

  3. A sorting nexin 17-binding domain within the LRP1 cytoplasmic tail mediates receptor recycling through the basolateral sorting endosome.

    PubMed

    Farfán, Pamela; Lee, Jiyeon; Larios, Jorge; Sotelo, Pablo; Bu, Guojun; Marzolo, María-Paz

    2013-07-01

    Sorting nexin 17 (SNX17) is an adaptor protein present in early endosomal antigen 1 (EEA1)-positive sorting endosomes that promotes the efficient recycling of low-density lipoprotein receptor-related protein 1 (LRP1) to the plasma membrane through recognition of the first NPxY motif in the cytoplasmic tail of this receptor. The interaction of LRP1 with SNX17 also regulates the basolateral recycling of the receptor from the basolateral sorting endosome (BSE). In contrast, megalin, which is apically distributed in polarized epithelial cells and localizes poorly to EEA1-positive sorting endosomes, does not interact with SNX17, despite containing three NPxY motifs, indicating that this motif is not sufficient for receptor recognition by SNX17. Here, we identified a cluster of 32 amino acids within the cytoplasmic domain of LRP1 that is both necessary and sufficient for SNX17 binding. To delineate the function of this SNX17-binding domain, we generated chimeric proteins in which the SNX17-binding domain was inserted into the cytoplasmic tail of megalin. This insertion mediated the binding of megalin to SNX17 and modified the cell surface expression and recycling of megalin in non-polarized cells. However, the polarized localization of chimeric megalin was not modified in polarized Madin-Darby canine kidney cells. These results provide evidence regarding the molecular and cellular mechanisms underlying the specificity of SNX17-binding receptors and the restricted function of SNX17 in the BSE.

  4. Replication-coupled chromatin assembly of newly synthesized histones: distinct functions for the histone tail domains.

    PubMed

    Ejlassi-Lassallette, Aïda; Thiriet, Christophe

    2012-02-01

    The maintenance of the genome during replication requires the assembly of nucleosomes with newly synthesized histones. Achieving the deposition of newly synthesized histones in chromatin implies their transport from the cytoplasm to the nucleus at the replication sites. Several lines of evidence have revealed critical functions of the histone tail domains in these conserved cellular processes. In this review, we discuss the role of the amino termini of the nucleosome building blocks, H2A/H2B and H3/H4, in different model systems. The experimental data showed that H2A/H2B tails and H3/H4 tails display distinct functions in nuclear import and chromatin assembly. Furthermore, we describe recent studies exploiting the unique properties of the slime mold, Physarum polycephalum , that have advanced understanding of the function of the highly conserved replication-dependent diacetylation of H4.

  5. Reclamation of acidic copper mine tailings using municipal biosolids

    SciTech Connect

    Rogers, M.T.; Thompson, T.L.; Bengson, S.A.

    1998-12-31

    Reclamation of copper mine tailings in a cost effective, successful, and sustainable manner is an ongoing area of evaluation in the arid southwest. A study was initiated in September, 1996 near Hayden, Arizona to evaluate the use of municipal biosolids for reclaiming acidic copper mine tailings (pH of 2.5 to 4.0). The main objectives of the study were to (1) define an appropriate level of biosolids application for optimum plant growth, and (2) evaluate the effects of green waste and lime amendments. The experiment was a randomized complete block design with four biosolid rates of 20, 70, 100 and 135 dry tons/acre, three amendment treatments (none, green waste, and green waste plus lime); with three replications. Non-replicated controls (no treatment, green waste only and lime only) were included for comparison. Shortly after biosolids incorporation to a depth of 10--12 inches, composite soil samples (0--12 inches) of each plot were taken. Biosolids incorporation increased the pH of the tailings (>5.75) and additional increases in pH were noted with lime application. In January 1997, the plots were seeded and sprinkler irrigation was commenced. A total of 4.47 inches of rainfall and 3.8 inches of irrigation were applied until harvest in May 1997. Data from the first growing season indicates optimum growth (>66 lbs/acre) at biosolids rates of 70--100 dry tons/acre. There was a significant positive effect on growth of green waste and lime amendments. Surface NO{sub 3}-N concentrations in biosolids amended plots were greatly reduced (from 23 to 6 mg/kg) by addition of green waste. There was no evidence for NO{sub 3}N leaching below 12 inches.

  6. A missense mutation in desmin tail domain linked to human dilated cardiomyopathy promotes cleavage of the head domain and abolishes its Z-disc localization

    PubMed Central

    Mavroidis, Manolis; Panagopoulou, Panagiota; Kostavasili, Ioanna; Weisleder, Noah; Capetanaki, Yassemi

    2008-01-01

    A missense mutation (Ile 451 to Met) at the tail domain of the muscle-specific intermediate filament protein desmin has been suggested to be a genetic cause of dilated cardiomyopathy. The Ile451Met mutation is located inside a conserved motif in the desmin tail domain, believed to have a potential role in the lateral packing of type III intermediate filaments. Nevertheless, the role of the type III intermediate filament tail domain remains elusive. To further study the role of this domain in the function of cardiomyocytes and in the development of cardiomyopathy, we generated transgenic mice expressing the mutant desmin(I451M) in the cardiac tissue. Analysis of hearts from transgenic animals revealed that mutant desmin loses its Z-disc localization but it can still associate with the intercalated discs, which, however, have an altered architecture, resembling other examples of dilated cardiomyoplathy. This is the first report demonstrating a critical role of the desmin head and tail domains in the formation of the IF scaffold around Z discs. It is further suggested that in cardiomyocytes, an interplay between desmin tail and head domains is taking place, which potentially protects the amino terminus of desmin from specific proteases. The fact that the association with intercalated discs seems unchanged suggests that this association must take place through the desmin tail, in contrast to the head domain that is most possibly involved in the Z-disc binding.—Mavroidis, M., Panagopoulou, P., Kostavasili, I., Weisleder, N., Capetanaki, Y. A missense mutation in desmin tail domain linked to human dilated cardiomyopathy promotes cleavage of the head domain and abolishes its Z-disc localization. PMID:18539904

  7. Mutations in the tail domain of DYNC1H1 cause dominant spinal muscular atrophy

    PubMed Central

    Harms, M.B.; Ori-McKenney, K.M.; Scoto, M.; Tuck, E.P.; Bell, S.; Ma, D.; Masi, S.; Allred, P.; Al-Lozi, M.; Reilly, M.M.; Miller, L.J.; Jani-Acsadi, A.; Pestronk, A.; Shy, M.E.; Muntoni, F.; Vallee, R.B.

    2012-01-01

    Objective: To identify the gene responsible for 14q32-linked dominant spinal muscular atrophy with lower extremity predominance (SMA-LED, OMIM 158600). Methods: Target exon capture and next generation sequencing was used to analyze the 73 genes in the 14q32 linkage interval in 3 SMA-LED family members. Candidate gene sequencing in additional dominant SMA families used PCR and pooled target capture methods. Patient fibroblasts were biochemically analyzed. Results: Regional exome sequencing of all candidate genes in the 14q32 interval in the original SMA-LED family identified only one missense mutation that segregated with disease state—a mutation in the tail domain of DYNC1H1 (I584L). Sequencing of DYNC1H1 in 32 additional probands with lower extremity predominant SMA found 2 additional heterozygous tail domain mutations (K671E and Y970C), confirming that multiple different mutations in the same domain can cause a similar phenotype. Biochemical analysis of dynein purified from patient-derived fibroblasts demonstrated that the I584L mutation dominantly disrupted dynein complex stability and function. Conclusions: We demonstrate that mutations in the tail domain of the heavy chain of cytoplasmic dynein (DYNC1H1) cause spinal muscular atrophy and provide experimental evidence that a human DYNC1H1 mutation disrupts dynein complex assembly and function. DYNC1H1 mutations were recently found in a family with Charcot-Marie-Tooth disease (type 2O) and in a child with mental retardation. Both of these phenotypes show partial overlap with the spinal muscular atrophy patients described here, indicating that dynein dysfunction is associated with a range of phenotypes in humans involving neuronal development and maintenance. PMID:22459677

  8. The host-binding domain of the P2 phage tail spike reveals a trimeric iron-binding structure

    PubMed Central

    Yamashita, Eiki; Nakagawa, Atsushi; Takahashi, Junichi; Tsunoda, Kin-ichi; Yamada, Seiko; Takeda, Shigeki

    2011-01-01

    The adsorption and infection of bacteriophage P2 is mediated by tail fibres and tail spikes. The tail spikes on the tail baseplate are used to irreversibly adsorb to the host cells. Recently, a P2 phage tail-spike protein, gpV, was purified and it was shown that a C-terminal domain, Ser87–Leu211, is sufficient for the binding of gpV to host Escherichia coli membranes [Kageyama et al. (2009 ▶), Biochemistry, 48, 10129–10135]. In this paper, the crystal structure of the C-terminal domain of P2 gpV is reported. The structure is a triangular pyramid and looks like a spearhead composed of an intertwined β-­sheet, a triple β-helix and a metal-binding region containing iron, calcium and chloride ions. PMID:21821878

  9. IEDA: Making Small Data BIG Through Interdisciplinary Partnerships Among Long-tail Domains

    NASA Astrophysics Data System (ADS)

    Lehnert, K. A.; Carbotte, S. M.; Arko, R. A.; Ferrini, V. L.; Hsu, L.; Song, L.; Ghiorso, M. S.; Walker, D. J.

    2014-12-01

    The Big Data world in the Earth Sciences so far exists primarily for disciplines that generate massive volumes of observational or computed data using large-scale, shared instrumentation such as global sensor networks, satellites, or high-performance computing facilities. These data are typically managed and curated by well-supported community data facilities that also provide the tools for exploring the data through visualization or statistical analysis. In many other domains, especially those where data are primarily acquired by individual investigators or small teams (known as 'Long-tail data'), data are poorly shared and integrated, lacking a community-based data infrastructure that ensures persistent access, quality control, standardization, and integration of data, as well as appropriate tools to fully explore and mine the data within the context of broader Earth Science datasets. IEDA (Integrated Earth Data Applications, www.iedadata.org) is a data facility funded by the US NSF to develop and operate data services that support data stewardship throughout the full life cycle of observational data in the solid earth sciences, with a focus on the data management needs of individual researchers. IEDA builds on a strong foundation of mature disciplinary data systems for marine geology and geophysics, geochemistry, and geochronology. These systems have dramatically advanced data resources in those long-tail Earth science domains. IEDA has strengthened these resources by establishing a consolidated, enterprise-grade infrastructure that is shared by the domain-specific data systems, and implementing joint data curation and data publication services that follow community standards. In recent years, other domain-specific data efforts have partnered with IEDA to take advantage of this infrastructure and improve data services to their respective communities with formal data publication, long-term preservation of data holdings, and better sustainability. IEDA hopes to

  10. Two-dimensional electron gases at head-to-head and tail-to-tail domain walls in ferroelectric thin films

    NASA Astrophysics Data System (ADS)

    García-Fernández, Pablo; Íñiguez, Jorge; Junquera, Javier

    Symmetry breaking at ferroelectric domain walls gives rise to new physical properties, offering the opportunity to use the domain walls themselves as a functional separate object in a device. One example is the appearance of an enhanced conductivity at the boundaries between ferroelectric domains in oxides. A realistic first-principles simulation of the domains walls is limited to highly-symmetric cleanly-cut walls in order to keep the number of atoms in the simulation box small. Here we use a recently developed second-principles method that treats all the lattice degrees of freedom and the relevant electronic ones on the same foot with high accuracy at a modest computational cost. We apply it to the demading physical problem of head-to-head (HH) and tail-to-tail (TT) domain walls in ferroelectric PbTiO3 thin films. These interfaces present a large and unfavourable electrostatic energy due to the polarization-induced bound charge at the domain wall. An accurate simulation should capture eventual charge transfers between the walls, and the concomitant electron-lattice coupling. We show how the polarization discontinuity in HH and TT domain walls in PbTiO3 thin films can be effectively screened by the formation of two-dimensional electron gases of electrons and holes. Finantial support from MINECO Grant No. FIS2012-37549-C05-04.

  11. Long single [alpha]-helical tail domains bridge the gap between structure and function of myosin VI

    SciTech Connect

    Spink, Benjamin J.; Sivaramakrishnan, Sivaraj; Lipfert, Jan; Doniach, Sebastian; Spudich, James A.

    2008-09-29

    Myosin VI has challenged the lever arm hypothesis of myosin movement because of its ability to take {approx}36-nm steps along actin with a canonical lever arm that seems to be too short to allow such large steps. Here we demonstrate that the large step of dimeric myosin VI is primarily made possible by a medial tail in each monomer that forms a rare single {alpha}-helix of {approx}10 nm, which is anchored to the calmodulin-bound IQ domain by a globular proximal tail. With the medial tail contributing to the {approx}36-nm step, rather than dimerizing as previously proposed, we show that the cargo binding domain is the dimerization interface. Furthermore, the cargo binding domain seems to be folded back in the presence of the catalytic head, constituting a potential regulatory mechanism that inhibits dimerization.

  12. Ubiquitination of the Dishevelled DIX domain blocks its head-to-tail polymerization

    PubMed Central

    Madrzak, Julia; Fiedler, Marc; Johnson, Christopher M.; Ewan, Richard; Knebel, Axel; Bienz, Mariann; Chin, Jason W.

    2015-01-01

    Dishevelled relays Wnt signals from the plasma membrane to different cytoplasmic effectors. Its signalling activity depends on its DIX domain, which undergoes head-to-tail polymerization to assemble signalosomes. The DIX domain is ubiquitinated in vivo at multiple lysines, which can be antagonized by various deubiquitinases (DUBs) including the CYLD tumour suppressor that attenuates Wnt signalling. Here, we generate milligram quantities of pure human Dvl2 DIX domain mono-ubiquitinated at two lysines (K54 and K58) by genetically encoded orthogonal protection with activated ligation (GOPAL), to investigate their effect on DIX polymerization. We show that the ubiquitination of DIX at K54 blocks its polymerization in solution, whereas DIX58-Ub remains oligomerization-competent. DUB profiling identified 28 DUBs that cleave DIX-ubiquitin conjugates, half of which prefer, or are specific for, DIX54-Ub, including Cezanne and CYLD. These DUBs thus have the potential to promote Dvl polymerization and signalosome formation, rather than antagonize it as previously thought for CYLD. PMID:25907794

  13. Site-specific study on stabilization of acid-generating mine tailings using coal fly ash

    SciTech Connect

    Shang, J.Q.; Wang, H.L.; Kovac, V.; Fyfe, J.

    2006-03-15

    A site-specific study on stabilizing acid-generating mine tailings from Sudbury Mine using a coal fly ash from Nanticoke Generating Station is presented in this paper. The objective of the study is to evaluate the feasibility of codisposal of the fly ash and mine tailings to reduce environmental impacts of Sudbury tailings disposal sites. The study includes three phases, i.e., characterization of the mine tailings, and coal fly ash, oxidation tests on the mine tailings and kinetic column permeation tests. The results of the experiments indicate that when permeated with acid mine drainage, the hydraulic conductivity of Nanticoke coal fly ash decreased more than three orders of magnitude (from 1 x 10{sup -6} to 1 x 10{sup -9} cm/s), mainly due to chemical reactions between the ash solids and acid mine drainage. Furthermore, the hydraulic gradient required for acid mine drainage to break through the coal fly ash is increased up to ten times (from 17 to 150) as compared with that for water. The results also show that the leachate from coal fly ash neutralizes the acidic pore fluid of mine tailings. The concentrations of trace elements in effluents from all kinetic column permeation tests indicated that coplacement of coal fly ash with mine tailings has the benefit of immobilizing trace elements, especially heavy metals. All regulated element concentrations from effluent during testing are well below the leachate quality criteria set by the local regulatory authority.

  14. The Tail-Elicited Tail Withdrawal Reflex of "Aplysia" Is Mediated Centrally at Tail Sensory-Motor Synapses and Exhibits Sensitization across Multiple Temporal Domains

    ERIC Educational Resources Information Center

    Philips, Gary T.; Sherff, Carolyn M.; Menges, Steven A.; Carew, Thomas J.

    2011-01-01

    The defensive withdrawal reflexes of "Aplysia californica" have provided powerful behavioral systems for studying the cellular and molecular basis of memory formation. Among these reflexes the (T-TWR) has been especially useful. In vitro studies examining the monosynaptic circuit for the T-TWR, the tail sensory-motor (SN-MN) synapses, have…

  15. Groundwater leaching of neutralized and untreated acid-leached uranium-mill tailings

    SciTech Connect

    Gee, G.W.; Begej, C.W.; Campbell, A.C.; Sauter, N.N.; Opitz, B.E.; Sherwood, D.R.

    1981-01-01

    Tailings neutralization was examined to determine the effect of neutralization on contaminant release. Column leaching of acid extracted uranium mill tailings from Exxon Highland Mill, Wyoming, Pathfinder Gas Hills Mill, Wyoming, and the Dawn Midnite Mill, Washington, resulted in the flushing of high concentrations of salts in the first four pore volumes of leachate, followed by a steady decrease to the original groundwater salt concentrations. Neutralization decreased the concentration of salts and radionuclides leaching from the tailings and decreased the volume of solution required to return the solution to the groundwater pH and EC. Radium-226 and uranium-238 leached quickly from the tailings in the initial pore volumes of both neutralized and unneutralized tailings, and then decreased significantly. 6 figures, 5 tables.

  16. Geophysical delineation of acidity and salinity in the Central Manitoba gold mine tailings pile, Manitoba, Canada

    NASA Astrophysics Data System (ADS)

    Tycholiz, C.; Ferguson, I. J.; Sherriff, B. L.; Cordeiro, M.; Sri Ranjan, R.; Pérez-Flores, M. A.

    2016-08-01

    Surface electrical and electromagnetic geophysical methods can map enhanced electrical conductivity caused by acid mine drainage in mine tailings piles. In this case study, we investigate quantitative relationships between geophysical responses and the electrical conductivity, acidity and salinity of tailing samples at the Central Manitoba Mine tailings in Manitoba, Canada. Previous electromagnetic surveys at the site identified zones of enhanced conductivity that were hypothesized to be caused by acid mine drainage. In the present study, high-resolution EM31 and DC-resistivity measurements were made on a profile through a zone of enhanced conductivity and laboratory measurements of salinity and pH were made on saturation paste extracts from an array of tailing samples collected from the upper 2 m of tailings along the profile. Observed spatial correlation of pH and pore-fluid salinity in the tailings samples confirms that the enhanced conductivity in the Central Manitoba Mine tailings is due to acid mine drainage. Contoured cross-sections of the data indicate that the acid mine drainage is concentrated near the base of the oxidized zone in the thicker parts of the tailings pile. The zone of increased acidity extends to the surface on sloping margins causing an increase in apparent conductivity in shallow penetrating geophysical responses. The quantitative relationship between measured pH and salinity shows that the conductivity increase associated with the acid mine drainage is due only in part to conduction by ions produced from dissociation of sulfuric acid. Comparison of the observations with fluid conductivity estimates based on statistical relationships of pH and ion concentrations in water samples from across the tailings pile shows that Ca2 + and Mg2 + ions also make significant contributions to the conductivity at all values of pH and Cu2 +, Al3 + and Fe3 + ions make additional contributions at low pH. Variability in the measured conductivity at constant

  17. The internalization signal in the cytoplasmic tail of lysosomal acid phosphatase consists of the hexapeptide PGYRHV.

    PubMed Central

    Lehmann, L E; Eberle, W; Krull, S; Prill, V; Schmidt, B; Sander, C; von Figura, K; Peters, C

    1992-01-01

    Lysosomal acid phosphatase (LAP) is rapidly internalized from the cell surface due to a tyrosine-containing internalization signal in its 19 amino acid cytoplasmic tail. Measuring the internalization of a series of LAP cytoplasmic tail truncation and substitution mutants revealed that the N-terminal 12 amino acids of the cytoplasmic tail are sufficient for rapid endocytosis and that the hexapeptide 411-PGYRHV-416 is the tyrosine-containing internalization signal. Truncation and substitution mutants of amino acid residues following Val416 can prevent internalization even though these residues do not belong to the internalization signal. It was shown recently that part of the LAP cytoplasmic tail peptide corresponding to 410-PPGY-413 forms a well-ordered beta turn structure in solution. Two-dimensional NMR spectroscopy of two modified LAP tail peptides, in which the single tyrosine was substituted either by phenylalanine or by alanine, revealed that the tendency to form a beta turn is reduced by 25% in the phenylalanine-containing peptide and by approximately 50% in the alanine-containing mutant peptide. Our results suggest, that in the short cytoplasmic tail of LAP tyrosine is required for stabilization of the right turn and that the aromatic ring system of the tyrosine residue is a contact point to the putative cytoplasmic receptor. Images PMID:1425575

  18. Crystal structures of the coil 2B fragment and the globular tail domain of human lamin B1

    SciTech Connect

    Ruan, Jianbin; Xu, Chao; Bian, Chuanbing; Lam, Robert; Wang, Jia-Pey; Kania, Joanna; Min, Jinrong; Zang, Jianye

    2012-07-18

    We present here the crystal structures of human lamin B1 globular tail domain and coiled 2B domain, which adopt similar folds to Ig-like domain and coiled-coil domain of lamin A, respectively. Despite the overall similarity, we found an extra intermolecular disulfide bond in the lamin B1 coil 2B domain, which does not exist in lamin A/C. In addition, the structural analysis indicates that interactions at the lamin B1 homodimer interface are quite different from those of lamin A/C. Thus our research not only reveals the diversely formed homodimers among lamin family members, but also sheds light on understanding the important roles of lamin B1 in forming the nuclear lamina matrix.

  19. Long-term stability of earthen materials in contact with acidic tailings solutions

    SciTech Connect

    Peterson, S.R.; Erikson, R.L.; Gee, G.W.

    1982-11-01

    The objectives of the studies documented in this report were to use experimental and geochemical computer modeling tools to assess the long-term environmental impact of leachate movement from acidic uranium mill tailings. Liner failure (i.e., an increase in the permeability of the liner material) was not found to be a problem when various acidic tailings solutions leached through liner materials for periods up to 3 years. On the contrary, materials that contained over 30% clay showed a decrease in permeability with time in the laboratory columns. The high clay materials tested appear suitable for lining tailings impoundment ponds. The decreases in permeability are attributed to pore plugging resulting from the precipitation of minerals and solids. This precipitation takes place due to the increase in pH of the tailings solution brought about by the buffering capacity of the soil. Geochemical modeling predicts, and x-ray characterization confirms, that precipitation of solids from solution is occurring in the acidic tailings solution/liner interactions studied. In conclusion the same mineralogical changes and contaminant reactions predicted by geochemical modeling and observed in laboratory studies were found at a drained evaporation pond (Lucky Mc in Wyoming) with a 4 year history of acid attack.

  20. Column leaching test to evaluate the use of alkaline industrial wastes to neutralize acid mine tailings

    SciTech Connect

    Doye, I.; Duchesne, J.

    2005-08-01

    Acid mine drainage is a serious environmental problem caused by the oxidation of sulfide minerals that releases highly acidic, sulfate, and metals-rich drainage. In this study, alkaline industrial wastes were mixed with acid mine tailings in order to obtain neutral conditions. A series of column leaching tests were performed to evaluate the behavior of reactive mine tailings amended with alkaline-additions under dynamic conditions. Column tests were conducted of oxidized mine tailings combined with cement kiln dust, red mud bauxite, and mixtures of cement kiln dust with red mud bauxite. The pH results show the addition of 10% of alkaline materials permits the maintenance of near neutral conditions. In the presence of 10% alkaline material, the concentration of toxic metals such as Al, Cu, Fe, Zn are significantly reduced as well as the number of viable cells (Thiobacillus ferrooxidans) compared to control samples.

  1. PDZ Binding Domains, Structural Disorder and Phosphorylation: A Menage-a-trois Tailing Dcp2 mRNA Decapping Enzymes.

    PubMed

    Gunawardana, Dilantha

    2016-01-01

    Diverse cellular activities are mediated through the interaction of protein domains and their binding partners. One such protein domain widely distributed in the higher metazoan world is the PDZ domain, which facilitates abundant protein-protein interactions. The PDZ domain-PDZ binding domain interaction has been implicated in several pathologies including Alzheimer's disease, Parkinson's disease and Down syndrome. PDZ domains bind to C-terminal peptides/proteins which have either of the following combinations: S/T-X-hydrophobic-COOH for type I, hydrophobic-Xhydrophobic- COOH for type II, and D/E-X-hydrophobic-COOH for type III, although hydrophobicity in the termini form the key characteristic of the PDZ-binding domains. We identified and characterized a Dcp2 type mRNA decapping enzyme from Arabidopsis thaliana, a protein containing a putative PDZ-binding domain using mutagenesis and protein biochemistry. Now we are using bioinformatics to study the Cterminal end of mRNA decapping enzymes from complex metazoans with the aim of (1) identifying putative PDZ-binding domains (2) Correlating structural disorder with PDZ binding domains and (3) Demonstrating the presence of phosphorylation sites in C-terminal extremities of Dcp2 type mRNA decapping enzymes. It is proposed here that the trinity of PDZbinding domains, structural disorder and phosphorylation-susceptible sites are a feature of the Dcp2 family of decapping enzymes and perhaps is a wider trick in protein evolution where scaffolding/tethering is a requirement for localization and function. It is critical though laboratory-based supporting evidence is sought to back-up this bioinformatics exploration into tail regions of mRNA decapping enzymes. PMID:27151193

  2. Role of organic acids in promoting colloidal transport of mercury from mine tailings

    USGS Publications Warehouse

    Slowey, A.J.; Johnson, S.B.; Rytuba, J.J.; Brown, Gordon E.

    2005-01-01

    A number of factors affect the transport of dissolved and paniculate mercury (Hg) from inoperative Hg mines, including the presence of organic acids in the rooting zone of vegetated mine waste. We examined the role of the two most common organic acids in soils (oxalic and citric acid) on Hg transport from such waste by pumping a mixed organic acid solution (pH 5.7) at 1 mL/min through Hg mine tailings columns. For the two total organic acid concentrations investigated (20 ??M and 1 mM), particle-associated Hg was mobilized, with the onset of paniculate Hg transport occurring later for the lower organic acid concentration. Chemical analyses of column effluent indicate that 98 wt % of Hg mobilized from the column was paniculate. Hg speciation was determined using extended X-ray absorption fine structure spectroscopy and transmission electron microscopy, showing that HgS minerals are dominant in the mobilized particles. Hg adsorbed to colloids is another likely mode of transport due to the abundance of Fe-(oxyhydr)oxides, Fe-sulfides, alunite, and jarosite in the tailings to which Hg(II) adsorbs. Organic acids produced by plants are likely to enhance the transport of colloid-associated Hg from vegetated Hg mine tailings by dissolving cements to enable colloid release. ?? 2005 American Chemical Society.

  3. Role of organic acids in promoting colloidal transport of mercury from mine tailings.

    PubMed

    Slowey, Aaron J; Johnson, Stephen B; Rytuba, James J; Brown, Gordon E

    2005-10-15

    A number of factors affect the transport of dissolved and particulate mercury (Hg) from inoperative Hg mines, including the presence of organic acids in the rooting zone of vegetated mine waste. We examined the role of the two most common organic acids in soils (oxalic and citric acid) on Hg transport from such waste by pumping a mixed organic acid solution (pH 5.7) at 1 mL/min through Hg mine tailings columns. For the two total organic acid concentrations investigated (20 microM and 1 mM), particle-associated Hg was mobilized, with the onset of particulate Hg transport occurring later for the lower organic acid concentration. Chemical analyses of column effluent indicate that 98 wt % of Hg mobilized from the column was particulate. Hg speciation was determined using extended X-ray absorption fine structure spectroscopy and transmission electron microscopy, showing that HgS minerals are dominant in the mobilized particles. Hg adsorbed to colloids is another likely mode of transport due to the abundance of Fe-(oxyhydr)oxides, Fe-sulfides, alunite, and jarosite in the tailings to which Hg(II) adsorbs. Organic acids produced by plants are likely to enhance the transport of colloid-associated Hg from vegetated Hg mine tailings by dissolving cements to enable colloid release.

  4. Revegetation of non-Acid-generating, thickened tailings with boreal trees: a greenhouse study.

    PubMed

    Larchevêque, Marie; Desrochers, Annie; Bussière, Bruno; Cartier, Hélène; David, Jean-Sébastien

    2013-01-01

    Tree planting presents clear advantages for mine reclamation that is aimed at achieving rapid reclamation of forested landscapes. A greenhouse study was conducted to evaluate the capacity of non-acid-generating, thickened tailings to support six boreal tree species during two growing seasons. One treatment was thickened tailings alone fertilized with inorganic N, P, and K fertilizer or chicken () manure. A thin layer of overburden topsoil was used to cover the tailings and was compared with topsoil alone, where normal tree growth was expected. Two amendments were also tested: overburden topsoil and vermicompost from food wastes. The presence of alkaline thickened tailings under the thin layer of acidic topsoil had a positive effect on tree height and root biomass (broadleaved and jack pine [ Lamb.]) by increasing topsoil pH and available Ca concentrations, which decreased Al, Zn, and Mn phytoavailability to trees; however, root contact with the tailings also increased their Cu concentrations. In thickened tailings that were mixed with topsoil, C/N ratios increased along the experiment from 21 to 40, a value where N immobilization by microorganisms occurred, as suggested by low N concentrations in tree tissues. In consequence, tree height growth (broadleaved) and biomass (conifers) were reduced. Amendment with compost raised the electrical conductivity (3.4 dS cm) to thresholds limiting broadleaved survival, while conifers showed a generalized decrease in biomass production. No trace metal contamination of the trees occurred in the mixtures, probably due to the near-neutral pH conferred by the tailings. PMID:23673827

  5. Deletion of the sequence encoding the tail domain of the bone morphogenetic protein type 2 receptor reveals a bone morphogenetic protein 7-specific gain of function.

    PubMed

    Leyton, Patricio A; Beppu, Hideyuki; Pappas, Alexandra; Martyn, Trejeeve M; Derwall, Matthias; Baron, David M; Galdos, Rita; Bloch, Donald B; Bloch, Kenneth D

    2013-01-01

    The bone morphogenetic protein (BMP) type II receptor (BMPR2) has a long cytoplasmic tail domain whose function is incompletely elucidated. Mutations in the tail domain of BMPR2 are found in familial cases of pulmonary arterial hypertension. To investigate the role of the tail domain of BMPR2 in BMP signaling, we generated a mouse carrying a Bmpr2 allele encoding a non-sense mediated decay-resistant mutant receptor lacking the tail domain of Bmpr2. We found that homozygous mutant mice died during gastrulation, whereas heterozygous mice grew normally without developing pulmonary arterial hypertension. Using pulmonary artery smooth muscle cells (PaSMC) from heterozygous mice, we determined that the mutant receptor was expressed and retained its ability to transduce BMP signaling. Heterozygous PaSMCs exhibited a BMP7‑specific gain of function, which was transduced via the mutant receptor. Using siRNA knockdown and cells from conditional knockout mice to selectively deplete BMP receptors, we observed that the tail domain of Bmpr2 inhibits Alk2‑mediated BMP7 signaling. These findings suggest that the tail domain of Bmpr2 is essential for normal embryogenesis and inhibits Alk2‑mediated BMP7 signaling in PaSMCs.

  6. Nucleic acids encoding a cellulose binding domain

    DOEpatents

    Shoseyov, O.; Shpiegl, I.; Goldstein, M.A.; Doi, R.H.

    1996-03-05

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques. 15 figs.

  7. Nucleic acids encoding a cellulose binding domain

    DOEpatents

    Shoseyov, Oded; Shpiegl, Itai; Goldstein, Marc A.; Doi, Roy H.

    1996-01-01

    A cellulose binding domain (CBD) having a high affinity for crystalline cellulose and chitin is disclosed, along with methods for the molecular cloning and recombinant production thereof. Fusion products comprising the CBD and a second protein are likewise described. A wide range of applications are contemplated for both the CBD and the fusion products, including drug delivery, affinity separations, and diagnostic techniques.

  8. G-protein-coupled receptors for neurotransmitter amino acids: C-terminal tails, crowded signalosomes.

    PubMed Central

    El Far, Oussama; Betz, Heinrich

    2002-01-01

    G-protein-coupled receptors (GPCRs) represent a superfamily of highly diverse integral membrane proteins that transduce external signals to different subcellular compartments, including nuclei, via trimeric G-proteins. By differential activation of diffusible G(alpha) and membrane-bound G(beta)gamma subunits, GPCRs might act on both cytoplasmic/intracellular and plasma-membrane-bound effector systems. The coupling efficiency and the plasma membrane localization of GPCRs are regulated by a variety of interacting proteins. In this review, we discuss recently disclosed protein interactions found with the cytoplasmic C-terminal tail regions of two types of presynaptic neurotransmitter receptors, the group III metabotropic glutamate receptors and the gamma-aminobutyric acid type-B receptors (GABA(B)Rs). Calmodulin binding to mGluR7 and other group III mGluRs may provide a Ca(2+)-dependent switch for unidirectional (G(alpha)) versus bidirectional (G(alpha) and G(beta)gamma) signalling to downstream effector proteins. In addition, clustering of mGluR7 by PICK1 (protein interacting with C-kinase 1), a polyspecific PDZ (PSD-95/Dlg1/ZO-1) domain containing synaptic organizer protein, sheds light on how higher-order receptor complexes with regulatory enzymes (or 'signalosomes') could be formed. The interaction of GABA(B)Rs with the adaptor protein 14-3-3 and the transcription factor ATF4 (activating transcription factor 4) suggests novel regulatory pathways for G-protein signalling, cytoskeletal reorganization and nuclear gene expression: processes that may all contribute to synaptic plasticity. PMID:12006104

  9. Direct binding of the Kex2p cytosolic tail to the VHS domain of yeast Gga2p facilitates TGN to prevacuolar compartment transport and is regulated by phosphorylation

    PubMed Central

    De, Mithu; Abazeed, Mohamed E.; Fuller, Robert S.

    2013-01-01

    Human Golgi-localized, γ-ear–containing, ADP-ribosylation factor–binding proteins (Ggas) bind directly to acidic dileucine sorting motifs in the cytosolic tails (C-tails) of intracellular receptors. Despite evidence for a role in recruiting ubiquitinated cargo, it remains unclear whether yeast Ggas also function by binding peptide-sorting signals directly. Two-hybrid analysis shows that the Gga1p and Gga2p Vps27, Hrs, Stam (VHS) domains both bind a site in the Kex2p C-tail and that the Gga2p VHS domain binds a site in the Vps10p C-tail. Binding requires deletion of an apparently autoinhibitory sequence in the Gga2p hinge. Ser780 in the Kex2p C-tail is crucial for binding: an Ala substitution blocks but an Asp substitution permits binding. Biochemical assays using purified Gga2p VHS–GGA and TOM1 (GAT) and glutathione S-transferase–Kex2p C-tail fusions show that Gga2p binds directly to the Kex2p C-tail, with relative affinities Asp780 > Ser780 > Ala780. Affinity-purified antibody against a peptide containing phospho-Ser­780 recognizes wild-type Kex2p but not S780A Kex2p, showing that Ser780 is phosphorylated in vivo; phosphorylation of Ser780 is up-regulated by cell wall–damaging drugs. Finally, mutation of Ser780 alters trafficking of Kex2p both in vivo and in cell-free trans-Golgi network (TGN)–prevacuolar compartment (PVC) transport. Thus yeast Gga adaptors facilitate TGN–PVC transport by direct binding of noncanonical phosphoregulated Gga-binding sites in cargo molecules. PMID:23408788

  10. Microbial diversity at the moderate acidic stage in three different sulfidic mine tailings dumps generating acid mine drainage.

    PubMed

    Korehi, Hananeh; Blöthe, Marco; Schippers, Axel

    2014-11-01

    In freshly deposited sulfidic mine tailings the pH is alkaline or circumneutral. Due to pyrite or pyrrhotite oxidation the pH is dropping over time to pH values <3 at which acidophilic iron- and sulfur-oxidizing prokaryotes prevail and accelerate the oxidation processes, well described for several mine waste sites. The microbial communities at the moderate acidic stage in mine tailings are only scarcely studied. Here we investigated the microbial diversity via 16S rRNA gene sequence analysis in eight samples (pH range 3.2-6.5) from three different sulfidic mine tailings dumps in Botswana, Germany and Sweden. In total 701 partial 16S rRNA gene sequences revealed a divergent microbial community between the three sites and at different tailings depths. Proteobacteria and Firmicutes were overall the most abundant phyla in the clone libraries. Acidobacteria, Actinobacteria, Bacteroidetes, and Nitrospira occurred less frequently. The found microbial communities were completely different to microbial communities in tailings at

  11. Terahertz time-domain spectroscopy of four hydroxycinnamic acid derivatives.

    PubMed

    Ge, Min; Zhao, Hongwei; Wang, Wenfeng; Zhang, Zengyan; Yu, Xiaohan; Li, Wenxin

    2006-11-01

    The well-resolved absorption spectra of the hydroxycinnamic acid (HCA) derivatives, caffeic acid, ferulic acid, sinapic acid and chlorogenic acid, were measured over the frequency region from 0.3 to 2.0 THz at 294 K with terahertz time-domain spectroscopy (THz-TDS). Theoretical calculation was applied to assist the analysis and assignment of the individual THz absorption spectra of the HCA derivatives with density functional theory (DFT). The distinctive spectral features were originated from the collective motion of molecules held together by hydrogen bonds. The real and imaginary parts of dielectric function of the four HCA derivatives were also obtained. PMID:19669446

  12. Terahertz Time-Domain Spectroscopy of Four Hydroxycinnamic Acid Derivatives

    PubMed Central

    Ge, Min; Zhao, Hongwei; Zhang, Zengyan; Yu, Xiaohan; Li, Wenxin

    2006-01-01

    The well-resolved absorption spectra of the hydroxycinnamic acid (HCA) derivatives, caffeic acid, ferulic acid, sinapic acid and chlorogenic acid, were measured over the frequency region from 0.3 to 2.0 THz at 294 K with terahertz time-domain spectroscopy (THz-TDS). Theoretical calculation was applied to assist the analysis and assignment of the individual THz absorption spectra of the HCA derivatives with density functional theory (DFT). The distinctive spectral features were originated from the collective motion of molecules held together by hydrogen bonds. The real and imaginary parts of dielectric function of the four HCA derivatives were also obtained. PMID:19669446

  13. The Thumb Domain Mediates Acid-sensing Ion Channel Desensitization.

    PubMed

    Krauson, Aram J; Carattino, Marcelo D

    2016-05-20

    Acid-sensing ion channels (ASICs) are cation-selective proton-gated channels expressed in neurons that participate in diverse physiological processes, including nociception, synaptic plasticity, learning, and memory. ASIC subunits contain intracellular N and C termini, two transmembrane domains that constitute the pore, and a large extracellular loop with defined domains termed the finger, β-ball, thumb, palm, and knuckle. Here we examined the contribution of the finger, β-ball, and thumb domains to activation and desensitization through the analysis of chimeras and the assessment of the effect of covalent modification of introduced Cys at the domain-domain interfaces. Our studies with ASIC1a-ASIC2a chimeras showed that swapping the thumb domain between subunits results in faster channel desensitization. Likewise, the covalent modification of Cys residues at selected positions in the β-ball-thumb interface accelerates the desensitization of the mutant channels. Studies of accessibility with thiol-reactive reagents revealed that the β-ball and thumb domains reside apart in the resting state but that they become closer to each other in response to extracellular acidification. We propose that the thumb domain moves upon continuous exposure to an acidic extracellular milieu, assisting with the closing of the pore during channel desensitization. PMID:27015804

  14. Structure of the Receptor-Binding Carboxy-Terminal Domain of the Bacteriophage T5 L-Shaped Tail Fibre with and without Its Intra-Molecular Chaperone

    PubMed Central

    Garcia-Doval, Carmela; Castón, José R.; Luque, Daniel; Granell, Meritxell; Otero, José M.; Llamas-Saiz, Antonio L.; Renouard, Madalena; Boulanger, Pascale; van Raaij, Mark J.

    2015-01-01

    Bacteriophage T5, a Siphovirus belonging to the order Caudovirales, has a flexible, three-fold symmetric tail, to which three L-shaped fibres are attached. These fibres recognize oligo-mannose units on the bacterial cell surface prior to infection and are composed of homotrimers of the pb1 protein. Pb1 has 1396 amino acids, of which the carboxy-terminal 133 residues form a trimeric intra-molecular chaperone that is auto-proteolyzed after correct folding. The structure of a trimer of residues 970–1263 was determined by single anomalous dispersion phasing using incorporated selenomethionine residues and refined at 2.3 Å resolution using crystals grown from native, methionine-containing, protein. The protein inhibits phage infection by competition. The phage-distal receptor-binding domain resembles a bullet, with the walls formed by partially intertwined beta-sheets, conferring stability to the structure. The fold of the domain is novel and the topology unique to the pb1 structure. A site-directed mutant (Ser1264 to Ala), in which auto-proteolysis is impeded, was also produced, crystallized and its 2.5 Å structure solved by molecular replacement. The additional chaperone domain (residues 1263–1396) consists of a central trimeric alpha-helical coiled-coil flanked by a mixed alpha-beta domain. Three long beta-hairpin tentacles, one from each chaperone monomer, extend into long curved grooves of the bullet-shaped domain. The chaperone-containing mutant did not inhibit infection by competition. PMID:26670244

  15. Oil sands thickened froth treatment tailings exhibit acid rock drainage potential during evaporative drying.

    PubMed

    Kuznetsov, Petr; Kuznetsova, Alsu; Foght, Julia M; Siddique, Tariq

    2015-02-01

    Bitumen extraction from oil sands ores after surface mining produces different tailings waste streams: 'froth treatment tailings' are enriched in pyrite relative to other streams. Tailings treatment can include addition of organic polymers to produce thickened tailings (TT). TT may be further de-watered by deposition into geotechnical cells for evaporative drying to increase shear strength prior to reclamation. To examine the acid rock drainage (ARD) potential of TT, we performed predictive analyses and laboratory experiments on material from field trials of two types of thickened froth treatment tailings (TT1 and TT2). Acid-base accounting (ABA) of initial samples showed that both TT1 and TT2 initially had net acid-producing potential, with ABA values of -141 and -230 t CaCO₃ equiv. 1000 t(-1) of TT, respectively. In long-term kinetic experiments, duplicate ~2-kg samples of TT were incubated in shallow trays and intermittently irrigated under air flow for 459 days to simulate evaporative field drying. Leachates collected from both TT samples initially had pH~6.8 that began decreasing after ~50 days (TT2) or ~250 days (TT1), stabilizing at pH~2. Correspondingly, the redox potential of leachates increased from 100-200 mV to 500-580 mV and electrical conductivity increased from 2-5 dS m(-1) to 26 dS m(-1), indicating dissolution of minerals during ARD. The rapid onset and prolonged ARD observed with TT2 is attributed to its greater pyrite (13.4%) and lower carbonate (1.4%) contents versus the slower onset of ARD in TT1 (initially 6.0% pyrite and 2.5% carbonates). 16S rRNA gene pyrosequencing analysis revealed rapid shift in microbial community when conditions became strongly acidic (pH~2) favoring the enrichment of Acidithiobacillus and Sulfobacillus bacteria in TT. This is the first report showing ARD potential of TT and the results have significant implications for effective management of pyrite-enriched oil sands tailings streams/deposits. PMID:25306090

  16. Oil sands thickened froth treatment tailings exhibit acid rock drainage potential during evaporative drying.

    PubMed

    Kuznetsov, Petr; Kuznetsova, Alsu; Foght, Julia M; Siddique, Tariq

    2015-02-01

    Bitumen extraction from oil sands ores after surface mining produces different tailings waste streams: 'froth treatment tailings' are enriched in pyrite relative to other streams. Tailings treatment can include addition of organic polymers to produce thickened tailings (TT). TT may be further de-watered by deposition into geotechnical cells for evaporative drying to increase shear strength prior to reclamation. To examine the acid rock drainage (ARD) potential of TT, we performed predictive analyses and laboratory experiments on material from field trials of two types of thickened froth treatment tailings (TT1 and TT2). Acid-base accounting (ABA) of initial samples showed that both TT1 and TT2 initially had net acid-producing potential, with ABA values of -141 and -230 t CaCO₃ equiv. 1000 t(-1) of TT, respectively. In long-term kinetic experiments, duplicate ~2-kg samples of TT were incubated in shallow trays and intermittently irrigated under air flow for 459 days to simulate evaporative field drying. Leachates collected from both TT samples initially had pH~6.8 that began decreasing after ~50 days (TT2) or ~250 days (TT1), stabilizing at pH~2. Correspondingly, the redox potential of leachates increased from 100-200 mV to 500-580 mV and electrical conductivity increased from 2-5 dS m(-1) to 26 dS m(-1), indicating dissolution of minerals during ARD. The rapid onset and prolonged ARD observed with TT2 is attributed to its greater pyrite (13.4%) and lower carbonate (1.4%) contents versus the slower onset of ARD in TT1 (initially 6.0% pyrite and 2.5% carbonates). 16S rRNA gene pyrosequencing analysis revealed rapid shift in microbial community when conditions became strongly acidic (pH~2) favoring the enrichment of Acidithiobacillus and Sulfobacillus bacteria in TT. This is the first report showing ARD potential of TT and the results have significant implications for effective management of pyrite-enriched oil sands tailings streams/deposits.

  17. The acidic domains of the Toc159 chloroplast preprotein receptor family are intrinsically disordered protein domains

    PubMed Central

    2009-01-01

    Background The Toc159 family of proteins serve as receptors for chloroplast-destined preproteins. They directly bind to transit peptides, and exhibit preprotein substrate selectivity conferred by an unknown mechanism. The Toc159 receptors each include three domains: C-terminal membrane, central GTPase, and N-terminal acidic (A-) domains. Although the function(s) of the A-domain remains largely unknown, the amino acid sequences are most variable within these domains, suggesting they may contribute to the functional specificity of the receptors. Results The physicochemical properties of the A-domains are characteristic of intrinsically disordered proteins (IDPs). Using CD spectroscopy we show that the A-domains of two Arabidopsis Toc159 family members (atToc132 and atToc159) are disordered at physiological pH and temperature and undergo conformational changes at temperature and pH extremes that are characteristic of IDPs. Conclusions Identification of the A-domains as IDPs will be important for determining their precise function(s), and suggests a role in protein-protein interactions, which may explain how these proteins serve as receptors for such a wide variety of preprotein substrates. PMID:20042108

  18. Amino acid residues 201-205 in C-terminal acidic tail region plays a crucial role in antibacterial activity of HMGB1

    PubMed Central

    2009-01-01

    Background Antibacterial activity is a novel function of high-mobility group box 1 (HMGB1). However, the functional site for this new effect is presently unknown. Methods and Results In this study, recombinant human HMGB1 A box and B box (rHMGB1 A box, rHMGB1 B box), recombinant human HMGB1 (rHMGB1) and the truncated C-terminal acidic tail mutant (tHMGB1) were prepared by the prokaryotic expression system. The C-terminal acidic tail (C peptide) was synthesized, which was composed of 30 amino acid residues. Antibacterial assays showed that both the full length rHMGB1 and the synthetic C peptide alone could efficiently inhibit bacteria proliferation, but rHMGB1 A box and B box, and tHMGB1 lacking the C-terminal acidic tail had no antibacterial function. These results suggest that C-terminal acidic tail is the key region for the antibacterial activity of HMGB1. Furthermore, we prepared eleven different deleted mutants lacking several amino acid residues in C-terminal acidic tail of HMGB1. Antibacterial assays of these mutants demonstrate that the amino acid residues 201-205 in C-terminal acidic tail region is the core functional site for the antibacterial activity of the molecule. Conclusion In sum, these results define the key region and the crucial site in HMGB1 for its antibacterial function, which is helpful to illustrating the antibacterial mechanisms of HMGB1. PMID:19751520

  19. Pollution in acid mine drainage from mine tailings in Svalbard Norwegian Arctic

    NASA Astrophysics Data System (ADS)

    Holm, E. B.; Brandvik, P. J.; Steinnes, E.

    2003-05-01

    Throughout the summer season of 2000 samples of acid mine drainage (AMD) were collected from areas below tailing deposits from the coal mining in Svalbard, Norwegian Arctic. The water was analysed for pH, oxygen, conductivity, 9 sulfate and various metals. Oxygen, pH and conductivity were measured by standard electrodes, sulphate was determined gravimetrically and metals were determined by flame/graphite furnace AAS. The AMD was found to contain heavy metals and sulphate in high concentrations, causing damage to the local tundra vegetation. Large spatial variation however was observed in pH (2.5-9.5) as well as in metal concentrations in the AMD, indicating strongly inhomogeneous distribution of sulphide minerais in the tailing deposits.

  20. Laboratory evaluation of limestone and lime neutralization of acidic uranium mill tailings solution. Progress report

    SciTech Connect

    Opitz, B.E.; Dodson, M.E.; Serne, R.J.

    1984-02-01

    Experiments were conducted to evaluate a two-step neutralization scheme for treatment of acidic uranium mill tailings solutions. Tailings solutions from the Lucky Mc Mill and Exxon Highland Mill, both in Wyoming, were neutralized with limestone, CaCO/sub 3/, to an intermediate pH of 4.0 or 5.0, followed by lime, Ca(OH)/sub 2/, neutralization to pH 7.3. The combination limestone/lime treatment methods, CaCO/sub 3/ neutralization to pH 4 followed by neutralization with Ca(OH)/sub 2/ to pH 7.3 resulted in the highest quality effluent solution with respect to EPA's water quality guidelines. The combination method is the most cost-effective treatment procedure tested in our studies. Neutralization experiments to evaluate the optimum solution pH for contaminant removal were performed on the same two tailings solutions using only lime Ca(OH)/sub 2/ as the neutralizing agent. The data indicate solution neutralization above pH 7.3 does not significantly increase removal of pH dependent contaminants from solution. Column leaching experiments were performed on the neutralized sludge material (the precipitated solid material which forms as the acidic tailings solutions are neutralized to pH 4 or above). The sludges were contacted with laboratory prepared synthetic ground water until several effluent pore volumes were collected. Effluent solutions were analyzed for macro ions, trace metals and radionuclides in an effort to evaluate the long term effectiveness of attenuating contaminants in sludges formed during solution neutralization. Neutralized sludge leaching experiments indicate that Ca, Na, Mg, Se, Cl, and SO/sub 4/ are the only constituents which show solution concentrations significantly higher than the synthetic ground water in the early pore volumes of long-term leaching studies.

  1. Evolution of plant colonization in acid and alkaline mine tailing ponds after amendments and microorganisms application

    NASA Astrophysics Data System (ADS)

    Acosta, Jose Alberto; Faz, Ángel; Kabas, Sebla; Zornoza, Raúl; Martínez-Martínez, Silvia

    2014-05-01

    Intense mining activities in the past were carried out in Cartagena-La Unión mining district, SE Spain, and caused excessive accumulation of toxic metals in tailing ponds which poses a high environmental and ecological risk. One of the remediation options gaining considerable interest in recent years is the in situ immobilization of metals. A corresponding reduction in the plant-available metal fraction allows re-vegetation and ecosystem restoration of the heavily contaminated sites. In addition, the use of microorganisms to improve the soil condition is a new tool used to increase spontaneous plant colonization. The aim of this research was to assess the effect of amendments (pig manure, sewage sludge, and lime) and microorganisms on plant cover establishment, as a consequence of metal immobilization and the improvement of soil properties. The study was carried out in two mine ponds (acid and alkaline). Twenty seven square field plots, each one consisting of 4 m2, were located in each pond. Four different doses of microorganism (0 ml, 20 ml, 100 ml and 200 ml of microorganism solution in each plot) and one dose of pig manure (5 kg per plot), sewage sludge (4 kg per plot) and lime (22 kg per plot) were used. Organic amendment doses were calculated according to European nitrogen legislations, and lime dose was calculated according with the potential acid production through total sulphur oxidation. Three replicates of each treatment (organic amendment + lime + microorganism dose 0, 1, 2, or 3) and control soil (with no amendments) were carried out. Plots were left to the semi-arid climate conditions after the addition of amendments to simulate real potential applications of the results. Identification of plant species and biodiversity was determined on each plot, after 2, 4, 6 and 8 months of amendment addition. The results showed that, in those plots without application of microorganism, 8 months after applications the number of species and individuals of each

  2. Phytostabilization potential of Jatropha curcas L. in polymetallic acid mine tailings.

    PubMed

    Wu, Qihang; Wang, Shizhong; Thangavel, Palaniswamy; Li, Qingfei; Zheng, Han; Bai, Jun; Qiu, Rongliang

    2011-09-01

    Greenhouse pot experiments were conducted to determine the growth response, metal tolerance, and phytostabilization potential of Jatropha curcas L The plants were grown on different degrees of multi-metal contaminated acid mine soils (T0, control; T1, moderately and T2, highly contaminated soils) with or without limestone amendments. The order of metal accumulation in J. curcas was roots>stems>leaves. The higher tolerance index (>90%) with no phytotoxic symptoms and growth reduction in T1 showed that this plant has the ability to tolerate polymetallic acid mine tailings. Further, various enzymatic and non-enzymatic antioxidants also actively involved in metal defense mechanism in J. curcas. On the other hand, to alleviate the predominant phytoavailable toxic metals such as Al, Cu, and Pb, different rates (0.1, 0.25, 0.50, and 1%) of limestone amendments were added in both T1 and T2 soils. The growth performance of J. curcas was improved due to the increase in soil pH and decrease in phytoavailable soil A1 (95%), Zn (approximately 75%), and Cu (approximately 65%) contents at 0.50% of lime addition. Based on the inherent tolerance ability of J. curcas in existing adverse environmental conditions without liming, it could be used as a suitable candidate for phytostabilization in acid mine tailings. PMID:21972519

  3. Mechanical changes in rat tail tendons induced by dibasic amino acids as a function of age.

    PubMed

    Reihsner, R; Menzel, E J

    1994-01-01

    Rat tail tendons from 54-day-old and 900-day-old animals were incubated with different concentrations of the dibasic amino acids, lysine and arginine. We observed a significant incorporation of these amino acids into the tendons. Uniaxial tension tests and relaxation experiments were performed at strain levels within the linear portion of the stress-strain relationship. The incorporation of the amino acids resulted in a decrease of ultimate stress and maximum Young's modulus and, after separation of the elastic and viscous stress components, in a decrease of the elastic fraction. The incorporation of amino acids and the resulting mechanical alterations were more pronounced in the young animals. The reversibility of the effects induced by the amino acids was tested. After the glycosaminoglycan chains were digested with chondroitinase ABC, we showed that the dibasic amino acids bind predominantly to the proteoglycan matrix. A possible analogy to the effects of amino acid incorporation on biomechanics and swelling with a monovalent cation such as Na+ is discussed.

  4. Efficient degradation of Acid Orange 7 in aqueous solution by iron ore tailing Fenton-like process.

    PubMed

    Zheng, Jianming; Gao, Zhanqi; He, Huan; Yang, Shaogui; Sun, Cheng

    2016-05-01

    An effective method based on iron ore tailing Fenton-like process was studied for removing an azo dye, Acid Orange 7 (AO7) in aqueous solution. Five tailings were characterized by X-ray fluorescence spectroscope (XFS), Brunner-Emmet-Teller (BET) measurement, and Scanning Electron Microscope (SEM). The result of XFS showed that Fe, Si and Ca were the most abundant elements and some toxic heavy metals were also present in the studied tailings. The result of BET analysis indicated that the studied tailings had very low surface areas (0.64-5.68 m(2) g(-1)). The degradation efficiencies of AO7 were positively correlated with the content of iron oxide and cupric oxide, and not related with the BET surface area of the tailings. The co-existing metal elements, particularly Cu, might accelerate the heterogeneous Fenton-like reaction. The effects of other parameters on heterogeneous Fenton-like degradation of AO7 by a converter slag iron tailing (tailing E) which contains highest iron oxide were also investigated. The tailing could be reused 10 times without significant decrease of the catalytic capacity. Very low amount of iron species and almost undetectable toxic elements were leached in the catalytic degradation of AO7 by the tailing E. The reaction products were identified by gas chromatography-mass spectrometry and a possible pathway of AO7 degradation was proposed. This study not only provides an effective method for removing azo dyes in polluted water by employing waste tailings as Fenton-like catalysts, but also uses waste tailings as the secondary resource. PMID:26891355

  5. Metabolism of branched-chain amino acids in leg muscles from tail-cast suspended intact and adrenalectomized rats

    NASA Technical Reports Server (NTRS)

    Jaspers, Stephen R.; Henriksen, Erik; Jacob, Stephan; Tischler, Marc E.

    1989-01-01

    The effects of muscle unloading, adrenalectomy, and cortisol treatment on the metabolism of branched-chain amino acids in the soleus and extensor digitorum longus of tail-cast suspended rats were investigated using C-14-labeled lucine, isoleucine, and valine in incubation studies. It was found that, compared to not suspended controls, the degradation of branched-chain amino acids in hind limb muscles was accelerated in tail-cast suspended rats. Adrenalectomy was found to abolish the aminotransferase flux and to diminish the dehydrogenase flux in the soleus. The data also suggest that cortisol treatment increases the rate of metabolism of branched-chain amino acids at the dehydrogenase step.

  6. The Role of the CAI-1 Fatty Acid Tail in the Vibrio cholerae Quorum Sensing Response

    PubMed Central

    Perez, Lark J.; Ng, Wai-Leung; Marano, Paul; Brook, Karolina; Bassler, Bonnie L.; Semmelhack, Martin F.

    2013-01-01

    Quorum sensing is a mechanism of chemical communication among bacteria that enables collective behaviors. In V. cholerae, the etiological agent of the disease cholera, quorum sensing controls group behaviors including virulence factor production and biofilm formation. The major V. cholerae quorum-sensing system consists of the extracellular signal molecule called CAI-1 and its cognate membrane bound receptor called CqsS. Here, the ligand binding activity of CqsS is probed with structural analogs of the natural signal. Enabled by our discovery of a structurally simplified analog of CAI-1, we prepared and analyzed a focused library. The molecules were designed to probe the effects of conformational and structural changes along the length of the fatty acid tail of CAI-1. Our results, combined with pharmacophore modeling, suggest a molecular basis for signal molecule recognition and receptor fidelity with respect to the fatty acid tail portion of CAI-1. These efforts provide novel probes to enhance discovery of anti-virulence agents for the treatment of V. cholerae. PMID:23092313

  7. Nutritional restriction and acid-base balance in white-tailed deer

    USGS Publications Warehouse

    DelGiudice, G.D.; Mech, L.D.; Seal, U.S.

    1994-01-01

    We examined the effect of progressive nutritional restriction on acid-base balance in seven captive, adult white-tailed deer (Odocoileus virginianus) from 4 February to 5 May 1988 in north central Minnesota (USA). Metabolic acidosis was indicated by low mean blood pH (7.25 to 7.33) in deer throughout the study. Mean urinary pH values declined (P = 0.020) from a mean (+SE) baseline of 8.3 +0.1 to 6.7 + 0.3 as restriction progressed. Acidemia and aciduria were associated with significant variations in mean blood CO2 (P = 0.006) and pO2 (P = 0.032), serum potassium (P = 0.004) concentrations, and with a significant (P = 0.104) handling date times group interaction in urinary potassium:creatinine values. Mean bicarbonate:carbonic acid ratios were consistently below 20:1 during nutritional restriction. Mean packed cell volume increased (P = 0.019) and serum total protein decreased (P = 0.001); thus there was evidence for progressive dehydration and net protein catabolism, respectively. Blood pCO2, serum sodium, and urinary sodium:creatinine were stable throughout the study. We propose that acidosis and aciduria are metabolic complications associated with nutritional restriction of white-tailed deer.

  8. Response of key soil parameters during compost-assisted phytostabilization in extremely acidic tailings: effect of plant species.

    PubMed

    Solís-Dominguez, Fernando A; White, Scott A; Hutter, Travis Borrillo; Amistadi, Mary Kay; Root, Robert A; Chorover, Jon; Maier, Raina M

    2012-01-17

    Phytostabilization of mine tailings acts to mitigate both eolian dispersion and water erosion events which can disseminate barren tailings over large distances. This technology uses plants to establish a vegetative cover to permanently immobilize contaminants in the rooting zone, often requiring addition of an amendment to assist plant growth. Here we report the results of a greenhouse study that evaluated the ability of six native plant species to grow in extremely acidic (pH ∼ 2.5) metalliferous (As, Pb, Zn: 2000-3000 mg kg(-1)) mine tailings from Iron King Mine Humboldt Smelter Superfund site when amended with a range of compost concentrations. Results revealed that three of the six plant species tested (buffalo grass, mesquite, and catclaw acacia) are good candidates for phytostabilization at an optimum level of 15% compost (w/w) amendment showing good growth and minimal shoot accumulation of metal(loid)s. A fourth candidate, quailbush, also met all criteria except for exceeding the domestic animal toxicity limit for shoot accumulation of zinc. A key finding of this study was that the plant species that grew most successfully on these tailings significantly influenced key tailings parameters; direct correlations between plant biomass and both increased tailings pH and neutrophilic heterotrophic bacterial counts were observed. We also observed decreased iron oxidizer counts and decreased bioavailability of metal(loid)s mainly as a result of compost amendment. Taken together, these results suggest that the phytostabilization process reduced tailings toxicity as well as the potential for metal(loid) mobilization. This study provides practical information on plant and tailings characteristics that is critically needed for successful implementation of assisted phytostabilization on acidic, metalliferous mine tailings sites. PMID:22191663

  9. Response of key soil parameters during compost-assisted phytostabilization in extremely acidic tailings: effect of plant species.

    PubMed

    Solís-Dominguez, Fernando A; White, Scott A; Hutter, Travis Borrillo; Amistadi, Mary Kay; Root, Robert A; Chorover, Jon; Maier, Raina M

    2012-01-17

    Phytostabilization of mine tailings acts to mitigate both eolian dispersion and water erosion events which can disseminate barren tailings over large distances. This technology uses plants to establish a vegetative cover to permanently immobilize contaminants in the rooting zone, often requiring addition of an amendment to assist plant growth. Here we report the results of a greenhouse study that evaluated the ability of six native plant species to grow in extremely acidic (pH ∼ 2.5) metalliferous (As, Pb, Zn: 2000-3000 mg kg(-1)) mine tailings from Iron King Mine Humboldt Smelter Superfund site when amended with a range of compost concentrations. Results revealed that three of the six plant species tested (buffalo grass, mesquite, and catclaw acacia) are good candidates for phytostabilization at an optimum level of 15% compost (w/w) amendment showing good growth and minimal shoot accumulation of metal(loid)s. A fourth candidate, quailbush, also met all criteria except for exceeding the domestic animal toxicity limit for shoot accumulation of zinc. A key finding of this study was that the plant species that grew most successfully on these tailings significantly influenced key tailings parameters; direct correlations between plant biomass and both increased tailings pH and neutrophilic heterotrophic bacterial counts were observed. We also observed decreased iron oxidizer counts and decreased bioavailability of metal(loid)s mainly as a result of compost amendment. Taken together, these results suggest that the phytostabilization process reduced tailings toxicity as well as the potential for metal(loid) mobilization. This study provides practical information on plant and tailings characteristics that is critically needed for successful implementation of assisted phytostabilization on acidic, metalliferous mine tailings sites.

  10. Solution structure and membrane-binding property of the N-terminal tail domain of human annexin I.

    PubMed

    Yoon, M K; Park, S H; Won, H S; Na, D S; Lee, B J

    2000-11-10

    The conformational preferences of AnxI(N26), a peptide corresponding to residues 2-26 of human annexin I, were investigated using CD and NMR spectroscopy. CD results showed that AnxI(N26) adopts a mainly alpha-helical conformation in membrane-mimetic environments, TFE/water and SDS micelles, while a predominantly random structure with slight helical propensity in aqueous buffer. The helical region of AnxI(N26) showed a nearly identical conformation between in TFE/water and in SDS micelles, except for the orientation of the Trp-12 side-chain, which was quite different between the two. The N-terminal region of the AnxI(N26) helix showed a typical amphipathic nature, which could be stabilized by the neighboring hydrophobic cluster. The helical stability of the peptide in SDS micelles was increased by addition of calcium ions. These results suggest that the N-terminal tail domain of human annexin I interacts with biological membranes in a partially calcium-dependent manner.

  11. Enhanced mobilization of arsenic and heavy metals from mine tailings by humic acid.

    PubMed

    Wang, Suiling; Mulligan, Catherine N

    2009-01-01

    Arsenic and heavy metal mobilization from mine tailings is an issue of concern as it might pose potential groundwater or ecological risks. Increasing attention recently has been focused on the effects of natural organic matter on the mobility behavior of the toxicants in the environment. Column experiments were carried out in this research study to evaluate the feasibility of using humic acid (HA) to mobilize arsenic and heavy metals (i.e., Cu, Pb and Zn) from an oxidized Pb-Zn mine tailings sample collected from Bathurst, New Brunswick, Canada. Capillary electrophoresis analyses indicated that arsenate [As(V)] was the only extractable arsenic species in the mine tailings and the addition of HA at pH 11 did not incur the oxidation-reduction or methylation reactions of arsenic. A 0.1% HA solution with an initial pH adjusted to 11 was selected as the flushing solution, while distilled water (initial pH adjusted to 11) was used as the control to account for the mobilization of arsenic and the heavy metals by physical mixing and the effect of pH. It was found that the HA could significantly enhance the mobilization of arsenic and heavy metals simultaneously from the mine tailings. After a 70-pore-volume-flushing, the mobilization of arsenic, copper, lead and zinc reached 97, 35, 838 and 224 mg kg(-1), respectively. The mobilization of arsenic and the heavy metals was found to be positively correlated with the mobilization of Fe in the presence of the HA. Moreover, the mobilization of arsenic was also correlated well with that of the heavy metals. The mobilization of co-existing metals to some extent might enhance arsenic mobilization in the presence of the HA by helping incorporate it into soluble aqueous organic complexes through metal-bridging mechanisms. Use of HA in arsenic and heavy metal remediation may be developed as an environmentally benign and possible effective remedial option to reduce and avoid further contamination.

  12. Efficient inhibition of heavy metal release from mine tailings against acid rain exposure by triethylenetetramine intercalated montmorillonite (TETA-Mt).

    PubMed

    Gong, Beini; Wu, Pingxiao; Huang, Zhujian; Li, Yuanyuan; Yang, Shanshan; Dang, Zhi; Ruan, Bo; Kang, Chunxi

    2016-11-15

    The potential application of triethylenetetramine intercalated montmorillonite (TETA-Mt) in mine tailings treatment and AMD (acid mine drainage) remediation was investigated with batch experiments. The structural and morphological characteristics of TETA-Mt were analyzed with XRD, FTIR, DTG-TG and SEM. The inhibition efficiencies of TETA-Mt against heavy metal release from mine tailings when exposed to acid rain leaching was examined and compared with that of triethylenetetramine (TETA) and Mt. Results showed that the overall inhibition by TETA-Mt surpassed that by TETA or Mt for various heavy metal ions over an acid rain pH range of 3-5.6 and a temperature range of 25-40°C. When mine tailings were exposed to acid rain of pH 4.8 (the average rain pH of the mining site where the mine tailings were from), TETA-Mt achieved an inhibition efficiency of over 90% for Cu(2+), Zn(2+), Cd(2+) and Mn(2+) release, and 70% for Pb(2+) at 25°C. It was shown that TETA-Mt has a strong buffering capacity. Moreover, TETA-Mt was able to adsorb heavy metal ions and the adsorption process was fast, suggesting that coordination was mainly responsible. These results showed the potential of TETA-Mt in AMD mitigation, especially in acid rain affected mining area. PMID:27450331

  13. Efficient inhibition of heavy metal release from mine tailings against acid rain exposure by triethylenetetramine intercalated montmorillonite (TETA-Mt).

    PubMed

    Gong, Beini; Wu, Pingxiao; Huang, Zhujian; Li, Yuanyuan; Yang, Shanshan; Dang, Zhi; Ruan, Bo; Kang, Chunxi

    2016-11-15

    The potential application of triethylenetetramine intercalated montmorillonite (TETA-Mt) in mine tailings treatment and AMD (acid mine drainage) remediation was investigated with batch experiments. The structural and morphological characteristics of TETA-Mt were analyzed with XRD, FTIR, DTG-TG and SEM. The inhibition efficiencies of TETA-Mt against heavy metal release from mine tailings when exposed to acid rain leaching was examined and compared with that of triethylenetetramine (TETA) and Mt. Results showed that the overall inhibition by TETA-Mt surpassed that by TETA or Mt for various heavy metal ions over an acid rain pH range of 3-5.6 and a temperature range of 25-40°C. When mine tailings were exposed to acid rain of pH 4.8 (the average rain pH of the mining site where the mine tailings were from), TETA-Mt achieved an inhibition efficiency of over 90% for Cu(2+), Zn(2+), Cd(2+) and Mn(2+) release, and 70% for Pb(2+) at 25°C. It was shown that TETA-Mt has a strong buffering capacity. Moreover, TETA-Mt was able to adsorb heavy metal ions and the adsorption process was fast, suggesting that coordination was mainly responsible. These results showed the potential of TETA-Mt in AMD mitigation, especially in acid rain affected mining area.

  14. Peptides derived from human galectin-3 N-terminal tail interact with its carbohydrate recognition domain in a phosphorylation-dependent manner

    SciTech Connect

    Berbís, M. Álvaro; André, Sabine; Cañada, F. Javier; Pipkorn, Rüdiger; Ippel, Hans; Mayo, Kevin H.; Kübler, Dieter; Gabius, Hans-Joachim; Jiménez-Barbero, Jesús

    2014-01-03

    Highlights: •Galectin-3 is composed of a carbohydrate recognition domain and an N-terminal tail. •Synthetic peptides derived from the tail are shown to interact with the CRD. •This interaction is modulated by Ser- and Tyr-phosphorylation of the peptides. -- Abstract: Galectin-3 (Gal-3) is a multi-functional effector protein that functions in the cytoplasm and the nucleus, as well as extracellularly following non-classical secretion. Structurally, Gal-3 is unique among galectins with its carbohydrate recognition domain (CRD) attached to a rather long N-terminal tail composed mostly of collagen-like repeats (nine in the human protein) and terminating in a short non-collagenous terminal peptide sequence unique in this lectin family and not yet fully explored. Although several Ser and Tyr sites within the N-terminal tail can be phosphorylated, the physiological significance of this post-translational modification remains unclear. Here, we used a series of synthetic (phospho)peptides derived from the tail to assess phosphorylation-mediated interactions with {sup 15}N-labeled Gal-3 CRD. HSQC-derived chemical shift perturbations revealed selective interactions at the backface of the CRD that were attenuated by phosphorylation of Tyr 107 and Tyr 118, while phosphorylation of Ser 6 and Ser 12 was essential. Controls with sequence scrambling underscored inherent specificity. Our studies shed light on how phosphorylation of the N-terminal tail may impact on Gal-3 function and prompt further studies using phosphorylated full-length protein.

  15. Trace metal mobilization from oil sands froth treatment thickened tailings exhibiting acid rock drainage.

    PubMed

    Kuznetsova, Alsu; Kuznetsov, Petr; Foght, Julia M; Siddique, Tariq

    2016-11-15

    Froth treatment thickened tailings (TT) are a waste product of bitumen extraction from surface-mined oil sands ores. When incubated in a laboratory under simulated moist oxic environmental conditions for ~450d, two different types of TT (TT1 and TT2) exhibited the potential to generate acid rock drainage (ARD) by producing acid leachate after 250 and 50d, respectively. We report here the release of toxic metals from TT via ARD, which could pose an environmental threat if oil sands TT deposits are not properly managed. Trace metal concentrations in leachate samples collected periodically revealed that Mn and Sr were released immediately even before the onset of ARD. Spikes in Co and Ni concentrations were observed both pre-ARD and during active ARD, particularly in TT1. For most elements measured (Fe, Cr, V, As, Cu, Pb, Zn, Cd, and Se), leaching was associated with ARD production. Though equivalent acidification (pH2) was achieved in leachate from both TT types, greater metal release was observed from TT2 where concentrations reached 10,000ppb for Ni, 5000ppb for Co, 3000ppb for As, 2000ppb for V, and 1000ppb for Cr. Generally, metal concentrations decreased in leachate with time during ARD and became negligible by the end of incubation (~450d) despite appreciable metals remaining in the leached TT. These results suggest that using TT for land reclamation purposes or surface deposition for volume reduction may unfavorably impact the environment, and warrants application of appropriate strategies for management of pyrite-enriched oil sands tailings streams. PMID:27443453

  16. Trace metal mobilization from oil sands froth treatment thickened tailings exhibiting acid rock drainage.

    PubMed

    Kuznetsova, Alsu; Kuznetsov, Petr; Foght, Julia M; Siddique, Tariq

    2016-11-15

    Froth treatment thickened tailings (TT) are a waste product of bitumen extraction from surface-mined oil sands ores. When incubated in a laboratory under simulated moist oxic environmental conditions for ~450d, two different types of TT (TT1 and TT2) exhibited the potential to generate acid rock drainage (ARD) by producing acid leachate after 250 and 50d, respectively. We report here the release of toxic metals from TT via ARD, which could pose an environmental threat if oil sands TT deposits are not properly managed. Trace metal concentrations in leachate samples collected periodically revealed that Mn and Sr were released immediately even before the onset of ARD. Spikes in Co and Ni concentrations were observed both pre-ARD and during active ARD, particularly in TT1. For most elements measured (Fe, Cr, V, As, Cu, Pb, Zn, Cd, and Se), leaching was associated with ARD production. Though equivalent acidification (pH2) was achieved in leachate from both TT types, greater metal release was observed from TT2 where concentrations reached 10,000ppb for Ni, 5000ppb for Co, 3000ppb for As, 2000ppb for V, and 1000ppb for Cr. Generally, metal concentrations decreased in leachate with time during ARD and became negligible by the end of incubation (~450d) despite appreciable metals remaining in the leached TT. These results suggest that using TT for land reclamation purposes or surface deposition for volume reduction may unfavorably impact the environment, and warrants application of appropriate strategies for management of pyrite-enriched oil sands tailings streams.

  17. Manganese ore tailing: optimization of acid leaching conditions and recovery of soluble manganese.

    PubMed

    Santos, Olívia de Souza Heleno; Carvalho, Cornélio de Freitas; Silva, Gilmare Antônia da; Santos, Cláudio Gouvêa Dos

    2015-01-01

    Manganese recovery from industrial ore processing waste by means of leaching with sulfuric acid was the objective of this study. Experimental conditions were optimized by multivariate experimental design approaches. In order to study the factors affecting leaching, a screening step was used involving a full factorial design with central point for three variables in two levels (2(3)). The three variables studied were leaching time, concentration of sulfuric acid and sample amount. The three factors screened were shown to be relevant and therefore a Doehlert design was applied to determine the best working conditions for leaching and to build the response surface. By applying the best leaching conditions, the concentrations of 12.80 and 13.64 %w/w of manganese for the global sample and for the fraction -44 + 37 μm, respectively, were found. Microbeads of chitosan were tested for removal of leachate acidity and recovering of soluble manganese. Manganese recovery from the leachate was 95.4%. Upon drying the leachate, a solid containing mostly manganese sulfate was obtained, showing that the proposed optimized method is efficient for manganese recovery from ore tailings.

  18. Separation of Main and Tail Rotor Noise Sources from Ground-Based Acoustic Measurements Using Time-Domain De-Dopplerization

    NASA Technical Reports Server (NTRS)

    Greenwood, Eric II; Schmitz, Fredric H.

    2009-01-01

    A new method of separating the contributions of helicopter main and tail rotor noise sources is presented, making use of ground-based acoustic measurements. The method employs time-domain de-Dopplerization to transform the acoustic pressure time-history data collected from an array of ground-based microphones to the equivalent time-history signals observed by an array of virtual inflight microphones traveling with the helicopter. The now-stationary signals observed by the virtual microphones are then periodically averaged with the main and tail rotor once per revolution triggers. The averaging process suppresses noise which is not periodic with the respective rotor, allowing for the separation of main and tail rotor pressure time-histories. The averaged measurements are then interpolated across the range of directivity angles captured by the microphone array in order to generate separate acoustic hemispheres for the main and tail rotor noise sources. The new method is successfully applied to ground-based microphone measurements of a Bell 206B3 helicopter and demonstrates the strong directivity characteristics of harmonic noise radiation from both the main and tail rotors of that helicopter.

  19. The Venus Fly Trap domain of the extracellular Ca2+ -sensing receptor is required for L-amino acid sensing.

    PubMed

    Mun, Hee-Chang; Franks, Alison H; Culverston, Emma L; Krapcho, Karen; Nemeth, Edward F; Conigrave, Arthur D

    2004-12-10

    We previously demonstrated that the human calcium-sensing receptor (CaR) is allosterically activated by L-amino acids (Conigrave, A. D., Quinn, S. J., and Brown, E. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 4814-4819). However, the domain-based location of amino acid binding has been uncertain. We now show that the Venus Fly Trap (VFT) domain of CaR, but none of its other major domains, is required for amino acid sensing. Several constructs were informative when expressed in HEK293 cells. First, the wild-type CaR exhibited allosteric activation by L-amino acids as previously observed. Second, two CaR-mGlu chimeric receptor constructs that retained the VFT domain of CaR, one containing the extracellular Cys-rich region of CaR and the other containing the Cys-rich region of the rat metabotropic glutamate type-1 (mGlu-1) receptor, together with the rat mGlu-1 transmembrane region and C-terminal tail, retained amino acid sensing. Third, a CaR lacking residues 1-599 of the N-terminal extracellular head but retaining an intact CaR transmembrane region and a functional but truncated C terminus (headless-T903 CaR) failed to respond to L-amino acids but retained responsiveness to the type-II calcimimetic NPS R-467. Finally, a T903 CaR control that retained an intact N terminus also retained L-amino acid sensing. Taken together, the data indicate that the VFT domain of CaR is necessary for L-amino acid sensing and are consistent with the hypothesis that the VFT domain is the site of L-amino acid binding. The findings support the concept that the mGlu-1 amino acid binding site for L-glutamate is conserved as an L-amino acid binding site in its homolog, the CaR.

  20. Insight into the Unfolding Properties of Chd64, a Small, Single Domain Protein with a Globular Core and Disordered Tails

    PubMed Central

    Dobryszycki, Piotr; Kaus-Drobek, Magdalena; Dadlez, Michał; Ożyhar, Andrzej

    2015-01-01

    Two major lipophilic hormones, 20-hydroxyecdysone (20E) and juvenile hormone (JH), govern insect development and growth. While the mode of action of 20E is well understood, some understanding of JH-dependent signalling has been attained only in the past few years, and the crosstalk of the two hormonal pathways remains unknown. Two proteins, the calponin-like Chd64 and immunophilin FKBP39 proteins, have recently been found to play pivotal roles in the formation of dynamic, multiprotein complex that cross-links these two signalling pathways. However, the molecular mechanism of the interaction remains unexplored. The aim of this work was to determine structural elements of Chd64 to provide an understanding of molecular basis of multiple interactions. We analysed Chd64 in two unrelated insect species, Drosophila melanogaster (DmChd64) and Tribolium castaneum (TcChd64). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS), we showed that both Chd64 proteins have disordered tails that outflank the globular core. The folds of the globular cores of both Chd64 resemble the calponin homology (CH) domain previously resolved by crystallography. Monitoring the unfolding of DmChd64 and TcChd64 by far-ultraviolet (UV) circular dichroism (CD) spectroscopy, fluorescence spectroscopy and size-exclusion chromatography (SEC) revealed a highly complex process. Chd64 unfolds and forms of a molten globule (MG)—like intermediate state. Furthermore, our data indicate that in some conditions, Chd64 may exists in discrete structural forms, indicating that the protein is pliable and capable of easily acquiring different conformations. The plasticity of Chd64 and the existence of terminal intrinsically disordered regions (IDRs) may be crucial for multiple interactions with many partners. PMID:26325194

  1. Comparison of the meat quality and fatty acid composition of traditional fat-tailed (Chall) and tailed (Zel) Iranian sheep breeds.

    PubMed

    Yousefi, Ali Reza; Kohram, Hamid; Zare Shahneh, Ahmad; Nik-khah, Ali; Campbell, Anna W

    2012-12-01

    The aim of this study was to compare the meat quality of a traditional fat-tailed breed, Chall, to a tailed Iranian sheep breed, Zel. Lambs were grazed on pasture until weaning, and then were finished until slaughter at 10-12 months. Meat quality traits were measured on the longissimus dorsi (LD) muscle. Zel lambs accumulated more intramuscular fat (IMF) (p<0.01) and had lower shear force and drip loss than Chall lambs (p<0.05). The meat color of Zel lambs was higher for both a* (p<0.001) and b* (p<0.01) compared to Chall lambs. Meat from Zel lambs was more tender (p<0.01) and more juicy (p<0.05) than Chall lambs. The PUFA:SFA fatty acid ratio (P:S) was higher (p<0.05) and the n-6:n-3 PUFA ratio was lower in Chall compared to Zel lambs (p<0.05). Overall, these results show that the eating quality of Zel lambs was better, but that this was at the cost of less favorable fatty acid profiles and poorer meat color. PMID:22652069

  2. Binding the Atypical RA Domain of Ste50p to the Unfolded Opy2p Cytoplasmic Tail Is Essential for the High-Osmolarity Glycerol Pathway

    PubMed Central

    Ekiel, Irena; Sulea, Traian; Jansen, Gregor; Kowalik, Maria; Minailiuc, Ovidiu; Cheng, Jing; Harcus, Doreen; Cygler, Miroslaw; Whiteway, Malcolm

    2009-01-01

    Activation of the high-osmolarity glycerol (HOG) pathway for osmoregulation in the yeast Saccharomyces cerevisiae involves interaction of the adaptor Ste50p with the cytoplasmic tail of single-transmembrane protein Opy2p. We have determined the solution structure of the Ste50p-RA (Ras association) domain, and it shows an atypical RA fold lacking the β1 and β2 strands of the canonical motif. Although the core of the RA domain is fully functional in the pheromone response, an additional region is required for the HOG pathway activation. Two peptide motifs within the intrinsically disordered cytoplasmic tail of Opy2p defined by NMR spectroscopy physically interact with the Step50p-RA domain. These Opy2p-derived peptides bind overlapping regions of the Step50p-RA domain with similarly weak affinities, suggesting a multivalent interaction of these proteins as a crucial point of control of the HOG pathway. As well, overall selection of signaling pathways depends on functionally distinct regions of the Ste50p-RA domain, implicating this element in the control of global regulatory decisions. PMID:19846660

  3. The tail domain of myosin M catalyses nucleotide exchange on Rac1 GTPases and can induce actin-driven surface protrusions.

    PubMed

    Geissler, H; Ullmann, R; Soldati, T

    2000-05-01

    Members of the myosin superfamily play crucial roles in cellular processes including management of the cortical cytoskeleton, organelle transport and signal transduction. GTPases of the Rho family act as key control elements in the reorganization of the actin cytoskeleton in response to growth factors, and other functions such as membrane trafficking, transcriptional regulation, growth control and development. Here, we describe a novel unconventional myosin from Dictyostelium discoideum, MyoM. Primary sequence analysis revealed that it has the appearance of a natural chimera between a myosin motor domain and a guanine nucleotide exchange factor (GEF) domain for Rho GTPases. The functionality of both domains was established. Binding of the motor domain to F-actin was ATP-dependent and potentially regulated by phosphorylation. The GEF domain displayed selective activity on Rac1-related GTPases. Overexpression, rather than absence of MyoM, affected the cell morphology and viability. Particularly in response to hypo-osmotic stress, cells overexpressing the MyoM tail domain extended massive actin-driven protrusions. The GEF was enriched at the tip of growing protuberances, probably through its pleckstrin homology domain. MyoM is the first unconventional myosin containing an active Rac-GEF domain, suggesting a role at the interface of Rac-mediated signal transduction and remodeling of the actin cytoskeleton. PMID:11208126

  4. Use of poly(lactic acid) amendments to promote the bacterial fixation of metals in zinc smelter tailings.

    PubMed

    Edenborn, H M

    2004-04-01

    The ability of poly(lactic acid) (PLA) to serve as a long-term source of lactic acid for bacterial sulfate reduction activity in zinc smelter tailings was investigated. Solid PLA polymers mixed in water hydrolyzed abiotically to release lactic acid into solution over an extended period of time. The addition of both PLA and gypsum was required for indigenous bacteria to lower redox potential, raise pH, and stimulate sulfate reduction activity in highly oxidized smelter tailings after one year of treatment. Bioavailable cadmium, copper, lead and zinc were all lowered significantly in PLA/gypsum treated soil, but PLA amendments alone increased the bioavailability of lead, nickel and zinc. Similar PLA amendments may be useful in constructed wetlands and reactive barrier walls for the passive treatment of mine drainage, where enhanced rates of bacterial sulfate reduction are desirable.

  5. Elongation of the Poly-γ-glutamate Tail of F420 Requires Both Domains of the F420:γ-Glutamyl Ligase (FbiB) of Mycobacterium tuberculosis.

    PubMed

    Bashiri, Ghader; Rehan, Aisyah M; Sreebhavan, Sreevalsan; Baker, Heather M; Baker, Edward N; Squire, Christopher J

    2016-03-25

    Cofactor F420is an electron carrier with a major role in the oxidoreductive reactions ofMycobacterium tuberculosis, the causative agent of tuberculosis. A γ-glutamyl ligase catalyzes the final steps of the F420biosynthesis pathway by successive additions ofl-glutamate residues to F420-0, producing a poly-γ-glutamate tail. The enzyme responsible for this reaction in archaea (CofE) comprises a single domain and produces F420-2 as the major species. The homologousM. tuberculosisenzyme, FbiB, is a two-domain protein and produces F420with predominantly 5-7l-glutamate residues in the poly-γ-glutamate tail. The N-terminal domain of FbiB is homologous to CofE with an annotated γ-glutamyl ligase activity, whereas the C-terminal domain has sequence similarity to an FMN-dependent family of nitroreductase enzymes. Here we demonstrate that full-length FbiB adds multiplel-glutamate residues to F420-0in vitroto produce F420-5 after 24 h; communication between the two domains is critical for full γ-glutamyl ligase activity. We also present crystal structures of the C-terminal domain of FbiB in apo-, F420-0-, and FMN-bound states, displaying distinct sites for F420-0 and FMN ligands that partially overlap. Finally, we discuss the features of a full-length structural model produced by small angle x-ray scattering and its implications for the role of N- and C-terminal domains in catalysis. PMID:26861878

  6. Localization of Daucus carota NMCP1 to the nuclear periphery: the role of the N-terminal region and an NLS-linked sequence motif, RYNLRR, in the tail domain.

    PubMed

    Kimura, Yuta; Fujino, Kaien; Ogawa, Kana; Masuda, Kiyoshi

    2014-01-01

    Recent ultrastructural studies revealed that a structure similar to the vertebrate nuclear lamina exists in the nuclei of higher plants. However, plant genomes lack genes for lamins and intermediate-type filament proteins, and this suggests that plant-specific nuclear coiled-coil proteins make up the lamina-like structure in plants. NMCP1 is a protein, first identified in Daucus carota cells, that localizes exclusively to the nuclear periphery in interphase cells. It has a tripartite structure comprised of head, rod, and tail domains, and includes putative nuclear localization signal (NLS) motifs. We identified the functional NLS of DcNMCP1 (carrot NMCP1) and determined the protein regions required for localizing to the nuclear periphery using EGFP-fused constructs transiently expressed in Apium graveolens epidermal cells. Transcription was driven under a CaMV35S promoter, and the genes were introduced into the epidermal cells by a DNA-coated microprojectile delivery system. Of the NLS motifs, KRRRK and RRHK in the tail domain were highly functional for nuclear localization. Addition of the N-terminal 141 amino acids from DcNMCP1 shifted the localization of a region including these NLSs from the entire nucleus to the nuclear periphery. Using this same construct, the replacement of amino acids in RRHK or its preceding sequence, YNL, with alanine residues abolished localization to the nuclear periphery, while replacement of KRRRK did not affect localization. The sequence R/Q/HYNLRR/H, including YNL and the first part of the sequence of RRHK, is evolutionarily conserved in a subclass of NMCP1 sequences from many plant species. These results show that NMCP1 localizes to the nuclear periphery by a combined action of a sequence composed of R/Q/HYNLRR/H, NLS, and the N-terminal region including the head and a portion of the rod domain, suggesting that more than one binding site is implicated in localization of NMCP1.

  7. Crystal structure of FAS thioesterase domain with polyunsaturated fatty acyl adduct and inhibition by dihomo-[gamma]-linolenic acid

    SciTech Connect

    Zhang, Wei; Chakravarty, Bornali; Zheng, Fei; Gu, Ziwei; Wu, Hongmei; Mao, Jianqiang; Wakil, Salih J.; Quiocho, Florante A.

    2012-05-29

    Human fatty acid synthase (hFAS) is a homodimeric multidomain enzyme that catalyzes a series of reactions leading to the de novo biosynthesis of long-chain fatty acids, mainly palmitate. The carboxy-terminal thioesterase (TE) domain determines the length of the fatty acyl chain and its ultimate release by hydrolysis. Because of the upregulation of hFAS in a variety of cancers, it is a target for antiproliferative agent development. Dietary long-chain polyunsaturated fatty acids (PUFAs) have been known to confer beneficial effects on many diseases and health conditions, including cancers, inflammations, diabetes, and heart diseases, but the precise molecular mechanisms involved have not been elucidated. We report the crystal structure of the hFAS TE domain covalently modified and inactivated by methyl {gamma}-linolenylfluorophosphonate. Whereas the structure confirmed the phosphorylation by the phosphonate head group of the active site serine, it also unexpectedly revealed the binding of the 18-carbon polyunsaturated {gamma}-linolenyl tail in a long groove-tunnel site, which itself is formed mainly by the emergence of an {alpha} helix (the 'helix flap'). We then found inhibition of the TE domain activity by the PUFA dihomo-{gamma}-linolenic acid; {gamma}- and {alpha}-linolenic acids, two popular dietary PUFAs, were less effective. Dihomo-{gamma}-linolenic acid also inhibited fatty acid biosynthesis in 3T3-L1 preadipocytes and selective human breast cancer cell lines, including SKBR3 and MDAMB231. In addition to revealing a novel mechanism for the molecular recognition of a polyunsaturated fatty acyl chain, our results offer a new framework for developing potent FAS inhibitors as therapeutics against cancers and other diseases.

  8. Soil acidity determines the effectiveness of an organic amendment and a native bacterium for increasing soil stabilisation in semiarid mine tailings.

    PubMed

    Carrasco, L; Caravaca, F; Azcón, R; Roldán, A

    2009-01-01

    Unstable mine tailings are vulnerable to water and air erosion, so it is important to promote their surface stabilisation in order to avoid the spread of heavy metals. In a greenhouse experiment, we assessed the effect of the addition of Aspergillus niger-treated sugar beet waste and inoculation with a native bacterium, Bacillus cereus, on the stabilisation of soil aggregates of two acidic, semiarid mine tailings, with different acidity degree, during watering and drying periods. Organic amendment raised the pH of both the moderately and highly acidic tailings, whereas the bacterial inoculation increased this parameter in the former. Only the amendment addition increased soil water-soluble carbon in both tailings compared with their controls, under either watering or drying conditions. Both the amendment and B. cereus enhanced water-soluble carbohydrates. Both treatments increased dehydrogenase activity and aggregate stability, particularly in the moderately acidic tailing under drying conditions. After soil drying, aggregate stability was increased by the amendment (about 66% higher than the control soil) and by the bacterium (about 45% higher than the control soil) in the moderately acidic tailing. The effectiveness of these treatments as structure-stabilisation methods for degraded, semiarid mine ecosystems appears to be restricted to tailings of moderate acidity. PMID:18954889

  9. Soil acidity determines the effectiveness of an organic amendment and a native bacterium for increasing soil stabilisation in semiarid mine tailings.

    PubMed

    Carrasco, L; Caravaca, F; Azcón, R; Roldán, A

    2009-01-01

    Unstable mine tailings are vulnerable to water and air erosion, so it is important to promote their surface stabilisation in order to avoid the spread of heavy metals. In a greenhouse experiment, we assessed the effect of the addition of Aspergillus niger-treated sugar beet waste and inoculation with a native bacterium, Bacillus cereus, on the stabilisation of soil aggregates of two acidic, semiarid mine tailings, with different acidity degree, during watering and drying periods. Organic amendment raised the pH of both the moderately and highly acidic tailings, whereas the bacterial inoculation increased this parameter in the former. Only the amendment addition increased soil water-soluble carbon in both tailings compared with their controls, under either watering or drying conditions. Both the amendment and B. cereus enhanced water-soluble carbohydrates. Both treatments increased dehydrogenase activity and aggregate stability, particularly in the moderately acidic tailing under drying conditions. After soil drying, aggregate stability was increased by the amendment (about 66% higher than the control soil) and by the bacterium (about 45% higher than the control soil) in the moderately acidic tailing. The effectiveness of these treatments as structure-stabilisation methods for degraded, semiarid mine ecosystems appears to be restricted to tailings of moderate acidity.

  10. Characterization and quantification of mining-related "naphthenic acids" in groundwater near a major oil sands tailings pond.

    PubMed

    Ahad, Jason M E; Pakdel, Hooshang; Savard, Martine M; Calderhead, Angus I; Gammon, Paul R; Rivera, Alfonso; Peru, Kerry M; Headley, John V

    2013-05-21

    The high levels of acid extractable organics (AEOs) containing naphthenic acids (NAs) found in oil sands process-affected waters (OSPW) are a growing concern in monitoring studies of aquatic ecosystems in the Athabasca oil sands region. The complexity of these compounds has substantially hindered their accurate analysis and quantification. Using a recently developed technique which determines the intramolecular carbon isotope signature of AEOs generated by online pyrolysis (δ(13)Cpyr), natural abundance radiocarbon, and high resolution Orbitrap mass spectrometry analyses, we evaluated the sources of AEOs along a groundwater flow path from a major oil sands tailings pond to the Athabasca River. OSPW was characterized by a δ(13)Cpyr value of approximately -21‰ and relatively high proportions of O₂ and O₂S species classes. In contrast, AEO samples located furthest down-gradient from the tailings pond and from the Athabasca River were characterized by a δ(13)Cpyr value of around -29‰, a greater proportion of highly oxygenated and N-containing compound classes, and a significant component of nonfossil and, hence, non-bitumen-derived carbon. The groundwater concentrations of mining-related AEOs determined using a two end-member isotopic mass balance were between 1.6 and 9.3 mg/L lower than total AEO concentrations, implying that a less discriminating approach to quantification would have overestimated subsurface levels of OSPW. This research highlights the need for accurate characterization of "naphthenic acids" in order to quantify potential seepage from tailings ponds. PMID:23607666

  11. Characterization and quantification of mining-related "naphthenic acids" in groundwater near a major oil sands tailings pond.

    PubMed

    Ahad, Jason M E; Pakdel, Hooshang; Savard, Martine M; Calderhead, Angus I; Gammon, Paul R; Rivera, Alfonso; Peru, Kerry M; Headley, John V

    2013-05-21

    The high levels of acid extractable organics (AEOs) containing naphthenic acids (NAs) found in oil sands process-affected waters (OSPW) are a growing concern in monitoring studies of aquatic ecosystems in the Athabasca oil sands region. The complexity of these compounds has substantially hindered their accurate analysis and quantification. Using a recently developed technique which determines the intramolecular carbon isotope signature of AEOs generated by online pyrolysis (δ(13)Cpyr), natural abundance radiocarbon, and high resolution Orbitrap mass spectrometry analyses, we evaluated the sources of AEOs along a groundwater flow path from a major oil sands tailings pond to the Athabasca River. OSPW was characterized by a δ(13)Cpyr value of approximately -21‰ and relatively high proportions of O₂ and O₂S species classes. In contrast, AEO samples located furthest down-gradient from the tailings pond and from the Athabasca River were characterized by a δ(13)Cpyr value of around -29‰, a greater proportion of highly oxygenated and N-containing compound classes, and a significant component of nonfossil and, hence, non-bitumen-derived carbon. The groundwater concentrations of mining-related AEOs determined using a two end-member isotopic mass balance were between 1.6 and 9.3 mg/L lower than total AEO concentrations, implying that a less discriminating approach to quantification would have overestimated subsurface levels of OSPW. This research highlights the need for accurate characterization of "naphthenic acids" in order to quantify potential seepage from tailings ponds.

  12. Determining Phthalic Acid Esters Using Terahertz Time Domain Spectroscopy

    NASA Astrophysics Data System (ADS)

    Liu, L.; Shen, L.; Yang, F.; Han, F.; Hu, P.; Song, M.

    2016-09-01

    In this report terahertz time domain spectroscopy (THz-TDS) is applied for determining phthalic acid esters (PAEs) in standard materials. We reported the THz transmission spectrum in the frequency range of 0.2 to 2.0 THz for three PAEs: di-n-butyl phthalate (DBP), di-isononyl phthalate (DINP), and di-2-ethylhexyl phthalate ester (DEHP). The study provided the refractive indices and absorption features of these materials. The absorption spectra of three PAEs were simulated by using Gaussian software with Density Functional Theory (DFT) methods. For pure standard PAEs, the values of the refractive indices changed between 1.50 and 1.60. At 1.0 THz, the refractive indices were 1.524, 1.535, and 1.563 for DINP, DEHP, and DBP, respectively. In this experiment different concentrations of DBP were investigated using THz-TDS. Changes were measured in the low THz frequency range for refractive indices and characteristic absorption. The results indicated that THz-TDS is promising as a new method in determining PAEs in many materials. The results of this study could be used to support the practical application of THz-TDS in quality detection and food monitoring. In particular, this new technique could be used in detecting hazardous materials and other substances present in wine or foods.

  13. Regulation of the electric charge in phosphatidic acid domains.

    PubMed

    Wang, Wenjie; Anderson, Nathaniel A; Travesset, Alex; Vaknin, David

    2012-06-21

    Although a minor component of the lipidome, phosphatidic acid (PA) plays a crucial role in nearly all signaling pathways involving cell membranes, in part because of its variable electrical charge in response to environmental conditions. To investigate how charge is regulated in domains of PA, we applied surface-sensitive X-ray reflectivity and fluorescence near-total-reflection techniques to determine the binding of divalent ions (Ca(2+) at various pH values) to 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA) and to the simpler lipid dihexadecyl phosphate (DHDP) spread as monolayers at the air/water interface. We found that the protonation state of PA is controlled not only by the pK(a) and local pH but also by the strong affinity to PA driven by electrostatic correlations from divalent ions and the cooperative effect of the two dissociable protons, which dramatically enhance the surface charge. A precise theoretical model is presented providing a general framework to predict the protonation state of PA. Implications for recent experiments on charge regulation by hydrogen bonding and the role of pH in PA signaling are discussed in detail.

  14. Bio-physicochemical effects of gamma irradiation treatment for naphthenic acids in oil sands fluid fine tailings.

    PubMed

    Boudens, Ryan; Reid, Thomas; VanMensel, Danielle; Prakasan M R, Sabari; Ciborowski, Jan J H; Weisener, Christopher G

    2016-01-01

    Naphthenic acids (NAs) are persistent compounds that are components of most petroleum, including those found in the Athabasca oil sands. Their presence in freshly processed tailings is of significant environmental concern due to their toxicity to aquatic organisms. Gamma irradiation (GI) was used to reduce the toxicity and concentration of NAs in oil sands process water (OSPW) and fluid fine tailings (FFT). This investigation systematically studied the impact of GI on the biogeochemical development and progressive reduction of toxicity using laboratory incubations of fresh and aged tailings under anoxic and oxic conditions. GI reduced NA concentrations in OSPW by up to 97% in OSPW and in FFT by 85%. The GI-treated FFT exhibited increased rates of biogeochemical change, dependent on the age of the tailings source. Dissolved oxygen (DO) flux was enhanced in GI-treated FFT from fresh and aged source materials, whereas hydrogen sulfide (HS(-)) flux was stimulated only in the fresh FFT. Acute toxicity to Vibrio fischeri was immediately reduced following GI treatment of fresh OSPW. GI treatment followed by 4-week incubation reduced toxicity of aged OSPW to V. fischeri. PMID:26356184

  15. Determination of thermodynamic and transport parameters of naphthenic acids and organic process chemicals in oil sand tailings pond water.

    PubMed

    Wang, Xiaomeng; Robinson, Lisa; Wen, Qing; Kasperski, Kim L

    2013-07-01

    Oil sand tailings pond water contains naphthenic acids and process chemicals (e.g., alkyl sulphates, quaternary ammonium compounds, and alkylphenol ethoxylates). These chemicals are toxic and can seep through the foundation of the tailings pond to the subsurface, potentially affecting the quality of groundwater. As a result, it is important to measure the thermodynamic and transport parameters of these chemicals in order to study the transport behavior of contaminants through the foundation as well as underground. In this study, batch adsorption studies and column experiments were performed. It was found that the transport parameters of these chemicals are related to their molecular structures and other properties. The computer program (CXTFIT) was used to further evaluate the transport process in the column experiments. The results from this study show that the transport of naphthenic acids in a glass column is an equilibrium process while the transport of process chemicals seems to be a non-equilibrium process. At the end of this paper we present a real-world case study in which the transport of the contaminants through the foundation of an external tailings pond is calculated using the lab-measured data. The results show that long-term groundwater monitoring of contaminant transport at the oil sand mining site may be necessary to avoid chemicals from reaching any nearby receptors. PMID:23736740

  16. Determination of thermodynamic and transport parameters of naphthenic acids and organic process chemicals in oil sand tailings pond water.

    PubMed

    Wang, Xiaomeng; Robinson, Lisa; Wen, Qing; Kasperski, Kim L

    2013-07-01

    Oil sand tailings pond water contains naphthenic acids and process chemicals (e.g., alkyl sulphates, quaternary ammonium compounds, and alkylphenol ethoxylates). These chemicals are toxic and can seep through the foundation of the tailings pond to the subsurface, potentially affecting the quality of groundwater. As a result, it is important to measure the thermodynamic and transport parameters of these chemicals in order to study the transport behavior of contaminants through the foundation as well as underground. In this study, batch adsorption studies and column experiments were performed. It was found that the transport parameters of these chemicals are related to their molecular structures and other properties. The computer program (CXTFIT) was used to further evaluate the transport process in the column experiments. The results from this study show that the transport of naphthenic acids in a glass column is an equilibrium process while the transport of process chemicals seems to be a non-equilibrium process. At the end of this paper we present a real-world case study in which the transport of the contaminants through the foundation of an external tailings pond is calculated using the lab-measured data. The results show that long-term groundwater monitoring of contaminant transport at the oil sand mining site may be necessary to avoid chemicals from reaching any nearby receptors.

  17. The Effect of Lactic Acid Bacteria-fermented Soybean Milk Products on Carrageenan-induced Tail Thrombosis in Rats

    PubMed Central

    KAMIYA, Seitaro; OGASAWARA, Masayoshi; ARAKAWA, Masayuki; HAGIMORI, Masayori

    2013-01-01

    Thrombosis is characterized by congenital and acquired procatarxis. Lactic acid bacteria-fermented soybean milk products (FS-LAB) inhibit hepatic lipid accumulation and prevent atherosclerotic plaque formation. However, the therapeutic efficacy of FS-LAB against thrombosis has yet to be investigated. In this study, FS-LAB were administered subcutaneously into the tails of rats, with the subsequent intravenous administration of κ-carrageenan 12 hr after the initial injection. In general, administration of κ-carrageenan induces thrombosis. The length of the infarcted tail regions was significantly shorter in the rats administered a single-fold or double-fold concentration of the FS-LAB solution compared with the region in control rats. Therefore, FS-LAB exhibited significant antithrombotic effects. Our study is the first to characterize the properties of FS-LAB and, by testing their efficacy on an in vivo rat model of thrombosis, demonstrate the potency of their antithrombotic effect. PMID:24936368

  18. Geochemical characterisation of seepage and drainage water quality from two sulphide mine tailings impoundments: Acid mine drainage versus neutral mine drainage

    USGS Publications Warehouse

    Heikkinen, P.M.; Raisanen, M.L.; Johnson, R.H.

    2009-01-01

    Seepage water and drainage water geochemistry (pH, EC, O2, redox, alkalinity, dissolved cations and trace metals, major anions, total element concentrations) were studied at two active sulphide mine tailings impoundments in Finland (the Hitura Ni mine and Luikonlahti Cu mine/talc processing plant). The data were used to assess the factors influencing tailings seepage quality and to identify constraints for water treatment. Changes in seepage water quality after equilibration with atmospheric conditions were evaluated based on geochemical modelling. At Luikonlahti, annual and seasonal changes were also studied. Seepage quality was largely influenced by the tailings mineralogy, and the serpentine-rich, low sulphide Hitura tailings produced neutral mine drainage with high Ni. In contrast, drainage from the high sulphide, multi-metal tailings of Luikonlahti represented typical acid mine drainage with elevated contents of Zn, Ni, Cu, and Co. Other factors affecting the seepage quality included weathering of the tailings along the seepage flow path, process water input, local hydrological settings, and structural changes in the tailings impoundment. Geochemical modelling showed that pH increased and some heavy metals were adsorbed to Fe precipitates after net alkaline waters equilibrated with the atmosphere. In the net acidic waters, pH decreased and no adsorption occurred. A combination of aerobic and anaerobic treatments is proposed for Hitura seepages to decrease the sulphate and metal loading. For Luikonlahti, prolonged monitoring of the seepage quality is suggested instead of treatment, since the water quality is still adjusting to recent modifications to the tailings impoundment.

  19. Inhibition of acid mine drainage and immobilization of heavy metals from copper flotation tailings using a marble cutting waste

    NASA Astrophysics Data System (ADS)

    Tozsin, Gulsen

    2016-01-01

    Acid mine drainage (AMD) with high concentrations of sulfates and metals is generated by the oxidation of sulfide bearing wastes. CaCO3-rich marble cutting waste is a residual material produced by the cutting and polishing of marble stone. In this study, the feasibility of using the marble cutting waste as an acid-neutralizing agent to inhibit AMD and immobilize heavy metals from copper flotation tailings (sulfide- bearing wastes) was investigated. Continuous-stirring shake-flask tests were conducted for 40 d, and the pH value, sulfate content, and dissolved metal content of the leachate were analyzed every 10 d to determine the effectiveness of the marble cutting waste as an acid neutralizer. For comparison, CaCO3 was also used as a neutralizing agent. The average pH value of the leachate was 2.1 at the beginning of the experiment ( t = 0). In the experiment employing the marble cutting waste, the pH value of the leachate changed from 6.5 to 7.8, and the sulfate and iron concentrations decreased from 4558 to 838 mg/L and from 536 to 0.01 mg/L, respectively, after 40 d. The marble cutting waste also removed more than 80wt% of heavy metals (Cd, Cr, Cu, Ni, Pb, and Zn) from AMD generated by copper flotation tailings.

  20. Structural and Functional Studies of gpX of Escherichia coli Phage P2 Reveal a Widespread Role for LysM Domains in the Baseplates of Contractile-Tailed Phages

    PubMed Central

    Fatehi Hassanabad, Mostafa; Chang, Tom; Pirani, Nawaz; Bona, Diane; Edwards, Aled M.

    2013-01-01

    A variety of bacterial pathogenicity determinants, including the type VI secretion system and the virulence cassettes from Photorhabdus and Serratia, share an evolutionary origin with contractile-tailed myophages. The well-characterized Escherichia coli phage P2 provides an excellent system for studies related to these systems, as its protein composition appears to represent the “minimal” myophage tail. In this study, we used nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of gpX, a 68-residue tail baseplate protein. Although the sequence and structure of gpX are similar to those of LysM domains, which are a large family associated with peptidoglycan binding, we did not detect a peptidoglycan-binding activity for gpX. However, bioinformatic analysis revealed that half of all myophages, including all that possess phage T4-like baseplates, encode a tail protein with a LysM-like domain, emphasizing a widespread role for this domain in baseplate function. While phage P2 gpX comprises only a single LysM domain, many myophages display LysM domain fusions with other tail proteins, such as the DNA circulation protein found in Mu-like phages and gp53 of T4-like phages. Electron microscopy of P2 phage particles with an incorporated gpX-maltose binding protein fusion revealed that gpX is located at the top of the baseplate, near the junction of the baseplate and tail tube. gpW, the orthologue of phage T4 gp25, was also found to localize to this region. A general colocalization of LysM-like domains and gpW homologues in diverse phages is supported by our bioinformatic analysis. PMID:24097944

  1. Contact of clay-liner materials with acidic tailings. II. Chemical modeling

    SciTech Connect

    Peterson, S.R.; Krupka, K.M.

    1981-09-01

    The ion speciation-solubility model WATEQ3 was used to model original aqueous solutions and solutions resulting from liner materials contacted with uranium mill tailings, synthetic mill tailings or H/sub 2/SO/sub 4/. The modeling results indicate solution species which are in apparent equilibrium with respect to particular solids. These solids provide potential solubility controls for their corresponding dissolved constituents. The disequilibrium indices computed by WATEQ3 indicate amorphic Fe(OH)/sub 3/(A), Al0HO/sub 4/, alunite (KA1/sub 3/(SO/sub 4/)/sub 2/(OH)/sub 6/), gypsum (CaSO/sub 4/ . 2H/sub 2/O), celestite (SrSO/sub 4/), anglesite (PbSO/sub 4/) and MnHPO/sub 4/ may have precipitated in the contacted liner materials and may also provide solubility controls for their dissolved constituents. The disequilibrium indices also show that the solutions resulting from the interaction of Highland Mill tailings are oversaturated with K-, H-, and Na-jarosites ((K,H,Na)Fe/sub 3/(SO/sub 4/)/sub 2/(OH)/sub 6/). Because jarosite has been identified by x-ray diffraction as a precipitate in these reacted liner materials, it would appear that there is a kinetic barrier which prohibits jarosite from being an effective solubility control. Results of this study also show that the solubilities of many solid phases were pH dependent. This exploratory use of geochemical modeling has demonstrated its capability to test solubility hypotheses for clay liners reacted with tailings solutions and to guide the analyses of important constituents and parameters for these solutions. Geochemical modeling can be used, in parallel with characterization techniques for the solid phases, to support the presence of the solid phase and to guide the search for further solid phases. Geochemical modeling is also an effective tool in delineating the chemical causes for changes in permeability of liner materials.

  2. Crystallogenesis of bacteriophage P22 tail accessory factor gp26 at acidic and neutral pH

    SciTech Connect

    Cingolani, Gino Andrews, Dewan; Casjens, Sherwood

    2006-05-01

    The crystallogenesis of bacteriophage P22 tail-fiber gp26 is described. To study possible pH-induced conformational changes in gp26 structure, native trimeric gp26 has been crystallized at acidic pH (4.6) and a chimera of gp26 fused to maltose-binding protein (MBP-gp26) has been crystallized at neutral and alkaline pH (7-10). Gp26 is one of three phage P22-encoded tail accessory factors essential for stabilization of viral DNA within the mature capsid. In solution, gp26 exists as an extended triple-stranded coiled-coil protein which shares profound structural similarities with class I viral membrane-fusion protein. In the cryo-EM reconstruction of P22 tail extracted from mature virions, gp26 forms an ∼220 Å extended needle structure emanating from the neck of the tail, which is likely to be brought into contact with the cell’s outer membrane when the viral DNA-injection process is initiated. To shed light on the potential role of gp26 in cell-wall penetration and DNA injection, gp26 has been crystallized at acidic, neutral and alkaline pH. Crystals of native gp26 grown at pH 4.6 diffract X-rays to 2.0 Å resolution and belong to space group P2{sub 1}, with a dimer of trimeric gp26 molecules in the asymmetric unit. To study potential pH-induced conformational changes in the gp26 structure, a chimera of gp26 fused to maltose-binding protein (MBP-gp26) was generated. Hexagonal crystals of MBP-gp26 were obtained at neutral and alkaline pH using the high-throughput crystallization robot at the Hauptman–Woodward Medical Research Institute, Buffalo, NY, USA. These crystals diffract X-rays to beyond 2.0 Å resolution. Structural analysis of gp26 crystallized at acidic, neutral and alkaline pH is in progress.

  3. A sting in the tail: the N-terminal domain of the androgen receptor as a drug target

    PubMed Central

    Monaghan, Amy E; McEwan, Iain J

    2016-01-01

    The role of androgen receptor (AR) in the initiation and progression of prostate cancer (PCa) is well established. Competitive inhibition of the AR ligand-binding domain (LBD) has been the staple of antiandrogen therapies employed to combat the disease in recent years. However, their efficacy has often been limited by the emergence of resistance, mediated through point mutations, and receptor truncations. As a result, the prognosis for patients with malignant castrate resistant disease remains poor. The amino-terminal domain (NTD) of the AR has been shown to be critical for AR function. Its modular activation function (AF-1) is important for both gene regulation and participation in protein-protein interactions. However, due to the intrinsically disordered structure of the domain, its potential as a candidate for therapeutic intervention has been dismissed in the past. The recent emergence of the small molecule EPI-001 has provided evidence that AR-NTD can be targeted therapeutically, independent of the LBD. Targeting of AR-NTD has the potential to disrupt multiple intermolecular interactions between AR and its coregulatory binding partners, in addition to intramolecular cross-talk between the domains of the AR. Therapeutics targeting these protein-protein interactions or NTD directly should also have efficacy against emerging AR splice variants which may play a role in PCa progression. This review will discuss the role of intrinsic disorder in AR function and illustrate how emerging therapies might target NTD in PCa. PMID:27212126

  4. In situ biodegradation of naphthenic acids in oil sands tailings pond water using indigenous algae-bacteria consortium.

    PubMed

    Mahdavi, Hamed; Prasad, Vinay; Liu, Yang; Ulrich, Ania C

    2015-01-01

    In this study, the biodegradation of total acid-extractable organics (TAOs), commonly called naphthenic acids (NAs), was investigated. An indigenous microbial culture containing algae and bacteria was taken from the surface of a tailings pond and incubated over the course of 120days. The influence of light, oxygen and the presence of indigenous algae and bacteria, and a diatom (Navicula pelliculosa) on the TAO removal rate were elucidated. The highest biodegradation rate was observed with bacteria growth only (without light exposure) with a half-life (t(1/2)) of 203days. The algae-bacteria consortium enhanced the detoxification process, however, bacterial biomass played the main role in toxicity reduction. Principal component analysis (PCA) conducted on FT-IR spectra, identified functional groups and bonds (representing potential markers for biotransformation of TAOs) as follows: hydroxyl, carboxyl and amide groups along with CH, arylH, arylOH and NH bonds.

  5. TACN-based cationic lipids with amino acid backbone and double tails: materials for non-viral gene delivery.

    PubMed

    Wang, Bing; Yi, Wen-Jing; Zhang, Ji; Zhang, Qin-Fang; Xun, Miao-Miao; Yu, Xiao-Qi

    2014-04-01

    Cationic lipids have become an efficient type of non-viral vectors for gene delivery. In this Letter, four cationic lipids containing 1,4,7-triazacyclononane (TACN) headgroup, glutamic/aspartic acid backbone and dioleyl tails were designed and synthesized. The TACN headgroup gives these lipids excellent pH buffering capacities, which were higher than branched 25 kDa PEI. Cationic liposomes prepared from these lipids and DOPE showed good DNA affinity, and full DNA condensation was found at N/P ratio of 3 via agarose gel electrophoresis. The lipoplexes were characterized by dynamic light scattering (DLS) assay, which gave proper particle sizes and zeta-potentials for transfection. In vitro gene transfection results in two cell lines reveal that TAN (with aspartic acid and amide bond in the structure) shows the best transfection efficiency, which is close to commercially available transfection agent Lipofectamine 2000.

  6. Improvement on the thermal stability and activity of plant cytosolic ascorbate peroxidase 1 by tailing hyper-acidic fusion partners.

    PubMed

    Zhang, Mengru; Gong, Ming; Yang, Yumei; Li, Xujuan; Wang, Haibo; Zou, Zhurong

    2015-04-01

    Cytosolic ascorbate peroxidase 1 (APX1) plays a crucial role in regulating the level of plant cellular reactive oxygen species and its thermolability is proposed to cause plant heat-susceptibility. Herein, several hyper-acidic fusion partners, such as the C-terminal peptide tails, were evaluated for their effects on the thermal stability and activity of APX1 from Jatropha curcas and Arabidopsis. The hyper-acidic fusion partners efficiently improved the thermostability and prevented thermal inactivation of APX1 in both plant species with an elevated heat tolerance of at least 2 °C. These hyper-acidified thermostable APX1 fusion variants are of considerable biotechnological potential and can provide a new route to enhance the heat tolerance of plant species especially of inherent thermo-sensitivity.

  7. Complete thermodynamic and kinetic characterization of the isomer-specific interaction between Pin1-WW domain and the amyloid precursor protein cytoplasmic tail phosphorylated at Thr668.

    PubMed

    De, Soumya; Greenwood, Alexander I; Rogals, Monique J; Kovrigin, Evgenii L; Lu, Kun Ping; Nicholson, Linda K

    2012-10-30

    Peptidyl prolyl cis-trans isomerization acts as an effective molecular timer that plays significant roles in biological and pathological processes. Enzymes such as Pin1 catalyze cis-trans isomerization, accelerating the otherwise slow isomerization rate into time scales relevant for cellular signaling. Here we have combined NMR line shape analysis, fluorescence spectroscopy, and isothermal titration calorimetry to determine the kinetic and thermodynamic parameters describing the trans-specific interaction between the binding domain of Pin1 (WW domain) and a key cis-trans molecular switch in the amyloid precursor protein cytoplasmic tail. A three-state model, in which the cis-trans isomerization equilibrium is coupled to the binding equilibrium through the trans isomer, was found to fit the data well. The trans isomer binds the WW domain with ∼22 μM affinity via very fast association (approaching the diffusion limit) and dissociation rates. The common structural and electrostatic characteristics of Pin1 substrates, which contain a phosphorylated serine/threonine-proline motif, suggest that very rapid binding kinetics are a general feature of Pin1 interactions with other substrates. The fast binding kinetics of the WW domain allows rapid response of Pin1 to the dynamic events of phosphorylation and dephosphorylation in the cell that alter the relative populations of diverse Pin1 substrates. Furthermore, our results also highlight the vastly different rates at which slow uncatalyzed cis-trans isomerization and fast isomer-specific binding events occur. These results, along with the experimental methods presented herein, should guide future experiments aimed at the thermodynamic and kinetic characterization of cis-trans molecular switches and isomer-specific interactions involved in various biological processes.

  8. Activation of transcription by PU.1 requires both acidic and glutamine domains.

    PubMed Central

    Klemsz, M J; Maki, R A

    1996-01-01

    The B-lymphocyte- and macrophage-specific transcription factor PU.1 is a member of the ets family of proteins. To understand how PU.1 functions as a transcription factor, we initiated a series of experiments to define its activation domain. Using deletion analysis, we showed that the activation domain of PU.1 is located in the amino-terminal half of the protein. Within this region, we identified three acidic subdomains and one glutamine-rich subdomain. The deletion of any of these subdomains resulted in a significant loss in the ability of PU.1 to transactivate in cotransfection studies. Amino acid substitution analysis showed that the activation of transcription by PU.1 requires acidic residues between amino acids 7 and 74 and a group of glutamine residues between amino acids 75 and 84. These data show that PU.1 contains two types of known activation domains and that both are required for maximal transactivation. PMID:8524320

  9. Enumeration of Thiobacilli within pH-Neutral and Acidic Mine Tailings and Their Role in the Development of Secondary Mineral Soil

    PubMed Central

    Southam, G.; Beveridge, T. J.

    1992-01-01

    The Lemoine tailings of Chibougamau, Quebec, Canada, were deposited as a pH-neutral mineral conglomerate consisting of aluminum-silicates, iron-aluminum-silicates, pyrite, chalcopyrite, and sphalerite. These tailings are colonized by an active population of Thiobacillus ferrooxidans which is localized to an acid zone occupying 40% of the tailings' surface. This population peaked at 7 × 108 most probable number per gram of tailings during July and August 1990 and extended to a depth of 40 cm from the surface. Examination of samples over this depth profile by transmission electron microscopy and electron dispersive spectroscopy revealed a microbially mediated mineral transition from sulfides (below 40 cm) to chlorides and phosphates (at the surface). Silicate minerals were unaltered by microbial action. Transmission electron microscopy showed a tight association between Thiobacillus species and the sulfide minerals, which helps account for their prominence in tailings environments. Accurate enumeration of T. ferrooxidans from tailings required the disruption of their bonding to the mineral interface. Vortexing of a 10% aqueous suspension of the tailings material prior to most-probable-number analysis best facilitated this release. Even though heavy metals were highly mobile under acidic conditions at the Lemoine tailings, it was evident by transmission electron microscopy and electron dispersive spectroscopy that they were being immobilized as bona fide fine-grain minerals containing iron, copper, chlorine, phosphorus, and oxygen on bacterial surfaces and exopolymers. This biomineralization increased with increasing bacterial numbers and was most evident in the upper 3 cm of the acidic zone. Images PMID:16348721

  10. Structures of a minimal human CFTR first nucleotide-binding domain as a monomer, head-to-tail homodimer, and pathogenic mutant

    SciTech Connect

    Atwell, Shane; Brouillette, Christie G.; Conners, Kris; Emtage, Spencer; Gheyi, Tarun; Guggino, William B.; Hendle, Jorg; Hunt, John F.; Lewis, Hal A.; Lu, Frances; Protasevich, Irina I.; Rodgers, Logan A.; Romero, Rich; Wasserman, Stephen R.; Weber, Patricia C.; Wetmore, Diana; Zhang, Feiyu F.; Zhao, Xun

    2010-04-26

    Upon removal of the regulatory insert (RI), the first nucleotide binding domain (NBD1) of human cystic fibrosis transmembrane conductance regulator (CFTR) can be heterologously expressed and purified in a form that remains stable without solubilizing mutations, stabilizing agents or the regulatory extension (RE). This protein, NBD1 387-646({Delta}405-436), crystallizes as a homodimer with a head-to-tail association equivalent to the active conformation observed for NBDs from symmetric ATP transporters. The 1.7-{angstrom} resolution X-ray structure shows how ATP occupies the signature LSGGQ half-site in CFTR NBD1. The {Delta}F508 version of this protein also crystallizes as a homodimer and differs from the wild-type structure only in the vicinity of the disease-causing F508 deletion. A slightly longer construct crystallizes as a monomer. Comparisons of the homodimer structure with this and previously published monomeric structures show that the main effect of ATP binding at the signature site is to order the residues immediately preceding the signature sequence, residues 542-547, in a conformation compatible with nucleotide binding. These residues likely interact with a transmembrane domain intracellular loop in the full-length CFTR channel. The experiments described here show that removing the RI from NBD1 converts it into a well-behaved protein amenable to biophysical studies yielding deeper insights into CFTR function.

  11. Microtubule-associated protein-like binding of the kinesin-1 tail to microtubules.

    PubMed

    Seeger, Mark A; Rice, Sarah E

    2010-03-12

    The kinesin-1 molecular motor contains an ATP-dependent microtubule-binding site in its N-terminal head domain and an ATP-independent microtubule-binding site in its C-terminal tail domain. Here we demonstrate that a kinesin-1 tail fragment associates with microtubules with submicromolar affinity. Binding is largely electrostatic in nature, and is facilitated by a region of basic amino acids in the tail and the acidic E-hook at the C terminus of tubulin. The tail binds to a site on tubulin that is independent of the head domain-binding site but overlaps with the binding site of the microtubule-associated protein Tau. Surprisingly, the kinesin tail domain stimulates microtubule assembly and stability in a manner similar to Tau. The biological function of this strong kinesin tail-microtubule interaction remains to be seen, but it is likely to play an important role in kinesin regulation due to the close proximity of the microtubule-binding region to the conserved regulatory and cargo-binding domains of the tail. PMID:20071331

  12. The double PHD finger domain of MOZ/MYST3 induces α-helical structure of the histone H3 tail to facilitate acetylation and methylation sampling and modification.

    PubMed

    Dreveny, Ingrid; Deeves, Sian E; Fulton, Joel; Yue, Baigong; Messmer, Marie; Bhattacharya, Amit; Collins, Hilary M; Heery, David M

    2014-01-01

    Histone tail modifications control many nuclear processes by dictating the dynamic exchange of regulatory proteins on chromatin. Here we report novel insights into histone H3 tail structure in complex with the double PHD finger (DPF) of the lysine acetyltransferase MOZ/MYST3/KAT6A. In addition to sampling H3 and H4 modification status, we show that the DPF cooperates with the MYST domain to promote H3K9 and H3K14 acetylation, although not if H3K4 is trimethylated. Four crystal structures of an extended DPF alone and in complex with unmodified or acetylated forms of the H3 tail reveal the molecular basis of crosstalk between H3K4me3 and H3K14ac. We show for the first time that MOZ DPF induces α-helical conformation of H3K4-T11, revealing a unique mode of H3 recognition. The helical structure facilitates sampling of H3K4 methylation status, and proffers H3K9 and other residues for modification. Additionally, we show that a conserved double glycine hinge flanking the H3 tail helix is required for a conformational change enabling docking of H3K14ac with the DPF. In summary, our data provide the first observations of extensive helical structure in a histone tail, revealing the inherent ability of the H3 tail to adopt alternate conformations in complex with chromatin regulators.

  13. The double PHD finger domain of MOZ/MYST3 induces α-helical structure of the histone H3 tail to facilitate acetylation and methylation sampling and modification

    PubMed Central

    Dreveny, Ingrid; Deeves, Sian E.; Fulton, Joel; Yue, Baigong; Messmer, Marie; Bhattacharya, Amit; Collins, Hilary M.; Heery, David M.

    2014-01-01

    Histone tail modifications control many nuclear processes by dictating the dynamic exchange of regulatory proteins on chromatin. Here we report novel insights into histone H3 tail structure in complex with the double PHD finger (DPF) of the lysine acetyltransferase MOZ/MYST3/KAT6A. In addition to sampling H3 and H4 modification status, we show that the DPF cooperates with the MYST domain to promote H3K9 and H3K14 acetylation, although not if H3K4 is trimethylated. Four crystal structures of an extended DPF alone and in complex with unmodified or acetylated forms of the H3 tail reveal the molecular basis of crosstalk between H3K4me3 and H3K14ac. We show for the first time that MOZ DPF induces α-helical conformation of H3K4-T11, revealing a unique mode of H3 recognition. The helical structure facilitates sampling of H3K4 methylation status, and proffers H3K9 and other residues for modification. Additionally, we show that a conserved double glycine hinge flanking the H3 tail helix is required for a conformational change enabling docking of H3K14ac with the DPF. In summary, our data provide the first observations of extensive helical structure in a histone tail, revealing the inherent ability of the H3 tail to adopt alternate conformations in complex with chromatin regulators. PMID:24150941

  14. Structure of Human Acid Sphingomyelinase Reveals the Role of the Saposin Domain in Activating Substrate Hydrolysis.

    PubMed

    Xiong, Zi-Jian; Huang, Jingjing; Poda, Gennady; Pomès, Régis; Privé, Gilbert G

    2016-07-31

    Acid sphingomyelinase (ASM) is a lysosomal phosphodiesterase that catalyzes the hydrolysis of sphingomyelin to produce ceramide and phosphocholine. While other lysosomal sphingolipid hydrolases require a saposin activator protein for full activity, the ASM polypeptide incorporates a built-in N-terminal saposin domain and does not require an external activator protein. Here, we report the crystal structure of human ASM and describe the organization of the three main regions of the enzyme: the N-terminal saposin domain, the proline-rich connector, and the catalytic domain. The saposin domain is tightly associated along an edge of the large, bowl-shaped catalytic domain and adopts an open form that exposes a hydrophobic concave surface approximately 30Å from the catalytic center. The calculated electrostatic potential of the enzyme is electropositive at the acidic pH of the lysosome, consistent with the strict requirement for the presence of acidic lipids in target membranes. Docking studies indicate that sphingomyelin binds with the ceramide-phosphate group positioned at the binuclear zinc center and molecular dynamic simulations indicate that the intrinsic flexibility of the saposin domain is important for monomer-dimer exchange and for membrane interactions. Overall, ASM uses a combination of electrostatic and hydrophobic interactions to cause local disruptions of target bilayers in order to bring the lipid headgroup to the catalytic center in a membrane-bound reaction. PMID:27349982

  15. Structure of Human Acid Sphingomyelinase Reveals the Role of the Saposin Domain in Activating Substrate Hydrolysis.

    PubMed

    Xiong, Zi-Jian; Huang, Jingjing; Poda, Gennady; Pomès, Régis; Privé, Gilbert G

    2016-07-31

    Acid sphingomyelinase (ASM) is a lysosomal phosphodiesterase that catalyzes the hydrolysis of sphingomyelin to produce ceramide and phosphocholine. While other lysosomal sphingolipid hydrolases require a saposin activator protein for full activity, the ASM polypeptide incorporates a built-in N-terminal saposin domain and does not require an external activator protein. Here, we report the crystal structure of human ASM and describe the organization of the three main regions of the enzyme: the N-terminal saposin domain, the proline-rich connector, and the catalytic domain. The saposin domain is tightly associated along an edge of the large, bowl-shaped catalytic domain and adopts an open form that exposes a hydrophobic concave surface approximately 30Å from the catalytic center. The calculated electrostatic potential of the enzyme is electropositive at the acidic pH of the lysosome, consistent with the strict requirement for the presence of acidic lipids in target membranes. Docking studies indicate that sphingomyelin binds with the ceramide-phosphate group positioned at the binuclear zinc center and molecular dynamic simulations indicate that the intrinsic flexibility of the saposin domain is important for monomer-dimer exchange and for membrane interactions. Overall, ASM uses a combination of electrostatic and hydrophobic interactions to cause local disruptions of target bilayers in order to bring the lipid headgroup to the catalytic center in a membrane-bound reaction.

  16. Characterization of ERM transactivation domain binding to the ACID/PTOV domain of the Mediator subunit MED25.

    PubMed

    Landrieu, Isabelle; Verger, Alexis; Baert, Jean-Luc; Rucktooa, Prakash; Cantrelle, François-Xavier; Dewitte, Frédérique; Ferreira, Elisabeth; Lens, Zoé; Villeret, Vincent; Monté, Didier

    2015-08-18

    The N-terminal acidic transactivation domain (TAD) of ERM/ETV5 (ERM38-68), a PEA3 group member of Ets-related transcription factors, directly interacts with the ACID/PTOV domain of the Mediator complex subunit MED25. Molecular details of this interaction were investigated using nuclear magnetic resonance (NMR) spectroscopy. The TAD is disordered in solution but has a propensity to adopt local transient secondary structure. We show that it folds upon binding to MED25 and that the resulting ERM-MED25 complex displays characteristics of a fuzzy complex. Mutational analysis further reveals that two aromatic residues in the ERM TAD (F47 and W57) are involved in the binding to MED25 and participate in the ability of ERM TAD to activate transcription. Mutation of a key residue Q451 in the VP16 H1 binding pocket of MED25 affects the binding of ERM. Furthermore, competition experiments show that ERM and VP16 H1 share a common binding interface on MED25. NMR data confirms the occupancy of this binding pocket by ERM TAD. Based on these experimental data, a structural model of a functional interaction is proposed. This study provides mechanistic insights into the Mediator-transactivator interactions. PMID:26130716

  17. Characterization of ERM transactivation domain binding to the ACID/PTOV domain of the Mediator subunit MED25

    PubMed Central

    Landrieu, Isabelle; Verger, Alexis; Baert, Jean-Luc; Rucktooa, Prakash; Cantrelle, François-Xavier; Dewitte, Frédérique; Ferreira, Elisabeth; Lens, Zoé; Villeret, Vincent; Monté, Didier

    2015-01-01

    The N-terminal acidic transactivation domain (TAD) of ERM/ETV5 (ERM38–68), a PEA3 group member of Ets-related transcription factors, directly interacts with the ACID/PTOV domain of the Mediator complex subunit MED25. Molecular details of this interaction were investigated using nuclear magnetic resonance (NMR) spectroscopy. The TAD is disordered in solution but has a propensity to adopt local transient secondary structure. We show that it folds upon binding to MED25 and that the resulting ERM–MED25 complex displays characteristics of a fuzzy complex. Mutational analysis further reveals that two aromatic residues in the ERM TAD (F47 and W57) are involved in the binding to MED25 and participate in the ability of ERM TAD to activate transcription. Mutation of a key residue Q451 in the VP16 H1 binding pocket of MED25 affects the binding of ERM. Furthermore, competition experiments show that ERM and VP16 H1 share a common binding interface on MED25. NMR data confirms the occupancy of this binding pocket by ERM TAD. Based on these experimental data, a structural model of a functional interaction is proposed. This study provides mechanistic insights into the Mediator–transactivator interactions. PMID:26130716

  18. Head-to-tail interactions of the coiled-coil domains regulate ClpB activity and cooperation with Hsp70 in protein disaggregation

    PubMed Central

    Carroni, Marta; Kummer, Eva; Oguchi, Yuki; Wendler, Petra; Clare, Daniel K; Sinning, Irmgard; Kopp, Jürgen; Mogk, Axel; Bukau, Bernd; Saibil, Helen R

    2014-01-01

    The hexameric AAA+ chaperone ClpB reactivates aggregated proteins in cooperation with the Hsp70 system. Essential for disaggregation, the ClpB middle domain (MD) is a coiled-coil propeller that binds Hsp70. Although the ClpB subunit structure is known, positioning of the MD in the hexamer and its mechanism of action are unclear. We obtained electron microscopy (EM) structures of the BAP variant of ClpB that binds the protease ClpP, clearly revealing MD density on the surface of the ClpB ring. Mutant analysis and asymmetric reconstructions show that MDs adopt diverse positions in a single ClpB hexamer. Adjacent, horizontally oriented MDs form head-to-tail contacts and repress ClpB activity by preventing Hsp70 interaction. Tilting of the MD breaks this contact, allowing Hsp70 binding, and releasing the contact in adjacent subunits. Our data suggest a wavelike activation of ClpB subunits around the ring. DOI: http://dx.doi.org/10.7554/eLife.02481.001 PMID:24843029

  19. Trace element uptake by Eleocharis equisetina (spike rush) in an abandoned acid mine tailings pond, northeastern Australia: implications for land and water reclamation in tropical regions.

    PubMed

    Lottermoser, Bernd G; Ashley, Paul M

    2011-10-01

    This study was conducted to determine the uptake of trace elements by the emergent wetland plant species Eleocharis equisetina at the historic Jumna tin processing plant, tropical Australia. The perennial emergent sedge was found growing in acid waters (pH 2.45) and metal-rich tailings (SnAsCuPbZn). E. equisetina displayed a pronounced acid tolerance and tendency to exclude environmentally significant elements (Al, As, Cd, Ce, Co, Cu, Fe, La, Ni, Pb, Se, Th, U, Y, Zn) from its above-substrate biomass. This study demonstrates that geobotanical and biogeochemical examinations of wetland plants at abandoned mined lands of tropical areas can reveal pioneering, metal-excluding macrophytes. Such aquatic macrophytes are of potential use in the remediation of acid mine waters and sulfidic tailings and the reclamation of disturbed acid sulfate soils in subtropical and tropical regions.

  20. Trace element uptake by Eleocharis equisetina (spike rush) in an abandoned acid mine tailings pond, northeastern Australia: implications for land and water reclamation in tropical regions.

    PubMed

    Lottermoser, Bernd G; Ashley, Paul M

    2011-10-01

    This study was conducted to determine the uptake of trace elements by the emergent wetland plant species Eleocharis equisetina at the historic Jumna tin processing plant, tropical Australia. The perennial emergent sedge was found growing in acid waters (pH 2.45) and metal-rich tailings (SnAsCuPbZn). E. equisetina displayed a pronounced acid tolerance and tendency to exclude environmentally significant elements (Al, As, Cd, Ce, Co, Cu, Fe, La, Ni, Pb, Se, Th, U, Y, Zn) from its above-substrate biomass. This study demonstrates that geobotanical and biogeochemical examinations of wetland plants at abandoned mined lands of tropical areas can reveal pioneering, metal-excluding macrophytes. Such aquatic macrophytes are of potential use in the remediation of acid mine waters and sulfidic tailings and the reclamation of disturbed acid sulfate soils in subtropical and tropical regions. PMID:21550704

  1. WHIM syndrome caused by a single amino acid substitution in the carboxy-tail of chemokine receptor CXCR4

    PubMed Central

    Liu, Qian; Chen, Haoqian; Ojode, Teresa; Gao, Xiangxi; Anaya-O'Brien, Sandra; Turner, Nicholas A.; Ulrick, Jean; DeCastro, Rosamma; Kelly, Corin; Cardones, Adela R.; Gold, Stuart H.; Hwang, Eugene I.; Wechsler, Daniel S.; Malech, Harry L.; Murphy, Philip M.

    2012-01-01

    WHIM syndrome is a rare, autosomal dominant, immunodeficiency disorder so-named because it is characterized by warts, hypogammaglobulinemia, infections, and myelokathexis (defective neutrophil egress from the BM). Gain-of-function mutations that truncate the C-terminus of the chemokine receptor CXCR4 by 10-19 amino acids cause WHIM syndrome. We have identified a family with autosomal dominant inheritance of WHIM syndrome that is caused by a missense mutation in CXCR4, E343K (1027G → A). This mutation is also located in the C-terminal domain, a region responsible for negative regulation of the receptor. Accordingly, like CXCR4R334X, the most common truncation mutation in WHIM syndrome, CXCR4E343K mediated approximately 2-fold increased signaling in calcium flux and chemotaxis assays relative to wild-type CXCR4; however, CXCR4E343K had a reduced effect on blocking normal receptor down-regulation from the cell surface. Therefore, in addition to truncating mutations in the C-terminal domain of CXCR4, WHIM syndrome may be caused by a single charge-changing amino acid substitution in this domain, E343K, that results in increased receptor signaling. PMID:22596258

  2. Structural Domains Underlying the Activation of Acid-Sensing Ion Channel 2a

    PubMed Central

    Schuhmacher, Laura-Nadine; Srivats, Shyam; Smith, Ewan St. John

    2015-01-01

    The acid-sensing ion channels (ASICs) are a family of ion channels expressed throughout the mammalian nervous system. The principal activator of ASICs is extracellular protons, and ASICs have been demonstrated to play a significant role in many physiologic and pathophysiologic processes, including synaptic transmission, nociception, and fear. However, not all ASICs are proton-sensitive: ASIC2a is activated by acid, whereas its splice variant ASIC2b is not. We made a series of chimeric ASIC2 proteins, and using whole-cell electrophysiology we have identified the minimal region of the ASIC2a extracellular domain that is required for ASIC2 proton activation: the first 87 amino acids after transmembrane domain 1. We next examined the function of different domains within the ASIC2b N-terminus and identified a region proximal to the first transmembrane domain that confers tachyphylaxis upon ASIC2a. We have thus identified domains of ASIC2 that are crucial to channel function and may be important for the function of other members of the ASIC family. PMID:25583083

  3. Tip-induced domain structures and polarization switching in ferroelectric amino acid glycine

    NASA Astrophysics Data System (ADS)

    Seyedhosseini, E.; Bdikin, I.; Ivanov, M.; Vasileva, D.; Kudryavtsev, A.; Rodriguez, B. J.; Kholkin, A. L.

    2015-08-01

    Bioorganic ferroelectrics and piezoelectrics are becoming increasingly important in view of their intrinsic compatibility with biological environment and biofunctionality combined with strong piezoelectric effect and a switchable polarization at room temperature. Here, we study tip-induced domain structures and polarization switching in the smallest amino acid β-glycine, representing a broad class of non-centrosymmetric amino acids. We show that β-glycine is indeed a room-temperature ferroelectric and polarization can be switched by applying a bias to non-polar cuts via a conducting tip of atomic force microscope (AFM). Dynamics of these in-plane domains is studied as a function of an applied voltage and pulse duration. The domain shape is dictated by polarization screening at the domain boundaries and mediated by growth defects. Thermodynamic theory is applied to explain the domain propagation induced by the AFM tip. Our findings suggest that the properties of β-glycine are controlled by the charged domain walls which in turn can be manipulated by an external bias.

  4. Tip-induced domain structures and polarization switching in ferroelectric amino acid glycine

    SciTech Connect

    Seyedhosseini, E. Ivanov, M.; Bdikin, I.; Vasileva, D.; Kudryavtsev, A.; Rodriguez, B. J.; Kholkin, A. L.

    2015-08-21

    Bioorganic ferroelectrics and piezoelectrics are becoming increasingly important in view of their intrinsic compatibility with biological environment and biofunctionality combined with strong piezoelectric effect and a switchable polarization at room temperature. Here, we study tip-induced domain structures and polarization switching in the smallest amino acid β-glycine, representing a broad class of non-centrosymmetric amino acids. We show that β-glycine is indeed a room-temperature ferroelectric and polarization can be switched by applying a bias to non-polar cuts via a conducting tip of atomic force microscope (AFM). Dynamics of these in-plane domains is studied as a function of an applied voltage and pulse duration. The domain shape is dictated by polarization screening at the domain boundaries and mediated by growth defects. Thermodynamic theory is applied to explain the domain propagation induced by the AFM tip. Our findings suggest that the properties of β-glycine are controlled by the charged domain walls which in turn can be manipulated by an external bias.

  5. Human fatty acid synthase: Structure and substrate selectivity of the thioesterase domain

    PubMed Central

    Chakravarty, Bornali; Gu, Ziwei; Chirala, Subrahmanyam S.; Wakil, Salih J.; Quiocho, Florante A.

    2004-01-01

    Human fatty acid synthase is a large homodimeric multifunctional enzyme that synthesizes palmitic acid. The unique carboxyl terminal thioesterase domain of fatty acid synthase hydrolyzes the growing fatty acid chain and plays a critical role in regulating the chain length of fatty acid released. Also, the up-regulation of human fatty acid synthase in a variety of cancer makes the thioesterase a candidate target for therapeutic treatment. The 2.6-Å resolution structure of human fatty acid synthase thioesterase domain reported here is comprised of two dissimilar subdomains, A and B. The smaller subdomain B is composed entirely of α-helices arranged in an atypical fold, whereas the A subdomain is a variation of the α/β hydrolase fold. The structure revealed the presence of a hydrophobic groove with a distal pocket at the interface of the two subdomains, which constitutes the candidate substrate binding site. The length and largely hydrophobic nature of the groove and pocket are consistent with the high selectivity of the thioesterase for palmitoyl acyl substrate. The structure also set the identity of the Asp residue of the catalytic triad of Ser, His, and Asp located in subdomain A at the proximal end of the groove. PMID:15507492

  6. Bile acids modulate signaling by functional perturbation of plasma membrane domains.

    PubMed

    Zhou, Yong; Maxwell, Kelsey N; Sezgin, Erdinc; Lu, Maryia; Liang, Hong; Hancock, John F; Dial, Elizabeth J; Lichtenberger, Lenard M; Levental, Ilya

    2013-12-13

    Eukaryotic cell membranes are organized into functional lipid and protein domains, the most widely studied being membrane rafts. Although rafts have been associated with numerous plasma membrane functions, the mechanisms by which these domains themselves are regulated remain undefined. Bile acids (BAs), whose primary function is the solubilization of dietary lipids for digestion and absorption, can affect cells by interacting directly with membranes. To investigate whether these interactions affected domain organization in biological membranes, we assayed the effects of BAs on biomimetic synthetic liposomes, isolated plasma membranes, and live cells. At cytotoxic concentrations, BAs dissolved synthetic and cell-derived membranes and disrupted live cell plasma membranes, implicating plasma membrane damage as the mechanism for BA cellular toxicity. At subtoxic concentrations, BAs dramatically stabilized domain separation in Giant Plasma Membrane Vesicles without affecting protein partitioning between coexisting domains. Domain stabilization was the result of BA binding to and disordering the nonraft domain, thus promoting separation by enhancing domain immiscibility. Consistent with the physical changes observed in synthetic and isolated biological membranes, BAs reorganized intact cell membranes, as evaluated by the spatial distribution of membrane-anchored Ras isoforms. Nanoclustering of K-Ras, related to nonraft membrane domains, was enhanced in intact plasma membranes, whereas the organization of H-Ras was unaffected. BA-induced changes in Ras lateral segregation potentiated EGF-induced signaling through MAPK, confirming the ability of BAs to influence cell signal transduction by altering the physical properties of the plasma membrane. These observations suggest general, membrane-mediated mechanisms by which biological amphiphiles can produce their cellular effects.

  7. The Lipid domain Phase diagram in a Dipalmitoyl-PC/Docosahaexnoic Acid-PE/Cholesterol System

    NASA Astrophysics Data System (ADS)

    Lor, Chai; Hirst, Linda

    2011-03-01

    Lipid domains in bilayer membrane and polyunsaturated fatty acids (PUFAs) are thought to play an important role in cellular activities. In particular, lipids containing docosahaexnoic acid are an interesting class of PUFAs due to their health benefits. In this project, we perform oxidation measurements of DHA-PE to determine the rate of oxidation in combination with antioxidants. A ternary diagram of DPPC/DHA-PE/cholesterol is mapped out to identify phase separation phenomena using atomic force microscope (AFM). Fluorescence microscopy is also used to image lipid domains in a flat bilayer with fluorescent labels. As expected, we observe the phase, shape, and size of lipid domains changes with varying composition. Moreover, we find that the roughness of the domains changes possibly due to overpacking of cholesterol in domains. This model study provides further understanding of the role of cholesterol in the bilayer membrane leading towards a better understanding of cell membranes. NSF award # DMR 0852791, ``CAREER: Self-Assembly of Polyunsaturated Lipids and Cholesterol In The Cell Membrane.''

  8. Dynamin at actin tails.

    PubMed

    Lee, Eunkyung; De Camilli, Pietro

    2002-01-01

    Dynamin, the product of the shibire gene of Drosophila, is a GTPase critically required for endocytosis. Some studies have suggested a functional link between dynamin and the actin cytoskeleton. This link is of special interest, because there is evidence implicating actin dynamics in endocytosis. Here we show that endogenous dynamin 2, as well as green fluorescence protein fusion proteins of both dynamin 1 and 2, is present in actin comets generated by Listeria or by type I PIP kinase (PIPK) overexpression. In PIPK-induced tails, dynamin is further enriched at the interface between the tails and the moving organelles. Dynamin mutants harboring mutations in the GTPase domain inhibited nucleation of actin tails induced by PIPK and moderately reduced their speed. Although dynamin localization to the tails required its proline-rich domain, expression of a dynamin mutant lacking this domain also diminished tail formation. In addition, this mutant disrupted a membrane-associated actin scaffold (podosome rosette) previously shown to include dynamin. These findings suggest that dynamin is part of a protein network that controls nucleation of actin from membranes. At endocytic sites, dynamin may couple the fission reaction to the polymerization of an actin pool that functions in the separation of the endocytic vesicles from the plasma membrane. PMID:11782545

  9. Transcriptional activation by the acidic domain of Vmw65 requires the integrity of the domain and involves additional determinants distinct from those necessary for TFIIB binding.

    PubMed Central

    Walker, S; Greaves, R; O'Hare, P

    1993-01-01

    In this work we have examined the requirements for activity of the acidic domain of Vmw65 (VP16) by deletion and site-directed mutagenesis of the region in the context of GAL4 fusion proteins. The results indicate that the present interpretation of what actually constitutes the activation domain is not correct. We demonstrate, using a promoter with one target site which is efficiently activated by the wild-type (wt) fusion protein, that amino acids distal to residue 453 are critical for activity. Truncation of the domain or substitution of residues in the distal region almost completely abrogate activity. However, inactivating mutations within the distal region are complemented by using a promoter containing multiple target sites. Moreover, duplication of the proximal region, but not the distal region, restores the ability to activate a promoter with a single target site. These results indicate some distinct qualitative difference between the proximal and distal regions. We have also examined the binding of nuclear proteins to the wt domain and to a variant with the distal region inactivated by mutation. The lack of activity of this variant is not explained by a lack of binding of TFIIB, a protein previously reported to be the likely target of the acidic domain. Therefore some additional function is involved in transcriptional activation by the acid domain, and determinants distinct from those involved in TFIIB binding are required for this function. Analysis of the total protein profiles binding to the wt and mutant domains has demonstrated the selective binding to the wt domain of a 135-kDa polypeptide, which is therefore a candidate component involved in this additional function. This is the first report to provide evidence for the proposal of a multiplicity of interactions within the acidic domain, by uncoupling requirements for one function from those for another. Images PMID:8395001

  10. Lysophosphatidic acid stimulates thrombomodulin lectin-like domain shedding in human endothelial cells

    SciTech Connect

    Wu Hualin; Lin ChiIou; Huang Yuanli; Chen, Pin-Shern; Kuo, Cheng-Hsiang; Chen, Mei-Shing; Wu, G.C.-C.; Shi, G.-Y.; Yang, H.-Y.; Lee Hsinyu

    2008-02-29

    Thrombomodulin (TM) is an anticoagulant glycoprotein highly expressed on endothelial cell surfaces. Increased levels of soluble TM in circulation have been widely accepted as an indicator of endothelial damage or dysfunction. Previous studies indicated that various proinflammatory factors stimulate TM shedding in various cell types such as smooth muscle cells and epithelial cells. Lysophosphatidic acid (LPA) is a bioactive lipid mediator present in biological fluids during endothelial damage or injury. In the present study, we first observed that LPA triggered TM shedding in human umbilical vein endothelial cells (HUVECs). By Cyflow analysis, we showed that the LPA-induced accessibility of antibodies to the endothelial growth factor (EGF)-like domain of TM is independent of matrix metalloproteinases (MMPs), while LPA-induced TM lectin-like domain shedding is MMP-dependent. Furthermore, a stable cell line expressing TM without its lectin-like domain exhibited a higher cell proliferation rate than a stable cell line expressing full-length TM. These results imply that LPA induces TM lectin-like domain shedding, which might contribute to the exposure of its EGF-like domain for EGF receptor (EGFR) binding, thereby stimulating subsequent cell proliferation. Based on our findings, we propose a novel mechanism for the exposure of TM EGF-like domain, which possibly mediates LPA-induced EGFR transactivation.

  11. Effect of arbuscular mycorrhizal fungi on plant biomass and the rhizosphere microbial community structure of mesquite grown in acidic lead/zinc mine tailings.

    PubMed

    Solís-Domínguez, Fernando A; Valentín-Vargas, Alexis; Chorover, Jon; Maier, Raina M

    2011-02-15

    Mine tailings in arid and semi-arid environments are barren of vegetation and subject to eolian dispersion and water erosion. Revegetation is a cost-effective strategy to reduce erosion processes and has wide public acceptance. A major cost of revegetation is the addition of amendments, such as compost, to allow plant establishment. In this paper we explore whether arbuscular mycorrhizal fungi (AMF) can help support plant growth in tailings at a reduced compost concentration. A greenhouse experiment was performed to determine the effects of three AMF inocula on biomass, shoot accumulation of heavy metals, and changes in the rhizosphere microbial community structure of the native plant Prosopis juliflora (mesquite). Plants were grown in an acidic lead/zinc mine tailings amended with 10% (w/w) compost amendment, which is slightly sub-optimal for plant growth in these tailings. After two months, AMF-inoculated plants showed increased dry biomass and root length (p<0.05) and effective AMF colonization compared to controls grown in uninoculated compost-amended tailings. Mesquite shoot tissue lead and zinc concentrations did not exceed domestic animal toxicity limits regardless of whether AMF inoculation was used. The rhizosphere microbial community structure was assessed using denaturing gradient gel electrophoresis (DGGE) profiles of the small subunit RNA gene for bacteria and fungi. Canonical correspondence analysis (CCA) of DGGE profiles showed that the rhizosphere fungal community structure at the end of the experiment was significantly different from the community structure in the tailings, compost, and AMF inocula prior to planting. Further, CCA showed that AMF inoculation significantly influenced the development of both the fungal and bacterial rhizosphere community structures after two months. The changes observed in the rhizosphere microbial community structure may be either a direct effect of the AMF inocula, caused by changes in plant physiology induced by

  12. Effect of arbuscular mycorrhizal fungi on plant biomass and the rhizosphere microbial community structure of mesquite grown in acidic lead/zinc mine tailings.

    PubMed

    Solís-Domínguez, Fernando A; Valentín-Vargas, Alexis; Chorover, Jon; Maier, Raina M

    2011-02-15

    Mine tailings in arid and semi-arid environments are barren of vegetation and subject to eolian dispersion and water erosion. Revegetation is a cost-effective strategy to reduce erosion processes and has wide public acceptance. A major cost of revegetation is the addition of amendments, such as compost, to allow plant establishment. In this paper we explore whether arbuscular mycorrhizal fungi (AMF) can help support plant growth in tailings at a reduced compost concentration. A greenhouse experiment was performed to determine the effects of three AMF inocula on biomass, shoot accumulation of heavy metals, and changes in the rhizosphere microbial community structure of the native plant Prosopis juliflora (mesquite). Plants were grown in an acidic lead/zinc mine tailings amended with 10% (w/w) compost amendment, which is slightly sub-optimal for plant growth in these tailings. After two months, AMF-inoculated plants showed increased dry biomass and root length (p<0.05) and effective AMF colonization compared to controls grown in uninoculated compost-amended tailings. Mesquite shoot tissue lead and zinc concentrations did not exceed domestic animal toxicity limits regardless of whether AMF inoculation was used. The rhizosphere microbial community structure was assessed using denaturing gradient gel electrophoresis (DGGE) profiles of the small subunit RNA gene for bacteria and fungi. Canonical correspondence analysis (CCA) of DGGE profiles showed that the rhizosphere fungal community structure at the end of the experiment was significantly different from the community structure in the tailings, compost, and AMF inocula prior to planting. Further, CCA showed that AMF inoculation significantly influenced the development of both the fungal and bacterial rhizosphere community structures after two months. The changes observed in the rhizosphere microbial community structure may be either a direct effect of the AMF inocula, caused by changes in plant physiology induced by

  13. Effect of Arbuscular Mycorrhizal Fungi on Plant Biomass and the Rhizosphere Microbial Community Structure of Mesquite Grown in Acidic Lead/Zinc Mine Tailings

    PubMed Central

    Solís-Domínguez, Fernando A.; Valentín-Vargas, Alexis; Chorover, Jon; Maier, Raina M.

    2011-01-01

    Mine tailings in arid and semi-arid environments are barren of vegetation and subject to eolian dispersion and water erosion. Revegetation is a cost-effective strategy to reduce erosion processes and has wide public acceptance. A major cost of revegetation is the addition of amendments, such as compost, to allow plant establishment. In this paper we explore whether arbuscular mycorrhizal fungi (AMF) can help support plant growth in tailings at a reduced compost concentration. A greenhouse experiment was performed to determine the effects of three AMF inocula on biomass, shoot accumulation of heavy metals, and changes in the rhizosphere microbial community structure of the native plant Prosopis juliflora (mesquite). Plants were grown in an acidic lead/zinc mine tailings amended with 10% (w/w) compost amendment, which is slightly sub-optimal for plant growth in these tailings. After two months, AMF-inoculated plants showed increased dry biomass and root length (p < 0.05) and effective AMF colonization compared to controls grown in uninoculated compost-amended tailings. Mesquite shoot tissue lead and zinc concentrations did not exceed domestic animal toxicity limits regardless of whether AMF inoculation was used. The rhizosphere microbial community structure was assessed using denaturing gradient gel electrophoresis (DGGE) profiles of the small subunit RNA gene for bacteria and fungi. Canonical correspondence analysis (CCA) of DGGE profiles showed that the rhizosphere fungal community structure at the end of the experiment was significantly different from the community structure in the tailings, compost, and AMF inocula prior to planting. Further, CCA showed that AMF inoculation significantly influenced the development of both the fungal and bacterial rhizosphere community structures after two months. The changes observed in the rhizosphere microbial community structure may be either a direct effect of the AMF inocula, caused by changes in plant physiology induced by

  14. The Arabidopsis abscisic acid response locus ABI4 encodes an APETALA 2 domain protein.

    PubMed Central

    Finkelstein, R R; Wang, M L; Lynch, T J; Rao, S; Goodman, H M

    1998-01-01

    Arabidopsis abscisic acid (ABA)-insensitive abi4 mutants have pleiotropic defects in seed development, including decreased sensitivity to ABA inhibition of germination and altered seed-specific gene expression. This phenotype is consistent with a role for ABI4 in regulating seed responses to ABA and/or seed-specific signals. We isolated the ABI4 gene by positional cloning and confirmed its identity by complementation analysis. The predicted protein product shows homology to a plant-specific family of transcriptional regulators characterized by a conserved DNA binding domain, the APETALA 2 domain. The single mutant allele identified has a single base pair deletion, resulting in a frameshift that should disrupt the C-terminal half of the protein but leave the presumed DNA binding domain intact. Expression analyses showed that despite the seed-specific nature of the mutant phenotype, ABI4 expression is not seed specific. PMID:9634591

  15. Incorporation of small BN domains in graphene during CVD using methane, boric acid and nitrogen gas.

    PubMed

    Bepete, George; Voiry, Damien; Chhowalla, Manish; Chiguvare, Zivayi; Coville, Neil J

    2013-07-21

    Chemical doping of graphene with small boron nitride (BN) domains has been shown to be an effective way of permanently modulating the electronic properties in graphene. Herein we show a facile method of growing large area graphene doped with small BN domains on copper foils using a single step CVD route with methane, boric acid powder and nitrogen gas as the carbon, boron and nitrogen sources respectively. This facile and safe process avoids the use of boranes and ammonia. Optical microscopy confirmed that continuous films were grown and Raman spectroscopy confirmed changes in the electronic structure of the grown BN doped graphene. Using XPS studies we find that both B and N can be substituted into the graphene structure in the form of small BN domains to give a B-N-C system. A novel structure for the BN doped graphene is proposed. PMID:23759928

  16. Incorporation of small BN domains in graphene during CVD using methane, boric acid and nitrogen gas.

    PubMed

    Bepete, George; Voiry, Damien; Chhowalla, Manish; Chiguvare, Zivayi; Coville, Neil J

    2013-07-21

    Chemical doping of graphene with small boron nitride (BN) domains has been shown to be an effective way of permanently modulating the electronic properties in graphene. Herein we show a facile method of growing large area graphene doped with small BN domains on copper foils using a single step CVD route with methane, boric acid powder and nitrogen gas as the carbon, boron and nitrogen sources respectively. This facile and safe process avoids the use of boranes and ammonia. Optical microscopy confirmed that continuous films were grown and Raman spectroscopy confirmed changes in the electronic structure of the grown BN doped graphene. Using XPS studies we find that both B and N can be substituted into the graphene structure in the form of small BN domains to give a B-N-C system. A novel structure for the BN doped graphene is proposed.

  17. Incorporation of small BN domains in graphene during CVD using methane, boric acid and nitrogen gas

    NASA Astrophysics Data System (ADS)

    Bepete, George; Voiry, Damien; Chhowalla, Manish; Chiguvare, Zivayi; Coville, Neil J.

    2013-06-01

    Chemical doping of graphene with small boron nitride (BN) domains has been shown to be an effective way of permanently modulating the electronic properties in graphene. Herein we show a facile method of growing large area graphene doped with small BN domains on copper foils using a single step CVD route with methane, boric acid powder and nitrogen gas as the carbon, boron and nitrogen sources respectively. This facile and safe process avoids the use of boranes and ammonia. Optical microscopy confirmed that continuous films were grown and Raman spectroscopy confirmed changes in the electronic structure of the grown BN doped graphene. Using XPS studies we find that both B and N can be substituted into the graphene structure in the form of small BN domains to give a B-N-C system. A novel structure for the BN doped graphene is proposed.

  18. Selection on synthesis cost affects interprotein amino acid usage in all three domains of life.

    PubMed

    Swire, Jonathan

    2007-05-01

    Most investigations of the forces shaping protein evolution have focused on protein function. However, cells are typically 50%-75% protein by dry weight, with protein expression levels distributed over five orders of magnitude. Cells may, therefore, be under considerable selection pressure to incorporate amino acids that are cheap to synthesize into proteins that are highly expressed. Such selection pressure has been demonstrated to alter amino acid usage in a few organisms, but whether "cost selection" is a general phenomenon remains unknown. One reason for this is that reliable protein expression level data is not available for most organisms. Accordingly, I have developed a new method for detecting cost selection. This method depends solely on interprotein gradients in amino acid usage. Applying it to an analysis of 43 whole genomes from all three domains of life, I show that selection on the synthesis cost of amino acids is a pervasive force in shaping the composition of proteins. Moreover, some amino acids have different price tags for different organisms--the cost of amino acids is changed for organisms living in hydrothermal vents compared with those living at the sea surface or for organisms that have difficulty acquiring elements such as nitrogen compared with those that do not--so I also investigated whether differences between organisms in amino acid usage might reflect differences in synthesis or acquisition costs. The results suggest that organisms evolve to alter amino acid usage in response to environmental conditions.

  19. Cold shock domain protein from Philosamia ricini prefers single-stranded nucleic acids binding.

    PubMed

    Mani, Ashutosh; Yadava, P K; Gupta, Dwijendra K

    2012-01-01

    The cold shock proteins are evolutionarily conserved nucleic acid-binding proteins. Their eukaryotic homologs are present as cold shock domain (CSD) in Y-box proteins. CSDs too share striking similarity among different organisms and show nucleic acid binding properties. The purpose of the study was to investigate the preferential binding affinity of CSD protein for nucleic acids in Philosamia ricini. We have cloned and sequenced the first cDNA coding for Y-box protein in P. ricini; the sequence has been deposited in GenBank. Comparative genomics and phylogenetic analytics further confirmed that the deduced amino acid sequence belongs to the CSD protein family. A comparative study employing molecular docking was performed with P. ricini CSD, human CSD, and bacterial cold shock protein with a range of nucleic acid entities. The results indicate that CSD per se exhibits preferential binding affinity for single-stranded RNA and DNA. Possibly, the flanking N- and C-terminal domains are additionally involved in interactions with dsDNA or in conferring extra stability to CSD for improved binding.

  20. Guidelines for the use of protein domains in acidic phospholipid imaging

    PubMed Central

    Platre, Matthieu Pierre; Jaillais, Yvon

    2015-01-01

    Acidic phospholipids are minor membrane lipids but critically important for signaling events. The main acidic phospholipids are phosphatidylinositol phosphates (PIPs also known as phosphoinositides), phosphatidylserine (PS) and phosphatidic acid (PA). Acidic phospholipids are precursors of second messengers of key signaling cascades or are second messengers themselves. They regulate the localization and activation of many proteins, and are involved in virtually all membrane trafficking events. As such, it is crucial to understand the subcellular localization and dynamics of each of these lipids within the cell. Over the years, several techniques have emerged in either fixed or live cells to analyze the subcellular localization and dynamics of acidic phospholipids. In this chapter, we review one of them: the use of genetically encoded biosensors that are based on the expression of specific lipid binding domains (LBDs) fused to fluorescent proteins. We discuss how to design such sensors, including the criteria for selecting the lipid binding domains of interest and to validate them. We also emphasize the care that must be taken during data analysis as well as the main limitations and advantages of this approach. PMID:26552684

  1. Expression of dehydratase domains from a polyunsaturated fatty acid synthase increases the production of fatty acids in Escherichia coli

    PubMed Central

    Oyola-Robles, Delise; Rullán-Lind, Carlos; Carballeira, Néstor M.; Baerga-Ortiz, Abel

    2014-01-01

    Increasing the production of fatty acids by microbial fermentation remains an important step towards the generation of biodiesel and other portable liquid fuels. In this work, we report an Escherichia coli strain engineered to overexpress a fragment consisting of four dehydratase domains from the polyunsaturated fatty acid (PUFA) synthase enzyme complex from the deep-sea bacterium, Photobacterium profundum. The DH1-DH2-UMA enzyme fragment was excised from its natural context within a multi-enzyme PKS and expressed as a stand-alone protein. Fatty acids were extracted from the cell pellet, esterified with methanol and quantified by GC-MS analysis. Results show that the E. coli strain expressing the DH tetradomain fragment was capable of producing up to a 5-fold increase (80.31 mg total FA/L culture) in total fatty acids over the negative control strain lacking the recombinant enzyme. The enhancement in production was observed across the board for all the fatty acids that are typically made by E. coli. The overexpression of the DH tetradomain did not affect E. coli cell growth, thus showing that the observed enhancement in fatty acid production was not a result of effects associated with cell density. The observed enhancement was more pronounced at lower temperatures (3.8-fold at 16 °C, 3.5-fold at 22 °C and 1.5-fold at 30 °C) and supplementation of the media with 0.4% glycerol did not result in an increase in fatty acid production. All these results taken together suggest that either the dehydration of fatty acid intermediates are a limiting step in the E. coli fatty acid biosynthesis machinery, or that the recombinant dehydratase domains used in this study are also capable of catalyzing thioester hydrolysis of the final products. The enzyme in this report is a new tool which could be incorporated into other existing strategies aimed at improving fatty acid production in bacterial fermentations towards accessible biodiesel precursors. PMID:24411456

  2. Bpur, the Lyme disease spirochete's PUR domain protein: identification as a transcriptional modulator and characterization of nucleic acid interactions.

    PubMed

    Jutras, Brandon L; Chenail, Alicia M; Carroll, Dustin W; Miller, M Clarke; Zhu, Haining; Bowman, Amy; Stevenson, Brian

    2013-09-01

    The PUR domain is a nucleic acid-binding motif found in critical regulatory proteins of higher eukaryotes and in certain species of bacteria. During investigations into mechanisms by which the Lyme disease spirochete controls synthesis of its Erp surface proteins, it was discovered that the borrelial PUR domain protein, Bpur, binds with high affinity to double-stranded DNA adjacent to the erp transcriptional promoter. Bpur was found to enhance the effects of the erp repressor protein, BpaB. Bpur also bound single-stranded DNA and RNA, with relative affinities RNA > double-stranded DNA > single-stranded DNA. Rational site-directed mutagenesis of Bpur identified amino acid residues and domains critical for interactions with nucleic acids, and it revealed that the PUR domain has a distinct mechanism of interaction with each type of nucleic acid ligand. These data shed light on both gene regulation in the Lyme spirochete and functional mechanisms of the widely distributed PUR domain.

  3. Sulfide oxidation and acid mine drainage formation within two active tailings impoundments in the Golden Quadrangle of the Apuseni Mountains, Romania.

    PubMed

    Sima, Mihaela; Dold, Bernhard; Frei, Linda; Senila, Marin; Balteanu, Dan; Zobrist, Jurg

    2011-05-30

    Sulfidic mine tailings have to be classified as one of the major source of hazardous materials leading to water contamination. This study highlights the processes leading to sulfide oxidation and acid mine drainage (AMD) formation in the active stage of two tailings impoundments located in the southern part of the Apuseni Mountains, in Romania, a well-known region for its long-term gold-silver and metal mining activity. Sampling was undertaken when both impoundments were still in operation in order to assess their actual stage of oxidation and long-term behavior in terms of the potential for acid mine drainage generation. Both tailings have high potential for AMD formation (2.5 and 3.7 wt.% of pyrite equivalent, respectively) with lesser amount of carbonates (5.6 and 3.6 wt.% of calcite equivalent) as neutralization potential (ABA=-55.6 and -85.1 tCaCO(3)/1000 t ) and showed clear signs of sulfide oxidation yet during operation. Sequential extraction results indicate a stronger enrichment and mobility of elements in the oxidized tailings: Fe as Fe(III) oxy-hydroxides and oxides (transformation from sulfide minerals, leaching in oxidation zone), Ca mainly in water soluble and exchangeable form where gypsum and calcite are dissolved and higher mobility of Cu for Ribita and Pb for Mialu. Two processes leading to the formation of mine drainage at this stage could be highlighted (1) a neutral Fe(II) plume forming in the impoundment with ferrihydrite precipitation at its outcrop and (2) acid mine drainage seeping in the unsaturated zone of the active dam, leading to the formation of schwertmannite at its outcrop. PMID:21316846

  4. Negatively Charged Amino Acids Near and in Transient Receptor Potential (TRP) Domain of TRPM4 Channel Are One Determinant of Its Ca2+ Sensitivity*

    PubMed Central

    Yamaguchi, Soichiro; Tanimoto, Akira; Otsuguro, Ken-ichi; Hibino, Hiroshi; Ito, Shigeo

    2014-01-01

    Transient receptor potential (TRP) channel melastatin subfamily member 4 (TRPM4) is a broadly expressed nonselective monovalent cation channel. TRPM4 is activated by membrane depolarization and intracellular Ca2+, which is essential for the activation. The Ca2+ sensitivity is known to be regulated by calmodulin and membrane phosphoinositides, such as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Although these regulators must play important roles in controlling TRPM4 activity, mutation analyses of the calmodulin-binding sites have suggested that Ca2+ binds to TRPM4 directly. However, the intrinsic binding sites in TRPM4 remain to be elucidated. Here, by using patch clamp and molecular biological techniques, we show that there are at least two functionally different divalent cation-binding sites, and the negatively charged amino acids near and in the TRP domain in the C-terminal tail of TRPM4 (Asp-1049 and Glu-1062 of rat TRPM4) are required for maintaining the normal Ca2+ sensitivity of one of the binding sites. Applications of Co2+, Mn2+, or Ni2+ to the cytosolic side potentiated TRPM4 currents, increased the Ca2+ sensitivity, but were unable to evoke TRPM4 currents without Ca2+. Mutations of the acidic amino acids near and in the TRP domain, which are conserved in TRPM2, TRPM5, and TRPM8, deteriorated the Ca2+ sensitivity in the presence of Co2+ or PI(4,5)P2 but hardly affected the sensitivity to Co2+ and PI(4,5)P2. These results suggest a novel role of the TRP domain in TRPM4 as a site responsible for maintaining the normal Ca2+ sensitivity. These findings provide more insights into the molecular mechanisms of the regulation of TRPM4 by Ca2+. PMID:25378404

  5. Evolution of soil properties and metals in acid and alkaline mine tailing ponds after amendments and microorganisms application

    NASA Astrophysics Data System (ADS)

    Acosta, Jose A.; Faz, Ángel; Zornoza, Raúl; Martínez-Martínez, Silvia; Bech, Jaume

    2015-04-01

    Intense mining activities in the past were carried out in Cartagena-La Unión mining district, SE Spain, and caused excessive accumulation of toxic metals in tailing ponds which poses a high environmental and ecological risk. One of the remediation options gaining considerable interest in recent years is the in situ immobilization of metals. A corresponding reduction in the plant-available metal fraction allows re-vegetation and ecosystem restoration of the heavily contaminated sites. In addition, the use of microorganisms to improve the soil condition is a new tool used to increase spontaneous plant colonization. The aim of this research was to assess the effect of amendments (pig manure, sewage sludge, and lime) and microorganisms on the evolution of soil properties and metals in acid and alkaline tailing ponds and to evaluate the content of metals in Zygophylum fabago one year after amendments application. The study was carried out in two mine ponds (acid and alkaline). Twenty seven square field plots, each one consisting of 4 m2, were located in each pond. Four different doses of microorganism (EM) (0 ml, 20 ml, 100 ml and 200 ml of microorganism solution in each plot) and one dose of pig manure (5 kg per plot), sewage sludge (4 kg per plot) and lime (22 kg per plot) were used. Organic amendment doses were calculated according to European nitrogen legislations, and lime dose was calculated according with the potential acid production through total sulphur oxidation. Three replicates of each treatment (organic amendment + lime + microorganism dose 0, 1, 2, or 3) and control soil (with no amendments) were carried out. Plots were left to the semi-arid climate conditions after the addition of amendments to simulate real potential applications of the results. Soil samples was collected every 4 month from each plot during one year, after this time Zygophylum fabago plants were sampled from each plots. Soil properties including: pH, salinity, total, inorganic and

  6. Differential Occurrence of Interactions and Interaction Domains in Proteins Containing Homopolymeric Amino Acid Repeats.

    PubMed

    Pelassa, Ilaria; Fiumara, Ferdinando

    2015-01-01

    Homopolymeric amino acids repeats (AARs), which are widespread in proteomes, have often been viewed simply as spacers between protein domains, or even as "junk" sequences with no obvious function but with a potential to cause harm upon expansion as in genetic diseases associated with polyglutamine or polyalanine expansions, including Huntington disease and cleidocranial dysplasia. A growing body of evidence indicates however that at least some AARs can form organized, functional protein structures, and can regulate protein function. In particular, certain AARs can mediate protein-protein interactions, either through homotypic AAR-AAR contacts or through heterotypic contacts with other protein domains. It is still unclear however, whether AARs may have a generalized, proteome-wide role in shaping protein-protein interaction networks. Therefore, we have undertaken here a bioinformatics screening of the human proteome and interactome in search of quantitative evidence of such a role. We first identified the sets of proteins that contain repeats of any one of the 20 amino acids, as well as control sets of proteins chosen at random in the proteome. We then analyzed the connectivity between the proteins of the AAR-containing protein sets and we compared it with that observed in the corresponding control networks. We find evidence for different degrees of connectivity in the different AAR-containing protein networks. Indeed, networks of proteins containing polyglutamine, polyglutamate, polyproline, and other AARs show significantly increased levels of connectivity, whereas networks containing polyleucine and other hydrophobic repeats show lower degrees of connectivity. Furthermore, we observed that numerous protein-protein, -nucleic acid, and -lipid interaction domains are significantly enriched in specific AAR protein groups. These findings support the notion of a generalized, combinatorial role of AARs, together with conventional protein interaction domains, in shaping

  7. In situ bioremediation of naphthenic acids contaminated tailing pond waters in the athabasca oil sands region--demonstrated field studies and plausible options: a review.

    PubMed

    Quagraine, E K; Peterson, H G; Headley, J V

    2005-01-01

    Currently, there are three industrial plants that recover oil from the lower Athabasca oil sands area, and there are plans in the future for several additional mines. The extraction procedures produce large volumes of slurry wastes contaminated with naphthenic acids (NAs). Because of a "zero discharge" policy the oil sands companies do not release any extraction wastes from their leases. The process-affected waters and fluid tailings contaminated with NAs are contained on-site primarily in large settling ponds. These fluid wastes from the tailing ponds can be acutely and chronically toxic to aquatic organisms, and NAs have been associated with this toxicity. The huge tailings containment area must ultimately be reclaimed, and this is of major concern to the oil sands industry. Some reclamation options have been investigated by both pioneering industries (Syncrude Energy Inc. and Suncor Inc.) with mixed results. The bioremediation techniques have limited success to date in biodegrading NAs to levels below 19 mg/L. Some tailing pond waters have been stored for more than 10 years, and it appears that the remaining high molecular weight NAs are refractory to the natural biodegradation process in the ponds. Some plausible options to further degrade the NAs in the tailings pond water include: bioaugmentation with bacteria selected to degrade the more refractory classes of NAs; the use of attachment materials such as clays to concentrate both the NA and the NA-degrading bacteria in their surfaces and/or pores; synergistic association between algae and bacteria consortia to promote efficient aerobic degradation; and biostimulation with nutrients to promote the growth and activity of the microorganisms. PMID:15756978

  8. Listeria phage and phage tail induction triggered by components of bacterial growth media (phosphate, LiCl, nalidixic acid, and acriflavine).

    PubMed

    Lemaître, Jean-Paul; Duroux, Amandine; Pimpie, Romain; Duez, Jean-Marie; Milat, Marie-Louise

    2015-03-01

    The detection of Listeria monocytogenes from food is currently carried out using a double enrichment. For the ISO methodology, this double enrichment is performed using half-Fraser and Fraser broths, in which the overgrowth of L. innocua can occur in samples where both species are present. In this study, we analyzed the induction of phages and phage tails of Listeria spp. in these media and in two brain heart infusion (BHI) broths (BHIM [bioMérieux] and BHIK [Biokar]) to identify putative effectors. It appears that Na2HPO4 at concentrations ranging from 1 to 40 g/liter with an initial pH of 7.5 can induce phage or phage tail production of Listeria spp., especially with 10 g/liter of Na2HPO4 and a pH of 7.5, conditions present in half-Fraser and Fraser broths. Exposure to LiCl in BHIM (18 to 21 g/liter) can also induce phage and phage tail release, but in half-Fraser and Fraser broths, the concentration of LiCl is much lower (3 g/liter). Low phage titers were induced by acriflavine and/or nalidixic acid. We also show that the production of phages and phage tails can occur in half-Fraser and Fraser broths. This study points out that induction of phages and phage tails could be triggered by compounds present in enrichment media. This could lead to a false-negative result for the detection of L. monocytogenes in food products.

  9. Clustering amino acid contents of protein domains: biochemical functions of proteins and implications for origin of biological macromolecules.

    PubMed

    Torshin, I Y

    2001-04-01

    Structural classes of protein domains correlate with their amino acid compositions. Several successful algorithms (that use only amino acid composition) have been elaborated for the prediction of structural class or potential biochemical significance. This work deals with dynamic classification (clustering) of the domains on the basis of their amino acid composition. Amino acid contents of domains from a non-redundant PDB set were clustered in 20-dimensional space of amino acid contents. Despite the variations of an empirical parameter and non-redundancy of the set, only one large cluster (tens-hundreds of proteins) surrounded by hundreds of small clusters (1-5 proteins), was identified. The core of the largest cluster contains at least 64% DNA (nucleotide)-interacting protein domains from various sources. About 90% of the proteins of the core are intracellular proteins. 83% of the DNA/nucleotide interacting domains in the core belong to the mixed alpha-beta folds (a+b, a/b), 14% are all-alpha (mostly helices) and all-beta (mostly beta-strands) proteins. At the same time, when core domains that belong to one organism (E.coli) are considered, over 80% of them prove to be DNA/nucleotide interacting proteins. The core is compact: amino acid contents of domains from the core lie in relatively narrow and specific ranges. The core also contains several Fe-S cluster-binding domains, amino acid contents of the core overlap with ferredoxin and CO-dehydrogenase clusters, the oldest known proteins. As Fe-S clusters are thought to be the first biocatalysts, the results are discussed in relation to contemporary experiments and models dealing with the origin of biological macromolecules. The origin of most primordial proteins is considered here to be a result of co-adsorption of nucleotides and amino acids on specific clays, followed by en-block polymerization of the adsorbed mixtures of amino acids.

  10. Change in oligomerization specificity of the p53 tetramerization domain by hydrophobic amino acid substitutions.

    PubMed Central

    Stavridi, E. S.; Chehab, N. H.; Caruso, L. C.; Halazonetis, T. D.

    1999-01-01

    The tumor suppressor function of the wild-type p53 protein is transdominantly inhibited by tumor-derived mutant p53 proteins. Such transdominant inhibition limits the prospects for gene therapy approaches that aim to introduce wild-type p53 into cancer cells. The molecular mechanism for transdominant inhibition involves sequestration of wild-type p53 subunits into inactive wild-type/mutant hetero-tetramers. Thus, p53 proteins, whose oligomerization specificity is altered so they cannot interact with tumor-derived mutant p53, would escape transdominant inhibition. Aided by the known three-dimensional structure of the p53 tetramerization domain and by trial and error we designed a novel domain with seven amino acid substitutions in the hydrophobic core. A full-length p53 protein bearing this novel domain formed homo-tetramers and had tumor suppressor function, but did not hetero-oligomerize with tumor-derived mutant p53 and resisted transdominant inhibition. Thus, hydrophobic core residues influence the oligomerization specificity of the p53 tetramerization domain. PMID:10493578

  11. Chaperone-protein interactions that mediate assembly of the bacteriophage lambda tail to the correct length

    PubMed Central

    Xu, Jun; Hendrix, Roger W.; Duda, Robert L.

    2013-01-01

    Bacteriophage λ makes two proteins with overlapping amino acid sequences that are essential for tail assembly. These two proteins, gpG and gpGT, are related by a programmed translational frameshift that is conserved among diverse phages and functions in λ to ensure that gpG and the frameshift product gpGT are made in a molar ratio of approximately 30:1. Although both proteins are required and must be present in the correct ratio for assembly of functional tails, neither is present in mature tails. During λ tail assembly major tail protein gpV polymerizes to form a long tube whose length is controlled by tape measure protein gpH. We show that the ‘G’ domains of gpG and gpGT bind to all or parts of tail length tape measure protein gpH, that the ‘T’ domain of gpGT binds to major tail shaft subunit gpV, and present a model for how gpG and gpGT chaperone gpH and direct the polymerization of gpV to form a tail of the correct length. PMID:23911548

  12. Effects of organic acids on the photosynthetic and antioxidant properties and accumulations of heavy metals of Melilotus officinalis grown in Cu tailing.

    PubMed

    Han, Yulin; Wu, Xue; Gu, Jiguang; Zhao, Jiuzhou; Huang, Suzhen; Yuan, Haiyan; Fu, Jiajia

    2016-09-01

    The effect of citric acid (CA), acetic acid (Ac), and ethylene diamine tetraacetic acid (EDTA) on the photosynthetic and antioxidant properties and the accumulation of some heavy metals (HMs) of Melilotus officinalis seedling growing in Cu mine tailings for 25 days were studied. Results showed that the formation of photosynthesizing cells of M. officinalis was inhibited by EDTA at 2 mmol/kg. Photosynthetic pigment contents under EDTA of 2 mmol/kg were reduced by 26, 40, and 19 %, respectively, compared to the control. The proline contents in aboveground and underground parts increased as the level of EDTA was enhanced. CA and Ac enhanced the activities of superoxide dismutase (SOD) and peroxidase (POD) in the aboveground parts and EDTA inhibited the activity of POD in the underground parts. The addition of CA promoted significantly the growth of M. officinalis, while the biomass decreased significantly under 2 mmol/kg EDTA. Cu contents in the aboveground parts treated with 0.5 and 2.0 mmol/kg EDTA reached 175.50 and 265.17 μg/g dry weight, respectively. Ac and EDTA treatments promoted Cd to translocate from root to aboveground parts. The result indicated that M. officinalis was a tolerant species of Cu tailing and can be used to remediate Cu contaminated environment, and rationally utilization of organic acids, especially EDTA, in the phytoremediation can improve the growth and metals accumulation of M. officinalis. PMID:27255310

  13. Effects of organic acids on the photosynthetic and antioxidant properties and accumulations of heavy metals of Melilotus officinalis grown in Cu tailing.

    PubMed

    Han, Yulin; Wu, Xue; Gu, Jiguang; Zhao, Jiuzhou; Huang, Suzhen; Yuan, Haiyan; Fu, Jiajia

    2016-09-01

    The effect of citric acid (CA), acetic acid (Ac), and ethylene diamine tetraacetic acid (EDTA) on the photosynthetic and antioxidant properties and the accumulation of some heavy metals (HMs) of Melilotus officinalis seedling growing in Cu mine tailings for 25 days were studied. Results showed that the formation of photosynthesizing cells of M. officinalis was inhibited by EDTA at 2 mmol/kg. Photosynthetic pigment contents under EDTA of 2 mmol/kg were reduced by 26, 40, and 19 %, respectively, compared to the control. The proline contents in aboveground and underground parts increased as the level of EDTA was enhanced. CA and Ac enhanced the activities of superoxide dismutase (SOD) and peroxidase (POD) in the aboveground parts and EDTA inhibited the activity of POD in the underground parts. The addition of CA promoted significantly the growth of M. officinalis, while the biomass decreased significantly under 2 mmol/kg EDTA. Cu contents in the aboveground parts treated with 0.5 and 2.0 mmol/kg EDTA reached 175.50 and 265.17 μg/g dry weight, respectively. Ac and EDTA treatments promoted Cd to translocate from root to aboveground parts. The result indicated that M. officinalis was a tolerant species of Cu tailing and can be used to remediate Cu contaminated environment, and rationally utilization of organic acids, especially EDTA, in the phytoremediation can improve the growth and metals accumulation of M. officinalis.

  14. Hydroxycinnamic acid-derived polymers constitute the polyaromatic domain of suberin

    NASA Technical Reports Server (NTRS)

    Bernards, M. A.; Lopez, M. L.; Zajicek, J.; Lewis, N. G.

    1995-01-01

    Suberin is an abundant, complex, intractable, plant cell wall polymeric network that forms both protective and wound-healing layers. Its function is, therefore, critical to the survival of all vascular plants. Its chemical structure and biosynthesis are poorly defined, although it is known to consist of both aromatic and aliphatic domains. While the composition of the aliphatic component has been fairly well characterized, that of the phenolic component has not. Using a combination of specific carbon-13 labeling techniques, and in situ solid state 13C NMR spectroscopic analysis, we now provide the first direct evidence for the nature of the phenolic domain of suberin and report here that it is almost exclusively comprised of a covalently linked, hydroxycinnamic acid-derived polymeric matrix.

  15. Amino acid coevolution reveals three-dimensional structure and functional domains of insect odorant receptors

    PubMed Central

    Hopf, Thomas A.; Morinaga, Satoshi; Ihara, Sayoko; Touhara, Kazushige; Marks, Debora S.; Benton, Richard

    2015-01-01

    Insect Odorant Receptors (ORs) comprise an enormous protein family that translates environmental chemical signals into neuronal electrical activity. These heptahelical receptors are proposed to function as ligand-gated ion channels and/or to act metabotropically as G protein-coupled receptors (GPCRs). Resolving their signalling mechanism has been hampered by the lack of tertiary structural information and primary sequence similarity to other proteins. We use amino acid evolutionary covariation across these ORs to define restraints on structural proximity of residue pairs, which permit de novo generation of three-dimensional models. The validity of our analysis is supported by the location of functionally important residues in highly constrained regions of the protein. Importantly, insect OR models exhibit a distinct transmembrane domain packing arrangement to that of canonical GPCRs, establishing the structural unrelatedness of these receptor families. The evolutionary couplings and models predict odour binding and ion conduction domains, and provide a template for rationale structure-activity dissection. PMID:25584517

  16. The acidic domain of the endothelial membrane protein GPIHBP1 stabilizes lipoprotein lipase activity by preventing unfolding of its catalytic domain

    PubMed Central

    Mysling, Simon; Kristensen, Kristian Kølby; Larsson, Mikael; Beigneux, Anne P; Gårdsvoll, Henrik; Fong, Loren G; Bensadouen, André; Jørgensen, Thomas JD; Young, Stephen G; Ploug, Michael

    2016-01-01

    GPIHBP1 is a glycolipid-anchored membrane protein of capillary endothelial cells that binds lipoprotein lipase (LPL) within the interstitial space and shuttles it to the capillary lumen. The LPL•GPIHBP1 complex is responsible for margination of triglyceride-rich lipoproteins along capillaries and their lipolytic processing. The current work conceptualizes a model for the GPIHBP1•LPL interaction based on biophysical measurements with hydrogen-deuterium exchange/mass spectrometry, surface plasmon resonance, and zero-length cross-linking. According to this model, GPIHBP1 comprises two functionally distinct domains: (1) an intrinsically disordered acidic N-terminal domain; and (2) a folded C-terminal domain that tethers GPIHBP1 to the cell membrane by glycosylphosphatidylinositol. We demonstrate that these domains serve different roles in regulating the kinetics of LPL binding. Importantly, the acidic domain stabilizes LPL catalytic activity by mitigating the global unfolding of LPL's catalytic domain. This study provides a conceptual framework for understanding intravascular lipolysis and GPIHBP1 and LPL mutations causing familial chylomicronemia. DOI: http://dx.doi.org/10.7554/eLife.12095.001 PMID:26725083

  17. The acidic domain of the endothelial membrane protein GPIHBP1 stabilizes lipoprotein lipase activity by preventing unfolding of its catalytic domain.

    PubMed

    Mysling, Simon; Kristensen, Kristian Kølby; Larsson, Mikael; Beigneux, Anne P; Gårdsvoll, Henrik; Fong, Loren G; Bensadouen, André; Jørgensen, Thomas Jd; Young, Stephen G; Ploug, Michael

    2016-01-01

    GPIHBP1 is a glycolipid-anchored membrane protein of capillary endothelial cells that binds lipoprotein lipase (LPL) within the interstitial space and shuttles it to the capillary lumen. The LPL•GPIHBP1 complex is responsible for margination of triglyceride-rich lipoproteins along capillaries and their lipolytic processing. The current work conceptualizes a model for the GPIHBP1•LPL interaction based on biophysical measurements with hydrogen-deuterium exchange/mass spectrometry, surface plasmon resonance, and zero-length cross-linking. According to this model, GPIHBP1 comprises two functionally distinct domains: (1) an intrinsically disordered acidic N-terminal domain; and (2) a folded C-terminal domain that tethers GPIHBP1 to the cell membrane by glycosylphosphatidylinositol. We demonstrate that these domains serve different roles in regulating the kinetics of LPL binding. Importantly, the acidic domain stabilizes LPL catalytic activity by mitigating the global unfolding of LPL's catalytic domain. This study provides a conceptual framework for understanding intravascular lipolysis and GPIHBP1 and LPL mutations causing familial chylomicronemia. PMID:26725083

  18. The acidic domain of the endothelial membrane protein GPIHBP1 stabilizes lipoprotein lipase activity by preventing unfolding of its catalytic domain.

    PubMed

    Mysling, Simon; Kristensen, Kristian Kølby; Larsson, Mikael; Beigneux, Anne P; Gårdsvoll, Henrik; Fong, Loren G; Bensadouen, André; Jørgensen, Thomas Jd; Young, Stephen G; Ploug, Michael

    2016-01-03

    GPIHBP1 is a glycolipid-anchored membrane protein of capillary endothelial cells that binds lipoprotein lipase (LPL) within the interstitial space and shuttles it to the capillary lumen. The LPL•GPIHBP1 complex is responsible for margination of triglyceride-rich lipoproteins along capillaries and their lipolytic processing. The current work conceptualizes a model for the GPIHBP1•LPL interaction based on biophysical measurements with hydrogen-deuterium exchange/mass spectrometry, surface plasmon resonance, and zero-length cross-linking. According to this model, GPIHBP1 comprises two functionally distinct domains: (1) an intrinsically disordered acidic N-terminal domain; and (2) a folded C-terminal domain that tethers GPIHBP1 to the cell membrane by glycosylphosphatidylinositol. We demonstrate that these domains serve different roles in regulating the kinetics of LPL binding. Importantly, the acidic domain stabilizes LPL catalytic activity by mitigating the global unfolding of LPL's catalytic domain. This study provides a conceptual framework for understanding intravascular lipolysis and GPIHBP1 and LPL mutations causing familial chylomicronemia.

  19. Domain-level rocking motion within a polymerase that translocates on single-stranded nucleic acid

    SciTech Connect

    Li, Huiyung; Li, Changzheng; Zhou, Sufeng; Poulos, Thomas L.; Gershon, Paul David

    2013-04-01

    An X-ray crystallographic structure is described for unliganded Vaccinia virus poly(A) polymerase monomer (VP55), showing the first domain-level structural isoforms among either VP55, it’s processivity factor VP39, or the VP55-VP39 heterodimer. The occurrence of domain-level motion specifically in monomeric VP55 is consistent with the finding that the monomeric protein undergoes saltatory translocation whereas the heterodimer does not. Vaccinia virus poly(A) polymerase (VP55) is the only known polymerase that can translocate independently with respect to single-stranded nucleic acid (ssNA). Previously, its structure has only been solved in the context of the VP39 processivity factor. Here, a crystal structure of unliganded monomeric VP55 has been solved to 2.86 Å resolution, showing the first backbone structural isoforms among either VP55 or its processivity factor (VP39). Backbone differences between the two molecules of VP55 in the asymmetric unit indicated that unliganded monomeric VP55 can undergo a ‘rocking’ motion of the N-terminal domain with respect to the other two domains, which may be ‘rigidified’ upon VP39 docking. This observation is consistent with previously demonstrated experimental molecular dynamics of the monomer during translocation with respect to nucleic acid and with different mechanisms of translocation in the presence and absence of processivity factor VP39. Side-chain conformational changes in the absence of ligand were observed at a key primer contact site and at the catalytic center of VP55. The current structure completes the trio of possible structural forms for VP55 and VP39, namely the VP39 monomer, the VP39–VP55 heterodimer and the VP55 monomer.

  20. Myometrial relaxation of mice via expression of two pore domain acid sensitive K(+) (TASK-2) channels.

    PubMed

    Kyeong, Kyu-Sang; Hong, Seung Hwa; Kim, Young Chul; Cho, Woong; Myung, Sun Chul; Lee, Moo Yeol; You, Ra Young; Kim, Chan Hyung; Kwon, So Yeon; Suzuki, Hikaru; Park, Yeon Jin; Jeong, Eun-Hwan; Kim, Hak Soon; Kim, Heon; Lim, Seung Woon; Xu, Wen-Xie; Lee, Sang Jin; Ji, Il Woon

    2016-09-01

    Myometrial relaxation of mouse via expression of two-pore domain acid sensitive (TASK) channels was studied. In our previous report, we suggested that two-pore domain acid-sensing K(+) channels (TASK-2) might be one of the candidates for the regulation of uterine circular smooth muscles in mice. In this study, we tried to show the mechanisms of relaxation via TASK-2 channels in marine myometrium. Isometric contraction measurements and patch clamp technique were used to verify TASK conductance in murine myometrium. Western blot and immunehistochemical study under confocal microscopy were used to investigate molecular identity of TASK channel. In this study, we showed that TEA and 4-AP insensitive non-inactivating outward K(+) current (NIOK) may be responsible for the quiescence of murine pregnant longitudinal myometrium. The characteristics of NIOK coincided with two-pore domain acid-sensing K(+) channels (TASK-2). NIOK in the presence of K(+) channel blockers was inhibited further by TASK inhibitors such as quinidine, bupivacaine, lidocaine, and extracellular acidosis. Furthermore, oxytocin and estrogen inhibited NIOK in pregnant myometrium. When compared to non-pregnant myometrium, pregnant myometrium showed stronger inhibition of NIOK by quinidine and increased immunohistochemical expression of TASK-2. Finally, TASK-2 inhibitors induced strong myometrial contraction even in the presence of L-methionine, a known inhibitor of stretch-activated channels in the longitudinal myometrium of mouse. Activation of TASK-2 channels seems to play an essential role for relaxing uterus during pregnancy and it might be one of the alternatives for preventing preterm delivery.

  1. Myometrial relaxation of mice via expression of two pore domain acid sensitive K+ (TASK-2) channels

    PubMed Central

    Kyeong, Kyu-Sang; Hong, Seung Hwa; Cho, Woong; Myung, Sun Chul; Lee, Moo Yeol; You, Ra Young; Kim, Chan Hyung; Kwon, So Yeon; Suzuki, Hikaru; Park, Yeon Jin; Jeong, Eun-Hwan; Kim, Hak Soon; Kim, Heon; Lim, Seung Woon; Xu, Wen-Xie; Lee, Sang Jin

    2016-01-01

    Myometrial relaxation of mouse via expression of two-pore domain acid sensitive (TASK) channels was studied. In our previous report, we suggested that two-pore domain acid-sensing K+ channels (TASK-2) might be one of the candidates for the regulation of uterine circular smooth muscles in mice. In this study, we tried to show the mechanisms of relaxation via TASK-2 channels in marine myometrium. Isometric contraction measurements and patch clamp technique were used to verify TASK conductance in murine myometrium. Western blot and immunehistochemical study under confocal microscopy were used to investigate molecular identity of TASK channel. In this study, we showed that TEA and 4-AP insensitive non-inactivating outward K+ current (NIOK) may be responsible for the quiescence of murine pregnant longitudinal myometrium. The characteristics of NIOK coincided with two-pore domain acid-sensing K+ channels (TASK-2). NIOK in the presence of K+ channel blockers was inhibited further by TASK inhibitors such as quinidine, bupivacaine, lidocaine, and extracellular acidosis. Furthermore, oxytocin and estrogen inhibited NIOK in pregnant myometrium. When compared to non-pregnant myometrium, pregnant myometrium showed stronger inhibition of NIOK by quinidine and increased immunohistochemical expression of TASK-2. Finally, TASK-2 inhibitors induced strong myometrial contraction even in the presence of L-methionine, a known inhibitor of stretch-activated channels in the longitudinal myometrium of mouse. Activation of TASK-2 channels seems to play an essential role for relaxing uterus during pregnancy and it might be one of the alternatives for preventing preterm delivery. PMID:27610042

  2. Myometrial relaxation of mice via expression of two pore domain acid sensitive K(+) (TASK-2) channels.

    PubMed

    Kyeong, Kyu-Sang; Hong, Seung Hwa; Kim, Young Chul; Cho, Woong; Myung, Sun Chul; Lee, Moo Yeol; You, Ra Young; Kim, Chan Hyung; Kwon, So Yeon; Suzuki, Hikaru; Park, Yeon Jin; Jeong, Eun-Hwan; Kim, Hak Soon; Kim, Heon; Lim, Seung Woon; Xu, Wen-Xie; Lee, Sang Jin; Ji, Il Woon

    2016-09-01

    Myometrial relaxation of mouse via expression of two-pore domain acid sensitive (TASK) channels was studied. In our previous report, we suggested that two-pore domain acid-sensing K(+) channels (TASK-2) might be one of the candidates for the regulation of uterine circular smooth muscles in mice. In this study, we tried to show the mechanisms of relaxation via TASK-2 channels in marine myometrium. Isometric contraction measurements and patch clamp technique were used to verify TASK conductance in murine myometrium. Western blot and immunehistochemical study under confocal microscopy were used to investigate molecular identity of TASK channel. In this study, we showed that TEA and 4-AP insensitive non-inactivating outward K(+) current (NIOK) may be responsible for the quiescence of murine pregnant longitudinal myometrium. The characteristics of NIOK coincided with two-pore domain acid-sensing K(+) channels (TASK-2). NIOK in the presence of K(+) channel blockers was inhibited further by TASK inhibitors such as quinidine, bupivacaine, lidocaine, and extracellular acidosis. Furthermore, oxytocin and estrogen inhibited NIOK in pregnant myometrium. When compared to non-pregnant myometrium, pregnant myometrium showed stronger inhibition of NIOK by quinidine and increased immunohistochemical expression of TASK-2. Finally, TASK-2 inhibitors induced strong myometrial contraction even in the presence of L-methionine, a known inhibitor of stretch-activated channels in the longitudinal myometrium of mouse. Activation of TASK-2 channels seems to play an essential role for relaxing uterus during pregnancy and it might be one of the alternatives for preventing preterm delivery. PMID:27610042

  3. Myometrial relaxation of mice via expression of two pore domain acid sensitive K+ (TASK-2) channels

    PubMed Central

    Kyeong, Kyu-Sang; Hong, Seung Hwa; Cho, Woong; Myung, Sun Chul; Lee, Moo Yeol; You, Ra Young; Kim, Chan Hyung; Kwon, So Yeon; Suzuki, Hikaru; Park, Yeon Jin; Jeong, Eun-Hwan; Kim, Hak Soon; Kim, Heon; Lim, Seung Woon; Xu, Wen-Xie; Lee, Sang Jin

    2016-01-01

    Myometrial relaxation of mouse via expression of two-pore domain acid sensitive (TASK) channels was studied. In our previous report, we suggested that two-pore domain acid-sensing K+ channels (TASK-2) might be one of the candidates for the regulation of uterine circular smooth muscles in mice. In this study, we tried to show the mechanisms of relaxation via TASK-2 channels in marine myometrium. Isometric contraction measurements and patch clamp technique were used to verify TASK conductance in murine myometrium. Western blot and immunehistochemical study under confocal microscopy were used to investigate molecular identity of TASK channel. In this study, we showed that TEA and 4-AP insensitive non-inactivating outward K+ current (NIOK) may be responsible for the quiescence of murine pregnant longitudinal myometrium. The characteristics of NIOK coincided with two-pore domain acid-sensing K+ channels (TASK-2). NIOK in the presence of K+ channel blockers was inhibited further by TASK inhibitors such as quinidine, bupivacaine, lidocaine, and extracellular acidosis. Furthermore, oxytocin and estrogen inhibited NIOK in pregnant myometrium. When compared to non-pregnant myometrium, pregnant myometrium showed stronger inhibition of NIOK by quinidine and increased immunohistochemical expression of TASK-2. Finally, TASK-2 inhibitors induced strong myometrial contraction even in the presence of L-methionine, a known inhibitor of stretch-activated channels in the longitudinal myometrium of mouse. Activation of TASK-2 channels seems to play an essential role for relaxing uterus during pregnancy and it might be one of the alternatives for preventing preterm delivery.

  4. Matrix Domain Modulates HIV-1 Gag's Nucleic Acid Chaperone Activity via Inositol Phosphate Binding ▿

    PubMed Central

    Jones, Christopher P.; Datta, Siddhartha A. K.; Rein, Alan; Rouzina, Ioulia; Musier-Forsyth, Karin

    2011-01-01

    Retroviruses replicate by reverse transcribing their single-stranded RNA genomes into double-stranded DNA using specific cellular tRNAs to prime cDNA synthesis. In HIV-1, human tRNA3Lys serves as the primer and is packaged into virions during assembly. The viral Gag protein is believed to chaperone tRNA3Lys placement onto the genomic RNA primer binding site; however, the timing and possible regulation of this event are currently unknown. Composed of the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains, the multifunctional HIV-1 Gag polyprotein orchestrates the highly coordinated process of virion assembly, but the contribution of these domains to tRNA3Lys annealing is unclear. Here, we show that NC is absolutely essential for annealing and that the MA domain inhibits Gag's tRNA annealing capability. During assembly, MA specifically interacts with inositol phosphate (IP)-containing lipids in the plasma membrane (PM). Surprisingly, we find that IPs stimulate Gag-facilitated tRNA annealing but do not stimulate annealing in Gag variants lacking the MA domain or containing point mutations involved in PM binding. Moreover, we find that IPs prevent MA from binding to nucleic acids but have little effect on NC or Gag. We propose that Gag binds to RNA either with both NC and MA domains or with NC alone and that MA-IP interactions alter Gag's binding mode. We propose that MA's interactions with the PM trigger the switch between these two binding modes and stimulate Gag's chaperone function, which may be important for the regulation of events such as tRNA primer annealing. PMID:21123373

  5. Tail Buffeting

    NASA Technical Reports Server (NTRS)

    Abdrashitov, G.

    1943-01-01

    An approximate theory of buffeting is here presented, based on the assumption of harmonic disturbing forces. Two cases of buffeting are considered: namely, for a tail angle of attack greater and less than the stalling angle, respectively. On the basis of the tests conducted and the results of foreign investigators, a general analysis is given of the nature of the forced vibrations the possible load limits on the tail, and the methods of elimination of buffeting.

  6. Quantitative analyses of tartaric acid based on terahertz time domain spectroscopy

    NASA Astrophysics Data System (ADS)

    Cao, Binghua; Fan, Mengbao

    2010-10-01

    Terahertz wave is the electromagnetic spectrum situated between microwave and infrared wave. Quantitative analysis based on terahertz spectroscopy is very important for the application of terahertz techniques. But how to realize it is still under study. L-tartaric acid is widely used as acidulant in beverage, and other food, such as soft drinks, wine, candy, bread and some colloidal sweetmeats. In this paper, terahertz time-domain spectroscopy is applied to quantify the tartaric acid. Two methods are employed to process the terahertz spectra of different samples with different content of tartaric acid. The first one is linear regression combining correlation analysis. The second is partial least square (PLS), in which the absorption spectra in the 0.8-1.4THz region are used to quantify the tartaric acid. To compare the performance of these two principles, the relative error of the two methods is analyzed. For this experiment, the first method does better than the second one. But the first method is suitable for the quantitative analysis of materials which has obvious terahertz absorption peaks, while for material which has no obvious terahertz absorption peaks, the second one is more appropriate.

  7. The (unusual) aspartic acid in the metal coordination sphere of the prokaryotic zinc finger domain.

    PubMed

    D'Abrosca, Gianluca; Russo, Luigi; Palmieri, Maddalena; Baglivo, Ilaria; Netti, Fortuna; de Paola, Ivan; Zaccaro, Laura; Farina, Biancamaria; Iacovino, Rosa; Pedone, Paolo Vincenzo; Isernia, Carla; Fattorusso, Roberto; Malgieri, Gaetano

    2016-08-01

    The possibility of choices of protein ligands and coordination geometries leads to diverse Zn(II) binding sites in zinc-proteins, allowing a range of important biological roles. The prokaryotic Cys2His2 zinc finger domain (originally found in the Ros protein from Agrobacterium tumefaciens) tetrahedrally coordinates zinc through two cysteine and two histidine residues and it does not adopt a correct fold in the absence of the metal ion. Ros is the first structurally characterized member of a family of bacterial proteins that presents several amino acid changes in the positions occupied in Ros by the zinc coordinating residues. In particular, the second position is very often occupied by an aspartic acid although the coordination of structural zinc by an aspartate in eukaryotic zinc fingers is very unusual. Here, by appropriately mutating the protein Ros, we characterize the aspartate role within the coordination sphere of this family of proteins demonstrating how the presence of this residue only slightly perturbs the functional structure of the prokaryotic zinc finger domain while it greatly influences its thermodynamic properties. PMID:27238756

  8. Acidic domain in dentin phosphophoryn facilitates cellular uptake: implications in targeted protein delivery.

    PubMed

    Ravindran, Sriram; Snee, Preston T; Ramachandran, Amsaveni; George, Anne

    2013-05-31

    Dentin phosphophoryn is nature's most acidic protein found predominantly in the dentin extracellular matrix. Its unique amino acid composition containing Asp-Ser (DS)-rich repeats makes it highly anionic. It has a low isoelectric point (pI 1.1) and, therefore, tends to be negatively charged at physiological pH. Phosphophoryn is normally associated with matrix mineralization as it can bind avidly to Ca(2+). It is well known that several macromolecules present in the extracellular matrix can be internalized and localized to specific intracellular compartments. In this study we demonstrate that dentin phosphophoryn (DPP) is internalized by several cell types via a non-conventional endocytic process. Utilizing a DSS polypeptide derived from DPP, we demonstrate the repetitive DSS-rich domain facilitates that endocytosis. As a proof-of-concept, we further demonstrate the use of this polypeptide as a protein delivery vehicle by delivering the osteoblast transcription factor Runx2 to the nucleus of mesenchymal cells. The functionality of the endocytosed Runx2 protein was demonstrated by performing gene expression analysis of Runx2 target genes. Nuclear localization was also demonstrated with the fusion protein DSS-Runx2 conjugated to quantum dots in two- and three-dimensional culture models in vitro and in vivo. Overall, we demonstrate that the DSS domain of DPP functions as a novel cell-penetrating peptide, and these findings demonstrate new opportunities for intracellular delivery of therapeutic proteins and cell tracking in vivo.

  9. Eicosapentaenoic acid inhibits glucose-induced membrane cholesterol crystalline domain formation through a potent antioxidant mechanism.

    PubMed

    Mason, R Preston; Jacob, Robert F

    2015-02-01

    Lipid oxidation leads to endothelial dysfunction, inflammation, and foam cell formation during atherogenesis. Glucose also contributes to lipid oxidation and promotes pathologic changes in membrane structural organization, including the development of cholesterol crystalline domains. In this study, we tested the comparative effects of eicosapentaenoic acid (EPA), an omega-3 fatty acid indicated for the treatment of very high triglyceride (TG) levels, and other TG-lowering agents (fenofibrate, niacin, and gemfibrozil) on lipid oxidation in human low-density lipoprotein (LDL) as well as membrane lipid vesicles prepared in the presence of glucose (200 mg/dL). We also examined the antioxidant effects of EPA in combination with atorvastatin o-hydroxy (active) metabolite (ATM). Glucose-induced changes in membrane structural organization were measured using small angle x-ray scattering approaches and correlated with changes in lipid hydroperoxide (LOOH) levels. EPA was found to inhibit LDL oxidation in a dose-dependent manner (1.0-10.0 µM) and was distinguished from the other TG-lowering agents, which had no significant effect as compared to vehicle treatment alone. Similar effects were observed in membrane lipid vesicles exposed to hyperglycemic conditions. The antioxidant activity of EPA, as observed in glucose-treated vesicles, was significantly enhanced in combination with ATM. Glucose treatment produced highly-ordered, membrane-restricted, cholesterol crystalline domains, which correlated with increased LOOH levels. Of the agents tested in this study, only EPA inhibited glucose-induced cholesterol domain formation. These data demonstrate that EPA, at pharmacologic levels, inhibits hyperglycemia-induced changes in membrane lipid structural organization through a potent antioxidant mechanism associated with its distinct, physicochemical interactions with the membrane bilayer. PMID:25449996

  10. Sequence-dependent nucleosome structural and dynamic polymorphism. Potential involvement of histone H2B N-terminal tail proximal domain.

    PubMed

    Sivolob, Andrei; Lavelle, Christophe; Prunell, Ariel

    2003-02-01

    Relaxation of nucleosomes on an homologous series (pBR) of ca 350-370 bp DNA minicircles originating from plasmid pBR322 was recently used as a tool to study their structure and dynamics. These nucleosomes thermally fluctuated between three distinct DNA conformations within a histone N-terminal tail-modulated equilibrium: one conformation was canonical, with 1.75 turn wrapping and negatively crossed entering and exiting DNAs; another was also "closed", but with these DNAs positively crossed; and the third was "open", with a lower than 1.5 turn wrapping and uncrossed DNAs. In this work, a new minicircle series (5S) of similar size was used, which contained the 5S nucleosome positioning sequence. Results showed that DNA in pBR nucleosomes was untwisted by approximately 0.2 turn relative to 5S nucleosomes, which DNase I footprinting confirmed in revealing a approximately 1 bp untwisting at each of the two dyad-distal sites where H2B N-terminal tails pass between the two gyres. In contrast, both nucleosomes showed untwistings at the dyad-proximal sites, i.e. on the other gyre, which were also observed in the high-resolution crystal structure. 5S nucleosomes also differ with respect to their dynamics: they hardly accessed the positively crossed conformation, but had an easier access to the negatively crossed conformation. Simulation showed that such reverse effects on the conformational free energies could be simply achieved by slightly altering the trajectories of entering and exiting DNAs. We propose that this is accomplished by H2B tail untwisting at the distal sites through action at a distance ( approximately 20 bp) on H3-tail interactions with the small groove at the nucleosome entry-exit. These results may help to gain a first glimpse into the two perhaps most intriguing features of the high-resolution structure: the alignment of the grooves on the two gyres and the passage of H2B and H3 N-terminal tails between them. PMID:12547190

  11. Comparative Analysis of Barophily-Related Amino Acid Content in Protein Domains of Pyrococcus abyssi and Pyrococcus furiosus

    PubMed Central

    Yafremava, Liudmila S.; Di Giulio, Massimo; Caetano-Anollés, Gustavo

    2013-01-01

    Amino acid substitution patterns between the nonbarophilic Pyrococcus furiosus and its barophilic relative P. abyssi confirm that hydrostatic pressure asymmetry indices reflect the extent to which amino acids are preferred by barophilic archaeal organisms. Substitution patterns in entire protein sequences, shared protein domains defined at fold superfamily level, domains in homologous sequence pairs, and domains of very ancient and very recent origin now provide further clues about the environment that led to the genetic code and diversified life. The pyrococcal proteomes are very similar and share a very early ancestor. Relative amino acid abundance analyses showed that biases in the use of amino acids are due to their shared fold superfamilies. Within these repertoires, only two of the five amino acids that are preferentially barophilic, aspartic acid and arginine, displayed this preference significantly and consistently across structure and in domains appearing in the ancestor. The more primordial asparagine, lysine and threonine displayed a consistent preference for nonbarophily across structure and in the ancestor. Since barophilic preferences are already evident in ancient domains that are at least ~3 billion year old, we conclude that barophily is a very ancient trait that unfolded concurrently with genetic idiosyncrasies in convergence towards a universal code. PMID:24187517

  12. Visualization of ferroelectric domains in a hydrogen-bonded molecular crystal using emission of terahertz radiation

    SciTech Connect

    Sotome, M.; Kida, N. Okamoto, H.; Horiuchi, S.

    2014-07-28

    Using a terahertz-radiation imaging, visualizations of ferroelectric domains were made in a room-temperature organic ferroelectric, croconic acid. In as-grown crystals, observed are ferroelectric domains with sizes larger than 50-μm square, which are separated by both 180° and tail-to-tail domain walls (DWs). By applying an electric field along c axis (the polarization direction), a pair of 180° DWs is generated and an each 180° DW oppositely propagates along a axis, resulting in a single domain. By cyclic applications of electric fields, a pair of 180° DWs repeatedly emerges, while no tail-to-tail DWs appear. We discuss the usefulness of the terahertz-radiation imaging as well as the observed unique DW dynamics.

  13. Growth of Quailbush in Acidic, Metalliferous Desert Mine Tailings: Effect of Azospirillum brasilense Sp6 on Biomass Production and Rhizosphere Community Structure

    PubMed Central

    de-Bashan, Luz E.; Hernandez, Juan-Pablo; Nelson, Karis N.; Bashan, Yoav

    2010-01-01

    Mine tailing deposits in semiarid and arid environments frequently remain devoid of vegetation due to the toxicity of the substrate and the absence of a diverse soil microbial community capable of supporting seed germination and plant growth. The contribution of the plant growth promoting bacterium (PGPB) Azospirillum brasilense Sp6 to the growth of quailbush in compost-amended, moderately acidic, high-metal content mine tailings using an irrigation-based reclamation strategy was examined along with its influence on the rhizosphere bacterial community. Sp6 inoculation resulted in a significant (2.2-fold) increase in plant biomass production. The data suggest that the inoculum successfully colonized the root surface and persisted throughout the 60-day experiment in both the rhizosphere, as demonstrated by excision and sequencing of the appropriate denaturing gradient gel electrophoresis (DGGE) band, and the rhizoplane, as indicated by fluorescent in situ hybridization of root surfaces. Changes in rhizosphere community structure in response to Sp6 inoculation were evaluated after 15, 30, and 60 days using DGGE analysis of 16S rRNA polymerase chain reaction amplicons. A comparison of DGGE profiles using canonical correspondence analysis revealed a significant treatment effect (Sp6-inoculated vs. uninoculated plants vs. unplanted) on bacterial community structure at 15, 30, and 60 days (p<0.05). These data indicate that in an extremely stressed environment such as acid mine tailings, an inoculated plant growth promoting bacterium not only can persist and stimulate plant growth but also can directly or indirectly influence rhizobacterial community development. PMID:20632001

  14. Growth of quailbush in acidic, metalliferous desert mine tailings: effect of Azospirillum brasilense Sp6 on biomass production and rhizosphere community structure.

    PubMed

    de-Bashan, Luz E; Hernandez, Juan-Pablo; Nelson, Karis N; Bashan, Yoav; Maier, Raina M

    2010-11-01

    Mine tailing deposits in semiarid and arid environments frequently remain devoid of vegetation due to the toxicity of the substrate and the absence of a diverse soil microbial community capable of supporting seed germination and plant growth. The contribution of the plant growth promoting bacterium (PGPB) Azospirillum brasilense Sp6 to the growth of quailbush in compost-amended, moderately acidic, high-metal content mine tailings using an irrigation-based reclamation strategy was examined along with its influence on the rhizosphere bacterial community. Sp6 inoculation resulted in a significant (2.2-fold) increase in plant biomass production. The data suggest that the inoculum successfully colonized the root surface and persisted throughout the 60-day experiment in both the rhizosphere, as demonstrated by excision and sequencing of the appropriate denaturing gradient gel electrophoresis (DGGE) band, and the rhizoplane, as indicated by fluorescent in situ hybridization of root surfaces. Changes in rhizosphere community structure in response to Sp6 inoculation were evaluated after 15, 30, and 60 days using DGGE analysis of 16S rRNA polymerase chain reaction amplicons. A comparison of DGGE profiles using canonical correspondence analysis revealed a significant treatment effect (Sp6-inoculated vs. uninoculated plants vs. unplanted) on bacterial community structure at 15, 30, and 60 days (p < 0.05). These data indicate that in an extremely stressed environment such as acid mine tailings, an inoculated plant growth promoting bacterium not only can persist and stimulate plant growth but also can directly or indirectly influence rhizobacterial community development.

  15. Organic acids on the growth, anatomical structure, biochemical parameters and heavy metal accumulation of Iris lactea var. chinensis seedling growing in Pb mine tailings.

    PubMed

    Han, Yu-Lin; Huang, Su-Zhen; Yuan, Hai-Yan; Zhao, Jiu-Zhou; Gu, Ji-Guang

    2013-08-01

    The effect of citric acid (CA) and ethylene diamine tetraacetic acid (EDTA) on the growth, anatomical structure, physiological responses and lead (Pb) accumulation of Iris lactea var. chinensis seedling growing in Pb mine tailings for 30 days were studied. Results showed that the dry weights (DW) of roots decreased significantly under both levels of CA. The DWs of leaves and roots treated with 2 mmol/kg EDTA decreased significantly and were 23 and 54 %, respectively, lower than those of the control. The tolerant indexes of I. lactea var. chinensis under all treatments of organic acids were lower than control. The root tip anatomical structure was little affected under the treatments of 2 mmol/kg CA and 2 mmol/kg EDTA compared with control. However, the formation of photosynthesizing cells was inhibited by the treatment of 2 mmol/kg EDTA. The concentrations of chlorophyll a, chlorophyll b and total carotenoids in the leaves treated with 2 mmol/kg EDTA significantly decreased. Higher CA level and lower EDTA level could trigger the synthesis of ascorbic acid and higher level of EDTA could trigger the synthesis of glutathione. CA and EDTA could promote Pb accumulation of I. lactea var. chinensis and Pb concentration in the leaves and roots at 2 mmol/kg EDTA treatment increased significantly and reached to 160.44 and 936.08 μg/g DW, respectively, and 1.8 and 1.6 times higher than those of the control. The results indicated that I. lactea var. chinensis could be used to remediate Pb tailing and the role of EDTA in promoting Pb accumulation was better than CA did.

  16. Crystal structure of the thioesterase domain of human fatty acid synthase inhibited by orlistat

    SciTech Connect

    Pemble,C.; Johnson, L.; Kridel, S.; Lowther, W.

    2007-01-01

    Human fatty acid synthase (FAS) is uniquely expressed at high levels in many tumor types. Pharmacological inhibition of FAS therefore represents an important therapeutic opportunity. The drug Orlistat, which has been approved by the US Food and Drug Administration, inhibits FAS, induces tumor cell-specific apoptosis and inhibits the growth of prostate tumor xenografts. We determined the 2.3-{angstrom}-resolution crystal structure of the thioesterase domain of FAS inhibited by Orlistat. Orlistat was captured in the active sites of two thioesterase molecules as a stable acyl-enzyme intermediate and as the hydrolyzed product. The details of these interactions reveal the molecular basis for inhibition and suggest a mechanism for acyl-chain length discrimination during the FAS catalytic cycle. Our findings provide a foundation for the development of new cancer drugs that target FAS.

  17. Nucleic acid sequences encoding D1 and D1/D2 domains of human coxsackievirus and adenovirus receptor (CAR)

    DOEpatents

    Freimuth, Paul I.

    2010-04-06

    The invention provides recombinant human CAR (coxsackievirus and adenovirus receptor) polypeptides which bind adenovirus. Specifically, polypeptides corresponding to adenovirus binding domain D1 and the entire extracellular domain of human CAR protein comprising D1 and D2 are provided. In another aspect, the invention provides nucleic acid sequences encoding these domains and expression vectors for producing the domains and bacterial cells containing such vectors. The invention also includes an isolated fusion protein comprised of the D1 polypeptide fused to a polypeptide which facilitates folding of D1 when expressed in bacteria. The functional D1 domain finds application in a therapeutic method for treating a patient infected with a CAR D1-binding virus, and also in a method for identifying an antiviral compound which interferes with viral attachment. The invention also provides a method for specifically targeting a cell for infection by a virus which binds to D1.

  18. Evaluation of sulfidic mine tailings solidified/stabilized with cement kiln dust and fly ash to control acid mine drainage

    SciTech Connect

    Nehdi, M.; Tariq, A.

    2008-11-15

    In the present research, industrial byproducts, namely, cement kiln dust (CKD) and Class C fly ash (FAC) have been used as candidate materials along with the partial addition of sulfate-resistant cement (SRC) in the Stabilization/solidification of polymetallic sulfidic mine tailings (MT). The effectiveness of S/S was assessed by comparing laboratory experimental values obtained from unconfined compressive strength, hydraulic conductivity and leaching propensity tests of S/S samples with regulatory standards for safe surface disposal of such wastes. Despite general regulatory compliance of compressive strength and hydraulic conductivity, some solidified/stabilized-cured matrices were found unable to provide the required immobilization of pollutants. Solidified/stabilized and 90-day cured mine tailings specimens made with composite binders containing (10% CKD + 10% FAC), (5% SRC + 15% FAC) and (5% SRC + 5% CKD + 10% FAC) significantly impaired the solubility of all contaminants investigated and proved successful in fixing metals within the matrix, in addition to achieving adequate unconfined compressive strength and hydraulic conductivity values, thus satisfying USEPA regulations. Laboratory investigations revealed that, for polymetallic mining waste, leachate concentrations are the most critical factor in assessing the effectiveness of S/S technology.

  19. The ETS family member ERM contains an alpha-helical acidic activation domain that contacts TAFII60.

    PubMed Central

    Defossez, P A; Baert, J L; Monnot, M; de Launoit, Y

    1997-01-01

    Transcription factors are modular entities built up of discrete domains, some devoted to DNA binding and others permitting transcriptional modulation. The structure of DNA binding domains has been thoroughly investigated and structural classes clearly defined. In sharp contrast, the structural constraints put on transactivating regions, if any, are mostly unknown. Our investigations focus on ERM, a eukaryotic transcription factor of the ETS family. We have previously shown that ERM harbours two transactivating domains (TADs) with distinct functional features: AD1 lies in the first 72 amino acids of ERM, while AD2 sits in the last 62. Here we show that AD1 is a bona fide acidic TAD, for it activated transcription in yeast cells, while AD2 did not. AD1 contains a 20 amino acid stretch predicted to form an alpha-helix that is found unchanged in the related PEA3 and ER81 transcription factors. Circular dichroism analysis revealed that a 32 amino acid peptide encompassing this region is unstructured in water but folds into a helix when the hydrophobic solvent trifluoroethanol is added. The isolated helix was sufficient to activate transcription and mutations predicted to disrupt it dramatically affected AD1-driven transactivation, whereas mutations decreasing its acidity had more gentle effects. A phenylalanine residue within the helix was particularly sensitive to mutations. Finally, we observed that ERM bound TAFII60 via AD1 and bound TBP and TAFII40, presumably via other activation domains. PMID:9358152

  20. Interactions between the PDZ domains of Bazooka (Par-3) and phosphatidic acid: in vitro characterization and role in epithelial development.

    PubMed

    Yu, Cao Guo; Harris, Tony J C

    2012-09-01

    Bazooka (Par-3) is a conserved polarity regulator that organizes molecular networks in a wide range of cell types. In epithelia, it functions as a plasma membrane landmark to organize the apical domain. Bazooka is a scaffold protein that interacts with proteins through its three PDZ (postsynaptic density 95, discs large, zonula occludens-1) domains and other regions. In addition, Bazooka has been shown to interact with phosphoinositides. Here we show that the Bazooka PDZ domains interact with the negatively charged phospholipid phosphatidic acid immobilized on solid substrates or in liposomes. The interaction requires multiple PDZ domains, and conserved patches of positively charged amino acid residues appear to mediate the interaction. Increasing or decreasing levels of diacylglycerol kinase or phospholipase D-enzymes that produce phosphatidic acid-reveal a role for phosphatidic acid in Bazooka embryonic epithelial activity but not its localization. Mutating residues implicated in phosphatidic acid binding revealed a possible role in Bazooka localization and function. These data implicate a closer connection between Bazooka and membrane lipids than previously recognized. Bazooka polarity landmarks may be conglomerates of proteins and plasma membrane lipids that modify each other's activities for an integrated effect on cell polarity.

  1. Kinase Associated-1 Domains Drive MARK/PAR1 Kinases to Membrane Targets by Binding Acidic Phospholipids

    SciTech Connect

    Moravcevic, Katarina; Mendrola, Jeannine M.; Schmitz, Karl R.; Wang, Yu-Hsiu; Slochower, David; Janmey, Paul A.; Lemmon, Mark A.

    2011-09-28

    Phospholipid-binding modules such as PH, C1, and C2 domains play crucial roles in location-dependent regulation of many protein kinases. Here, we identify the KA1 domain (kinase associated-1 domain), found at the C terminus of yeast septin-associated kinases (Kcc4p, Gin4p, and Hsl1p) and human MARK/PAR1 kinases, as a membrane association domain that binds acidic phospholipids. Membrane localization of isolated KA1 domains depends on phosphatidylserine. Using X-ray crystallography, we identified a structurally conserved binding site for anionic phospholipids in KA1 domains from Kcc4p and MARK1. Mutating this site impairs membrane association of both KA1 domains and intact proteins and reveals the importance of phosphatidylserine for bud neck localization of yeast Kcc4p. Our data suggest that KA1 domains contribute to coincidence detection, allowing kinases to bind other regulators (such as septins) only at the membrane surface. These findings have important implications for understanding MARK/PAR1 kinases, which are implicated in Alzheimer's disease, cancer, and autism.

  2. Two Amino Acid Residues Confer Different Binding Affinities of Abelson Family Kinase Src Homology 2 Domains for Phosphorylated Cortactin*

    PubMed Central

    Gifford, Stacey M.; Liu, Weizhi; Mader, Christopher C.; Halo, Tiffany L.; Machida, Kazuya; Boggon, Titus J.; Koleske, Anthony J.

    2014-01-01

    The closely related Abl family kinases, Arg and Abl, play important non-redundant roles in the regulation of cell morphogenesis and motility. Despite similar N-terminal sequences, Arg and Abl interact with different substrates and binding partners with varying affinities. This selectivity may be due to slight differences in amino acid sequence leading to differential interactions with target proteins. We report that the Arg Src homology (SH) 2 domain binds two specific phosphotyrosines on cortactin, a known Abl/Arg substrate, with over 10-fold higher affinity than the Abl SH2 domain. We show that this significant affinity difference is due to the substitution of arginine 161 and serine 187 in Abl to leucine 207 and threonine 233 in Arg, respectively. We constructed Abl SH2 domains with R161L and S187T mutations alone and in combination and find that these substitutions are sufficient to convert the low affinity Abl SH2 domain to a higher affinity “Arg-like” SH2 domain in binding to a phospho-cortactin peptide. We crystallized the Arg SH2 domain for structural comparison to existing crystal structures of the Abl SH2 domain. We show that these two residues are important determinants of Arg and Abl SH2 domain binding specificity. Finally, we expressed Arg containing an “Abl-like” low affinity mutant Arg SH2 domain (L207R/T233S) and find that this mutant, although properly localized to the cell periphery, does not support wild type levels of cell edge protrusion. Together, these observations indicate that these two amino acid positions confer different binding affinities and cellular functions on the distinct Abl family kinases. PMID:24891505

  3. Analgesic and Anti-Inflammatory Properties of Gelsolin in Acetic Acid Induced Writhing, Tail Immersion and Carrageenan Induced Paw Edema in Mice

    PubMed Central

    Gupta, Ashok Kumar; Parasar, Devraj; Sagar, Amin; Choudhary, Vikas; Chopra, Bhupinder Singh; Garg, Renu; Ashish; Khatri, Neeraj

    2015-01-01

    Plasma gelsolin levels significantly decline in several disease conditions, since gelsolin gets scavenged when it depolymerizes and caps filamentous actin released in the circulation following tissue injury. It is well established that our body require/implement inflammatory and analgesic responses to protect against cell damage and injury to the tissue. This study was envisaged to examine analgesic and anti-inflammatory activity of exogenous gelsolin (8 mg/mouse) in mice models of pain and acute inflammation. Administration of gelsolin in acetic acid-induced writhing and tail immersion tests not only demonstrated a significant reduction in the number of acetic acid-induced writhing effects, but also exhibited an analgesic activity in tail immersion test in mice as compared to placebo treated mice. Additionally, anti-inflammatory function of gelsolin (8 mg/mouse) compared with anti-inflammatory drug diclofenac sodium (10 mg/kg)] was confirmed in the carrageenan injection induced paw edema where latter was measured by vernier caliper and fluorescent tomography imaging. Interestingly, results showed that plasma gelsolin was capable of reducing severity of inflammation in mice comparable to diclofenac sodium. Analysis of cytokines and histo-pathological examinations of tissue revealed administration of gelsolin and diclofenac sodium significantly reduced production of pro-inflammatory cytokines, TNF-α and IL-6. Additionally, carrageenan groups pretreated with diclofenac sodium or gelsolin showed a marked decrease in edema and infiltration of inflammatory cells in paw tissue. Our study provides evidence that administration of gelsolin can effectively reduce the pain and inflammation in mice model. PMID:26426535

  4. Binding of basal transcription factor TFIIH to the acidic activation domains of VP16 and p53.

    PubMed Central

    Xiao, H; Pearson, A; Coulombe, B; Truant, R; Zhang, S; Regier, J L; Triezenberg, S J; Reinberg, D; Flores, O; Ingles, C J

    1994-01-01

    Acidic transcriptional activation domains function well in both yeast and mammalian cells, and some have been shown to bind the general transcription factors TFIID and TFIIB. We now show that two acidic transactivators, herpes simplex virus VP16 and human p53, directly interact with the multisubunit human general transcription factor TFIIH and its Saccharomyces cerevisiae counterpart, factor b. The VP16- and p53-binding domains in these factors lie in the p62 subunit of TFIIH and in the homologous subunit, TFB1, of factor b. Point mutations in VP16 that reduce its transactivation activity in both yeast and mammalian cells weaken its binding to both yeast and human TFIIH. This suggests that binding of activation domains to TFIIH is an important aspect of transcriptional activation. Images PMID:7935417

  5. Catalytic residues are shared between two pseudosubunits of the dehydratase domain of the animal fatty acid synthase.

    PubMed

    Pasta, Saloni; Witkowski, Andrzej; Joshi, Anil K; Smith, Stuart

    2007-12-01

    Expression, characterization, and mutagenesis of a series of N-terminal fragments of an animal fatty acid synthase, containing the beta-ketoacyl synthase, acyl transferase, and dehydratase domains, demonstrate that the dehydratase domain consists of two pseudosubunits, derived from contiguous regions of the same polypeptide, in which a single active site is formed by the cooperation of the catalytic histidine 878 residue of the first pseudosubunit with aspartate 1032 of the second pseudosubunit. Mutagenesis and modeling studies revealed an essential role for glutamine 1036 in anchoring the position of the catalytic aspartate. These findings establish that sequence elements previously assigned to a central structural core region of the type I fatty acid synthases and some modular polyketide synthase counterparts play an essential catalytic role as part of the dehydratase domain.

  6. Amino acid residues in the laminin G domains of protein S involved in tissue factor pathway inhibitor interaction.

    PubMed

    Somajo, Sofia; Ahnström, Josefin; Fernandez-Recio, Juan; Gierula, Magdalena; Villoutreix, Bruno O; Dahlbäck, Björn

    2015-05-01

    Protein S functions as a cofactor for tissue factor pathway inhibitor (TFPI) and activated protein C (APC). The sex hormone binding globulin (SHBG)-like region of protein S, consisting of two laminin G-like domains (LG1 and LG2), contains the binding site for C4b-binding protein (C4BP) and TFPI. Furthermore, the LG-domains are essential for the TFPI-cofactor function and for expression of full APC-cofactor function. The aim of the current study was to localise functionally important interaction sites in the protein S LG-domains using amino acid substitutions. Four protein S variants were created in which clusters of surface-exposed amino acid residues within the LG-domains were substituted. All variants bound normally to C4BP and were fully functional as cofactors for APC in plasma and in pure component assays. Two variants, SHBG2 (E612A, I614A, F265A, V393A, H453A), involving residues from both LG-domains, and SHBG3 (K317A, I330A, V336A, D365A) where residues in LG1 were substituted, showed 50-60 % reduction in enhancement of TFPI in FXa inhibition assays. For SHBG3 the decreased TFPI cofactor function was confirmed in plasma based thrombin generation assays. Both SHBG variants bound to TFPI with decreased affinity in surface plasmon resonance experiments. The TFPI Kunitz 3 domain is known to contain the interaction site for protein S. Using in silico analysis and protein docking exercises, preliminary models of the protein S SHBG/TFPI Kunitz domain 3 complex were created. Based on a combination of experimental and in silico data we propose a binding site for TFPI on protein S, involving both LG-domains.

  7. Synthesis and anticoagulant activity of bioisosteric sulfonic-Acid analogues of the antithrombin-binding pentasaccharide domain of heparin.

    PubMed

    Herczeg, Mihály; Lázár, László; Bereczky, Zsuzsanna; Kövér, Katalin E; Timári, István; Kappelmayer, János; Lipták, András; Antus, Sándor; Borbás, Anikó

    2012-08-20

    Two pentasaccharide sulfonic acids that were related to the antithrombin-binding domain of heparin were prepared, in which two or three primary sulfate esters were replaced by sodium-sulfonatomethyl moieties. The sulfonic-acid groups were formed on a monosaccharide level and the obtained carbohydrate sulfonic-acid esters were found to be excellent donors and acceptors in the glycosylation reactions. Throughout the synthesis, the hydroxy groups to be methylated were masked in the form of acetates and the hydroxy groups to be sulfated were masked with benzyl groups. The disulfonic-acid analogue was prepared in a [2+3] block synthesis by using a trisaccharide disulfonic acid as an acceptor and a glucuronide disaccharide as a donor. For the synthesis of the pentasaccharide trisulfonic acid, a more-efficient approach, which involved elongation of the trisaccharide acceptor with a non-oxidized precursor of the glucuronic acid followed by post-glycosidation oxidation at the tetrasaccharide level and a subsequent [1+4] coupling reaction, was elaborated. In vitro evaluation of the anticoagulant activity of these new sulfonic-acid derivatives revealed that the disulfonate analogue inhibited the blood-coagulation-proteinase factor Xa with outstanding efficacy; however, the introduction of the third sulfonic-acid moiety resulted in a notable decrease in the anti-Xa activity. The difference in the biological activity of the disulfonic- and trisulfonic-acid counterparts could be explained by the different conformation of their L-iduronic-acid residues.

  8. The transcriptional activator GCN4 contains multiple activation domains that are critically dependent on hydrophobic amino acids.

    PubMed Central

    Drysdale, C M; Dueñas, E; Jackson, B M; Reusser, U; Braus, G H; Hinnebusch, A G

    1995-01-01

    GCN4 is a transcriptional activator in the bZIP family that regulates amino acid biosynthetic genes in the yeast Saccharomyces cerevisiae. Previous work suggested that the principal activation domain of GCN4 is a highly acidic segment of approximately 40 amino acids located in the center of the protein. We conducted a mutational analysis of GCN4 with a single-copy allele expressed under the control of the native promoter and translational control elements. Our results indicate that GCN4 contains two activation domains of similar potency that can function independently to promote high-level transcription of the target genes HIS3 and HIS4. One of these domains is coincident with the acidic activation domain defined previously; the other extends over the N-terminal one-third of the protein. Both domains are partially dependent on the coactivator protein ADA2. Each domain appears to be composed of two or more small subdomains that have additive effects on transcription and that can cooperate in different combinations to promote high-level expression of HIS3 and HIS4. At least three of these subdomains are critically dependent on bulky hydrophobic amino acids for their function. Five of the important hydrophobic residues, Phe-97, Phe-98, Met-107, Tyr-110, and Leu-113, fall within a region of proposed sequence homology between GCN4 and the herpesvirus acidic activator VP16. The remaining three residues, Trp-120, Leu-123, and Phe-124, are highly conserved between GCN4 and its Neurospora counterpart, cpc-1. Because of the functional redundancy in the activation domain, mutations at positions 97 and 98 must be combined with mutations at positions 120 to 124 to observe a substantial reduction in activation by full-length GCN4, and substitution of all eight hydrophobic residues was required to inactivate full-length GCN4. These hydrophobic residues may mediate important interactions between GCN4 and one or more of its target proteins in the transcription initiation complex

  9. A single amino acid change in the cytoplasmic domain of the simian immunodeficiency virus transmembrane molecule increases envelope glycoprotein expression on infected cells.

    PubMed Central

    LaBranche, C C; Sauter, M M; Haggarty, B S; Vance, P J; Romano, J; Hart, T K; Bugelski, P J; Marsh, M; Hoxie, J A

    1995-01-01

    We have described a virus termed CP-MAC, derived from the BK28 molecular clone of simian immunodeficiency virus, that was remarkable for its ability to infect Sup-T1 cells with rapid kinetics, cell fusion, and CD4 down-modulation (C. C. LaBranche, M. M. Sauter, B. S. Haggarty, P. J. Vance, J. Romano, T. K. Hart, P. J. Bugelski, and J. A. Hoxie, J. Virol. 68:5509-5522, 1994 [Erratum 68:7665-7667]). Compared with BK28, CP-MAC exhibited a number of changes in its envelope glycoproteins, including a highly stable association between the external (SU) and transmembrane (TM) molecules, a more rapid electrophoretic mobility of TM, and, of particular interest, a marked increase in the level of envelope protein expression on the surface of infected cells. These changes were shown to be associated with 11 coding mutations in the env gene (5 in SU and 6 in TM). In this report, we demonstrate that a single amino acid mutation of a Tyr to a Cys at position 723 (Y723C) in the TM cytoplasmic domain of CP-MAC is the principal determinant for the increased expression of envelope glycoproteins on the cell surface. When introduced into the env gene of BK28, the Y723C mutation produced up to a 25-fold increase in the levels of SU and TM on chronically infected cells, as determined by fluorescence-activated cell sorter analysis with monoclonal and polyclonal antibodies. A similar effect was observed when a Tyr-to-Cys change was introduced at the analogous position (amino acid 721) in the SIVmac239 molecular clone, which, unlike BK28 does not contain a premature stop codon in its TM cytoplasmic tail. Substituting other amino acids, including Ala, Ile, and Ser, at this position produced increases in surface envelope glycoproteins that were similar to that observed for the Cys substitution, while a Tyr-to-Phe mutation produced a smaller increase. These results could not be accounted for by differences in the kinetics or efficiency of envelope glycoprotein processing or by shedding of SU

  10. Comparison of amino acids physico-chemical properties and usage of late embryogenesis abundant proteins, hydrophilins and WHy domain.

    PubMed

    Jaspard, Emmanuel; Hunault, Gilles

    2014-01-01

    Late Embryogenesis Abundant proteins (LEAPs) comprise several diverse protein families and are mostly involved in stress tolerance. Most of LEAPs are intrinsically disordered and thus poorly functionally characterized. LEAPs have been classified and a large number of their physico-chemical properties have been statistically analyzed. LEAPs were previously proposed to be a subset of a very wide family of proteins called hydrophilins, while a domain called WHy (Water stress and Hypersensitive response) was found in LEAP class 8 (according to our previous classification). Since little is known about hydrophilins and WHy domain, the cross-analysis of their amino acids physico-chemical properties and amino acids usage together with those of LEAPs helps to describe some of their structural features and to make hypothesis about their function. Physico-chemical properties of hydrophilins and WHy domain strongly suggest their role in dehydration tolerance, probably by interacting with water and small polar molecules. The computational analysis reveals that LEAP class 8 and hydrophilins are distinct protein families and that not all LEAPs are a protein subset of hydrophilins family as proposed earlier. Hydrophilins seem related to LEAP class 2 (also called dehydrins) and to Heat Shock Proteins 12 (HSP12). Hydrophilins are likely unstructured proteins while WHy domain is structured. LEAP class 2, hydrophilins and WHy domain are thus proposed to share a common physiological role by interacting with water or other polar/charged small molecules, hence contributing to dehydration tolerance. PMID:25296175

  11. Arsenic as an Endocrine Disruptor: Arsenic Disrupts Retinoic Acid Receptor–and Thyroid Hormone Receptor–Mediated Gene Regulation and Thyroid Hormone–Mediated Amphibian Tail Metamorphosis

    PubMed Central

    Davey, Jennifer C.; Nomikos, Athena P.; Wungjiranirun, Manida; Sherman, Jenna R.; Ingram, Liam; Batki, Cavus; Lariviere, Jean P.; Hamilton, Joshua W.

    2008-01-01

    Background Chronic exposure to excess arsenic in drinking water has been strongly associated with increased risks of multiple cancers, diabetes, heart disease, and reproductive and developmental problems in humans. We previously demonstrated that As, a potent endocrine disruptor at low, environmentally relevant levels, alters steroid signaling at the level of receptor-mediated gene regulation for all five steroid receptors. Objectives The goal of this study was to determine whether As can also disrupt gene regulation via the retinoic acid (RA) receptor (RAR) and/or the thyroid hormone (TH) receptor (TR) and whether these effects are similar to previously observed effects on steroid regulation. Methods and results Human embryonic NT2 or rat pituitary GH3 cells were treated with 0.01–5 μM sodium arsenite for 24 hr, with or without RA or TH, respectively, to examine effects of As on receptor-mediated gene transcription. At low, noncytotoxic doses, As significantly altered RAR-dependent gene transcription of a transfected RAR response element–luciferase construct and the native RA-inducible cytochrome P450 CYP26A gene in NT2 cells. Likewise, low-dose As significantly altered expression of a transfected TR response element–luciferase construct and the endogenous TR-regulated type I deiodinase (DIO1) gene in a similar manner in GH3 cells. An amphibian ex vivo tail metamorphosis assay was used to examine whether endocrine disruption by low-dose As could have specific pathophysiologic consequences, because tail metamorphosis is tightly controlled by TH through TR. TH-dependent tail shrinkage was inhibited in a dose-dependent manner by 0.1– 4.0 μM As. Conclusions As had similar effects on RAR- and TR-mediated gene regulation as those previously observed for the steroid receptors, suggesting a common mechanism or action. Arsenic also profoundly affected a TR-dependent developmental process in a model animal system at very low concentrations. Because RAR and TH are

  12. The Amino Acid Specificity for Activation of Phenylalanine Hydroxylase Matches the Specificity for Stabilization of Regulatory Domain Dimers

    PubMed Central

    2016-01-01

    Liver phenylalanine hydroxylase is allosterically activated by phenylalanine. The structural changes that accompany activation have not been identified, but recent studies of the effects of phenylalanine on the isolated regulatory domain of the enzyme support a model in which phenylalanine binding promotes regulatory domain dimerization. Such a model predicts that compounds that stabilize the regulatory domain dimer will also activate the enzyme. Nuclear magnetic resonance spectroscopy and analytical ultracentrifugation were used to determine the ability of different amino acids and phenylalanine analogues to stabilize the regulatory domain dimer. The abilities of these compounds to activate the enzyme were analyzed by measuring their effects on the fluorescence change that accompanies activation and on the activity directly. At concentrations of 10–50 mM, d-phenylalanine, l-methionine, l-norleucine, and (S)-2-amino-3-phenyl-1-propanol were able to activate the enzyme to the same extent as 1 mM l-phenylalanine. Lower levels of activation were seen with l-4-aminophenylalanine, l-leucine, l-isoleucine, and 3-phenylpropionate. The ability of these compounds to stabilize the regulatory domain dimer agreed with their ability to activate the enzyme. These results support a model in which allosteric activation of phenylalanine hydroxylase is linked to dimerization of regulatory domains. PMID:26252467

  13. The Amino Acid Specificity for Activation of Phenylalanine Hydroxylase Matches the Specificity for Stabilization of Regulatory Domain Dimers.

    PubMed

    Zhang, Shengnan; Hinck, Andrew P; Fitzpatrick, Paul F

    2015-08-25

    Liver phenylalanine hydroxylase is allosterically activated by phenylalanine. The structural changes that accompany activation have not been identified, but recent studies of the effects of phenylalanine on the isolated regulatory domain of the enzyme support a model in which phenylalanine binding promotes regulatory domain dimerization. Such a model predicts that compounds that stabilize the regulatory domain dimer will also activate the enzyme. Nuclear magnetic resonance spectroscopy and analytical ultracentrifugation were used to determine the ability of different amino acids and phenylalanine analogues to stabilize the regulatory domain dimer. The abilities of these compounds to activate the enzyme were analyzed by measuring their effects on the fluorescence change that accompanies activation and on the activity directly. At concentrations of 10-50 mM, d-phenylalanine, l-methionine, l-norleucine, and (S)-2-amino-3-phenyl-1-propanol were able to activate the enzyme to the same extent as 1 mM l-phenylalanine. Lower levels of activation were seen with l-4-aminophenylalanine, l-leucine, l-isoleucine, and 3-phenylpropionate. The ability of these compounds to stabilize the regulatory domain dimer agreed with their ability to activate the enzyme. These results support a model in which allosteric activation of phenylalanine hydroxylase is linked to dimerization of regulatory domains.

  14. Innovative Approach to Prevent Acid Drainage from Uranium Mill Tailings Based on the Application of Na-Ferrate (VI)

    SciTech Connect

    Fernandes, H.M.; Reinhart, D.; Lettie, L.; Franklin, M.R.; Fernandes, H.M.; Franklin, M.R.; Daly, L.J.

    2006-07-01

    The operation of uranium mining and milling plants gives rise to huge amounts of wastes from both mining and milling operations. When pyrite is present in these materials, the generation of acid drainage can take place and result in the contamination of underground and surface waters through the leaching of heavy metals and radionuclides. To solve this problem, many studies have been conducted to find cost-effective solutions to manage acid mine drainage; however, no adequate strategy to deal with sulfide-ric h wastes is currently available. Ferrate (VI) is a powerful oxidizing agent in aqueous media. Under acidic conditions, the redox potential of the Ferrate (VI) ion is the highest of any other oxidant used in wastewater treatment processes. The standard half cell reduction potential of ferrate (VI) has been determined as +2.20 V to + 0.72 V in acidic and basic solutions, respectively. Ferrate (VI) exhibits a multitude of advantageous properties, including higher reactivity and selectivity than traditional oxidant alternatives, as well as disinfectant, flocculating, and coagulant properties. Despite numerous beneficial properties in environmental applications, ferrate (VI) has remained commercially unavailable. Starting in 1953, different methods for producing a high purity, powdered ferrate (VI) product were developed. However, producing this dry, stabilized ferrate (VI) product required numerous process steps which led to excessive synthesis costs (over $20/lb) thereby preventing bulk industrial use. Recently a novel synthesis method for the production of a liquid ferrate (VI) based on hypochlorite oxidation of ferric ion in strongly alkaline solutions has been discovered (USPTO 6,790,428; September 14, 2004). This on-site synthesis process dramatically reduces manufacturing cost for the production of ferrate (VI) by utilizing common commodity feedstocks. This breakthrough means that for the first time ferrate (VI) can be an economical alternative to treating

  15. The carboxy-terminal tail of pyruvate dehydrogenase kinase 2 is required for the kinase activity.

    PubMed

    Klyuyeva, Alla; Tuganova, Alina; Popov, Kirill M

    2005-10-18

    Pyruvate dehydrogenase kinase 2 (PDK2) is a prototypical mitochondrial protein kinase that regulates the activity of the pyruvate dehydrogenase complex. Recent structural studies have established that PDK2 consists of a catalytic core built of the B and K domains and the relatively long amino and carboxyl tails of unknown function. Here, we show that the carboxy-terminal truncation variants of PDK2 display a greatly diminished capacity for phosphorylation of holo-PDC. This effect is due largely to the inability of the transacetylase component of PDC to promote the phosphorylation reaction catalyzed by the truncated PDK2 variants. Furthermore, the truncated forms of PDK2 bind poorly to the lipoyl-bearing domain(s) provided by the transacetylase component. Taken together, these data strongly suggest that the carboxyl tails of PDK isozymes contribute to the lipoyl-bearing domain-binding site of the kinase molecule. We also show that the carboxyl tails derived from isozymes PDK1, PDK3, and PDK4 are capable of supporting the kinase activity of the kinase core derived from PDK2 as well as binding of the respective PDK2 chimeras to the lipoyl-bearing domain. Furthermore, the chimera carrying the carboxyl tail of PDK3 displays a stronger response to the addition of the transacetylase component along with a better binding to the lipoyl-bearing domain, suggesting that, at least in part, the differences in the amino acid sequences of the carboxyl tails account for the differences between PDK isozymes. PMID:16216081

  16. Rational Molecular Design of Potent PLK1 PBD Domain-binding Phosphopeptides Using Preferential Amino Acid Building Blocks.

    PubMed

    Mao, Xin-Li; Wang, Kui-Feng; Zhu, Feng; Pan, Zhao-Hu; Wu, Guo-Min; Zhu, Hong-Yuan

    2016-08-01

    Polo-like kinase 1 (PLK1) is an important regulator in diverse aspects of the cell cycle and proliferation. The protein has a highly conserved polo-box domain (PBD) present in C-terminal noncatalytic region, which exhibits a relatively broad sequence specificity in recognizing and binding phosphorylated substrates to control substrate phosphorylation by the kinase. In order to elucidate the structural basis, thermodynamic property, and biological implication underlying PBD-substrate recognition and association, a systematic amino acid preference profile of phosphopeptide interaction with PLK1 PBD domain was established via virtual mutagenesis analysis and mutation energy calculation, from which the contribution of different amino acids at each residue position of two reference phosphopeptides to domain-peptide binding was characterized comprehensively and quantitatively. With the profile, we are able to determine the favorable, neutral, and unfavorable amino acid types for each position of PBD-binding phosphopeptides, and we also explored the molecular origin of the broad sequence specificity in PBD-substrate recognition. To practice computational findings, the profile was further employed to guide rational design of potent PBD binders; three 6-mer phosphopeptides (i.e., IQSpSPC, LQSpTPF, and LNSpTPT) were successfully developed, which can efficiently target PBD domain with high affinity (Kd = 5.7 ± 1.1, 0.75 ± 0.18, and 7.2 ± 2.6 μm, resp.) as measured by a fluorescence anisotropy assay. The complex structure of PLK1 PBD domain with a newly designed, potent phosphopeptide LQSpTPF as well as diverse noncovalent chemical forces, such as H-bonds and hydrophobic interactions at the complex interface, were examined in detail to reveal the molecular mechanism of high affinity and stability of the complex system.

  17. Transmembrane domain V plays a stabilizing role in the function of human bile acid transporter SLC10A2.

    PubMed

    Moore, Robyn H; Chothe, Paresh; Swaan, Peter W

    2013-07-30

    The human apical sodium-dependent bile acid transporter (hASBT, SLC10A2), primarily expressed in the ileum, is involved in both the recycling of bile acids and cholesterol homeostasis. In this study, the structure-function relationship of transmembrane domain 5 (TM5) residues involved in transport is elucidated. Cysteine scanning mutagenesis of each consecutive residue on TM5 resulted in 96% of mutants having a significantly decreased transport activity, although each was expressed at the cell surface. Specifically, G197 and I208 were no longer functional, and G201 and G212 functioned at a level of <10% upon cysteine mutation. Interestingly, each of these exists along one face of the helix. Studies suggest that neither G201 nor G212 is on the substrate pathway. Conservative alanine mutations of the four residues displayed a higher activity in all but G197A, indicating its functional importance. G197 and G201 form a GxxxG motif, which has been found to be important in helix-helix interactions. According to our model, G197 and G201 face transmembrane domain 4 (TM4) residues G179 and P175, respectively. Similarly, G212 faces G237, which forms part of a GxxxG domain in transmembrane domain 6 (TM6). It is possible that these GxxxG domains and their interacting partners are responsible for maintaining the structure of the helices and their interactions with one another. I205 and I208 are both in positions to anchor the GxxxG domains and direct the change in interaction of TM5 from TM4 to TM6. Combined, the results suggest that residues along TM5 are critical for ASBT function but are not directly involved in substrate translocation.

  18. Response of amino acids in hindlimb muscles to recovery from hypogravity and unloading by tail-cast suspension

    NASA Technical Reports Server (NTRS)

    Tischler, M. E.; Henriksen, E. J.; Jacob, S.; Cook, P. H.

    1985-01-01

    Concentrations of glutamine, glutamate, aspartate (+ asparagine) and alanine were compared in hindlimb muscles of SL-3 and ground control rats. Alanine was lower in the soleus of flown rats but not of suspended animals, with no response in other muscles except a slight increase in the unloaded plantaris. With recovery, alanine in the soleus was elevated. Since no differences in alanine metabolism were found by isolated muscle, changes in muscle alanine are probably due to altered body use of this amino acid leading to varied plasma levels.

  19. A structure-specific nucleic acid-binding domain conserved among DNA repair proteins

    PubMed Central

    Mason, Aaron C.; Rambo, Robert P.; Greer, Briana; Pritchett, Michael; Tainer, John A.; Cortez, David; Eichman, Brandt F.

    2014-01-01

    SMARCAL1, a DNA remodeling protein fundamental to genome integrity during replication, is the only gene associated with the developmental disorder Schimke immuno-osseous dysplasia (SIOD). SMARCAL1-deficient cells show collapsed replication forks, S-phase cell cycle arrest, increased chromosomal breaks, hypersensitivity to genotoxic agents, and chromosomal instability. The SMARCAL1 catalytic domain (SMARCAL1CD) is composed of an SNF2-type double-stranded DNA motor ATPase fused to a HARP domain of unknown function. The mechanisms by which SMARCAL1 and other DNA translocases repair replication forks are poorly understood, in part because of a lack of structural information on the domains outside of the common ATPase motor. In the present work, we determined the crystal structure of the SMARCAL1 HARP domain and examined its conformation and assembly in solution by small angle X-ray scattering. We report that this domain is conserved with the DNA mismatch and damage recognition domains of MutS/MSH and NER helicase XPB, respectively, as well as with the putative DNA specificity motif of the T4 phage fork regression protein UvsW. Loss of UvsW fork regression activity by deletion of this domain was rescued by its replacement with HARP, establishing the importance of this domain in UvsW and demonstrating a functional complementarity between these structurally homologous domains. Mutation of predicted DNA-binding residues in HARP dramatically reduced fork binding and regression activities of SMARCAL1CD. Thus, this work has uncovered a conserved substrate recognition domain in DNA repair enzymes that couples ATP-hydrolysis to remodeling of a variety of DNA structures, and provides insight into this domain’s role in replication fork stability and genome integrity. PMID:24821763

  20. Identification of functionally important amino acids in the cellulose-binding domain of Trichoderma reesei cellobiohydrolase I.

    PubMed Central

    Linder, M.; Mattinen, M. L.; Kontteli, M.; Lindeberg, G.; Ståhlberg, J.; Drakenberg, T.; Reinikainen, T.; Pettersson, G.; Annila, A.

    1995-01-01

    Cellobiohydrolase I (CBHI) of Trichoderma reesei has two functional domains, a catalytic core domain and a cellulose binding domain (CBD). The structure of the CBD reveals two distinct faces, one of which is flat and the other rough. Several other fungal cellulolytic enzymes have similar two-domain structures, in which the CBDs show a conserved primary structure. Here we have evaluated the contributions of conserved amino acids in CBHI CBD to its binding to cellulose. Binding isotherms were determined for a set of six synthetic analogues in which conserved amino acids were substituted. Two-dimensional NMR spectroscopy was used to assess the structural effects of the substitutions by comparing chemical shifts, coupling constants, and NOEs of the backbone protons between the wild-type CBD and the analogues. In general, the structural effects of the substitutions were minor, although in some cases decreased binding could clearly be ascribed to conformational perturbations. We found that at least two tyrosine residues and a glutamine residue on the flat face were essential for tight binding of the CBD to cellulose. A change on the rough face had only a small effect on the binding and it is unlikely that this face interacts with cellulose directly. PMID:7549870

  1. Synergistic transcriptional enhancement does not depend on the number of acidic activation domains bound to the promoter.

    PubMed Central

    Oliviero, S; Struhl, K

    1991-01-01

    Many eukaryotic transcriptional activator proteins contain a DNA-binding domain that interacts with specific promoter sequences and an acidic activation region that is required to stimulate transcription. Transcriptional enhancement by such activator proteins is often synergistic and promiscuous; promoters containing multiple binding sites for an individual protein or even for unrelated proteins can be 10-100 times more active than promoters with single sites. It has been suggested that such synergy reflects a nonlinear response of the basic transcription machinery to the number and/or quality of acidic activation regions. Here, we determine the transcriptional activity of Jun-Fos heterodimers containing one or two GCN4 acidic activation regions on promoters containing one or two Ap-1 target sites. Surprisingly, heterodimers with one or two acidic regions activate transcription with similar efficiency and are equally synergistic (10- to 15-fold) on promoters containing two target sites. Thus, transcriptional synergy does not depend on the number of acidic activation regions but rather on the number of proteins bound to the promoter. This suggests that synergy is mediated either by cooperative DNA binding or by alternative mechanisms in which the DNA-binding domain plays a more direct role in transcription (e.g., changes in DNA structure, nucleosome displacement, or direct interactions with the transcriptional machinery). Images PMID:1898773

  2. Identification of acidic and aromatic residues in the Zta activation domain essential for Epstein-Barr virus reactivation.

    PubMed

    Deng, Z; Chen, C J; Zerby, D; Delecluse, H J; Lieberman, P M

    2001-11-01

    Epstein-Barr virus (EBV) lytic cycle transcription and DNA replication require the transcriptional activation function of the viral immediate-early protein Zta. We describe a series of alanine substitution mutations in the Zta activation domain that reveal two functional motifs based on amino acid composition. Alanine substitution of single or paired hydrophobic aromatic amino acid residues resulted in modest transcription activation defects, while combining four substitutions of aromatic residues (F22/F26/W74/F75) led to more severe transcription defects. Substitution of acidic amino acid residue E27, D35, or E54 caused severe transcription defects on most viral promoters. Promoter- and cell-specific defects were observed for some substitution mutants. Aromatic residues were required for Zta interaction with TFIIA-TFIID and the CREB-binding protein (CBP) and for stimulation of CBP histone acetyltransferase activity in vitro. In contrast, acidic amino acid substitution mutants interacted with TFIIA-TFIID and CBP indistinguishably from the wild type. The nuclear domain 10 (ND10) protein SP100 was dispersed by most Zta mutants, but acidic residue mutations led to reduced, while aromatic substitution mutants led to increased SP100 nuclear staining. Acidic residue substitution mutants had more pronounced defects in transcription activation of endogenous viral genes in latently infected cells and for viral replication, as measured by the production of infectious virus. One mutant, K12/F13, was incapable of stimulating EBV lytic replication but had only modest transcription defects. These results indicate that Zta stimulates viral reactivation through two nonredundant structural motifs, one of which interacts with general transcription factors and coactivators, and the other has an essential but as yet not understood function in lytic transcription.

  3. Responses of amino acids in hindlimb muscles to recovery from hypogravity and unloading by tail-cast suspension

    NASA Technical Reports Server (NTRS)

    Tischler, M. E.; Henriksen, E. J.; Jacob, S.; Cook, P. H.

    1985-01-01

    Amino acids were assayed in muscles from rats exposed to 7 days of hypogravity and 12 h of gravity (F) or 6 days of suspension with (R) or without (H) 12 h of loading. In these groups, lower aspartate was common only to the soleus (SOL) relative to control muscles, the smallest difference being in group R. This difference in aspartate for F and H, but not for R, correlated with lower malate suggesting diminution of citric acid cycle intermediates. The R SOL value was increased over the H SOL. Therefore desite 12 h of loading, the F SOL was more comparable to the H SOL. The role of stress in preventing recovery of the F SOL was apparent from the ratios of glutamine/glutamate. Synthesis of glutamine is enhanced by glucocorticoids and is reflected by an increased ratio. In 5 of the 6 F muscles studied, this ratio was greater than in controls. In contrast, the ratio in all R muscles was similar to controls and showed recovery from the values in H muscles. Hence the post-flight treatment of F rats may have produced additional stress. Despite this stress, in some respects the SOL responses to hypogravity were similar to its responses to unloding by suspension.

  4. Transcriptional activation by TFIIB mutants that are severely impaired in interaction with promoter DNA and acidic activation domains.

    PubMed Central

    Chou, S; Struhl, K

    1997-01-01

    Biochemical experiments indicate that the general transcription factor IIB (TFIIB) can interact directly with acidic activation domains and that activators can stimulate transcription by increasing recruitment of TFIIB to promoters. For promoters at which recruitment of TFIIB to promoters is limiting in vivo, one would predict that transcriptional activity should be particularly sensitive to TFIIB mutations that decrease the association of TFIIB with promoter DNA and/or with activation domains; i.e., such TFIIB mutations should exacerbate a limiting step that occurs in wild-type cells. Here, we describe mutations on the DNA-binding surface of TFIIB that severely affect both TATA-binding protein (TBP)-TFIIB-TATA complex formation and interaction with the VP16 activation domain in vitro. These TFIIB mutations affect the stability of the TBP-TFIIB-TATA complex in vivo because they are synthetically lethal in combination with TBP mutants impaired for TFIIB binding. Interestingly, these TFIIB derivatives support viability, and they efficiently respond to Gal4-VP16 and natural acidic activators in different promoter contexts. These results suggest that in vivo, recruitment of TFIIB is not generally a limiting step for acidic activators. However, one TFIIB derivative shows reduced transcription of GAL4, suggesting that TFIIB may be limiting at a subset of promoters in vivo. PMID:9372910

  5. Protein cold adaptation strategy via a unique seven-amino acid domain in the icefish (Chionodraco hamatus) PEPT1 transporter.

    PubMed

    Rizzello, Antonia; Romano, Alessandro; Kottra, Gabor; Acierno, Raffaele; Storelli, Carlo; Verri, Tiziano; Daniel, Hannelore; Maffia, Michele

    2013-04-23

    Adaptation of organisms to extreme environments requires proteins to work at thermodynamically unfavorable conditions. To adapt to subzero temperatures, proteins increase the flexibility of parts of, or even the whole, 3D structure to compensate for the lower thermal kinetic energy available at low temperatures. This may be achieved through single-site amino acid substitutions in regions of the protein that undergo large movements during the catalytic cycle, such as in enzymes or transporter proteins. Other strategies of cold adaptation involving changes in the primary amino acid sequence have not been documented yet. In Antarctic icefish (Chionodraco hamatus) peptide transporter 1 (PEPT1), the first transporter cloned from a vertebrate living at subzero temperatures, we came upon a unique principle of cold adaptation. A de novo domain composed of one to six repeats of seven amino acids (VDMSRKS), placed as an extra stretch in the cytosolic COOH-terminal region, contributed per se to cold adaptation. VDMSRKS was in a protein region uninvolved in transport activity and, notably, when transferred to the COOH terminus of a warm-adapted (rabbit) PEPT1, it conferred cold adaptation to the receiving protein. Overall, we provide a paradigm for protein cold adaptation that relies on insertion of a unique domain that confers greater affinity and maximal transport rates at low temperatures. Due to its ability to transfer a thermal trait, the VDMSRKS domain represents a useful tool for future cell biology or biotechnological applications. PMID:23569229

  6. Structure and Mutagenesis of Neural Cell Adhesion Molecule Domains Evidence for Flexibility in the Placement of Polysialic Acid Attachment Sites

    SciTech Connect

    Foley, Deirdre A.; Swartzentruber, Kristin G.; Lavie, Arnon; Colley, Karen J.

    2010-11-09

    The addition of {alpha}2,8-polysialic acid to the N-glycans of the neural cell adhesion molecule, NCAM, is critical for brain development and plays roles in synaptic plasticity, learning and memory, neuronal regeneration, and the growth and invasiveness of cancer cells. Our previous work indicates that the polysialylation of two N-glycans located on the fifth immunoglobulin domain (Ig5) of NCAM requires the presence of specific sequences in the adjacent fibronectin type III repeat (FN1). To understand the relationship of these two domains, we have solved the crystal structure of the NCAM Ig5-FN1 tandem. Unexpectedly, the structure reveals that the sites of Ig5 polysialylation are on the opposite face from the FN1 residues previously found to be critical for N-glycan polysialylation, suggesting that the Ig5-FN1 domain relationship may be flexible and/or that there is flexibility in the placement of Ig5 glycosylation sites for polysialylation. To test the latter possibility, new Ig5 glycosylation sites were engineered and their polysialylation tested. We observed some flexibility in glycosylation site location for polysialylation and demonstrate that the lack of polysialylation of a glycan attached to Asn-423 may be in part related to a lack of terminal processing. The data also suggest that, although the polysialyltransferases do not require the Ig5 domain for NCAM recognition, their ability to engage with this domain is necessary for polysialylation to occur on Ig5 N-glycans.

  7. Computational insight into the catalytic implication of head/tail-first orientation of arachidonic acid in human 5-lipoxygenase: consequences for the positional specificity of oxygenation.

    PubMed

    Saura, Patricia; Maréchal, Jean-Didier; Masgrau, Laura; Lluch, José M; González-Lafont, Àngels

    2016-08-17

    In the present work we have combined homology modeling, protein-ligand dockings, quantum mechanics/molecular mechanics calculations and molecular dynamics simulations to generate human 5-lipoxygenase (5-LOX):arachidonic acid (AA) complexes consistent with the 5-lipoxygenating activity (which implies hydrogen abstraction at the C7 position). Our results suggest that both the holo and the apo forms of human Stable 5-LOX could accommodate AA in a productive form for 5-lipoxygenation. The former, in a tail-first orientation, with the AA carboxylate end interacting with Lys409, gives the desired structures with C7 close to the Fe-OH(-) cofactor and suitable barrier heights for H7 abstraction. Only when using the apo form structure, a head-first orientation with the AA carboxylate close to His600 (a residue recently proposed as essential for AA positioning) is obtained in the docking calculations. However, the calculated barrier heights for this head-first orientation are in principle consistent with 5-LOX specificity, but also with 12/8 regioselectivity. Finally, long MD simulations give support to the recent hypothesis that the Phe177 + Tyr181 pair needs to close the active site access during the chemical reaction, and suggest that in the case of a head-first orientation Phe177 may be the residue interacting with the AA carboxylate. PMID:27489112

  8. Two Cytoplasmic Acylation Sites and an Adjacent Hydrophobic Residue, but No Other Conserved Amino Acids in the Cytoplasmic Tail of HA from Influenza A Virus Are Crucial for Virus Replication

    PubMed Central

    Siche, Stefanie; Brett, Katharina; Möller, Lars; Kordyukova, Larisa V.; Mintaev, Ramil R.; Alexeevski, Andrei V.; Veit, Michael

    2015-01-01

    Recruitment of the matrix protein M1 to the assembly site of the influenza virus is thought to be mediated by interactions with the cytoplasmic tail of hemagglutinin (HA). Based on a comprehensive sequence comparison of all sequences present in the database, we analyzed the effect of mutating conserved residues in the cytosol-facing part of the transmembrane region and cytoplasmic tail of HA (A/WSN/33 (H1N1) strain) on virus replication and morphology of virions. Removal of the two cytoplasmic acylation sites and substitution of a neighboring isoleucine by glutamine prevented rescue of infectious virions. In contrast, a conservative exchange of the same isoleucine, non-conservative exchanges of glycine and glutamine, deletion of the acylation site at the end of the transmembrane region and shifting it into the tail did not affect virus morphology and had only subtle effects on virus growth and on the incorporation of M1 and Ribo-Nucleoprotein Particles (RNPs). Thus, assuming that essential amino acids are conserved between HA subtypes we suggest that, besides the two cytoplasmic acylation sites (including adjacent hydrophobic residues), no other amino acids in the cytoplasmic tail of HA are indispensable for virus assembly and budding. PMID:26670246

  9. Two Cytoplasmic Acylation Sites and an Adjacent Hydrophobic Residue, but No Other Conserved Amino Acids in the Cytoplasmic Tail of HA from Influenza A Virus Are Crucial for Virus Replication.

    PubMed

    Siche, Stefanie; Brett, Katharina; Möller, Lars; Kordyukova, Larisa V; Mintaev, Ramil R; Alexeevski, Andrei V; Veit, Michael

    2015-12-01

    Recruitment of the matrix protein M1 to the assembly site of the influenza virus is thought to be mediated by interactions with the cytoplasmic tail of hemagglutinin (HA). Based on a comprehensive sequence comparison of all sequences present in the database, we analyzed the effect of mutating conserved residues in the cytosol-facing part of the transmembrane region and cytoplasmic tail of HA (A/WSN/33 (H1N1) strain) on virus replication and morphology of virions. Removal of the two cytoplasmic acylation sites and substitution of a neighboring isoleucine by glutamine prevented rescue of infectious virions. In contrast, a conservative exchange of the same isoleucine, non-conservative exchanges of glycine and glutamine, deletion of the acylation site at the end of the transmembrane region and shifting it into the tail did not affect virus morphology and had only subtle effects on virus growth and on the incorporation of M1 and Ribo-Nucleoprotein Particles (RNPs). Thus, assuming that essential amino acids are conserved between HA subtypes we suggest that, besides the two cytoplasmic acylation sites (including adjacent hydrophobic residues), no other amino acids in the cytoplasmic tail of HA are indispensable for virus assembly and budding. PMID:26670246

  10. The Bel1 protein of human foamy virus contains one positive and two negative control regions which regulate a distinct activation domain of 30 amino acids.

    PubMed Central

    Lee, C W; Chang, J; Lee, K J; Sung, Y C

    1994-01-01

    The Bel1 transactivator is essential for the replication of human foamy virus (HFV). To define the functional domains of HFV Bel1, we generated random missense mutations throughout the entire coding sequence of Bel1. Functional analyses of 24 missense mutations have revealed the presence of at least two functional domains in Bel1. One domain corresponds to a basic amino acid-rich motif which acts as a bipartite nuclear targeting sequence. A second, central domain corresponds to a presumed effector region which, when mutated, leads to dominant-negative mutants and/or lacks transactivating ability. In addition, deletion analyses and domain-swapping experiments further showed that Bel1 protein contains a strong carboxy-terminal activation domain. The activating region is also capable of functioning as a transcription-activating domain in yeast cells, although it does not bear any significant sequence homology to the well-characterized acidic activation domain which is known to function only in yeast and mammalian cells. We also demonstrated that the regions of Bel1 from residues 1 to 76 and from residues 153 to 225 repressed transcriptional activation exerted by the Bel1 activation domain. In contrast, the region from residues 82 to 150 appears to overcome an inhibitory effect. These results indicate that Bel1 contains one positive and two negative regulatory domains that modulate a distinct activation domain of Bel1. These regulatory domains of Bel1 cannot affect the function of the VP16 activation domain, suggesting that these domains specifically regulate the activation domain of Bel1. Furthermore, in vivo competition experiments showed that the positive regulatory domain acts in trans. Thus, our results demonstrate that Bel1-mediated transactivation appears to undergo a complex regulatory pathway which provides a novel mode of regulation for a transcriptional activation domain. Images PMID:8139046

  11. A perspective of stepwise utilisation of Bayer red mud: Step two--Extracting and recovering Ti from Ti-enriched tailing with acid leaching and precipitate flotation.

    PubMed

    Huang, Yanfang; Chai, Wencui; Han, Guihong; Wang, Wenjuan; Yang, Shuzhen; Liu, Jiongtian

    2016-04-15

    The extraction and recovery of Ti from Ti-enriched tailing with acid leaching and precipitate flotation, as one of the critical steps, was proposed for the stepwise utilization of red mud. The factors influencing acid leaching and precipitate flotation were examined by factorial design. The leaching thermodynamics, kinetics of Ti(4+), Al(3+) and Fe(3+), and the mechanism of selectively Fe(3+) removal using [Hbet][Tf2N] as precipitating reagent were discussed. The extracting of Ti(4+), Al(3+) and Fe(3+) in concentrated H2SO4 is controlled by diffusion reactions, depending mainly upon leaching time and temperature. The maximum extracting efficiency of Ti(4+) is approximately 92.3%, whereas Al(3+) and Fe(3+) leaching are respectively 75.8% and 84.2%. [Hbet][Tf2N], as a precipitating reagent, operates through a coordination mechanism in flotation. The pH value is the key factor influencing the flotation recovery of Ti(4+), whereas the dosage of precipitating reagent is that for Al(3+) recovery. The maximum flotation recovery of Ti(4+) is 92.7%, whereas the maximum Al(3+) recovery is 93.5%. The total recovery rate for extracting and recovering titanium is 85.5%. The liquor with Ti(4+) of 15.5g/L, Al(3+) of 30.4g/L and Fe(3+) of 0.48g/L was obtained for the following hydrolysis step in the integrated process for red mud utilisation. PMID:26799223

  12. A Segment of 97 Amino Acids within the Translocation Domain of Clostridium difficile Toxin B Is Essential for Toxicity

    PubMed Central

    Zhang, Yongrong; Shi, Lianfa; Li, Shan; Yang, Zhiyong; Standley, Clive; Yang, Zhong; ZhuGe, Ronghua; Savidge, Tor; Wang, Xiaoning; Feng, Hanping

    2013-01-01

    Clostridium difficile toxin B (TcdB) intoxicates target cells by glucosylating Rho GTPases. TcdB (269 kDa) consists of at least 4 functional domains including a glucosyltransferase domain (GTD), a cysteine protease domain (CPD), a translocation domain (TD), and a receptor binding domain (RBD). The function and molecular mode of action of the TD, which is the largest segment of TcdB and comprises nearly 50% of the protein, remain largely unknown. Here we show that a 97-amino-acid segment (AA1756 – 1852, designated as ?97 or D97), located in the C-terminus of the TD and adjacent to the RBD, is essential for the cellular activity of TcdB. Deletion of this segment in TcdB (designated as TxB-D97), did not adversely alter toxin enzymatic activities or its cellular binding and uptake capacity. TxB-D97 bound to and entered cells in a manner similar to TcdB holotoxin. Both wild type and mutant toxins released their GTDs similarly in the presence of inositol hexakisphosphate (InsP6), and showed a similar glucosyltransferase activity in a cell-free glucosylating assay. Despite these similarities, the cytotoxic activity of TxB-D97 was reduced by more than 5 logs compared to wild type toxin, supported by the inability of TxB-D97 to glucosylate Rac1 of target cells. Moreover, the mutant toxin failed to elicit tumor necrosis factor alpha (TNF-α) in macrophages, a process dependent on the glucosyltransferase activity of the toxin. Cellular fractionation of toxin-exposed cells revealed that TxB-D97 was unable to efficiently release the GTD into cytosol. Thereby, we conclude the 97-amino-acid region of the TD C-terminus of TcdB adjacent to the RBD, is essential for the toxicity of TcdB. PMID:23484044

  13. Nuclear magnetic resonance structure of the nucleic acid-binding domain of severe acute respiratory syndrome coronavirus nonstructural protein 3.

    PubMed

    Serrano, Pedro; Johnson, Margaret A; Chatterjee, Amarnath; Neuman, Benjamin W; Joseph, Jeremiah S; Buchmeier, Michael J; Kuhn, Peter; Wüthrich, Kurt

    2009-12-01

    The nuclear magnetic resonance (NMR) structure of a globular domain of residues 1071 to 1178 within the previously annotated nucleic acid-binding region (NAB) of severe acute respiratory syndrome coronavirus nonstructural protein 3 (nsp3) has been determined, and N- and C-terminally adjoining polypeptide segments of 37 and 25 residues, respectively, have been shown to form flexibly extended linkers to the preceding globular domain and to the following, as yet uncharacterized domain. This extension of the structural coverage of nsp3 was obtained from NMR studies with an nsp3 construct comprising residues 1066 to 1181 [nsp3(1066-1181)] and the constructs nsp3(1066-1203) and nsp3(1035-1181). A search of the protein structure database indicates that the globular domain of the NAB represents a new fold, with a parallel four-strand beta-sheet holding two alpha-helices of three and four turns that are oriented antiparallel to the beta-strands. Two antiparallel two-strand beta-sheets and two 3(10)-helices are anchored against the surface of this barrel-like molecular core. Chemical shift changes upon the addition of single-stranded RNAs (ssRNAs) identified a group of residues that form a positively charged patch on the protein surface as the binding site responsible for the previously reported affinity for nucleic acids. This binding site is similar to the ssRNA-binding site of the sterile alpha motif domain of the Saccharomyces cerevisiae Vts1p protein, although the two proteins do not share a common globular fold.

  14. Identification of an Amino Acid Domain Encoded by the Capsid Protein Gene of Porcine Circovirus Type 2 that Modulates Viral Protein Distribution During Replication

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous work showed that distinct amino acid motifs are encoded by the Rep, Cap and ORF3 genes of two subgroups of porcine circoviruses (PCV), PCV2a and PCV2b. At a specific location of the gene, a certain amino acid residue or sequence is preferred. Specifically, two amino acid domains located in ...

  15. Phage Display Based Cloning of Proteins Interacting with the Cytoplasmic Tail of Membrane Immunoglobulins

    PubMed Central

    Geisberger, Roland; Prlic, Martin; Achatz-Straussberger, Gertrude; Oberndorfer, Iris; Luger, Elke; Lamers, Marinus; Crameri, Reto; Appenzeller, Ulrich; Wienands, Jürgen; Breitenbach, Michael; Ferreira, Fatima; Achatz, Gernot

    2002-01-01

    The reduced quantity and quality of serum immunoglobulins (sIgs) in mutant mice expressing truncated cytoplasmic tails of IgE and IgG1 indicate an active role for the cytoplasmic domains of mIgG1 and mIgE. We used phage display technology to identify candidate proteins able to interact with the cytoplasmic tail of mIgE. Using a murine cDNA B cell library displayed on the surface of phage as prey and the 28 amino acid long cytoplasmic tail of IgE as bait, we isolated phage encoding the murine hematopoietic progenitor kinase 1 (HPK1). Surface plasmon resonance analysis measurements confirmed affinity of HPK1 to the mIgE cytoplasmic tail and revealed association to other immunoglobulin isotypes as well. Immunoprecipitation experiments, using lysates from two B cell lines expressing nitrophenyl (NP) specific mIgE molecules showed co-precipitation of IgE and HPK1. The interaction of HPK1 with the cytoplasmic domains of membrane immunoglobulins indicate an active role of the tails as part of an isotype specific signal transduction, independent from the Igα/Igβ heterodimers, and may represent a missing link to upstream regulatory elements of HPK1 activation. PMID:12885153

  16. The ATPase domain but not the acidic region of Cockayne syndrome group B gene product is essential for DNA repair.

    PubMed

    Brosh, R M; Balajee, A S; Selzer, R R; Sunesen, M; Proietti De Santis, L; Bohr, V A

    1999-11-01

    Cockayne syndrome (CS) is a human genetic disorder characterized by UV sensitivity, developmental abnormalities, and premature aging. Two of the genes involved, CSA and CSB, are required for transcription-coupled repair (TCR), a subpathway of nucleotide excision repair that removes certain lesions rapidly and efficiently from the transcribed strand of active genes. CS proteins have also been implicated in the recovery of transcription after certain types of DNA damage such as those lesions induced by UV light. In this study, site-directed mutations have been introduced to the human CSB gene to investigate the functional significance of the conserved ATPase domain and of a highly acidic region of the protein. The CSB mutant alleles were tested for genetic complementation of UV-sensitive phenotypes in the human CS-B homologue of hamster UV61. In addition, the CSB mutant alleles were tested for their ability to complement the sensitivity of UV61 cells to the carcinogen 4-nitroquinoline-1-oxide (4-NQO), which introduces bulky DNA adducts repaired by global genome repair. Point mutation of a highly conserved glutamic acid residue in ATPase motif II abolished the ability of CSB protein to complement the UV-sensitive phenotypes of survival, RNA synthesis recovery, and gene-specific repair. These data indicate that the integrity of the ATPase domain is critical for CSB function in vivo. Likewise, the CSB ATPase point mutant failed to confer cellular resistance to 4-NQO, suggesting that ATP hydrolysis is required for CSB function in a TCR-independent pathway. On the contrary, a large deletion of the acidic region of CSB protein did not impair the genetic function in the processing of either UV- or 4-NQO-induced DNA damage. Thus the acidic region of CSB is likely to be dispensable for DNA repair, whereas the ATPase domain is essential for CSB function in both TCR-dependent and -independent pathways. PMID:10564257

  17. Double chromodomains cooperate to recognize the methylated histone H3 tail

    SciTech Connect

    Flanagan, John F.; Mi, Li-Zhi; Chruszcz, Maksymilian; Cymborowski, Marcin; Clines, Katrina L.; Kim, Youngchang; Minor, Wladek; Rastinejad, Fraydoon; Khorasanizadeh, Sepideh

    2010-07-19

    Chromodomains are modules implicated in the recognition of lysine-methylated histone tails and nucleic acids. CHD (for chromo-ATPase/helicase-DNA-binding) proteins regulate ATP-dependent nucleosome assembly and mobilization through their conserved double chromodomains and SWI2/SNF2 helicase/ATPase domain. The Drosophila CHD1 localizes to the interbands and puffs of the polytene chromosomes, which are classic sites of transcriptional activity. Other CHD isoforms (CHD3/4 or Mi-2) are important for nucleosome remodelling in histone deacetylase complexes. Deletion of chromodomains impairs nucleosome binding and remodelling by CHD proteins. Here we describe the structure of the tandem arrangement of the human CHD1 chromodomains, and its interactions with histone tails. Unlike HP1 and Polycomb proteins that use single chromodomains to bind to their respective methylated histone H3 tails, the two chromodomains of CHD1 cooperate to interact with one methylated H3 tail. We show that the human CHD1 double chromodomains target the lysine 4-methylated histone H3 tail (H3K4me), a hallmark of active chromatin. Methylammonium recognition involves two aromatic residues, not the three-residue aromatic cage used by chromodomains of HP1 and Polycomb proteins. Furthermore, unique inserts within chromodomain 1 of CHD1 block the expected site of H3 tail binding seen in HP1 and Polycomb, instead directing H3 binding to a groove at the inter-chromodomain junction.

  18. AXIAL SKELETAL AND HOX EXPRESSION DOMAIN ALTERATIONS INDUCED BY RETINOIC ACID, VALPROIC ACID AND BROMOXYNIL DURING MURINE DEVELOPMENT

    EPA Science Inventory

    ABSTRACT

    Retinoic acid (RA) alters the developmental fate of the axial skeletal anlage. "Anteriorizations" or "posteriorizations", the assumption of characteristics of embryonic areas normally anterior or posterior to the affected tissues, are correlated with altered emb...

  19. Amino acid sequence and domain structure of entactin. Homology with epidermal growth factor precursor and low density lipoprotein receptor

    PubMed Central

    1988-01-01

    Entactin (nidogen), a 150-kD sulfated glycoprotein, is a major component of basement membranes and forms a highly stable noncovalent complex with laminin. The complete amino acid sequence of mouse entactin has been derived from sequencing of cDNA clones. The 5.9-kb cDNA contains a 3,735-bp open reading frame followed by a 3'- untranslated region of 2.2 kb. The open reading frame encodes a 1,245- residue polypeptide with an unglycosylated Mr of 136,500, a 28-residue signal peptide, two Asn-linked glycosylation sites, and two potential Ca2+-binding sites. Analysis of the deduced amino acid sequence predicts that the molecule consists of two globular domains of 70 and 36 kD separated by a cysteine-rich domain of 28 kD. The COOH-terminal globular domain shows homology to the EGF precursor and the low density lipoprotein receptor. Entactin contains six EGF-type cysteine-rich repeat units and one copy of a cysteine-repeat motif found in thyroglobulin. The Arg-Gly-Asp cell recognition sequence is present in one of the EGF-type repeats, and a synthetic peptide from the putative cell-binding site of entactin was found to promote the attachment of mouse mammary tumor cells. PMID:3264556

  20. Tail gut cyst.

    PubMed

    Rao, G Mallikarjuna; Haricharan, P; Ramanujacharyulu, S; Reddy, K Lakshmi

    2002-01-01

    The tail gut is a blind extension of the hindgut into the tail fold just distal to the cloacal membrane. Remnants of this structure may form tail gut cyst. We report a 14-year-old girl with tail gut cyst that presented as acute abdomen. The patient recovered after cyst excision.

  1. Tail biting in pigs.

    PubMed

    Schrøder-Petersen, D L; Simonsen, H B

    2001-11-01

    One of the costly and welfare-reducing problems in modern pig production is tail biting. Tail biting is an abnormal behaviour, characterized by one pig's dental manipulation of another pig's tail. Tail biting can be classified into two groups: the pre-injury stage, before any wound on the tail is present, and the injury stage, where the tail is wounded and bleeding. Tail biting in the injury stage will reduce welfare of the bitten pig and the possible spread of infection is a health as well as welfare problem. The pigs that become tail biters may also suffer, because they are frustrated due to living in a stressful environment. This frustration may result in an excessive motivation for biting the tails of pen mates. This review aims to summarize recent research and theories in relation to tail biting. PMID:11681870

  2. Acidic Residues Control the Dimerization of the N-terminal Domain of Black Widow Spiders’ Major Ampullate Spidroin 1

    PubMed Central

    Bauer, Joschka; Schaal, Daniel; Eisoldt, Lukas; Schweimer, Kristian; Schwarzinger, Stephan; Scheibel, Thomas

    2016-01-01

    Dragline silk is the most prominent amongst spider silks and comprises two types of major ampullate spidroins (MaSp) differing in their proline content. In the natural spinning process, the conversion of soluble MaSp into a tough fiber is, amongst other factors, triggered by dimerization and conformational switching of their helical amino-terminal domains (NRN). Both processes are induced by protonation of acidic residues upon acidification along the spinning duct. Here, the structure and monomer-dimer-equilibrium of the domain NRN1 of Latrodectus hesperus MaSp1 and variants thereof have been investigated, and the key residues for both could be identified. Changes in ionic composition and strength within the spinning duct enable electrostatic interactions between the acidic and basic pole of two monomers which prearrange into an antiparallel dimer. Upon naturally occurring acidification this dimer is stabilized by protonation of residue E114. A conformational change is independently triggered by protonation of clustered acidic residues (D39, E76, E81). Such step-by-step mechanism allows a controlled spidroin assembly in a pH- and salt sensitive manner, preventing premature aggregation of spider silk proteins in the gland and at the same time ensuring fast and efficient dimer formation and stabilization on demand in the spinning duct. PMID:27681031

  3. Acidic Residues Control the Dimerization of the N-terminal Domain of Black Widow Spiders’ Major Ampullate Spidroin 1

    NASA Astrophysics Data System (ADS)

    Bauer, Joschka; Schaal, Daniel; Eisoldt, Lukas; Schweimer, Kristian; Schwarzinger, Stephan; Scheibel, Thomas

    2016-09-01

    Dragline silk is the most prominent amongst spider silks and comprises two types of major ampullate spidroins (MaSp) differing in their proline content. In the natural spinning process, the conversion of soluble MaSp into a tough fiber is, amongst other factors, triggered by dimerization and conformational switching of their helical amino-terminal domains (NRN). Both processes are induced by protonation of acidic residues upon acidification along the spinning duct. Here, the structure and monomer-dimer-equilibrium of the domain NRN1 of Latrodectus hesperus MaSp1 and variants thereof have been investigated, and the key residues for both could be identified. Changes in ionic composition and strength within the spinning duct enable electrostatic interactions between the acidic and basic pole of two monomers which prearrange into an antiparallel dimer. Upon naturally occurring acidification this dimer is stabilized by protonation of residue E114. A conformational change is independently triggered by protonation of clustered acidic residues (D39, E76, E81). Such step-by-step mechanism allows a controlled spidroin assembly in a pH- and salt sensitive manner, preventing premature aggregation of spider silk proteins in the gland and at the same time ensuring fast and efficient dimer formation and stabilization on demand in the spinning duct.

  4. Structure and function of the interacting domains of Spire and Fmn-family formins

    SciTech Connect

    Vizcarra, Christina L.; Kreutz, Barry; Rodal, Avital A.; Toms, Angela V.; Lu, Jun; Zheng, Wei; Quinlan, Margot E.; Eck, Michael J.

    2012-07-11

    Evidence for cooperation between actin nucleators is growing. The WH2-containing nucleator Spire and the formin Cappuccino interact directly, and both are essential for assembly of an actin mesh during Drosophila oogenesis. Their interaction requires the kinase noncatalytic C-lobe domain (KIND) domain of Spire and the C-terminal tail of the formin. Here we describe the crystal structure of the KIND domain of human Spir1 alone and in complex with the tail of Fmn2, a mammalian ortholog of Cappuccino. The KIND domain is structurally similar to the C-lobe of protein kinases. The Fmn2 tail is coordinated in an acidic cleft at the base of the domain that appears to have evolved via deletion of a helix from the canonical kinase fold. Our functional analysis of Cappuccino reveals an unexpected requirement for its tail in actin assembly. In addition, we find that the KIND/tail interaction blocks nucleation by Cappuccino and promotes its displacement from filament barbed ends providing insight into possible modes of cooperation between Spire and Cappuccino.

  5. Numeral series hidden in the distribution of atomic mass of amino acids to codon domains in the genetic code.

    PubMed

    Wohlin, Åsa

    2015-03-21

    The distribution of codons in the nearly universal genetic code is a long discussed issue. At the atomic level, the numeral series 2x(2) (x=5-0) lies behind electron shells and orbitals. Numeral series appear in formulas for spectral lines of hydrogen. The question here was if some similar scheme could be found in the genetic code. A table of 24 codons was constructed (synonyms counted as one) for 20 amino acids, four of which have two different codons. An atomic mass analysis was performed, built on common isotopes. It was found that a numeral series 5 to 0 with exponent 2/3 times 10(2) revealed detailed congruency with codon-grouped amino acid side-chains, simultaneously with the division on atom kinds, further with main 3rd base groups, backbone chains and with codon-grouped amino acids in relation to their origin from glycolysis or the citrate cycle. Hence, it is proposed that this series in a dynamic way may have guided the selection of amino acids into codon domains. Series with simpler exponents also showed noteworthy correlations with the atomic mass distribution on main codon domains; especially the 2x(2)-series times a factor 16 appeared as a conceivable underlying level, both for the atomic mass and charge distribution. Furthermore, it was found that atomic mass transformations between numeral systems, possibly interpretable as dimension degree steps, connected the atomic mass of codon bases with codon-grouped amino acids and with the exponent 2/3-series in several astonishing ways. Thus, it is suggested that they may be part of a deeper reference system.

  6. Numeral series hidden in the distribution of atomic mass of amino acids to codon domains in the genetic code.

    PubMed

    Wohlin, Åsa

    2015-03-21

    The distribution of codons in the nearly universal genetic code is a long discussed issue. At the atomic level, the numeral series 2x(2) (x=5-0) lies behind electron shells and orbitals. Numeral series appear in formulas for spectral lines of hydrogen. The question here was if some similar scheme could be found in the genetic code. A table of 24 codons was constructed (synonyms counted as one) for 20 amino acids, four of which have two different codons. An atomic mass analysis was performed, built on common isotopes. It was found that a numeral series 5 to 0 with exponent 2/3 times 10(2) revealed detailed congruency with codon-grouped amino acid side-chains, simultaneously with the division on atom kinds, further with main 3rd base groups, backbone chains and with codon-grouped amino acids in relation to their origin from glycolysis or the citrate cycle. Hence, it is proposed that this series in a dynamic way may have guided the selection of amino acids into codon domains. Series with simpler exponents also showed noteworthy correlations with the atomic mass distribution on main codon domains; especially the 2x(2)-series times a factor 16 appeared as a conceivable underlying level, both for the atomic mass and charge distribution. Furthermore, it was found that atomic mass transformations between numeral systems, possibly interpretable as dimension degree steps, connected the atomic mass of codon bases with codon-grouped amino acids and with the exponent 2/3-series in several astonishing ways. Thus, it is suggested that they may be part of a deeper reference system. PMID:25623487

  7. Domains of the cucumber mosaic virus 2b silencing suppressor protein affecting inhibition of salicylic acid-induced resistance and priming of salicylic acid accumulation during infection

    PubMed Central

    Zhou, Tao; Murphy, Alex M.; Lewsey, Mathew G.; Westwood, Jack H.; Zhang, Heng-Mu; González, Inmaculada; Canto, Tomás

    2014-01-01

    The cucumber mosaic virus (CMV) 2b silencing suppressor protein allows the virus to overcome resistance to replication and local movement in inoculated leaves of plants treated with salicylic acid (SA), a resistance-inducing plant hormone. In Arabidopsis thaliana plants systemically infected with CMV, the 2b protein also primes the induction of SA biosynthesis during this compatible interaction. We found that CMV infection of susceptible tobacco (Nicotiana tabacum) also induced SA accumulation. Utilization of mutant 2b proteins expressed during infection of tobacco showed that the N- and C-terminal domains, which had previously been implicated in regulation of symptom induction, were both required for subversion of SA-induced resistance, while all mutants tested except those affecting the putative phosphorylation domain had lost the ability to prime SA accumulation and expression of the SA-induced marker gene PR-1. PMID:24633701

  8. Expression of a collagen-binding domain fusion protein: effect of amino acid supplementation, inducer type, and culture conditions.

    PubMed

    Fruchtl, McKinzie; Sakon, Joshua; Beitle, Robert

    2015-01-01

    Collagen binding domain fusion proteins are of significant importance because of their potential as therapeutic biomaterials. In this paper, we investigate the production of such therapeutic proteins via fermentation of Escherichia coli on both an undefined medium and a defined medium. Defined media with amino acid supplementation provided higher amounts of therapeutic protein than undefined media with no supplementation. Additionally, utilizing lactose instead of isopropyl-β-d-thio-galactoside (IPTG) for induction and extending batch time yielded higher amounts of the model therapeutic.

  9. Support Effects on Bronsted acid site densities and alcohol dehydration turnover rates on tungsten oxide domains

    SciTech Connect

    Macht, Josef; Baertsch, Chelsey D.; May-Lozano, Marcos; Soled, Stuart L.; Wang, Yong; Iglesia, Enrique

    2005-03-01

    Initial activity and acid site density of several WAl, WSi (MCM41) and one WSn sample were determined. Trans/cis 2-butene selectivity is dependent on the support. Presumably, these differences are due to subtle differences in base strengths. 2-Butanol dehydration rates (per W-atom) reached maximum values at intermediate WOx surface densities on WAl, as reported for 2-butanol dehydration reactions on WZr. Titration results indicate that Bronsted acid sites are required for 2-butanol dehydration on WAl, WSi and WSn. UV-visible studies suggest that WAl is much more difficult to reduce than WZr. The detection of reduced centers on WAl, the number of which correlates to Bronsted acid site density and catalyst activity, as well as the temperature dependence of Bronsted acid site density indicate the in-situ formation of these active sites. We infer that this mechanism is common among all supported WOx samples described in this study. Turnover rates are a function of Bronsted acid site density only. High acid site densities lead to high turnover rates. Higher active site densities may cause stronger conjugate bases, as a higher electron density has to be stabilized, and thus weaker acidity, enabling a faster rate of product desorption. The maximum achievable active site density is dependent on the support. WZr reaches a higher active site density than WAl.

  10. Effect of the amino acid substitution in the DNA-binding domain of the Fur regulator on production of pyoverdine.

    PubMed

    Valešová, Renáta; Palyzová, Andrea; Marešová, Helena; Stěpánek, Václav; Babiak, Peter; Kyslík, Pavel

    2013-07-01

    The ferric uptake regulator gene (fur), its promoter region and Fur box of pvdS gene involved in siderophore-mediated iron uptake system were sequenced in the parent strain Pseudomonas aeruginosa PAO1 and in the fur mutant FPA121 derived from the strain PAO1. We identified the gene fur 179 bearing a novel, single-point mutation that changed the amino acid residue Gln60Pro in the DNA-binding domain of the Fur protein. The synthesis of pyoverdine was studied in cultures of the strains PAO1 and FPA121 grown in iron-deplete and iron-replete (60 μmol/L FeIII) medium. The amino acid replacement in the regulatory Fur protein is responsible for the overproduction of pyoverdine in iron-deplete and iron-replete medium. No mutation was identified in the Fur box of the gene pvdS.

  11. Amino acid sequence and location of the disulfide bonds in bovine beta 2 glycoprotein I: the presence of five Sushi domains.

    PubMed

    Kato, H; Enjyoji, K

    1991-12-17

    beta 2 glycoprotein I is a plasma protein with the ability to bind with various kinds of negatively charged substances. The complete amino acid sequence and the location of all the disulfide bonds of bovine beta 2 glycoprotein I were determined. Bovine beta 2 glycoprotein I consists of 326 amino acid residues with five asparagine-linked carbohydrate chains. Homology with the human protein was calculated to be 83%. Eleven disulfide bonds in bovine beta 2 glycoprotein I constitute four characteristic domains, Sushi domains, and one modified form of a Sushi domain.

  12. Environmental factors influencing the structural dynamics of soil microbial communities during assisted phytostabilization of acid-generating mine tailings: a mesocosm experiment.

    PubMed

    Valentín-Vargas, Alexis; Root, Robert A; Neilson, Julia W; Chorover, Jon; Maier, Raina M

    2014-12-01

    Compost-assisted phytostabilization has recently emerged as a robust alternative for reclamation of metalliferous mine tailings. Previous studies suggest that root-associated microbes may be important for facilitating plant establishment on the tailings, yet little is known about the long-term dynamics of microbial communities during reclamation. A mechanistic understanding of microbial community dynamics in tailings ecosystems undergoing remediation is critical because these dynamics profoundly influence both the biogeochemical weathering of tailings and the sustainability of a plant cover. Here we monitor the dynamics of soil microbial communities (i.e. bacteria, fungi, archaea) during a 12-month mesocosm study that included 4 treatments: 2 unplanted controls (unamended and compost-amended tailings) and 2 compost-amended seeded tailings treatments. Bacterial, fungal and archaeal communities responded distinctively to the revegetation process and concurrent changes in environmental conditions and pore water chemistry. Compost addition significantly increased microbial diversity and had an immediate and relatively long-lasting buffering-effect on pH, allowing plants to germinate and thrive during the early stages of the experiment. However, the compost buffering capacity diminished after six months and acidification took over as the major factor affecting plant survival and microbial community structure. Immediate changes in bacterial communities were observed following plant establishment, whereas fungal communities showed a delayed response that apparently correlated with the pH decline. Fluctuations in cobalt pore water concentrations, in particular, had a significant effect on the structure of all three microbial groups, which may be linked to the role of cobalt in metal detoxification pathways. The present study represents, to our knowledge, the first documentation of the dynamics of the three major microbial groups during revegetation of compost

  13. Environmental Factors Influencing the Structural Dynamics of Soil Microbial Communities During Assisted Phytostabilization of Acid-Generating Mine Tailings: a Mesocosm Experiment

    PubMed Central

    Valentín-Vargas, Alexis; Root, Robert A.; Neilson, Julia W; Chorover, Jon; Maier, Raina M.

    2014-01-01

    Compost-assisted phytostabilization has recently emerged as a robust alternative for reclamation of metalliferous mine tailings. Previous studies suggest that root-associated microbes may be important for facilitating plant establishment on the tailings, yet little is known about the long-term dynamics of microbial communities during reclamation. A mechanistic understanding of microbial community dynamics in tailings ecosystems undergoing remediation is critical because these dynamics profoundly influence both the biogeochemical weathering of tailings and the sustainability of a plant cover. Here we monitor the dynamics of soil microbial communities (i.e. bacteria, fungi, archaea) during a 12-month mesocosm study that included 4 treatments: 2 unplanted controls (unamended and compost-amended tailings) and 2 compost-amended seeded tailings treatments. Bacterial, fungal and archaeal communities responded distinctively to the revegetation process and concurrent changes in environmental conditions and pore water chemistry. Compost addition significantly increased microbial diversity and had an immediate and relatively long-lasting buffering-effect on pH, allowing plants to germinate and thrive during the early stages of the experiment. However, the compost buffering capacity diminished after six months and acidification took over as the major factor affecting plant survival and microbial community structure. Immediate changes in bacterial communities were observed following plant establishment, whereas fungal communities showed a delayed response that apparently correlated with the pH decline. Fluctuations in cobalt pore water concentrations, in particular, had a significant effect on the structure of all three microbial groups, which may be linked to the role of cobalt in metal detoxification pathways. The present study represents, to our knowledge, the first documentation of the dynamics of the three major microbial groups during revegetation of compost

  14. Computational protein design with electrostatic focusing: experimental characterization of a conditionally folded helical domain with a reduced amino acid alphabet.

    PubMed

    Suárez-Diez, Maria; Pujol, Anaïs M; Matzapetakis, Manolis; Jaramillo, Alfonso; Iranzo, Olga

    2013-07-01

    Automated methodologies to design synthetic proteins from first principles use energy computations to estimate the ability of the sequences to adopt a targeted structure. This approach is still far from systematically producing native-like sequences, due, most likely, to inaccuracies when modeling the interactions between the protein and its aqueous environment. This is particularly challenging when engineering small protein domains (with less polar pair interactions than with the solvent). We have re-designed a three-helix bundle, domain B, using a fixed backbone and a four amino acid alphabet. We have enlarged the rotamer library with conformers that increase the weight of electrostatic interactions within the design process without altering the energy function used to compute the folding free energy. Our synthetic sequences show less than 15% similarity to any Swissprot sequence. We have characterized our sequences in different solvents using circular dichroism and nuclear magnetic resonance. The targeted structure achieved is dependent on the solvent used. This method can be readily extended to larger domains. Our method will be useful for the engineering of proteins that become active only in a given solvent and for designing proteins in the context of hydrophobic solvents, an important fraction of the situations in the cell.

  15. Cognition in ring-tailed lemurs.

    PubMed

    Kittler, Klara; Schnoell, Anna Viktoria; Fichtel, Claudia

    2015-01-01

    In order to better understand the evolution of cognitive abilities in primates, information on cognitive traits of the most basal living primates can provide important comparative baseline data. Compared to haplorhine primates, lemurs have relatively smaller brains and reduced abilities to solve problems in the technical and social domain. However, recent studies have suggested that some cognitive abilities of lemurs are qualitatively equal to those of haplorhines. Here, we review studies investigating cognitive abilities in the technical and social domain of ring-tailed lemur cognition. In the physical domain, ring-tailed lemurs exhibit similar qualitative cognitive skills as other lemurs but also haplorhine primates. In the social domain, ring-tailed lemurs appear to be more skilled in visual perspective taking than other lemurs. Compared to other lemurs, they also have highly elaborated communicative skills. Moreover, within-group coalitions have been observed in female ring-tailed lemurs during rare events of female evictions but not in other lemur species. However, in several other aspects of social cognition, such as reconciliation and social learning, ring-tailed lemurs' cognitive abilities are equal to those of other lemurs. Thus, additional systematic comparative studies in physical and social cognition are required for a more comprehensive understanding of the processes of cognitive evolution among primates. PMID:26022306

  16. The RRM Domain of Human Fused in Sarcoma Protein Reveals a Non-Canonical Nucleic Acid Binding Site

    PubMed Central

    Liu, Xuehui; Niu, Chunyan; Ren, Jintao; Zhang, Jiayu; Xie, Xiaodong; Zhu, Haining; Feng, Wei; Gong, Weimin

    2012-01-01

    Fused in sarcoma (FUS) is involved in many processes of RNA metabolism. FUS and another RNA binding protein, TDP-43, are implicated in amyotrophic lateral sclerosis (ALS). It is significant to characterize the RNA recognition motif (RRM) of FUS as its nucleic acid binding properties are unclear. More importantly, abolishing the RNA binding ability of the RRM domain of TDP43 was reported to suppress the neurotoxicity of TDP-43 in Drosophila. The sequence of FUS-RRM varies significantly from canonical RRMs, but the solution structure of FUS-RRM determined by NMR showed a similar overall folding as other RRMs. We found that FUS-RRM directly bound to RNA and DNA and the binding affinity was in the micromolar range as measured by surface plasmon resonance and NMR titration. The nucleic acid binding pocket in FUS-RRM is significantly distorted since several critical aromatic residues are missing. An exceptionally positively charged loop in FUS-RRM, which is not found in other RRMs, is directly involved in the RNA/DNA binding. Substituting the lysine residues in the unique KK loop impaired the nucleic acid binding and altered FUS subcellular localization. The results provide insights into the nucleic acid binding properties of FUS-RRM and its potential relevance to ALS. PMID:23200923

  17. Chlorogenic acid-arabinose hybrid domains in coffee melanoidins: Evidences from a model system.

    PubMed

    Moreira, Ana S P; Coimbra, Manuel A; Nunes, Fernando M; Passos, Cláudia P; Santos, Sónia A O; Silvestre, Armando J D; Silva, André M N; Rangel, Maria; Domingues, M Rosário M

    2015-10-15

    Arabinose from arabinogalactan side chains was hypothesized as a possible binding site for chlorogenic acids in coffee melanoidins. To investigate this hypothesis, a mixture of 5-O-caffeoylquinic acid (5-CQA), the most abundant chlorogenic acid in green coffee beans, and (α1 → 5)-L-arabinotriose, structurally related to arabinogalactan side chains, was submitted to dry thermal treatments. The compounds formed during thermal processing were identified by electrospray ionization mass spectrometry (ESI-MS) and characterized by tandem MS (ESI-MS(n)). Compounds composed by one or two CQAs covalently linked with pentose (Pent) residues (1-12) were identified, along with compounds bearing a sugar moiety but composed exclusively by the quinic or caffeic acid moiety of CQAs. The presence of isomers was demonstrated by liquid chromatography online coupled to ESI-MS and ESI-MS(n). Pent1-2CQA were identified in coffee samples. These results give evidence for a diversity of chlorogenic acid-arabinose hybrids formed during roasting, opening new perspectives for their identification in melanoidin structures. PMID:25952851

  18. Chlorogenic acid-arabinose hybrid domains in coffee melanoidins: Evidences from a model system.

    PubMed

    Moreira, Ana S P; Coimbra, Manuel A; Nunes, Fernando M; Passos, Cláudia P; Santos, Sónia A O; Silvestre, Armando J D; Silva, André M N; Rangel, Maria; Domingues, M Rosário M

    2015-10-15

    Arabinose from arabinogalactan side chains was hypothesized as a possible binding site for chlorogenic acids in coffee melanoidins. To investigate this hypothesis, a mixture of 5-O-caffeoylquinic acid (5-CQA), the most abundant chlorogenic acid in green coffee beans, and (α1 → 5)-L-arabinotriose, structurally related to arabinogalactan side chains, was submitted to dry thermal treatments. The compounds formed during thermal processing were identified by electrospray ionization mass spectrometry (ESI-MS) and characterized by tandem MS (ESI-MS(n)). Compounds composed by one or two CQAs covalently linked with pentose (Pent) residues (1-12) were identified, along with compounds bearing a sugar moiety but composed exclusively by the quinic or caffeic acid moiety of CQAs. The presence of isomers was demonstrated by liquid chromatography online coupled to ESI-MS and ESI-MS(n). Pent1-2CQA were identified in coffee samples. These results give evidence for a diversity of chlorogenic acid-arabinose hybrids formed during roasting, opening new perspectives for their identification in melanoidin structures.

  19. Comparison of backbone dynamics of the type III antifreeze protein and antifreeze-like domain of human sialic acid synthase.

    PubMed

    Choi, Yong-Geun; Park, Chin-Ju; Kim, Hee-Eun; Seo, Yeo-Jin; Lee, Ae-Ree; Choi, Seo-Ree; Lee, Shim Sung; Lee, Joon-Hwa

    2015-02-01

    Antifreeze proteins (AFPs) are found in a variety of cold-adapted (psychrophilic) organisms to promote survival at subzero temperatures by binding to ice crystals and decreasing the freezing temperature of body fluids. The type III AFPs are small globular proteins that consist of one α-helix, three 3(10)-helices, and two β-strands. Sialic acids play important roles in a variety of biological functions, such as development, recognition, and cell adhesion and are synthesized by conserved enzymatic pathways that include sialic acid synthase (SAS). SAS consists of an N-terminal catalytic domain and a C-terminal antifreeze-like (AFL) domain, which is similar to the type III AFPs. Despite having very similar structures, AFL and the type III AFPs exhibit very different temperature-dependent stability and activity. In this study, we have performed backbone dynamics analyses of a type III AFP (HPLC12 isoform) and the AFL domain of human SAS (hAFL) at various temperatures. We also characterized the structural/dynamic properties of the ice-binding surfaces by analyzing the temperature gradient of the amide proton chemical shift and its correlation with chemical shift deviation from random coil. The dynamic properties of the two proteins were very different from each other. While HPLC12 was mostly rigid with a few residues exhibiting slow motions, hAFL showed fast internal motions at low temperature. Our results provide insight into the molecular basis of thermostability and structural flexibility in homologous psychrophilic HPLC12 and mesophilic hAFL proteins.

  20. THE INTEGRITY OF THE α-HELICAL DOMAIN OF INTESTINAL FATTY ACID BINDING PROTEIN IS ESSENTIAL FOR THE COLLISION-MEDIATED TRANSFER OF FATTY ACIDS TO PHOSPHOLIPID MEMBRANES

    PubMed Central

    Franchini, G. R.; Storch, J.; Corsico, B.

    2015-01-01

    Summary Intestinal FABP (IFABP) and liver FABP (LFABP), homologous proteins expressed at high levels in intestinal absorptive cells, employ markedly different mechanisms of fatty acid transfer to acceptor model membranes. Transfer from IFABP occurs during protein-membrane-collisional interactions, while for LFABP transfer occurs by diffusion through the aqueous phase. In addition, transfer from IFABP is markedly faster than from LFABP. The overall goal of this study was to further explore the structural differences between IFABP and LFABP which underlie their large functional differences in ligand transport. In particular, we addressed the role of the αI-helix domain in the unique transport properties of intestinal FABP. A chimeric protein was engineered with the ‘body’ (ligand binding domain) of IFABP and the αI-helix of LFABP (α(I)LβIFABP), and the fatty acid transfer properties of the chimeric FABP were examined using a fluorescence resonance energy transfer assay. The results showed a significant decrease in the absolute rate of FA transfer from α(I)LβIFABP compared to IFABP. The results indicate that the αI-helix is crucial for IFABP collisional FA transfer, and further indicate the participation of the αII-helix in the formation of a protein-membrane “collisional complex”. Photo-crosslinking experiments with a photoactivable reagent demonstrated the direct interaction of IFABP with membranes and further supports the importance of the αI helix of IFABP in its physical interaction with membranes. PMID:18284926

  1. Amino acid mutations in the caldesmon COOH-terminal functional domain increase force generation in bladder smooth muscle.

    PubMed

    Deng, Maoxian; Boopathi, Ettickan; Hypolite, Joseph A; Raabe, Tobias; Chang, Shaohua; Zderic, Stephen; Wein, Alan J; Chacko, Samuel

    2013-11-15

    Caldesmon (CaD), a component of smooth muscle thin filaments, binds actin, tropomyosin, calmodulin, and myosin and inhibits actin-activated ATP hydrolysis by smooth muscle myosin. Internal deletions of the chicken CaD functional domain that spans from amino acids (aa) 718 to 731, which corresponds to aa 512-530 including the adjacent aa sequence in mouse CaD, lead to diminished CaD-induced inhibition of actin-activated ATP hydrolysis by myosin. Transgenic mice with mutations of five aa residues (Lys(523) to Gln, Val(524) to Leu, Ser(526) to Thr, Pro(527) to Cys, and Lys(529) to Ser), which encompass the ATPase inhibitory determinants located in exon 12, were generated by homologous recombination. Homozygous (-/-) animals did not develop, but heterozygous (+/-) mice carrying the expected mutations in the CaD ATPase inhibitory domain (CaD mutant) matured and reproduced normally. The peak force produced in response to KCl and electrical field stimulation by the detrusor smooth muscle from the CaD mutant was high compared with that of the wild type. CaD mutant mice revealed nonvoiding contractions during bladder filling on awake cystometry, suggesting that the CaD ATPase inhibitory domain suppresses force generation during the filling phase and this suppression is partially released by mutations in 50% of CaD in heterozygous. Our data show for the first time a functional phenotype, at the intact smooth muscle tissue and in vivo organ levels, following mutation of a functional domain at the COOH-terminal region of CaD.

  2. Modulation of cell surface transport and lipid raft localization by the cytoplasmic tail of the influenza virus hemagglutinin.

    PubMed

    Scolari, Silvia; Imkeller, Katharina; Jolmes, Fabian; Veit, Michael; Herrmann, Andreas; Schwarzer, Roland

    2016-01-01

    Viral glycoproteins are highly variable in their primary structure, but on the other hand feature a high functional conservation to fulfil their versatile tasks during the pathogenic life cycle. Typically, all protein domains are optimized in that indispensable functions can be assigned to small conserved motifs or even individual amino acids. The cytoplasmic tail of many viral spike proteins, although of particular relevance for the virus biology, is often only insufficiently characterized. Hemagglutinin (HA), the receptor-binding protein of the influenza virus comprises a short cytoplasmic tail of 13 amino acids that exhibits three highly conserved palmitoylation sites. However, the particular importance of these modifications and the tail in general for intracellular trafficking and lateral membrane organization remains elusive. In this study, we generated HA core proteins consisting of transmembrane domain, cytoplasmic tail and a minor part of the ectodomain, tagged with a yellow fluorescent protein. Different mutation and truncation variants of these chimeric proteins were investigated using confocal microscopy, to characterize the role of cytoplasmic tail and palmitoylation for the intracellular trafficking to plasma membrane and Golgi apparatus. In addition, we assessed raft partitioning of the variants by Foerster resonance energy transfer with an established raft marker. We revealed a substantial influence of the cytoplasmic tail length on the intracellular distribution and surface exposure of the proteins. A complete removal of the tail hampers a physiological trafficking of the protein, whereas a partial truncation can be compensated by cytoplasmic palmitoylations. Plasma membrane raft partitioning on the other hand was found to imperatively require palmitoylations, and the cysteine at position 551 turned out to be of most relevance. Our data shed further light on the tight interconnection between cytoplasmic elements and intracellular trafficking and

  3. Transmembrane domain II of the human bile acid transporter SLC10A2 coordinates sodium translocation.

    PubMed

    Sabit, Hairat; Mallajosyula, Sairam S; MacKerell, Alexander D; Swaan, Peter W

    2013-11-01

    Human apical sodium-dependent bile acid transporter (hASBT, SLC10A2) is responsible for intestinal reabsorption of bile acids and plays a key role in cholesterol homeostasis. We used a targeted and systematic approach to delineate the role of highly conserved transmembrane helix 2 on the expression and function of hASBT. Cysteine mutation significantly depressed transport activity for >60% of mutants without affecting cell surface localization of the transporter. All mutants were inaccessible toward chemical modification by membrane-impermeant MTSET reagent, strongly suggesting that transmembrane 2 (TM2) plays an indirect role in bile acid substrate translocation. Both bile acid uptake and sodium dependence of TM2 mutants revealed a distinct α-helical periodicity. Kinetic studies with conservative and non-conservative mutants of sodium sensitive residues further underscored the importance of Gln(75), Phe(76), Met(79), Gly(83), Leu(86), Phe(90), and Asp(91) in hASBT function. Computational analysis indicated that Asp(91) may coordinate with sodium during the transport cycle. Combined, our data propose that a consortium of sodium-sensitive residues along with previously reported residues (Thr(134), Leu(138), and Thr(149)) from TM3 may form the sodium binding and translocation pathway. Notably, residues Gln(75), Met(79), Thr(82), and Leu(86) from TM2 are highly conserved in TM3 of a putative remote bacterial homologue (ASBTNM), suggesting a universal mechanism for the SLC10A transporter family.

  4. Distinct pharmacology of rat and human histamine H3 receptors: role of two amino acids in the third transmembrane domain

    PubMed Central

    Ligneau, X; Morisset, S; Tardivel-Lacombe, J; Gbahou, F; Ganellin, C R; Stark, H; Schunack, W; Schwartz, J -C; Arrang, J -M

    2000-01-01

    Starting from the sequence of the human histamine H3 receptor (hH3R) cDNA, we have cloned the corresponding rat cDNA. Whereas the two deduced proteins show 93.5% overall homology and differ only by five amino acid residues at the level of the transmembrane domains (TMs), some ligands displayed distinct affinities. Thioperamide and ciproxifan were about 10 fold more potent at the rat than at the human receptor, whereas FUB 349 displayed a reverse preference. Histamine, (R)α-methylhistamine, proxyfan or clobenpropit were nearly equipotent at H3 receptors of both species. The inverse discrimination patterns of ciproxifan and FUB 349 were partially changed by mutation of one amino acid (V122A), and fully abolished by mutation of two amino acids (A119T and V122A), in TM3 of the rH3R located in the vicinity of Asp114 purported to salt-link the ammonium group of histamine. Therefore, these two residues appear to be responsible for the distinct pharmacology of the H3R in the two species. PMID:11090094

  5. NMR conformational properties of an Anthrax Lethal Factor domain studied by multiple amino acid-selective labeling

    SciTech Connect

    Vourtsis, Dionysios J.; Chasapis, Christos T.; Pairas, George; Bentrop, Detlef; Spyroulias, Georgios A.

    2014-07-18

    Highlights: • A polypeptide, N-ALF{sub 233}, was overexpressed in E. coli and successfully isolated. • We produced {sup 2}H/{sup 15}N/{sup 13}C labeled protein samples. • Amino acid selective approaches were applied. • We acquired several heteronuclear NMR spectra, to complete the backbone assignment. • Prediction of the secondary structure was performed. - Abstract: NMR-based structural biology urgently needs cost- and time-effective methods to assist both in the process of acquiring high-resolution NMR spectra and their subsequent analysis. Especially for bigger proteins (>20 kDa) selective labeling is a frequently used means of sequence-specific assignment. In this work we present the successful overexpression of a polypeptide of 233 residues, corresponding to the structured part of the N-terminal domain of Anthrax Lethal Factor, using Escherichia coli expression system. The polypeptide was subsequently isolated in pure, soluble form and analyzed structurally by solution NMR spectroscopy. Due to the non-satisfying quality and resolution of the spectra of this 27 kDa protein, an almost complete backbone assignment became feasible only by the combination of uniform and novel amino acid-selective labeling schemes. Moreover, amino acid-type selective triple-resonance NMR experiments proved to be very helpful.

  6. Electrodialytic remediation of copper mine tailings.

    PubMed

    Hansen, Henrik K; Rojo, Adrián; Ottosen, Lisbeth M

    2005-01-31

    Mining activities in Chile have generated large amounts of solid waste, which have been deposited in mine tailing impoundments. These impoundments cause concern to the communities due to dam failures or natural leaching to groundwater and rivers. This work shows the laboratory results of nine electrodialytic remediation experiments on copper mine tailings. The results show that electric current could remove copper from watery tailing if the potential gradient was higher than 2 V/cm during 21 days. With addition of sulphuric acid, the process was enhanced because the pH decreased to around 4, and the copper by this reason was released in the solution. Furthermore, with acidic tailing the potential gradient was less than 2 V/cm. The maximum copper removal reached in the anode side was 53% with addition of sulphuric acid in 21 days experiment at 20 V using approximately 1.8 kg mine tailing on dry basis. In addition, experiments with acidic tailing show that the copper removal is proportional with time.

  7. Acidianus Tailed Spindle Virus: a New Archaeal Large Tailed Spindle Virus Discovered by Culture-Independent Methods

    PubMed Central

    Hochstein, Rebecca A.; Amenabar, Maximiliano J.; Munson-McGee, Jacob H.; Boyd, Eric S.

    2016-01-01

    ABSTRACT The field of viral metagenomics has expanded our understanding of viral diversity from all three domains of life (Archaea, Bacteria, and Eukarya). Traditionally, viral metagenomic studies provide information about viral gene content but rarely provide knowledge about virion morphology and/or cellular host identity. Here we describe a new virus, Acidianus tailed spindle virus (ATSV), initially identified by bioinformatic analysis of viral metagenomic data sets from a high-temperature (80°C) acidic (pH 2) hot spring located in Yellowstone National Park, followed by more detailed characterization using only environmental samples without dependency on culturing. Characterization included the identification of the large tailed spindle virion morphology, determination of the complete 70.8-kb circular double-stranded DNA (dsDNA) viral genome content, and identification of its cellular host. Annotation of the ATSV genome revealed a potential three-domain gene product containing an N-terminal leucine-rich repeat domain, followed by a likely posttranslation regulatory region consisting of high serine and threonine content, and a C-terminal ESCRT-III domain, suggesting interplay with the host ESCRT system. The host of ATSV, which is most closely related to Acidianus hospitalis, was determined by a combination of analysis of cellular clustered regularly interspaced short palindromic repeat (CRISPR)/Cas loci and dual viral and cellular fluorescence in situ hybridization (viral FISH) analysis of environmental samples and confirmed by culture-based infection studies. This work provides an expanded pathway for the discovery, isolation, and characterization of new viruses using culture-independent approaches and provides a platform for predicting and confirming virus hosts. IMPORTANCE Virus discovery and characterization have been traditionally accomplished by using culture-based methods. While a valuable approach, it is limited by the availability of culturable hosts. In

  8. Effects of a mixture of fatty acids from sugar cane (Saccharum officinarum L.) wax oil in two models of inflammation: zymosan-induced arthritis and mice tail test of psoriasis.

    PubMed

    Ledón, N; Casacó, A; Remirez, D; González, A; Cruz, J; González, R; Capote, A; Tolón, Z; Rojas, E; Rodríguez, V J; Merino, N; Rodríguez, S; Ancheta, O; Cano, M C

    2007-10-01

    A mixture of fatty acids obtained from sugar cane (Saccharum officinarum L.) wax oil (FAM), in which the main constituents are palmitic, oleic, linoleic, and linolenic acids, was evaluated in two models of inflammation: zymosan-induced arthritis and in the tail test for psoriasis, both on mice. In the first model, FAM significantly reduced zymozan-induced increase of beta glucuronidase (DE(50) 90+/-7 mg/kg). Histopathological studies showed inhibition in cellular infiltration and reduction of synovial hyperplasia and synovitis, whereas in the second test, histopathological and ultrastructural studies showed that topical application of FAM induced orthokeratosis with the presence of keratohyalin granules in the previously parakeratotic adult mouse tail, and without effects on epidermal thickness. The ED(50) of FAM in this model was 155+/-10 mg. The results of our studies showed that topical application of FAM exerts an important anti-inflammatory activity in both tests without evidence of irritant effects. The anti-inflamatory effects exerted by FAM may be due to its inhibitory effects on arachidonic acid metabolism. To our knowledge, this is the first report on the anti-inflammatory effect of sugar cane by-products in experimental models of arthritis and psoriasis.

  9. A cholesterol recognition amino acid consensus domain in GP64 fusion protein facilitates anchoring of baculovirus to mammalian cells.

    PubMed

    Luz-Madrigal, Agustin; Asanov, Alexander; Camacho-Zarco, Aldo R; Sampieri, Alicia; Vaca, Luis

    2013-11-01

    Baculoviridae is a large family of double-stranded DNA viruses that selectively infect insects. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the best-studied baculovirus from the family. Many studies over the last several years have shown that AcMNPV can enter a wide variety of mammalian cells and deliver genetic material for foreign gene expression. While most animal viruses studied so far have developed sophisticated mechanisms to selectively infect specific cells and tissues in an organism, AcMNPV can penetrate and deliver foreign genes into most cells studied to this date. The details about the mechanisms of internalization have been partially described. In the present study, we have identified a cholesterol recognition amino acid consensus (CRAC) domain present in the AcMNPV envelope fusion protein GP64. We demonstrated the association of a CRAC domain with cholesterol, which is important to facilitate the anchoring of the virus at the mammalian cell membrane. Furthermore, this initial anchoring favors AcMNPV endocytosis via a dynamin- and clathrin-dependent mechanism. Under these conditions, efficient baculovirus-driven gene expression is obtained. In contrast, when cholesterol is reduced from the plasma membrane, AcMNPV enters the cell via a dynamin- and clathrin-independent mechanism. The result of using this alternative internalization pathway is a reduced level of baculovirus-driven gene expression. This study is the first to document the importance of a novel CRAC domain in GP64 and its role in modulating gene delivery in AcMNPV.

  10. A Cholesterol Recognition Amino Acid Consensus Domain in GP64 Fusion Protein Facilitates Anchoring of Baculovirus to Mammalian Cells

    PubMed Central

    Luz-Madrigal, Agustin; Asanov, Alexander; Camacho-Zarco, Aldo R.; Sampieri, Alicia

    2013-01-01

    Baculoviridae is a large family of double-stranded DNA viruses that selectively infect insects. Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is the best-studied baculovirus from the family. Many studies over the last several years have shown that AcMNPV can enter a wide variety of mammalian cells and deliver genetic material for foreign gene expression. While most animal viruses studied so far have developed sophisticated mechanisms to selectively infect specific cells and tissues in an organism, AcMNPV can penetrate and deliver foreign genes into most cells studied to this date. The details about the mechanisms of internalization have been partially described. In the present study, we have identified a cholesterol recognition amino acid consensus (CRAC) domain present in the AcMNPV envelope fusion protein GP64. We demonstrated the association of a CRAC domain with cholesterol, which is important to facilitate the anchoring of the virus at the mammalian cell membrane. Furthermore, this initial anchoring favors AcMNPV endocytosis via a dynamin- and clathrin-dependent mechanism. Under these conditions, efficient baculovirus-driven gene expression is obtained. In contrast, when cholesterol is reduced from the plasma membrane, AcMNPV enters the cell via a dynamin- and clathrin-independent mechanism. The result of using this alternative internalization pathway is a reduced level of baculovirus-driven gene expression. This study is the first to document the importance of a novel CRAC domain in GP64 and its role in modulating gene delivery in AcMNPV. PMID:23986592

  11. Poly(acrylic acid)-directed synthesis of colloidally stable single domain magnetite nanoparticles via partial oxidation

    NASA Astrophysics Data System (ADS)

    Altan, Cem L.; Gurten, Berna; Sadza, Roel; Yenigul, Elcin; Sommerdijk, Nico A. J. M.; Bucak, Seyda

    2016-10-01

    Octahedral, single domain magnetite nanoparticles with average size of ~55 nm were synthesized through oxidative aging of a ferrous hydroxide (Fe(OH)2) precursor at high pH in water. The synthesis was also carried out in the presence of the hydrophilic polymer poly(acrylic acid). Presence of the polymer changed the particle morphology from octahedral to spherical while average size decreased to 40-50 nm. Although these particles have a tendency to precipitate due to their high magnetic moment, dispersions of these particles were obtained in the presence of this particular polymer which made the particles stable in water for several days making them suitable for various biotechnological applications such as cell separation owing to their low toxicity.

  12. A C-terminal acidic domain regulates degradation of the transcriptional coactivator Bob1.

    PubMed

    Lindner, John M; Wong, Christina S F; Möller, Andreas; Nielsen, Peter J

    2013-12-01

    Bob1 (Obf-1 or OCA-B) is a 34-kDa transcriptional coactivator encoded by the Pou2af1 gene that is essential for normal B-cell development and immune responses in mice. During lymphocyte activation, Bob1 protein levels dramatically increase independently of mRNA levels, suggesting that the stability of Bob1 is regulated. We used a fluorescent protein-based reporter system to analyze protein stability in response to genetic and physiological perturbations and show that, while Bob1 degradation is proteasome mediated, it does not require ubiquitination of Bob1. Furthermore, degradation of Bob1 in B cells appears to be largely independent of the E3 ubiquitin ligase Siah. We propose a novel mechanism of Bob1 turnover in B cells, whereby an acidic region in the C terminus of Bob1 regulates the activity of degron signals elsewhere in the protein. Changes that make the C terminus more acidic, including tyrosine phosphorylation-mimetic mutations, stabilize the instable murine Bob1 protein, indicating that B cells may regulate Bob1 stability and activity via signaling pathways. Finally, we show that expressing a stable Bob1 mutant in B cells suppresses cell proliferation and induces changes in surface marker expression commonly seen during B-cell differentiation.

  13. Disentangling the perturbational effects of amino acid substitutions in the DNA-binding domain of p53.

    PubMed

    Wacey, A I; Cooper, D N; Liney, D; Hovig, E; Krawczak, M

    1999-01-01

    The spectrum of somatic cancer-associated missense mutations in the human TP53 gene was studied in order to assess the potential structural and functional importance of various intra-molecular properties associated with these substitutions. Relating the observed frequency of particular amino acid substitutions in the p53 DNA-binding domain to their expected frequency, as calculated from DNA sequence-dependent mutation rates, yielded estimates of their relative clinical observation likelihood (RCOL). Several biophysical properties were found to display significant covariation with RCOL values. Thus RCOL values were observed to decrease with increasing solvent accessibility of the substituted residue and with increasing distance from the p53 DNA-binding and Zn2+ -binding sites. The number of adverse steric interactions introduced by an amino acid replacement was found to be positively correlated with its RCOL value, irrespective of the magnitude of the interactions. A gain in hydrogen bond number was found to be only half as likely to come to clinical attention as mutations involving either a reduction or no change in hydrogen bond number. When the difference in potential energy between the wild-type and mutant DNA-binding domains was considered, RCOL values exhibited a minimum around changes of zero. Finally, classification of mutated residues in terms of their protein/solvent environment yielded, for somatic p53 mutations, RCOL values that resembled those previously determined for inherited mutations of human factor IX causing haemophilia B, suggesting that similar mechanisms may be responsible for the mutation-related perturbation of biological function in different protein folds.

  14. Docosahexaenoic acid promotes micron scale liquid-ordered domains. A comparison study of docosahexaenoic versus oleic acid containing phosphatidylcholine in raft-like mixtures.

    PubMed

    Georgieva, R; Chachaty, C; Hazarosova, R; Tessier, C; Nuss, P; Momchilova, A; Staneva, G

    2015-06-01

    The understanding of the functional role of the lipid diversity in biological membranes is a major challenge. Lipid models have been developed to address this issue by using lipid mixtures generating liquid-ordered (Lo)/liquid-disordered (Ld) immiscibility. The present study examined mixtures comprising Egg sphingomyelin (SM), cholesterol (chol) and phosphatidylcholine (PC) either containing docosahexaenoic (PDPC) or oleic acid (POPC). The mixtures were examined in terms of their capability to induce phase separation at the micron- and nano-scales. Fluorescence microscopy, electron spin resonance (ESR), X-ray diffraction (XRD) and calorimetry methods were used to analyze the lateral organization of the mixtures. Fluorescence microscopy of giant vesicles could show that the temperature of the micron-scale Lo/Ld miscibility is higher for PDPC than for POPC ternary mixtures. At 37°C, no micron-scale Lo/Ld phase separation could be identified in the POPC containing mixtures while it was evident for PDPC. In contrast, a phase separation was distinguished for both PC mixtures by ESR and XRD, indicative that PDPC and POPC mixtures differed in micron vs nano domain organization. Compared to POPC, the higher line tension of the Lo domains observed in PDPC mixtures is assumed to result from the higher difference in Lo/Ld order parameter rather than hydrophobic mismatch.

  15. Bacterial GRAS domain proteins throw new light on gibberellic acid response mechanisms

    PubMed Central

    Zhang, Dapeng; Iyer, Lakshminarayan M.; Aravind, L.

    2012-01-01

    Summary: Gibberellic acids (GAs) are key plant hormones, regulating various aspects of growth and development, which have been at the center of the ‘green revolution’. GRAS family proteins, the primary players in GA signaling pathways, remain poorly understood. Using sequence-profile searches, structural comparisons and phylogenetic analysis, we establish that the GRAS family first emerged in bacteria and belongs to the Rossmann fold methyltransferase superfamily. All bacterial and a subset of plant GRAS proteins are likely to function as small-molecule methylases. The remaining plant versions have lost one or more AdoMet (SAM)-binding residues while preserving their substrate-binding residues. We predict that GRAS proteins might either modify or bind small molecules such as GAs or their derivatives. Contact: aravind@ncbi.nlm.nih.gov Supplementary Information: Supplementary Material for this article is available at Bioinformatics online. PMID:22829623

  16. Human genes for complement components C1r and C1s in a close tail-to-tail arrangement.

    PubMed Central

    Kusumoto, H; Hirosawa, S; Salier, J P; Hagen, F S; Kurachi, K

    1988-01-01

    Complementary DNA clones for human C1s were isolated from cDNA libraries that were prepared with poly(A)+ RNAs of human liver and HepG2 cells. A clone with the largest cDNA insert of 2664 base pairs (bp) was analyzed for its complete nucleotide sequence. It contained 202 bp of a 5' untranslated region, 45 bp of coding for a signal peptide (15 amino acid residues), 2019 bp for complement component C1s zymogen (673 amino acid residues), 378 bp for a 3' untranslated region, a stop codon, and 17 bp of a poly(A) tail. The amino acid sequence of C1s was 40.5% identical to that of C1r, with excellent matches of tentative disulfide bond locations conserving the overall domain structure of C1r. DNA blotting and sequencing analyses of genomic DNA and of an isolated genomic DNA clone clearly showed that the human genes for C1r and C1s are closely located in a "tail-to-tail" arrangement at a distance of about 9.5 kilobases. Furthermore, RNA blot analyses showed that both C1r and C1s genes are primarily expressed in liver, whereas most other tissues expressed both C1r and C1s genes at much lower levels (less than 10% of that in liver). Multiple molecular sizes of specific mRNAs were observed in the RNA blot analyses for both C1r and C1s, indicating that alternative RNA processing(s), likely an alternative polyadenylylation, might take place for both genes. Images PMID:2459702

  17. Low-frequency vibrational modes of benzoic acid investigated by terahertz time-domain spectroscopy and theoretical simulations

    NASA Astrophysics Data System (ADS)

    Yan, Hui; Fan, Wen-hui; Zheng, Zhuan-ping

    2011-08-01

    In this paper, the low-frequency vibrational modes of crystalline benzoic acid (BA) have been investigated by terahertz time-domain spectroscopy (THz-TDS) and theoretical simulations based on the linearity combination of atomic orbital within the Density Functional Theory (DFT) as well as ab initio molecular orbital method at second-order Moller-Plesset Perturbation Theory (MP2) level for single molecule and dimer. Experimentally, a series of prominent absorption features of pure benzoic acid relevant to intra- and inter-molecular vibrational modes have been obtained below 4 THz at room temperature. For the theoretical simulations, geometry-optimization results of bond lengths and dihedral angles in both BA monomer and dimer are very close to experimental neutron diffraction measurements. Furthermore, the simulation results demonstrate absorption profile centered at 1.89 THz contains low-frequency modes of Ph-COOH twisting due to intramolecular motion and cogwheel owing to intermolecular motion. All the intra- and inter-molecular vibrational modes measured have also been assigned.

  18. Clean tailing reclamation: Tailing reprocessing for sulfide removal and vegetation establishment

    SciTech Connect

    Jennings, S.R.; Kruegar, J.

    1997-12-31

    Mine wastes exhibiting elevated heavy metal concentrations are widespread causes of resource degradation in the western US and elsewhere. This problem is further exacerbated by the presence of pyrite that oxidizes upon exposure to the atmosphere resulting in acid generation. Since pyrite was not recovered as a mineral of economic value during mining, it was disposed of in waste piles and tailing ponds that are now a source of acid generation and release of metals to the environment. Tailing cleaning, or sulfide mineral recovery through reprocessing, was evaluated as an innovative reclamation technology. Tailing materials, from both operational and abandoned mines, were collected to evaluate the feasibility of sulfide mineral recovery. Successful mineral separation was performed resulting in a low volume metal sulfide concentrate and a high volume cleaned silicate media. Total metal concentrations were decreased in the cleaned tailing material and elevated in the sulfide concentrate compared with the original tailing chemistry. In greenhouse trials, vegetation establishment in cleaned tailing material was compared with plant growth in topsoil and lime-amended tailings. While vegetation performance was best in the topsoil control, both lime-amended and cleaned tailings displayed adequate plant growth.

  19. A single amino acid substitution within the transmembrane domain of the human immunodeficiency virus type 1 Vpu protein renders simian-human immunodeficiency virus (SHIV{sub KU-1bMC33}) susceptible to rimantadine

    SciTech Connect

    Hout, David R.; Gomez, Lisa M.; Pacyniak, Erik; Miller, Jean-Marie; Hill, M. Sarah; Stephens, Edward B. . E-mail: estephen@kumc.edu

    2006-05-10

    Previous studies from our laboratory have shown that the transmembrane domain (TM) of the Vpu protein of human immunodeficiency virus type 1 (HIV-1) contributes to the pathogenesis of SHIV{sub KU-1bMC33} in macaques and that the TM domain of Vpu could be replaced with the M2 protein viroporin from influenza A virus. Recently, we showed that the replacement of the TM domain of Vpu with that of the M2 protein of influenza A virus resulted in a virus (SHIV{sub M2}) that was sensitive to rimantadine [Hout, D.R., Gomez, M.L., Pacyniak, E., Gomez, L.M., Inbody, S.H., Mulcahy, E.R., Culley, N., Pinson, D.M., Powers, M.F., Wong, S.W., Stephens, E.B., 2006. Substitution of the transmembrane domain of Vpu in simian human immunodeficiency virus (SHIV{sub KU-1bMC33}) with that of M2 of influenza A results in a virus that is sensitive to inhibitors of the M2 ion channel and is pathogenic for pig-tailed macaques. Virology 344, 541-558]. Based on previous studies of the M2 protein which have shown that the His-X-X-X-Trp motif within the M2 is essential to the function of the M2 proton channel, we have constructed a novel SHIV in which the alanine at position 19 of the TM domain was replaced with a histidine residue resulting in the motif His-Ile-Leu-Val-Trp. The SHIV{sub VpuA19H} replicated with similar kinetics as the parental SHIV{sub KU-1bMC33} and pulse-chase analysis revealed that the processing of viral proteins was similar to SHIV{sub KU-1bMC33}. This SHIV{sub VpuA19H} virus was found to be more sensitive to the M2 ion channel blocker rimantadine than SHIV{sub M2}. Electron microscopic examination of SHIV{sub VpuA19H}-infected cells treated with rimantadine revealed an accumulation of viral particles at the cell surface and within intracellular vesicles, which was similar to that previously observed to SHIV{sub M2}-infected cells treated with rimantadine. These data indicate that the Vpu protein of HIV-1 can be converted into a rimantadine-sensitive ion channel with the

  20. Identification of Domains and Amino Acids Essential to the Collagen Galactosyltransferase Activity of GLT25D1

    PubMed Central

    Perrin-Tricaud, Claire; Rutschmann, Christoph; Hennet, Thierry

    2011-01-01

    Collagen is modified by hydroxylation and glycosylation of hydroxylysine residues. This glycosylation is initiated by the β1,O galactosyltransferases GLT25D1 and GLT25D2. The structurally similar protein cerebral endothelial cell adhesion molecule CEECAM1 was previously reported to be inactive when assayed for collagen glycosyltransferase activity. To address the cause of the absent galactosyltransferase activity, we have generated several chimeric constructs between the active human GLT25D1 and inactive human CEECAM1 proteins. The assay of these chimeric constructs pointed to a short central region and a large C-terminal region of CEECAM1 leading to the loss of collagen galactosyltransferase activity. Examination of the three DXD motifs of the active GLT25D1 by site-directed mutagenesis confirmed the importance of the first (amino acids 166–168) and second motif (amino acids 461–463) for enzymatic activity, whereas the third one was dispensable. Since the second DXD motif is incomplete in CEECAM1, we have restored the motif by introducing the substitution S461D. This change did not restore the activity of the C-terminal region, thereby showing that additional amino acids were required in this C-terminal region to confer enzymatic activity. Finally, we have introduced the substitution Q471R-V472M-N473Q-P474V in the CEECAM1-C-terminal construct, which is found in most animal GLT25D1 and GLT25D2 isoforms but not in CEECAM1. This substitution was shown to partially restore collagen galactosyltransferase activity, underlining its importance for catalytic activity in the C-terminal domain. Because multiple mutations in different regions of CEECAM1 contribute to the lack of galactosyltransferase activity, we deduced that CEECAM1 is functionally different from the related GLT25D1 protein. PMID:22216269

  1. Yeast peroxisomal multifunctional enzyme: (3R)-hydroxyacyl-CoA dehydrogenase domains A and B are required for optimal growth on oleic acid.

    PubMed

    Qin, Y M; Marttila, M S; Haapalainen, A M; Siivari, K M; Glumoff, T; Hiltunen, J K

    1999-10-01

    The yeast peroxisomal (3R)-hydroxyacyl-CoA dehydrogenase/2-enoyl-CoA hydratase 2 (multifunctional enzyme type 2; MFE-2) has two N-terminal domains belonging to the short chain alcohol dehydrogenase/reductase superfamily. To investigate the physiological roles of these domains, here called A and B, Saccharomyces cerevisiae fox-2 cells (devoid of Sc MFE-2) were taken as a model system. Gly(16) and Gly(329) of the S. cerevisiae A and B domains, corresponding to Gly(16), which is mutated in the human MFE-2 deficiency, were mutated to serine and cloned into the yeast expression plasmid pYE352. In oleic acid medium, fox-2 cells transformed with pYE352:: ScMFE-2(aDelta) and pYE352::ScMFE-2(bDelta) grew slower than cells transformed with pYE352::ScMFE-2, whereas cells transformed with pYE352::ScMFE-2(aDeltabDelta) failed to grow. Candida tropicalis MFE-2 with a deleted hydratase 2 domain (Ct MFE- 2(h2Delta)) and mutational variants of the A and B domains (Ct MFE- 2(h2DeltaaDelta), Ct MFE- 2(h2DeltabDelta), and Ct MFE- 2(h2DeltaaDeltabDelta)) were overexpressed and characterized. All proteins were dimers with similar secondary structure elements. Both wild type domains were enzymatically active, with the B domain showing the highest activity with short chain and the A domain with medium and long chain (3R)-hydroxyacyl-CoA substrates. The data show that the dehydrogenase domains of yeast MFE-2 have different substrate specificities required to allow the yeast to propagate optimally on fatty acids as the carbon source.

  2. C-terminal Domains of N-Methyl-d-aspartic Acid Receptor Modulate Unitary Channel Conductance and Gating*

    PubMed Central

    Maki, Bruce A.; Aman, Teresa K.; Amico-Ruvio, Stacy A.; Kussius, Cassandra L.; Popescu, Gabriela K.

    2012-01-01

    NMDA receptors (NRs) are glutamate-gated calcium-permeable channels that are essential for normal synaptic transmssion and contribute to neurodegeneration. Tetrameric proteins consist of two obligatory GluN1 (N1) and two GluN2 (N2) subunits, of which GluN2A (2A) and GluN2B (2B) are prevalent in adult brain. The intracellularly located C-terminal domains (CTDs) make a significant portion of mass of the receptors and are essential for plasticity and excitotoxicity, but their functions are incompletely defined. Recent evidence shows that truncation of the N2 CTD alters channel kinetics; however, the mechanism by which this occurs is unclear. Here we recorded activity from individual NRs lacking the CTDs of N1, 2A, or 2B and determined the gating mechanisms of these receptors. Receptors lacking the N1 CTDs had larger unitary conductance and faster deactivation kinetics, receptors lacking the 2A or 2B CTDs had longer openings and longer desensitized intervals, and the first 100 amino acids of the N2 CTD were essential for these changes. In addition, receptors lacking the CTDs of either 2A or 2B maintained isoform-specific kinetic differences and swapping CTDs between 2A and 2B had no effect on single-channel properties. Based on these results, we suggest that perturbations in the CTD can modify the NR-mediated signal in a subunit-dependent manner, in 2A these effects are most likely mediated by membrane-proximal residues, and the isoform-specific biophysical properties conferred by 2A and 2B are CTD-independent. The kinetic mechanisms we developed afford a quantitative approach to understanding how the intracellular domains of NR subunits can modulate the responses of the receptor. PMID:22948148

  3. Histidine-41 of the cytochrome b5 domain of the borage delta6 fatty acid desaturase is essential for enzyme activity.

    PubMed

    Sayanova, O; Shewry, P R; Napier, J A

    1999-10-01

    Unlike most other plant microsomal desaturases, the Delta6-fatty acid desaturase from borage (Borago officinalis) contains an N-terminal extension that shows homology to the small hemoprotein cytochrome (Cyt) b5. To determine if this domain serves as a functional electron donor for the Delta6-fatty acid desaturase, mutagenesis and functional analysis by expression in transgenic Arabidopsis was carried out. Although expression of the wild-type borage Delta6-fatty acid desaturase resulted in the synthesis and accumulation of Delta6-unsaturated fatty acids, this was not observed in plants transformed with N-terminally deleted forms of the desaturase. Site-directed mutagenesis was used to disrupt one of the axial heme-binding residues (histidine-41) of the Cyt b5 domain; expression of this mutant form of the Delta6-desaturase in transgenic plants failed to produce Delta6-unsaturated fatty acids. These data indicate that the Cyt b5 domain of the borage Delta6-fatty acid desaturase is essential for enzymatic activity.

  4. Histidine-41 of the Cytochrome b5 Domain of the Borage Δ6 Fatty Acid Desaturase Is Essential for Enzyme Activity1

    PubMed Central

    Sayanova, Olga; Shewry, Peter R.; Napier, Johnathan A.

    1999-01-01

    Unlike most other plant microsomal desaturases, the Δ6-fatty acid desaturase from borage (Borago officinalis) contains an N-terminal extension that shows homology to the small hemoprotein cytochrome (Cyt) b5. To determine if this domain serves as a functional electron donor for the Δ6-fatty acid desaturase, mutagenesis and functional analysis by expression in transgenic Arabidopsis was carried out. Although expression of the wild-type borage Δ6-fatty acid desaturase resulted in the synthesis and accumulation of Δ6-unsaturated fatty acids, this was not observed in plants transformed with N-terminally deleted forms of the desaturase. Site-directed mutagenesis was used to disrupt one of the axial heme-binding residues (histidine-41) of the Cyt b5 domain; expression of this mutant form of the Δ6-desaturase in transgenic plants failed to produce Δ6-unsaturated fatty acids. These data indicate that the Cyt b5 domain of the borage Δ6-fatty acid desaturase is essential for enzymatic activity. PMID:10517856

  5. The Translocation Domain of Botulinum Neurotoxin A Moderates the Propensity of the Catalytic Domain to Interact with Membranes at Acidic pH

    PubMed Central

    Araye, Anne; Goudet, Amélie; Barbier, Julien; Pichard, Sylvain; Baron, Bruno; England, Patrick; Pérez, Javier; Zinn-Justin, Sophie; Chenal, Alexandre; Gillet, Daniel

    2016-01-01

    Botulinum neurotoxin A (BoNT/A) is composed of three domains: a catalytic domain (LC), a translocation domain (HN) and a receptor-binding domain (HC). Like most bacterial toxins BoNT/A is an amphitropic protein, produced in a soluble form that is able to interact, penetrate and/or cross a membrane to achieve its toxic function. During intoxication BoNT/A is internalized by the cell by receptor-mediated endocytosis. Then, LC crosses the membrane of the endocytic compartment and reaches the cytosol. This translocation is initiated by the low pH found in this compartment. It has been suggested that LC passes in an unfolded state through a transmembrane passage formed by HN. We report here that acidification induces no major conformational change in either secondary or tertiary structures of LC and HN of BoNT/A in solution. GdnHCl-induced denaturation experiments showed that the stability of LC and HN increases as pH drops, and that HN further stabilizes LC. Unexpectedly we found that LC has a high propensity to interact with and permeabilize anionic lipid bilayers upon acidification without the help of HN. This property is downplayed when LC is linked to HN. HN thus acts as a chaperone for LC by enhancing its stability but also as a moderator of the membrane interaction of LC. PMID:27070312

  6. DEFECTS IN CERVICAL VERTEBRAE IN BORIC ACID-EXPOSED RAT EMBRYOS ARE ASSOCIATED WITH ANTERIOR SHIFTS OF HOX GENE EXPRESSION DOMAINS

    EPA Science Inventory

    Defects in cervical vertebrae in boric acid-exposed rat embryos are associated with anterior shifts of hox gene expression domains

    Nathalie Wery,1 Michael G. Narotsky,2 Nathalie Pacico,1 Robert J. Kavlock,2 Jacques J. Picard,1 AND Francoise Gofflot,1*
    1Unit of Developme...

  7. The formation of right-handed and left-handed chiral nanopores within a single domain during amino acid self-assembly on Au(111).

    PubMed

    Yang, Sena; Jeon, Aram; Driver, Russell W; Kim, Yeonwoo; Jeon, Eun Hee; Kim, Sehun; Lee, Hee-Seung; Lee, Hangil

    2016-05-25

    We report the formation of both right- and left-handed chiral nanopores within a single domain during the self-assembly of an amino acid derivative on an inert Au(111) surface using STM. DFT calculations employed to rationalize this unusual result identified that intermolecular interactions between chiral, windmill-shaped tetramers are crucial for self-assembly.

  8. Incorporating significant amino acid pairs and protein domains to predict RNA splicing-related proteins with functional roles.

    PubMed

    Hsu, Justin Bo-Kai; Huang, Kai-Yao; Weng, Tzu-Ya; Huang, Chien-Hsun; Lee, Tzong-Yi

    2014-01-01

    Machinery of pre-mRNA splicing is carried out through the interaction of RNA sequence elements and a variety of RNA splicing-related proteins (SRPs) (e.g. spliceosome and splicing factors). Alternative splicing, which is an important post-transcriptional regulation in eukaryotes, gives rise to multiple mature mRNA isoforms, which encodes proteins with functional diversities. However, the regulation of RNA splicing is not yet fully elucidated, partly because SRPs have not yet been exhaustively identified and the experimental identification is labor-intensive. Therefore, we are motivated to design a new method for identifying SRPs with their functional roles in the regulation of RNA splicing. The experimentally verified SRPs were manually curated from research articles. According to the functional annotation of Splicing Related Gene Database, the collected SRPs were further categorized into four functional groups including small nuclear Ribonucleoprotein, Splicing Factor, Splicing Regulation Factor and Novel Spliceosome Protein. The composition of amino acid pairs indicates that there are remarkable differences among four functional groups of SRPs. Then, support vector machines (SVMs) were utilized to learn the predictive models for identifying SRPs as well as their functional roles. The cross-validation evaluation presents that the SVM models trained with significant amino acid pairs and functional domains could provide a better predictive performance. In addition, the independent testing demonstrates that the proposed method could accurately identify SRPs in mammals/plants as well as effectively distinguish between SRPs and RNA-binding proteins. This investigation provides a practical means to identifying potential SRPs and a perspective for exploring the regulation of RNA splicing.

  9. Active and accurate trans-translation requires distinct determinants in the C-terminal tail of SmpB protein and the mRNA-like domain of transfer messenger RNA (tmRNA).

    PubMed

    Camenares, Devin; Dulebohn, Daniel P; Svetlanov, Anton; Karzai, A Wali

    2013-10-18

    Unproductive ribosome stalling in eubacteria is resolved by the actions of SmpB protein and transfer messenger (tm) RNA. We examined the functional significance of conserved regions of SmpB and tmRNA to the trans-translation process. Our investigations reveal that the N-terminal 20 residues of SmpB, which are located near the ribosomal decoding center, are dispensable for all known SmpB activities. In contrast, a set of conserved residues that reside at the junction between the tmRNA-binding core and the C-terminal tail of SmpB play an important role in tmRNA accommodation. Our data suggest that the highly conserved glycine 132 acts as a flexible hinge that enables movement of the C-terminal tail, thus permitting proper positioning and establishment of the tmRNA open reading frame (ORF) as the surrogate template. To gain further insights into the function of the SmpB C-terminal tail, we examined the tagging activity of hybrid variants of tmRNA and the SmpB protein, in which the tmRNA ORF or the SmpB C-terminal tail was substituted with the equivalent but highly divergent sequences from Francisella tularensis. We observed that the hybrid tmRNA was active but resulted in less accurate selection of the resume codon. Cognate hybrid SmpB was necessary to restore activity. Furthermore, accurate tagging was observed when the identity of the resume codon was reverted from GGC to GCA. Taken together, these data suggest that the engagement of the tmRNA ORF and the selection of the correct translation resumption point are distinct activities that are influenced by independent tmRNA and SmpB determinants.

  10. Salicylic Acid Based Small Molecule Inhibitor for the Oncogenic Src Homology-2 Domain Containing Protein Tyrosine Phosphatase-2 (SHP2)

    SciTech Connect

    Zhang, Xian; He, Yantao; Liu, Sijiu; Yu, Zhihong; Jiang, Zhong-Xing; Yang, Zhenyun; Dong, Yuanshu; Nabinger, Sarah C.; Wu, Li; Gunawan, Andrea M.; Wang, Lina; Chan, Rebecca J.; Zhang, Zhong-Yin

    2010-08-13

    The Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) plays a pivotal role in growth factor and cytokine signaling. Gain-of-function SHP2 mutations are associated with Noonan syndrome, various kinds of leukemias, and solid tumors. Thus, there is considerable interest in SHP2 as a potential target for anticancer and antileukemia therapy. We report a salicylic acid based combinatorial library approach aimed at binding both active site and unique nearby subpockets for enhanced affinity and selectivity. Screening of the library led to the identification of a SHP2 inhibitor II-B08 (compound 9) with highly efficacious cellular activity. Compound 9 blocks growth factor stimulated ERK1/2 activation and hematopoietic progenitor proliferation, providing supporting evidence that chemical inhibition of SHP2 may be therapeutically useful for anticancer and antileukemia treatment. X-ray crystallographic analysis of the structure of SHP2 in complex with 9 reveals molecular determinants that can be exploited for the acquisition of more potent and selective SHP2 inhibitors.

  11. Distribution of eastern equine encephalomyelitis viral protein and nucleic acid within central nervous tissue lesions in white-tailed deer (Odocoileus virginianus).

    PubMed

    Kiupel, M; Fitzgerald, S D; Pennick, K E; Cooley, T M; O'Brien, D J; Bolin, S R; Maes, R K; Del Piero, F

    2013-11-01

    An outbreak of eastern equine encephalomyelitis (EEE) occurred in Michigan free-ranging white-tailed deer (Odocoileus virginianus) during late summer and fall of 2005. Brain tissue from 7 deer with EEE, as confirmed by reverse transcriptase polymerase chain reaction, was studied. Detailed microscopic examination, indirect immunohistochemistry (IHC), and in situ hybridization (ISH) were used to characterize the lesions and distribution of the EEE virus within the brain. The main lesion in all 7 deer was a polioencephalomyelitis with leptomeningitis, which was more prominent within the cerebral cortex, thalamus, hypothalamus, and brainstem. In 3 deer, multifocal microhemorrhages surrounded smaller vessels with or without perivascular cuffing, although vasculitis was not observed. Neuronal necrosis, associated with perineuronal satellitosis and neutrophilic neuronophagia, was most prominent in the thalamus and the brainstem. Positive IHC labeling was mainly observed in the perikaryon, axons, and dendrites of necrotic and intact neurons and, to a much lesser degree, in glial cells, a few neutrophils in the thalamus and the brainstem, and occasionally the cerebral cortex of the 7 deer. There was minimal IHC-based labeling in the cerebellum and hippocampus. ISH labeling was exclusively observed in the cytoplasm of neurons, with a distribution similar to IHC-positive neurons. Neurons positive by IHC and ISH were most prominent in the thalamus and brainstem. The neuropathology of EEE in deer is compared with other species. Based on our findings, EEE has to be considered a differential diagnosis for neurologic disease and meningoencephalitis in white-tailed deer.

  12. Crystallization and preliminary X-ray diffraction analysis of the sialic acid-binding domain (VP8*) of porcine rotavirus strain CRW-8

    SciTech Connect

    Scott, Stacy A.; Holloway, Gavan; Coulson, Barbara S.; Szyczew, Alex J.; Kiefel, Milton J.; Itzstein, Mark von; Blanchard, Helen

    2005-06-01

    The sialic acid-binding domain (VP8*) component of the porcine CRW-8 rotavirus spike protein has been overexpressed in E. coli, purified and co-crystallized with an N-acetylneuraminic acid derivative. X-ray diffraction data have been collected to 2.3 Å, which has enabled determination of the structure by molecular replacement. Rotavirus recognition and attachment to host cells involves interaction with the spike protein VP4 that projects outwards from the surface of the virus particle. An integral component of these spikes is the VP8* domain, which is implicated in the direct recognition and binding of sialic acid-containing cell-surface carbohydrates and facilitates subsequent invasion by the virus. The expression, purification, crystallization and preliminary X-ray diffraction analysis of VP8* from porcine CRW-8 rotavirus is reported. Diffraction data have been collected to 2.3 Å resolution, enabling the determination of the VP8* structure by molecular replacement.

  13. Indanylacetic acid derivatives carrying 4-thiazolyl-phenoxy tail groups, a new class of potent PPAR alpha/gamma/delta pan agonists: synthesis, structure-activity relationship, and in vivo efficacy.

    PubMed

    Rudolph, Joachim; Chen, Libing; Majumdar, Dyuti; Bullock, William H; Burns, Michael; Claus, Thomas; Dela Cruz, Fernando E; Daly, Michelle; Ehrgott, Frederick J; Johnson, Jeffrey S; Livingston, James N; Schoenleber, Robert W; Shapiro, Jeffrey; Yang, Ling; Tsutsumi, Manami; Ma, Xin

    2007-03-01

    Compounds that simultaneously activate the three peroxisome proliferator-activated receptor (PPAR) subtypes alpha, gamma, and delta hold potential to address the adverse metabolic and cardiovascular conditions associated with diabetes and the metabolic syndrome. We recently identified the indanylacetic acid moiety as a well-tunable PPAR agonist head group. Here we report the synthesis and structure-activity relationship (SAR) studies of novel aryl tail group derivatives that led to a new class of potent PPAR pan agonists. While most of the tail group modifications imparted potent PPAR delta agonist activity, improvement of PPAR alpha and gamma activity required the introduction of new heterocyclic substituents that were not known in the PPAR literature. Systematic optimization led to the discovery of 4-thiazolyl-phenyl derivatives with potent PPAR alpha/gamma/delta pan agonistic activity. The lead candidate from this series was found to exhibit excellent ADME properties and superior therapeutic potential compared to known PPAR gamma activating agents by favorably modulating lipid levels in hApoA1 mice and hyperlipidemic hamsters, while normalizing glucose levels in diabetic rodent models. PMID:17274610

  14. [Effect of mutations and modifications of amino acid residues on zinc-induced interaction of the metal-binding domain of β-amyloid with DNA].

    PubMed

    Khmeleva, S A; Mezentsev, Y V; Kozin, S A; Mitkevich, V A; Medvedev, A E; Ivanov, A S; Bodoev, N V; Makarov, A A; Radko, S P

    2015-01-01

    Interaction of intranuclear β-amyloid with DNA is considered to be a plausible mechanism of Alzheimer's disease pathogenesis. The interaction of single- and double-stranded DNA with synthetic peptides was analyzed using surface plasmon resonance. The peptides represent the metal-binding domain of β-amyloid (amino acids 1-16) and its variants with chemical modifications and point substitutions of amino acid residues which are associated with enhanced neurotoxicity of β-amyloid in cell tests. It has been shown that the presence of zinc ions is necessary for the interaction of the peptides with DNA in solution. H6R substitution has remarkably reduced the ability of domain 1-16 to bind DNA. This is in accordance with the supposition that the coordination of a zinc ion by amino acid residues His6, Glu11, His13, and His14 of the β-amyloid metal-binding domain results in the occurrence of an anion-binding site responsible for the interaction of the domain with DNA. Zinc-induced dimerization and oligomerization of domain 1-16 associated with phosphorylation of Ser8 and the presence of unblocked amino- and carboxy-terminal groups have resulted in a decrease of peptide concentrations required for detection of the peptide-DNA interaction. The presence of multiple anion-binding sites on the dimers and oligomers is responsible for the enhancement of the peptide-DNA interaction. A substitution of the negatively charged residue Asp7 for the neutral residue Asn in close proximity to the anion-binding site of the domain 1-16 of Aβ facilitates the electrostatic interaction between this site and phosphates of a polynucleotide chain, which enhances zinc-induced binding to DNA.

  15. Serum albumin complexation of acetylsalicylic acid metabolites.

    PubMed

    Jurkowski, Wiktor; Porebski, Grzegorz; Obtułowicz, Krystyna; Roterman, Irena

    2009-06-01

    One possible origin of the type I hypersensitivity reaction is reaction of drugs such as acetylsalicylic acid and its metabolites being complexed with human serum albumin. Albumin, being transporting molecule abundant in blood plasma is able to bind large array of ligands varying from small single carbon particles to long hydrophobic tailed lipidic acids (e.g. myristic acid). This non specificity is possible because of multi domain scaffold and large flexibility of inter-domain loops, which results in serious reorientation of domains. Hypothesis that acetylsalicylic acid metabolites may play indirect role in activation of allergic reaction has been tested. Binding of acetylsalicylic acid metabolites in intra-domain space causes significant increase of liability of domains IIIA and IIIB. One of metabolites, salicyluric acid, once is bound causes distortion and partial unfolding of helices in domains IA, IIB and IIIB. Changed are both directions and amplitude of relative motions as well as intra-domain distances. In result albumin is able to cross-link of adjacent IgE receptors which subsequently starts allergic reaction.

  16. Serum albumin complexation of acetylsalicylic acid metabolites.

    PubMed

    Jurkowski, Wiktor; Porebski, Grzegorz; Obtułowicz, Krystyna; Roterman, Irena

    2009-06-01

    One possible origin of the type I hypersensitivity reaction is reaction of drugs such as acetylsalicylic acid and its metabolites being complexed with human serum albumin. Albumin, being transporting molecule abundant in blood plasma is able to bind large array of ligands varying from small single carbon particles to long hydrophobic tailed lipidic acids (e.g. myristic acid). This non specificity is possible because of multi domain scaffold and large flexibility of inter-domain loops, which results in serious reorientation of domains. Hypothesis that acetylsalicylic acid metabolites may play indirect role in activation of allergic reaction has been tested. Binding of acetylsalicylic acid metabolites in intra-domain space causes significant increase of liability of domains IIIA and IIIB. One of metabolites, salicyluric acid, once is bound causes distortion and partial unfolding of helices in domains IA, IIB and IIIB. Changed are both directions and amplitude of relative motions as well as intra-domain distances. In result albumin is able to cross-link of adjacent IgE receptors which subsequently starts allergic reaction. PMID:19689242

  17. Five glutamic acid residues in the C-terminal domain of the ChlD subunit play a major role in conferring Mg(2+) cooperativity upon magnesium chelatase.

    PubMed

    Brindley, Amanda A; Adams, Nathan B P; Hunter, C Neil; Reid, James D

    2015-11-10

    Magnesium chelatase catalyzes the first committed step in chlorophyll biosynthesis by inserting a Mg(2+) ion into protoporphyrin IX in an ATP-dependent manner. The cyanobacterial (Synechocystis) and higher-plant chelatases exhibit a complex cooperative response to free magnesium, while the chelatases from Thermosynechococcus elongatus and photosynthetic bacteria do not. To investigate the basis for this cooperativity, we constructed a series of chimeric ChlD proteins using N-terminal, central, and C-terminal domains from Synechocystis and Thermosynechococcus. We show that five glutamic acid residues in the C-terminal domain play a major role in this process.

  18. The role of formin tails in actin nucleation, processive elongation, and filament bundling.

    PubMed

    Vizcarra, Christina L; Bor, Batbileg; Quinlan, Margot E

    2014-10-31

    Formins are multidomain proteins that assemble actin in a wide variety of biological processes. They both nucleate and remain processively associated with growing filaments, in some cases accelerating filament growth. The well conserved formin homology 1 and 2 domains were originally thought to be solely responsible for these activities. Recently a role in nucleation was identified for the Diaphanous autoinhibitory domain (DAD), which is C-terminal to the formin homology 2 domain. The C-terminal tail of the Drosophila formin Cappuccino (Capu) is conserved among FMN formins but distinct from other formins. It does not have a DAD domain. Nevertheless, we find that Capu-tail plays a role in filament nucleation similar to that described for mDia1 and other formins. Building on this, replacement of Capu-tail with DADs from other formins tunes nucleation activity. Capu-tail has low-affinity interactions with both actin monomers and filaments. Removal of the tail reduces actin filament binding and bundling. Furthermore, when the tail is removed, we find that processivity is compromised. Despite decreased processivity, the elongation rate of filaments is unchanged. Again, replacement of Capu-tail with DADs from other formins tunes the processive association with the barbed end, indicating that this is a general role for formin tails. Our data show a role for the Capu-tail domain in assembling the actin cytoskeleton, largely mediated by electrostatic interactions. Because of its multifunctionality, the formin tail is a candidate for regulation by other proteins during cytoskeletal rearrangements.

  19. The Tail of BPM

    NASA Astrophysics Data System (ADS)

    Kruba, Steve; Meyer, Jim

    Business process management suites (BPMS's) represent one of the fastest growing segments in the software industry as organizations automate their key business processes. As this market matures, it is interesting to compare it to Chris Anderson's 'Long Tail.' Although the 2004 "Long Tail" article in Wired magazine was primarily about the media and entertainment industries, it has since been applied (and perhaps misapplied) to other markets. Analysts describe a "Tail of BPM" market that is, perhaps, several times larger than the traditional BPMS product market. This paper will draw comparisons between the concepts in Anderson's article (and subsequent book) and the BPM solutions market.

  20. Jupiter's magnetic tail

    NASA Technical Reports Server (NTRS)

    Ness, N. F.; Acuna, M. H.; Lepping, R. P.; Behannon, K. W.; Burlaga, L. F.; Neubauer, F. M.

    1979-01-01

    Voyager 1 observations of the Jovian magnetosphere are discussed which are most naturally interpreted in terms of a well-developed magnetic tail on the nightside of the planet. It is shown that this tail, with a 'neutral sheet' separating the upper and lower lobes of opposite field polarity, is formed and controlled by external forces associated with the solar wind. The inner magnetosphere's current tail is found to merge with the magnetotail's neutral sheet. It is concluded that this configuration leads to a strong local-time control of the outer Jovian magnetosphere rather than planetary control.

  1. Domain structures and molecular evolution of class I and class II major histocompatibility gene complex (MHC) products deduced from amino acid and nucleotide sequence homologies

    NASA Astrophysics Data System (ADS)

    Ohnishi, Koji

    1984-12-01

    Domain structures of class I and class II MHC products were analyzed from a viewpoint of amino acid and nucleotide sequence homologies. Alignment statistics revealed that class I (transplantation) antigen H chains consist of four mutually homologous domains, and that class II (HLA-DR) antigen β and α chains are both composed of three mutually homologous ones. The N-terminal three and two domains of class I and class II (both β and α) gene products, respectively, all of which being ˜90 residues long, were concluded to be homologous to β2-microglobulin (β2M). The membraneembedded C-terminal shorter domains of these MHC products were also found to be homologous to one another and to the third domain of class I H chains. Class I H chains were found to be more closely related to class II α chains than to class II β chains. Based on these findings, an exon duplication history from a common ancestral gene encoding a β2M-like primodial protein of one-domain-length up to the contemporary MHC products was proposed.

  2. Perlecan domain I-conjugated, hyaluronic acid-based hydrogel particles for enhanced chondrogenic differentiation via BMP-2 release

    PubMed Central

    Jha, Amit K.; Yang, Weidong; Kirn-Safran, Catherine B.; Farach-Carson, Mary C.; Jia, Xinqiao

    2009-01-01

    We have developed a biomimetic growth factor delivery system that effectively stimulates the chondrogenic differentiation of the cultured mesenchymal stem cells via the controlled presentation of bone morphogenetic protein 2 (BMP-2). Hyaluronic acid (HA)-based, microscopic hydrogel particles (HGPs) with inherent nanopores and defined functional groups were synthesized by an inverse emulsion polymerization technique. Recombinantly produced, heparan sulfate (HS)-bearing perlecan domain I (PlnDI) was covalently immobilized to HA HGPs (HGP-P1) via a flexible poly(ethylene glycol) (PEG) linker through the lysine amines in the core protein of PlnDI employing reductive amination. Compared to HGP without PlnDI, HGP-P1 exhibited significantly (p<0.05) higher BMP-2 binding capacity and distinctly different BMP-2 release kinetics. Heparitinase treatment increased the amount of BMP-2 released from HGP-P1, confirming the HS-dependent BMP-2 binding. While BMP-2 was released from HGPs with a distinct burst release followed by a minimal cumulative release, its release from HGP-P1 exhibited a minimal burst release followed by linear release kinetics over 15 days. The bioactivity of the hydrogel particles was evaluated using micromass culture of multipotent mesenchymal stem cells (MSCs), and the chondrogenic differentiation was assessed by the production of glycosaminoglycan, aggrecan and collagen type II. Our results revealed that BMP-2 loaded HGP-P1 stimulates more robust cartilage specific ECM production as compared to BMP-2 loaded HGP, due to the ability of HGP-P1 to potentiate BMP-2 and modulate its release with a near zero-order release kinetics. The PlnDI conjugated, HA HGPs provide an improved BMP-2 delivery system for stimulating chondrogenic differentiation in vitro, with potential therapeutic application for cartilage repair and regeneration. PMID:19775743

  3. Uranium mill tailings neutralization: contaminant complexation and tailings leaching studies

    SciTech Connect

    Opitz, B.E.; Dodson, M.E.; Serne, R.J.

    1985-05-01

    Laboratory experiments were performed to compare the effectiveness of limestone (CaCO/sub 3/) and hydrated lime (Ca(OH)/sub 2/) for improving waste water quality through the neutralization of acidic uranium mill tailings liquor. The experiments were designed to also assess the effects of three proposed mechanisms - carbonate complexation, elevated pH, and colloidal particle adsorption - on the solubility of toxic contaminants found in a typical uranium mill waste solution. Of special interest were the effects each of these possible mechanisms had on the solution concentrations of trace metals such as Cd, Co, Mo, Zn, and U after neutralization. Results indicated that the neutralization of acidic tailings to a pH of 7.3 using hydrated lime provided the highest overall waste water quality. Both the presence of a carbonate source or elevating solution pH beyond pH = 7.3 resulted in a lowering of previously achieved water quality, while adsorption of contaminants onto colloidal particles was not found to affect the solution concentration of any constituent investigated. 24 refs., 8 figs., 19 tabs.

  4. Identification of a novel site in the tail of dynein heavy chain important for dynein function in vivo.

    PubMed

    Qiu, Rongde; Zhang, Jun; Xiang, Xin

    2013-01-25

    The minus end-directed microtubule motor cytoplasmic dynein is responsible for the intracellular movements of many organelles, including nuclei and endosomes. The dynein heavy chain contains a C-terminal motor domain and an N-terminal tail domain. The tail binds other dynein subunits and the cargo-interacting dynactin complex but is dispensable for movement of single dynein molecules in vitro. Here, we identified a mutation in the Aspergillus nidulans heavy chain tail domain, nudA(F208V), which causes obvious defects in dynein-mediated nuclear positioning and early endosome movement. Astonishingly, the nudA(F208I) mutation in the same position does not cause the same defects, suggesting that a subtle difference in the size of the amino acid side chain at this position has a significant consequence. Importantly, our biochemical analyses indicate that the nudA(F208V) mutation does not affect dynein subunit interactions and the mutant dynein is also able to bind dynactin and another dynein regulator, NUDF/LIS1. The mutant dynein is able to physically interact with the early endosome cargo, but dynein-mediated early endosome movement away from the hyphal tip occurs at a significantly reduced frequency. Within the small group of early endosomes that move away from the hyphal tip in the mutant, the average speed of movement is lower than that in the wild type. Given the dispensability of the dynein tail in dynein motility in vitro, our results support the notion that the structural integrity of the dynein tail is critical in vivo for the coordination of dynein force production and movement when the motor is heavily loaded.

  5. The Tail Suspension Test

    PubMed Central

    Terrillion, Chantelle E.; Piantadosi, Sean C.; Bhat, Shambhu; Gould, Todd D.

    2012-01-01

    The tail-suspension test is a mouse behavioral test useful in the screening of potential antidepressant drugs, and assessing of other manipulations that are expected to affect depression related behaviors. Mice are suspended by their tails with tape, in such a position that it cannot escape or hold on to nearby surfaces. During this test, typically six minutes in duration, the resulting escape oriented behaviors are quantified. The tail-suspension test is a valuable tool in drug discovery for high-throughput screening of prospective antidepressant compounds. Here, we describe the details required for implementation of this test with additional emphasis on potential problems that may occur and how to avoid them. We also offer a solution to the tail climbing behavior, a common problem that renders this test useless in some mouse strains, such as the widely used C57BL/6. Specifically, we prevent tail climbing behaviors by passing mouse tails through a small plastic cylinder prior to suspension. Finally, we detail how to manually score the behaviors that are manifested in this test. PMID:22315011

  6. In vivo biotinylation and incorporation of a photo-inducible unnatural amino acid to an antibody-binding domain improve site-specific labeling of antibodies.

    PubMed

    Kanje, Sara; Hober, Sophia

    2015-04-01

    Antibodies are important molecules in many research fields, where they play a key role in various assays. Antibody labeling is therefore of great importance. Currently, most labeling techniques take advantage of certain amino acid side chains that commonly appear throughout proteins. This makes it hard to control the position and exact degree of labeling of each antibody. Hence, labeling of the antibody may affect the antibody-binding site. This paper presents a novel protein domain based on the IgG-binding domain C2 of streptococcal protein G, containing the unnatural amino acid BPA, that can cross-link other molecules. This novel domain can, with improved efficiency compared to previously reported similar domains, site-specifically cross-link to IgG at the Fc region. An efficient method for simultaneous in vivo incorporation of BPA and specific biotinylation in a flask cultivation of Escherichia coli is described. In comparison to a traditionally labeled antibody sample, the C2-labeled counterpart proved to have a higher proportion of functional antibodies when immobilized on a solid surface and the same limit of detection in an ELISA. This method of labeling is, due to its efficiency and simplicity, of high interest for all antibody-based assays where it is important that labeling does not interfere with the antibody-binding site.

  7. Structure and function of the PWI motif: a novel nucleic acid-binding domain that facilitates pre-mRNA processing

    PubMed Central

    Szymczyna, Blair R.; Bowman, John; McCracken, Susan; Pineda-Lucena, Antonio; Lu, Ying; Cox, Brian; Lambermon, Mark; Graveley, Brenton R.; Arrowsmith, Cheryl H.; Blencowe, Benjamin J.

    2003-01-01

    The PWI motif is a highly conserved domain of unknown function in the SRm160 splicing and 3′-end cleavage-stimulatory factor, as well as in several other known or putative pre-mRNA processing components. We show here that the PWI motif is a new type of RNA/DNA-binding domain that has an equal preference for single- and double-stranded nucleic acids. Deletion of the motif prevents SRm160 from binding RNA and stimulating 3′-end cleavage, and its substitution with a heterologous RNA-binding domain restores these functions. The NMR solution structure of the SRm160-PWI motif reveals a novel, four-helix bundle and represents the first example of an α-helical fold that can bind single-stranded (ss)RNA. Structure-guided mutagenesis indicates that the same surface is involved in RNA and DNA binding and requires the cooperative action of a highly conserved, adjacent basic region. Thus, the PWI motif is a novel type of nucleic acid-binding domain that likely has multiple important functions in pre-mRNA processing, including SRm160-dependent stimulation of 3′-end formation. PMID:12600940

  8. The sea urchin mitochondrial transcription factor A binds and bends DNA efficiently despite its unusually short C-terminal tail.

    PubMed

    Malarkey, Christopher S; Lionetti, Claudia; Deceglie, Stefania; Roberti, Marina; Churchill, Mair E A; Cantatore, Palmiro; Loguercio Polosa, Paola

    2016-07-01

    Mitochondrial transcription factor A (TFAM) is a key component for the protection and transcription of the mitochondrial genome. TFAM belongs to the high mobility group (HMG) box family of DNA binding proteins that are able to bind to and bend DNA. Human TFAM (huTFAM) contains two HMG box domains separated by a linker region, and a 26 amino acid C-terminal tail distal to the second HMG box. Previous studies on huTFAM have shown that requisites for proper DNA bending and specific binding to the mitochondrial genome are specific intercalating residues and the C-terminal tail. We have characterized TFAM from the sea urchin Paracentrotus lividus (suTFAM). Differently from human, suTFAM contains a short 9 amino acid C-terminal tail, yet it still has the ability to specifically bind to mtDNA. To provide information on the mode of binding of the protein we used fluorescence resonance energy transfer (FRET) assays and found that, in spite of the absence of a canonical C-terminal tail, suTFAM distorts DNA at a great extent and recognizes specific target with high affinity. Site directed mutagenesis showed that the two Phe residues placed in corresponding position of the two intercalating Leu of huTFAM are responsible for the strong bending and the great binding affinity of suTFAM. PMID:27101895

  9. Geochemistry of Metals from mine tailing in Taxco Mexico

    NASA Astrophysics Data System (ADS)

    Morton-Bermea, O.; ARMIENTA, A.; BARRERA, M.; TALAVERA, O.; HERNANDEZ, E.

    2001-12-01

    The mining district of Taxco in Central Mexico has been exploited since prehispanic times. The processing of metals produced tailings with high heavy metal concentrations. Those tailings constitute a potential risk to the environment. To assess the effects of the mine tailing on water quality, tailing samples and water samples from rivers, wells and tailing effluents were collected and analyzed for Cu, Zn, As and Pb. Metals were analyzed with by ICP-MS. Tailing samples were leached with water to determine pH and sulfate concentration. The highest metal contents were found in the samples with a pH acid. As, Pb and Zn are over the drinking water standards in some of the water samples.

  10. Functional Analysis of a Rickettsial OmpA Homology Domain of Shigella flexneri IcsA

    PubMed Central

    Charles, Macarthur; Magdalena, Juana; Theriot, Julie A.; Goldberg, Marcia B.

    1999-01-01

    Shigella flexneri is a gram-negative bacterium that causes diarrhea and dysentery by invasion and spread through the colonic epithelium. Bacteria spread by assembling actin and other cytoskeletal proteins of the host into “actin tails” at the bacterial pole; actin tail assembly provides the force required to move bacteria through the cell cytoplasm and into adjacent cells. The 120-kDa S. flexneri outer membrane protein IcsA is essential for actin assembly. IcsA is anchored in the outer membrane by a carboxy-terminal domain (the β domain), such that the amino-terminal 706 amino acid residues (the α domain) are exposed on the exterior of the bacillus. The α domain is therefore likely to contain the domains that are important to interactions with host factors. We identify and characterize a domain of IcsA within the α domain that bears significant sequence similarity to two repeated domains of rickettsial OmpA, which has been implicated in rickettsial actin tail formation. Strains of S. flexneri and Escherichia coli that carry derivatives of IcsA containing deletions within this domain display loss of actin recruitment and increased accessibility to IcsA-specific antibody on the surface of intracytoplasmic bacteria. However, site-directed mutagenesis of charged residues within this domain results in actin assembly that is indistinguishable from that of the wild type, and in vitro competition of a polypeptide of this domain fused to glutathione S-transferase did not alter the motility of the wild-type construct. Taken together, our data suggest that the rickettsial homology domain of IcsA is required for the proper conformation of IcsA and that its disruption leads to loss of interactions of other IcsA domains within the amino terminus with host cytoskeletal proteins. PMID:9922250

  11. Functional analysis of conserved aromatic amino acids in the discoidin domain of Paenibacillus β-1,3-glucanase

    PubMed Central

    2009-01-01

    The 190-kDa Paenibacillus β-1,3-glucanase (LamA) contains a catalytic module of the glycoside hydrolase family 16 (GH16) and several auxiliary domains. Of these, a discoidin domain (DS domain), present in both eukaryotic and prokaryotic proteins with a wide variety of functions, exists at the carboxyl-terminus. To better understand the bacterial DS domain in terms of its structure and function, this domain alone was expressed in Escherichia coli and characterized. The results indicate that the DS domain binds various polysaccharides and enhances the biological activity of the GH16 module on composite substrates. We also investigated the importance of several conserved aromatic residues in the domain's stability and substrate-binding affinity. Both were affected by mutations of these residues; however, the effect on protein stability was more notable. In particular, the forces contributed by a sandwiched triad (W1688, R1756, and W1729) were critical for the presumable β-sandwich fold. PMID:19930717

  12. Comparative Modeling and Molecular Dynamics Simulation of Substrate Binding in Human Fatty Acid Synthase: Enoyl Reductase and β-Ketoacyl Reductase Catalytic Domains

    PubMed Central

    John, Arun; Krishnakumar, Subramanian

    2015-01-01

    Fatty acid synthase (FASN, EC 2.3.1.85), is a multi-enzyme dimer complex that plays a critical role in lipogenesis. This lipogenic enzyme has gained importance beyond its physiological role due to its implications in several clinical conditions-cancers, obesity, and diabetes. This has made FASN an attractive pharmacological target. Here, we have attempted to predict the theoretical models for the human enoyl reductase (ER) and β-ketoacyl reductase (KR) domains based on the porcine FASN crystal structure, which was the structurally closest template available at the time of this study. Comparative modeling methods were used for studying the structure-function relationships. Different validation studies revealed the predicted structures to be highly plausible. The respective substrates of ER and KR domains-namely, trans-butenoyl and β-ketobutyryl-were computationally docked into active sites using Glide in order to understand the probable binding mode. The molecular dynamics simulations of the apo and holo states of ER and KR showed stable backbone root mean square deviation trajectories with minimal deviation. Ramachandran plot analysis showed 96.0% of residues in the most favorable region for ER and 90.3% for the KR domain, respectively. Thus, the predicted models yielded significant insights into the substrate binding modes of the ER and KR catalytic domains and will aid in identifying novel chemical inhibitors of human FASN that target these domains. PMID:25873848

  13. Expression, purification, crystallization and preliminary X-ray diffraction analysis of the VP8* sialic acid-binding domain of porcine rotavirus strain OSU

    SciTech Connect

    Zhang, Yang-De Li, Hao; Liu, Hui; Pan, Yi-Feng

    2007-02-01

    Porcine rotavirus strain OSU VP8* domain has been expressed, purified and crystallized. X-ray diffraction data from different crystal forms of the VP8* domain have been collected to 2.65 and 2.2 Å resolution, respectively. The rotavirus outer capsid spike protein VP4 is utilized in the process of rotavirus attachment to and membrane penetration of host cells. VP4 is cleaved by trypsin into two domains: VP8* and VP5*. The VP8* domain is implicated in initial interaction with sialic acid-containing cell-surface carbohydrates and triggers subsequent virus invasion. The VP8* domain from porcine OSU rotavirus was cloned and expressed in Escherichia coli. Different crystal forms (orthorhombic P2{sub 1}2{sub 1}2{sub 1} and tetragonal P4{sub 1}2{sub 1}2) were harvested from two distinct crystallization conditions. Diffraction data have been collected to 2.65 and 2.2 Å resolution and the VP8*{sub 65–224} structure was determined by molecular replacement.

  14. On resin amino acid side chain attachment strategy for the head to tail synthesis of new glutamine containing gramicidin-S analogs and their antimicrobial activity.

    PubMed

    Derbal, Safa; Hensler, Mary; Fang, Weiqin; Nizet, Victor; Ghedira, Kamel; Nefzi, Adel

    2010-10-01

    The alarming increase in infections caused by multiple drug resistant bacteria including methicillin-resistant Staphylococcus aureus has prompted a desperate search for new antimicrobials. Augmenting the discoveries of completely new scaffolds with antimicrobial activity are efforts aimed at modifying existing molecules to optimize activity or reduce toxicity. We report herein the parallel solid-phase synthesis of analogues of the cationic antimicrobial peptide gramicidin S (GS) using amino acid side chain attachment strategy. The ornithine (Orn) residues were replaced by glutamine (Gln) and the aromatic D-phenylalanine (Phe) were replaced by different aromatic D-amino acids. Additional Gln containing GS analogues with all the possible combinations of the hydrophobic amino acids valine and leucine were also synthesized. In this work we also report the antibacterial activity of these analogs against several clinically-important drug-resistant Gram-positive and Gram-negative pathogens.

  15. Effects of amino acid mutations in the pore-forming domain of the hemolytic lectin CEL-III.

    PubMed

    Nagao, Tomonao; Masaki, Risa; Unno, Hideaki; Goda, Shuichiro; Hatakeyama, Tomomitsu

    2016-10-01

    The hemolytic lectin CEL-III forms transmembrane pores in the membranes of target cells. A study on the effect of site-directed mutation at Lys405 in domain 3 of CEL-III indicated that replacements of this residue by relatively smaller residues lead to a marked increase in hemolytic activity, suggesting that moderately destabilizing domain 3 facilitates formation of transmembrane pores through conformational changes.

  16. Effects of amino acid mutations in the pore-forming domain of the hemolytic lectin CEL-III.

    PubMed

    Nagao, Tomonao; Masaki, Risa; Unno, Hideaki; Goda, Shuichiro; Hatakeyama, Tomomitsu

    2016-10-01

    The hemolytic lectin CEL-III forms transmembrane pores in the membranes of target cells. A study on the effect of site-directed mutation at Lys405 in domain 3 of CEL-III indicated that replacements of this residue by relatively smaller residues lead to a marked increase in hemolytic activity, suggesting that moderately destabilizing domain 3 facilitates formation of transmembrane pores through conformational changes. PMID:27101707

  17. C2-domain abscisic acid-related proteins mediate the interaction of PYR/PYL/RCAR abscisic acid receptors with the plasma membrane and regulate abscisic acid sensitivity in Arabidopsis.

    PubMed

    Rodriguez, Lesia; Gonzalez-Guzman, Miguel; Diaz, Maira; Rodrigues, Americo; Izquierdo-Garcia, Ana C; Peirats-Llobet, Marta; Fernandez, Maria A; Antoni, Regina; Fernandez, Daniel; Marquez, Jose A; Mulet, Jose M; Albert, Armando; Rodriguez, Pedro L

    2014-12-01

    Membrane-delimited abscisic acid (ABA) signal transduction plays a critical role in early ABA signaling, but the molecular mechanisms linking core signaling components to the plasma membrane are unclear. We show that transient calcium-dependent interactions of PYR/PYL ABA receptors with membranes are mediated through a 10-member family of C2-domain ABA-related (CAR) proteins in Arabidopsis thaliana. Specifically, we found that PYL4 interacted in an ABA-independent manner with CAR1 in both the plasma membrane and nucleus of plant cells. CAR1 belongs to a plant-specific gene family encoding CAR1 to CAR10 proteins, and bimolecular fluorescence complementation and coimmunoprecipitation assays showed that PYL4-CAR1 as well as other PYR/PYL-CAR pairs interacted in plant cells. The crystal structure of CAR4 was solved, which revealed that, in addition to a classical calcium-dependent lipid binding C2 domain, a specific CAR signature is likely responsible for the interaction with PYR/PYL receptors and their recruitment to phospholipid vesicles. This interaction is relevant for PYR/PYL function and ABA signaling, since different car triple mutants affected in CAR1, CAR4, CAR5, and CAR9 genes showed reduced sensitivity to ABA in seedling establishment and root growth assays. In summary, we identified PYR/PYL-interacting partners that mediate a transient Ca(2+)-dependent interaction with phospholipid vesicles, which affects PYR/PYL subcellular localization and positively regulates ABA signaling. PMID:25465408

  18. C2-domain abscisic acid-related proteins mediate the interaction of PYR/PYL/RCAR abscisic acid receptors with the plasma membrane and regulate abscisic acid sensitivity in Arabidopsis.

    PubMed

    Rodriguez, Lesia; Gonzalez-Guzman, Miguel; Diaz, Maira; Rodrigues, Americo; Izquierdo-Garcia, Ana C; Peirats-Llobet, Marta; Fernandez, Maria A; Antoni, Regina; Fernandez, Daniel; Marquez, Jose A; Mulet, Jose M; Albert, Armando; Rodriguez, Pedro L

    2014-12-01

    Membrane-delimited abscisic acid (ABA) signal transduction plays a critical role in early ABA signaling, but the molecular mechanisms linking core signaling components to the plasma membrane are unclear. We show that transient calcium-dependent interactions of PYR/PYL ABA receptors with membranes are mediated through a 10-member family of C2-domain ABA-related (CAR) proteins in Arabidopsis thaliana. Specifically, we found that PYL4 interacted in an ABA-independent manner with CAR1 in both the plasma membrane and nucleus of plant cells. CAR1 belongs to a plant-specific gene family encoding CAR1 to CAR10 proteins, and bimolecular fluorescence complementation and coimmunoprecipitation assays showed that PYL4-CAR1 as well as other PYR/PYL-CAR pairs interacted in plant cells. The crystal structure of CAR4 was solved, which revealed that, in addition to a classical calcium-dependent lipid binding C2 domain, a specific CAR signature is likely responsible for the interaction with PYR/PYL receptors and their recruitment to phospholipid vesicles. This interaction is relevant for PYR/PYL function and ABA signaling, since different car triple mutants affected in CAR1, CAR4, CAR5, and CAR9 genes showed reduced sensitivity to ABA in seedling establishment and root growth assays. In summary, we identified PYR/PYL-interacting partners that mediate a transient Ca(2+)-dependent interaction with phospholipid vesicles, which affects PYR/PYL subcellular localization and positively regulates ABA signaling.

  19. C2-Domain Abscisic Acid-Related Proteins Mediate the Interaction of PYR/PYL/RCAR Abscisic Acid Receptors with the Plasma Membrane and Regulate Abscisic Acid Sensitivity in Arabidopsis[C][W

    PubMed Central

    Rodriguez, Lesia; Diaz, Maira; Rodrigues, Americo; Izquierdo-Garcia, Ana C.; Peirats-Llobet, Marta; Fernandez, Maria A.; Antoni, Regina; Fernandez, Daniel; Marquez, Jose A.; Mulet, Jose M.; Albert, Armando; Rodriguez, Pedro L.

    2014-01-01

    Membrane-delimited abscisic acid (ABA) signal transduction plays a critical role in early ABA signaling, but the molecular mechanisms linking core signaling components to the plasma membrane are unclear. We show that transient calcium-dependent interactions of PYR/PYL ABA receptors with membranes are mediated through a 10-member family of C2-domain ABA-related (CAR) proteins in Arabidopsis thaliana. Specifically, we found that PYL4 interacted in an ABA-independent manner with CAR1 in both the plasma membrane and nucleus of plant cells. CAR1 belongs to a plant-specific gene family encoding CAR1 to CAR10 proteins, and bimolecular fluorescence complementation and coimmunoprecipitation assays showed that PYL4-CAR1 as well as other PYR/PYL-CAR pairs interacted in plant cells. The crystal structure of CAR4 was solved, which revealed that, in addition to a classical calcium-dependent lipid binding C2 domain, a specific CAR signature is likely responsible for the interaction with PYR/PYL receptors and their recruitment to phospholipid vesicles. This interaction is relevant for PYR/PYL function and ABA signaling, since different car triple mutants affected in CAR1, CAR4, CAR5, and CAR9 genes showed reduced sensitivity to ABA in seedling establishment and root growth assays. In summary, we identified PYR/PYL-interacting partners that mediate a transient Ca2+-dependent interaction with phospholipid vesicles, which affects PYR/PYL subcellular localization and positively regulates ABA signaling. PMID:25465408

  20. Nuclear Magnetic Resonance Structure of the Nucleic Acid-Binding Domain of Severe Acute Respiratory Syndrome Coronavirus Nonstructural Protein 3▿

    PubMed Central

    Serrano, Pedro; Johnson, Margaret A.; Chatterjee, Amarnath; Neuman, Benjamin W.; Joseph, Jeremiah S.; Buchmeier, Michael J.; Kuhn, Peter; Wüthrich, Kurt

    2009-01-01

    The nuclear magnetic resonance (NMR) structure of a globular domain of residues 1071 to 1178 within the previously annotated nucleic acid-binding region (NAB) of severe acute respiratory syndrome coronavirus nonstructural protein 3 (nsp3) has been determined, and N- and C-terminally adjoining polypeptide segments of 37 and 25 residues, respectively, have been shown to form flexibly extended linkers to the preceding globular domain and to the following, as yet uncharacterized domain. This extension of the structural coverage of nsp3 was obtained from NMR studies with an nsp3 construct comprising residues 1066 to 1181 [nsp3(1066-1181)] and the constructs nsp3(1066-1203) and nsp3(1035-1181). A search of the protein structure database indicates that the globular domain of the NAB represents a new fold, with a parallel four-strand β-sheet holding two α-helices of three and four turns that are oriented antiparallel to the β-strands. Two antiparallel two-strand β-sheets and two 310-helices are anchored against the surface of this barrel-like molecular core. Chemical shift changes upon the addition of single-stranded RNAs (ssRNAs) identified a group of residues that form a positively charged patch on the protein surface as the binding site responsible for the previously reported affinity for nucleic acids. This binding site is similar to the ssRNA-binding site of the sterile alpha motif domain of the Saccharomyces cerevisiae Vts1p protein, although the two proteins do not share a common globular fold. PMID:19828617

  1. Phylogenetic analysis of evolutionary relationships of the planctomycete division of the domain bacteria based on amino acid sequences of elongation factor Tu.

    PubMed

    Jenkins, C; Fuerst, J A

    2001-05-01

    Sequences from the tuf gene coding for the elongation factor EF-Tu were amplified and sequenced from the genomic DNA of Pirellula marina and Isosphaera pallida, two species of bacteria within the order Planctomycetales. A near-complete (1140-bp) sequence was obtained from Pi. marina and a partial (759-bp) sequence was obtained for I. pallida. Alignment of the deduced Pi. marina EF-Tu amino acid sequence against reference sequences demonstrated the presence of a unique 11-amino acid sequence motif not present in any other division of the domain Bacteria. Pi. marina shared the highest percentage amino acid sequence identity with I. pallida but showed only a low percentage identity with other members of the domain Bacteria. This is consistent with the concept of the planctomycetes as a unique division of the Bacteria. Neither primary sequence comparison of EF-Tu nor phylogenetic analysis supports any close relationship between planctomycetes and the chlamydiae, which has previously been postulated on the basis of 16S rRNA. Phylogenetic analysis of aligned EF-Tu amino acid sequences performed using distance, maximum-parsimony, and maximum-likelihood approaches yielded contradictory results with respect to the position of planctomycetes relative to other bacteria. It is hypothesized that long-branch attraction effects due to unequal evolutionary rates and mutational saturation effects may account for some of the contradictions. PMID:11443344

  2. RapA2 Is a Calcium-binding Lectin Composed of Two Highly Conserved Cadherin-like Domains That Specifically Recognize Rhizobium leguminosarum Acidic Exopolysaccharides*

    PubMed Central

    Abdian, Patricia L.; Caramelo, Julio J.; Ausmees, Nora; Zorreguieta, Angeles

    2013-01-01

    In silico analyses have revealed a conserved protein domain (CHDL) widely present in bacteria that has significant structural similarity to eukaryotic cadherins. A CHDL domain was shown to be present in RapA, a protein that is involved in autoaggregation of Rhizobium cells, biofilm formation, and adhesion to plant roots as shown by us and others. Structural similarity to cadherins suggested calcium-dependent oligomerization of CHDL domains as a mechanistic basis for RapA action. Here we show by circular dichroism spectroscopy, light scattering, isothermal titration calorimetry, and other methods that RapA2 from Rhizobium leguminosarum indeed exhibits a cadherin-like β-sheet conformation and that its proper folding and stability are dependent on the binding of one calcium ion per protein molecule. By further in silico analysis we also reveal that RapA2 consists of two CHDL domains and expand the range of CHDL-containing proteins in bacteria and archaea. However, light scattering assays at various concentrations of added calcium revealed that RapA2 formed neither homo-oligomers nor hetero-oligomers with RapB (a distinct CHDL protein), indicating that RapA2 does not mediate cellular interactions through a cadherin-like mechanism. Instead, we demonstrate that RapA2 interacts specifically with the acidic exopolysaccharides (EPSs) produced by R. leguminosarum in a calcium-dependent manner, sustaining a role of these proteins in the development of the biofilm matrix made of EPS. Because EPS binding by RapA2 can only be attributed to its two CHDL domains, we propose that RapA2 is a calcium-dependent lectin and that CHDL domains in various bacterial and archaeal proteins confer carbohydrate binding activity to these proteins. PMID:23235153

  3. The FKBP-rapamycin binding domain of human TOR undergoes strong conformational changes in the presence of membrane mimetics with and without the regulator phosphatidic acid.

    PubMed

    Rodriguez Camargo, Diana C; Link, Nina M; Dames, Sonja A

    2012-06-19

    The Ser/Thr kinase target of rapamycin (TOR) is a central controller of cellular growth and metabolism. Misregulation of TOR signaling is involved in metabolic and neurological disorders and tumor formation. TOR can be inhibited by association of a complex of rapamycin and FKBP12 to the FKBP12-rapamycin binding (FRB) domain. This domain was further proposed to interact with phosphatidic acid (PA), a lipid second messenger present in cellular membranes. Because mammalian TOR has been localized at various cellular membranes and in the nucleus, the output of TOR signaling may depend on its localization, which is expected to be influenced by the interaction with complex partners and regulators in response to cellular signals. Here, we present a detailed characterization of the interaction of the FRB domain with PA and how it is influenced by the surrounding membrane environment. On the basis of nuclear magnetic resonance- and circular dichroism-monitored binding studies using different neutral and negatively charged lipids as well as different membrane mimetics (micelles, bicelles, and liposomes), the FRB domain may function as a conditional peripheral membrane protein. However, the data for the isolated domain just indicate an increased affinity for negatively charged lipids and membrane patches but no specific preference for PA or PA-enriched regions. The membrane-mimetic environment induces strong conformational changes that largely maintain the α-helical secondary structure content but presumably disperse the helices in the lipidic environment. Consistent with overlapping binding surfaces for different lipids and the FKBP12-rapamycin complex, binding of the inhibitor complex protects the FRB domain from interactions with membrane mimetics at lower lipid concentrations.

  4. Exploring the activity space of peptides binding to diverse SH3 domains using principal property descriptors derived from amino acid rotamers.

    PubMed

    He, Ping; Wu, Wei; Yang, Kang; Jing, Tao; Liao, Ke-Long; Zhang, Wei; Wang, Hai-Dong; Hua, Xing

    2011-01-01

    Although there were intensive works addressed on multivariate extraction of the informative components from numerous physicochemical parameters of amino acids in isolated state, the various conformational behaviors of amino acids in complicated biological context have long been underappreciated in the field of quantitative structure-activity relationship (QSAR). In this work, the amino acid rotamers, which were derived from statistical survey of protein crystal structures, were used to reproduce the conformational variety of amino acid side-chains in real condition. In this procedure, these rotamers were superposed into a nx x ny x nz lattice and an artificial probe was employed to detect four kinds of nonbonding field potentials (i.e., electrostatic, steric, hydrophobic, and hydrogen bonds) at each lattice point using a Gaussian-type potential function; the generated massive data were then subjected to a principal component analysis (PCA) treatment to obtain a set of few, informative amino acid descriptors. We used this set of descriptors, that we named principal property descriptors derived from amino acid rotamers (PDAR), to characterize over 13,000 peptides with known binding affinities to 10 types of SH3 domains. Genetic algorithm/ partial least square regression (GA/PLS) modeling and Monte Carlo cross-validation (MCCV) demonstrated that the correlation between the PDAR descriptors and the binding affinities of peptides are comparable with or even better than previously published models. Furthermore, from the PDAR-based QSAR models we concluded that the core motif of peptides, particularly the electrostatic property, hydrophobicity, and hydrogen bond at residue positions P3, P2, and/or P0, contribute significantly to the hAmph SH3 domain-peptide binding, whereas two ends of the peptides, such as P6, P4, P-4, and P5, only play a secondary role in the binding.

  5. The Aquaporin Splice Variant NbXIP1;1α Is Permeable to Boric Acid and Is Phosphorylated in the N-terminal Domain.

    PubMed

    Ampah-Korsah, Henry; Anderberg, Hanna I; Engfors, Angelica; Kirscht, Andreas; Norden, Kristina; Kjellstrom, Sven; Kjellbom, Per; Johanson, Urban

    2016-01-01

    Aquaporins (AQPs) are membrane channel proteins that transport water and uncharged solutes across different membranes in organisms in all kingdoms of life. In plants, the AQPs can be divided into seven different subfamilies and five of these are present in higher plants. The most recently characterized of these subfamilies is the XIP subfamily, which is found in most dicots but not in monocots. In this article, we present data on two different splice variants (α and β) of NbXIP1;1 from Nicotiana benthamiana. We describe the heterologous expression of NbXIP1;1α and β in the yeast Pichia pastoris, the subcellular localization of the protein in this system and the purification of the NbXIP1;1α protein. Furthermore, we investigated the functionality and the substrate specificity of the protein by stopped-flow spectrometry in P. pastoris spheroplasts and with the protein reconstituted in proteoliposomes. The phosphorylation status of the protein and localization of the phosphorylated amino acids were verified by mass spectrometry. Our results show that NbXIP1;1α is located in the plasma membrane when expressed in P. pastoris, that it is not permeable to water but to boric acid and that the protein is phosphorylated at several amino acids in the N-terminal cytoplasmic domain of the protein. A growth assay showed that the yeast cells expressing the N-terminally His-tagged NbXIP1;1α were more sensitive to boric acid as compared to the cells expressing the C-terminally His-tagged isoform. This might suggest that the N-terminal His-tag functionally mimics the phosphorylation of the N-terminal domain and that the N-terminal domain is involved in gating of the channel. PMID:27379142

  6. The Aquaporin Splice Variant NbXIP1;1α Is Permeable to Boric Acid and Is Phosphorylated in the N-terminal Domain.

    PubMed

    Ampah-Korsah, Henry; Anderberg, Hanna I; Engfors, Angelica; Kirscht, Andreas; Norden, Kristina; Kjellstrom, Sven; Kjellbom, Per; Johanson, Urban

    2016-01-01

    Aquaporins (AQPs) are membrane channel proteins that transport water and uncharged solutes across different membranes in organisms in all kingdoms of life. In plants, the AQPs can be divided into seven different subfamilies and five of these are present in higher plants. The most recently characterized of these subfamilies is the XIP subfamily, which is found in most dicots but not in monocots. In this article, we present data on two different splice variants (α and β) of NbXIP1;1 from Nicotiana benthamiana. We describe the heterologous expression of NbXIP1;1α and β in the yeast Pichia pastoris, the subcellular localization of the protein in this system and the purification of the NbXIP1;1α protein. Furthermore, we investigated the functionality and the substrate specificity of the protein by stopped-flow spectrometry in P. pastoris spheroplasts and with the protein reconstituted in proteoliposomes. The phosphorylation status of the protein and localization of the phosphorylated amino acids were verified by mass spectrometry. Our results show that NbXIP1;1α is located in the plasma membrane when expressed in P. pastoris, that it is not permeable to water but to boric acid and that the protein is phosphorylated at several amino acids in the N-terminal cytoplasmic domain of the protein. A growth assay showed that the yeast cells expressing the N-terminally His-tagged NbXIP1;1α were more sensitive to boric acid as compared to the cells expressing the C-terminally His-tagged isoform. This might suggest that the N-terminal His-tag functionally mimics the phosphorylation of the N-terminal domain and that the N-terminal domain is involved in gating of the channel.

  7. The Aquaporin Splice Variant NbXIP1;1α Is Permeable to Boric Acid and Is Phosphorylated in the N-terminal Domain

    PubMed Central

    Ampah-Korsah, Henry; Anderberg, Hanna I.; Engfors, Angelica; Kirscht, Andreas; Norden, Kristina; Kjellstrom, Sven; Kjellbom, Per; Johanson, Urban

    2016-01-01

    Aquaporins (AQPs) are membrane channel proteins that transport water and uncharged solutes across different membranes in organisms in all kingdoms of life. In plants, the AQPs can be divided into seven different subfamilies and five of these are present in higher plants. The most recently characterized of these subfamilies is the XIP subfamily, which is found in most dicots but not in monocots. In this article, we present data on two different splice variants (α and β) of NbXIP1;1 from Nicotiana benthamiana. We describe the heterologous expression of NbXIP1;1α and β in the yeast Pichia pastoris, the subcellular localization of the protein in this system and the purification of the NbXIP1;1α protein. Furthermore, we investigated the functionality and the substrate specificity of the protein by stopped-flow spectrometry in P. pastoris spheroplasts and with the protein reconstituted in proteoliposomes. The phosphorylation status of the protein and localization of the phosphorylated amino acids were verified by mass spectrometry. Our results show that NbXIP1;1α is located in the plasma membrane when expressed in P. pastoris, that it is not permeable to water but to boric acid and that the protein is phosphorylated at several amino acids in the N-terminal cytoplasmic domain of the protein. A growth assay showed that the yeast cells expressing the N-terminally His-tagged NbXIP1;1α were more sensitive to boric acid as compared to the cells expressing the C-terminally His-tagged isoform. This might suggest that the N-terminal His-tag functionally mimics the phosphorylation of the N-terminal domain and that the N-terminal domain is involved in gating of the channel. PMID:27379142

  8. A recombinant hepatitis B core antigen polypeptide with the protamine-like domain deleted self-assembles into capsid particles but fails to bind nucleic acids.

    PubMed Central

    Gallina, A; Bonelli, F; Zentilin, L; Rindi, G; Muttini, M; Milanesi, G

    1989-01-01

    We have cloned in Escherichia coli both the complete core gene of hepatitis B virus and a truncated version of it, leading to the synthesis of high levels of a core-antigen-equivalent polypeptide (r-p22) and of an e-antigen-equivalent polypeptide (r-p16), respectively. We then compared the structural and antigenic properties of the two polypeptides, as well as their ability to bind viral nucleic acids. r-p16 was found to self-assemble into capsid-like particles that appeared similar, when observed under the electron microscope, to those formed by r-p22. In r-p16 particles, disulfide bonds linked the truncated polypeptides in dimers, assembled in the particle by noncovalent interactions. In r-p22 capsids, further disulfide bonds, conceivably involving the carboxy-terminal cysteines of r-p22 polypeptides, joined the dimers together, converting the structure into a covalently closed lattice. The protamine-like domain was at least partly exposed on the surface of r-p22 particles, since it was accessible to selective proteolysis. Finally, r-p22, but not r-p16, was shown to bind native and denatured DNA as well as RNA. Taken together, these results suggest that the protamine-like domain in core polypeptides is a nucleic acid-binding domain and is dispensable for the correct folding and assembly of amino-terminal and central regions. Images PMID:2677399

  9. X-Ray Structure of the Amidase Domain of AtzF, the Allophanate Hydrolase from the Cyanuric Acid-Mineralizing Multienzyme Complex

    PubMed Central

    Balotra, Sahil; Newman, Janet; Cowieson, Nathan P.; French, Nigel G.; Campbell, Peter M.; Briggs, Lyndall J.; Warden, Andrew C.; Easton, Christopher J.; Peat, Thomas S.

    2014-01-01

    The activity of the allophanate hydrolase from Pseudomonas sp. strain ADP, AtzF, provides the final hydrolytic step for the mineralization of s-triazines, such as atrazine and cyanuric acid. Indeed, the action of AtzF provides metabolic access to two of the three nitrogens in each triazine ring. The X-ray structure of the N-terminal amidase domain of AtzF reveals that it is highly homologous to allophanate hydrolases involved in a different catabolic process in other organisms (i.e., the mineralization of urea). The smaller C-terminal domain does not appear to have a physiologically relevant catalytic function, as reported for the allophanate hydrolase of Kluyveromyces lactis, when purified enzyme was tested in vitro. However, the C-terminal domain does have a function in coordinating the quaternary structure of AtzF. Interestingly, we also show that AtzF forms a large, ca. 660-kDa, multienzyme complex with AtzD and AtzE that is capable of mineralizing cyanuric acid. The function of this complex may be to channel substrates from one active site to the next, effectively protecting unstable metabolites, such as allophanate, from solvent-mediated decarboxylation to a dead-end metabolic product. PMID:25362066

  10. Amino acid residues that flank core peptide epitopes and the extracellular domains of CD4 modulate differential signaling through the T cell receptor

    PubMed Central

    1994-01-01

    Hen egg lysozyme 52-61-specific CD4+ T cells responded by interleukin 2 (IL-2) secretion to any peptide containing this epitope regardless of length of NH2- and COOH-terminal composition. However, CD4- variants could only respond to peptides containing the two COOH-terminal tryptophans at positions 62 and 63. Substitutions at these positions defined patterns of reactivity that were specific for individual T cells inferring a T cell receptor (TCR)-based phenomenon. Thus, the fine specificity of major histocompatibility complex (MHC)-peptide recognition by the TCR was dramatically affected by CD4 and the COOH- terminal peptide composition. Peptides that failed to induce IL-2 secretion in the CD4- variants nevertheless induced strong tyrosine phosphorylation of CD3 zeta. Thus, whereas the TCR still recognized and bound to the MHC class II-peptide complex resulting in protein phosphorylation, this interaction failed to induce effective signal transduction manifested by IL-2 secretion. This provides a clear example of differential signaling mediated by peptides known to be naturally processed. In addition, the external domains of CD4, rather than its cytoplasmic tail, were critical in aiding TCR recognition of all peptides derived from a single epitope. These data suggest that the nested flanking residues, which are present on MHC class II but not class I bound peptides, are functionally relevant. PMID:7515103

  11. REAR PROFILE OF TAIL FROM SECOND LEVEL OF TAIL DOCK ...

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

    REAR PROFILE OF TAIL FROM SECOND LEVEL OF TAIL DOCK STAND, SHOWING AIRCRAFT NUMBER (319), HORIZONTAL STABILIZER, TAIL CONE AND COOLING CTS FOR THE AUXILIARY POWER UNIT (APU), MECHANIC PAUL RIDEOUT IS LOWERING THE BALANCE PANELS ON THE STABILIZERS FOR LUBRICATION AND INSPECTION. - Greater Buffalo International Airport, Maintenance Hangar, Buffalo, Erie County, NY

  12. Solution structure of histone chaperone ANP32B: interaction with core histones H3-H4 through its acidic concave domain.

    PubMed

    Tochio, Naoya; Umehara, Takashi; Munemasa, Yoshiko; Suzuki, Toru; Sato, Shin; Tsuda, Kengo; Koshiba, Seizo; Kigawa, Takanori; Nagai, Ryozo; Yokoyama, Shigeyuki

    2010-08-01

    Eukaryotic gene expression is regulated by histone deposition onto and eviction from nucleosomes, which are mediated by several chromatin-modulating factors. Among them, histone chaperones are key factors that facilitate nucleosome assembly. Acidic nuclear phosphoprotein 32B (ANP32B) belongs to the ANP32 family, which shares N-terminal leucine-rich repeats (LRRs) and a C-terminal variable anionic region. The C-terminal region functions as an inhibitor of histone acetylation, but the functional roles of the LRR domain in chromatin regulation have remained elusive. Here, we report that the LRR domain of ANP32B possesses histone chaperone activity and forms a curved structure with a parallel beta-sheet on the concave side and mostly helical elements on the convex side. Our analyses revealed that the interaction of ANP32B with the core histones H3-H4 occurs on its concave side, and both the acidic and hydrophobic residues that compose the concave surface are critical for histone binding. These results provide a structural framework for understanding the functional mechanisms of acidic histone chaperones.

  13. Pinpointing the putative heparin/sialic acid-binding residues in the 'sushi' domain 7 of factor H: a molecular modeling study.

    PubMed

    Ranganathan, S; Male, D A; Ormsby, R J; Giannakis, E; Gordon, D L

    2000-01-01

    Factor H, a secretory glycoprotein comprising 20 short consensus repeat (SCR) or 'sushi' domains of about 60 amino acids each, is a regulator of the complement system. The complement-regulatory functions of factor H are targeted by its binding to polyanions such as heparin/sialic acid, involving SCRs 7 and 20. Recently, the SCR 7 heparin-binding site was shown to be co-localized with the Streptococcus Group A M protein binding site on factor H (T.K. Blackmore et al., Infect. Immun. 66, 1427 (1998)). Using sequence analysis of all heparin-binding domains of factor H and its closest homologues, molecular modeling of SCRs 6 and 7, and surface electrostatic potential studies, the residues implicated in heparin/sialic acid binding to SCR 7 have been localized to four regions of sequence space containing stretches of basic as well as histidine residues. The heparin-binding site is spatially compact and lies near the interface between SCRs 6 and 7, with residues in the interdomain linker playing a significant role.

  14. A single amino acid in the second Ig-like domain of the human Fc gamma receptor II is critical for human IgG2 binding.

    PubMed

    Warmerdam, P A; van de Winkel, J G; Vlug, A; Westerdaal, N A; Capel, P J

    1991-08-15

    The low-affinity human Fc gamma RIIa is encoded by a single gene with allelic variation, defined by low-responder and high-responder alleles (LR and HR). The HR Fc gamma RIIa transcript interacts strongly with murine (m) IgG1 complexes, in contrast to the LR Fc gamma RIIa. Furthermore, the transcripts can be discriminated by mAb 41H16, which recognizes an epitope expressed on the HR Fc gamma RIIa molecule. We report that this receptor is also polymorphic in its reactivity with human (h) IgG2. Binding studies using well-defined hIgG dimers revealed that LR Fc gamma RIIa molecules can efficiently bind hIgG2, in contrast to HR Fc gamma RIIa. Previous work of others showed one amino acid difference between the allelic forms of Fc gamma RII. We, however, found a second amino acid difference between both allelic forms. In this study, hybrid Fc gamma RIIa molecules were constructed to determine the epitope for mAb 41H16 and the binding domain for mIgG1 and hIgG2 complexes. Our data point to the importance of the amino acid at position 131, located in the second Ig-like domain of Fc gamma RIIa. When an arginine residue is present at amino acid position 131, the receptor is recognized by mAb 41H16. Furthermore, the receptor can bind mIgG1-sensitized indicator E, but binds hIgG2 dimers only weakly. When a histidine residue is present at this amino acid position, hIgG2 dimers do bind efficiently to Fc gamma RII, whereas mIgG1-sensitized E and mAb 41H16 exhibit a strongly diminished binding.

  15. Magnetic domain wall gratings for magnetization reversal tuning and confined dynamic mode localization

    NASA Astrophysics Data System (ADS)

    Trützschler, Julia; Sentosun, Kadir; Mozooni, Babak; Mattheis, Roland; McCord, Jeffrey

    2016-08-01

    High density magnetic domain wall gratings are imprinted in ferromagnetic-antiferromagnetic thin films by local ion irradiation by which alternating head-to-tail-to-head-to-tail and head-to-head-to-tail-to-tail spatially overlapping domain wall networks are formed. Unique magnetic domain processes result from the interaction of anchored domain walls. Non-linear magnetization response is introduced by the laterally distributed magnetic anisotropy phases. The locally varying magnetic charge distribution gives rise to localized and guided magnetization spin-wave modes directly constrained by the narrow domain wall cores. The exchange coupled multiphase material structure leads to unprecedented static and locally modified dynamic magnetic material properties.

  16. Limited proteolysis and sequence analysis of the 2-oxo acid dehydrogenase complexes from Escherichia coli. Cleavage sites and domains in the dihydrolipoamide acyltransferase components.

    PubMed Central

    Packman, L C; Perham, R N

    1987-01-01

    The structures of the dihydrolipoamide acyltransferase (E2) components of the 2-oxo acid dehydrogenase complexes from Escherichia coli were investigated by limited proteolysis. Trypsin and Staphylococcus aureus V8 proteinase were used to excise the three lipoyl domains from the E2p component of the pyruvate dehydrogenase complex and the single lipoyl domain from the E2o component of the 2-oxoglutarate dehydrogenase complex. The principal sites of action of these enzymes on each E2 chain were determined by sequence analysis of the isolated lipoyl fragments and of the truncated E2p and E2o chains. Each of the numerous cleavage sites (12 in E2p, six in E2o) fell within similar segments of the E2 chains, namely stretches of polypeptide rich in alanine, proline and/or charged amino acids. These regions are clearly accessible to proteinases of Mr 24,000-28,000 and, on the basis of n.m.r. spectroscopy, some of them have previously been implicated in facilitating domain movements by virtue of their conformational flexibility. The limited proteolysis data suggest that E2p and E2o possess closer architectural similarities than would be predicted from inspection of their amino acid sequences. As a result of this work, an error was detected in the sequence of E2o inferred from the previously published sequence of the encoding gene, sucB. The relevant peptides from E2o were purified and sequenced by direct means; an amended sequence is presented. Images Fig. 1. Fig. 2. PMID:3297046

  17. Interaction of bacterial fatty-acid-displaced regulators with DNA is interrupted by tyrosine phosphorylation in the helix-turn-helix domain

    PubMed Central

    Derouiche, Abderahmane; Bidnenko, Vladimir; Grenha, Rosa; Pigonneau, Nathalie; Ventroux, Magali; Franz-Wachtel, Mirita; Nessler, Sylvie; Noirot-Gros, Marie-Françoise; Mijakovic, Ivan

    2013-01-01

    Bacteria possess transcription regulators (of the TetR family) specifically dedicated to repressing genes for cytochrome P450, involved in oxidation of polyunsaturated fatty acids. Interaction of these repressors with operator sequences is disrupted in the presence of fatty acids, and they are therefore known as fatty-acid-displaced regulators. Here, we describe a novel mechanism of inactivating the interaction of these proteins with DNA, illustrated by the example of Bacillus subtilis regulator FatR. FatR was found to interact in a two-hybrid assay with TkmA, an activator of the protein-tyrosine kinase PtkA. We show that FatR is phosphorylated specifically at the residue tyrosine 45 in its helix-turn-helix domain by the kinase PtkA. Structural modelling reveals that the hydroxyl group of tyrosine 45 interacts with DNA, and we show that this phosphorylation reduces FatR DNA binding capacity. Point mutants mimicking phosphorylation of FatR in vivo lead to a strong derepression of the fatR operon, indicating that this regulatory mechanism works independently of derepression by polyunsaturated fatty acids. Tyrosine 45 is a highly conserved residue, and PtkA from B. subtilis can phosphorylate FatR homologues from other bacteria. This indicates that phosphorylation of tyrosine 45 may be a general mechanism of switching off bacterial fatty-acid-displaced regulators. PMID:23939619

  18. Identification of the domains of cauliflower mosaic virus protein P6 responsible for suppression of RNA silencing and salicylic acid signalling

    PubMed Central

    Laird, Janet; McInally, Carol; Carr, Craig; Doddiah, Sowjanya; Yates, Gary; Chrysanthou, Elina; Khattab, Ahmed; Love, Andrew J.; Geri, Chiara; Sadanandom, Ari; Smith, Brian O.; Kobayashi, Kappei

    2013-01-01

    Cauliflower mosaic virus (CaMV) encodes a 520 aa polypeptide, P6, which participates in several essential activities in the virus life cycle including suppressing RNA silencing and salicylic acid-responsive defence signalling. We infected Arabidopsis with CaMV mutants containing short in-frame deletions within the P6 ORF. A deletion in the distal end of domain D-I (the N-terminal 112 aa) of P6 did not affect virus replication but compromised symptom development and curtailed the ability to restore GFP fluorescence in a GFP-silenced transgenic Arabidopsis line. A deletion in the minimum transactivator domain was defective in virus replication but retained the capacity to suppress RNA silencing locally. Symptom expression in CaMV-infected plants is apparently linked to the ability to suppress RNA silencing. When transiently co-expressed with tomato bushy stunt virus P19, an elicitor of programmed cell death in Nicotiana tabacum, WT P6 suppressed the hypersensitive response, but three mutants, two with deletions within the distal end of domain D-I and one involving the N-terminal nuclear export signal (NES), were unable to do so. Deleting the N-terminal 20 aa also abolished the suppression of pathogen-associated molecular pattern-dependent PR1a expression following agroinfiltration. However, the two other deletions in domain D-I retained this activity, evidence that the mechanisms underlying these functions are not identical. The D-I domain of P6 when expressed alone failed to suppress either cell death or PR1a expression and is therefore necessary but not sufficient for all three defence suppression activities. Consequently, concerns about the biosafety of genetically modified crops carrying truncated ORFVI sequences appear unfounded. PMID:24088344

  19. A full-coordinate model of the polymerase domain of HIV-1 reverse transcriptase and its interaction with a nucleic acid substrate

    NASA Technical Reports Server (NTRS)

    Setlik, R. F.; Meyer, D. J.; Shibata, M.; Roskwitalski, R.; Ornstein, R. L.; Rein, R.

    1994-01-01

    We present a full-coordinate model of residues 1-319 of the polymerase domain of HIV-I reverse transcriptase. This model was constructed from the x-ray crystallographic structure of Jacobo-Molina et al. (Jacobo-Molina et al., P.N.A.S. USA 90, 6320-6324 (1993)) which is currently available to the degree of C-coordinates. The backbone and side-chain atoms were constructed using the MAXSPROUT suite of programs (L. Holm and C. Sander, J. Mol. Biol. 218, 183-194 (1991)) and refined through molecular modeling. A seven base pair A-form dsDNA was positioned in the nucleic acid binding cleft to represent the template-primer complex. The orientation of the template-primer complex in the nucleic acid binding cleft was guided by the positions of phosphorus atoms in the crystal structure.

  20. Dataset for the NMR structure of the intrinsically disordered acidic region of XPC bound to the PH domain of TFIIH p62

    PubMed Central

    Okuda, Masahiko; Nishimura, Yoshifumi

    2016-01-01

    The global genome nucleotide excision repair factor XPC firstly detects DNA lesions and then recruits a ten-subunit complex TFIIH through binding to the subunit p62 to unwind the damaged DNA for excision repair. This data article contains detailed nuclear magnetic resonance (NMR) restraints (nuclear Overhauser enhancement (NOE)-derived distance restraints, dihedral angle restraints, and hydrogen bond restraints) used for the structure determination of the complex formed between the intrinsically disordered acidic region of XPC and the pleckstrin homology (PH) domain of TFIIH p62, related to the recent work entitled “Structural insight into the mechanism of TFIIH recognition by the acidic string of the nucleotide excision repair factor XPC.” [1]. PMID:26909369

  1. Cloning, purification, crystallization and X-ray crystallographic analysis of the periplasmic sensing domain of Pseudomonas fluorescens chemotactic transducer of amino acids type A (CtaA).

    PubMed

    Ud-Din, Abu Iftiaf Md Salah; Roujeinikova, Anna

    2016-09-01

    Chemotaxis towards nutrients plays a crucial role in root colonization by Pseudomonas fluorescens. The P. fluorescens chemotactic transducer of amino acids type A (CtaA) mediates movement towards amino acids present in root exudates. In this study, the periplasmic sensory domain of CtaA has been crystallized by the hanging-drop vapor diffusion method using ammonium sulfate as a precipitating agent. A complete data set was collected to 1.9 Å resolution using cryocooling conditions and synchrotron radiation. The crystals belong to space group I222 or I212121, with unit-cell parameters a = 67.2, b = 76.0, c = 113.3 Å. This is an important step towards elucidation of the structural basis of how CtaA recognizes its signal molecules and transduces the signal across the membrane. PMID:27251445

  2. Size does matter: 18 amino acids at the N-terminal tip of an amino acid transporter in Leishmania determine substrate specificity

    PubMed Central

    Schlisselberg, Doreen; Mazarib, Eldar; Inbar, Ehud; Rentsch, Doris; Myler, Peter J.; Zilberstein, Dan

    2015-01-01

    Long N-terminal tails of amino acid transporters are known to act as sensors of the internal pool of amino acids and as positive regulators of substrate flux rate. In this study we establish that N-termini of amino acid transporters can also determine substrate specificity. We show that due to alternative trans splicing, the human pathogen Leishmania naturally expresses two variants of the proline/alanine transporter, one 18 amino acid shorter than the other. We demonstrate that the longer variant (LdAAP24) translocates both proline and alanine, whereas the shorter variant (∆18LdAAP24) translocates just proline. Remarkably, co-expressing the hydrophilic N-terminal peptide of the long variant with ∆18LdAAP24 was found to recover alanine transport. This restoration of alanine transport could be mediated by a truncated N-terminal tail, though truncations exceeding half of the tail length were no longer functional. Taken together, the data indicate that the first 18 amino acids of the negatively charged N-terminal LdAAP24 tail are required for alanine transport and may facilitate the electrostatic interactions of the entire negatively charged N-terminal tail with the positively charged internal loops in the transmembrane domain, as this mechanism has been shown to underlie regulation of substrate flux rate for other transporters. PMID:26549185

  3. Requirement for the E1 Helicase C-Terminal Domain in Papillomavirus DNA Replication In Vivo

    PubMed Central

    Bergvall, Monika; Gagnon, David; Titolo, Steve; Lehoux, Michaël; D'Abramo, Claudia M.

    2016-01-01

    ABSTRACT The papillomavirus (PV) E1 helicase contains a conserved C-terminal domain (CTD), located next to its ATP-binding site, whose function in vivo is still poorly understood. The CTD is comprised of an alpha helix followed by an acidic region (AR) and a C-terminal extension termed the C-tail. Recent biochemical studies on bovine papillomavirus 1 (BPV1) E1 showed that the AR and C-tail regulate the oligomerization of the protein into a double hexamer at the origin. In this study, we assessed the importance of the CTD of human papillomavirus 11 (HPV11) E1 in vivo, using a cell-based DNA replication assay. Our results indicate that combined deletion of the AR and C-tail drastically reduces DNA replication, by 85%, and that further truncation into the alpha-helical region compromises the structural integrity of the E1 helicase domain and its interaction with E2. Surprisingly, removal of the C-tail alone or mutation of highly conserved residues within the domain still allows significant levels of DNA replication (55%). This is in contrast to the absolute requirement for the C-tail reported for BPV1 E1 in vitro and confirmed here in vivo. Characterization of chimeric proteins in which the AR and C-tail from HPV11 E1 were replaced by those of BPV1 indicated that while the function of the AR is transferable, that of the C-tail is not. Collectively, these findings define the contribution of the three CTD subdomains to the DNA replication activity of E1 in vivo and suggest that the function of the C-tail has evolved in a PV type-specific manner. IMPORTANCE While much is known about hexameric DNA helicases from superfamily 3, the papillomavirus E1 helicase contains a unique C-terminal domain (CTD) adjacent to its ATP-binding site. We show here that this CTD is important for the DNA replication activity of HPV11 E1 in vivo and that it can be divided into three functional subdomains that roughly correspond to the three conserved regions of the CTD: an alpha helix, needed

  4. Raf-1 kinase possesses distinct binding domains for phosphatidylserine and phosphatidic acid. Phosphatidic acid regulates the translocation of Raf-1 in 12-O-tetradecanoylphorbol-13-acetate-stimulated Madin-Darby canine kidney cells.

    PubMed

    Ghosh, S; Strum, J C; Sciorra, V A; Daniel, L; Bell, R M

    1996-04-01

    Previous studies demonstrated that the cysteine-rich amino-terminal domain of Raf-1 kinase interacts selectively with phosphatidylserine (Ghosh, S., Xie, W. Q., Quest, A. F. G., Mabrouk, G. M., Strum, J. C., and Bell, R. M. (1994) J. Biol. Chem. 269, 10000-10007). Further analysis showed that full-length Raf-1 bound to both phosphatidylserine and phosphatidic acid (PA). Specifically, a carboxyl-terminal domain of Raf-1 kinase (RafC; residues 295 648 of human Raf-1) interacted strongly with phosphatidic acid. The binding of RafC to PA displayed positive cooperativity with Hill numbers between 3.3 and 6.2; the apparent Kd ranged from 4.9 +/- 0.6 to 7.8 +/- 0.9 mol % PA. The interaction of RafC with PA displayed a pH dependence distinct from the interaction between the cysteine-rich domain of Raf-1 and PA. Also, the RafC-PA interaction was unaffected at high ionic strength. Of all the lipids tested, only PA and cardiolipin exhibited high affinity binding; other acidic lipids were either ineffective or weakly effective. By deletion mutagenesis, the PA binding site within RafC was narrowed down to a 35-amino acid segment between residues 389 and 423. RafC did not bind phosphatidyl alcohols; also, inhibition of PA formation in Madin-Darby canine kidney cells by treatment with 1% ethanol significantly reduced the translocation of Raf-1 from the cytosol to the membrane following stimulation with 12-O-tetradecanoylphorbol-13-acetate. These results suggest a potential role of the lipid second messenger, PA, in the regulation of translocation and subsequent activation of Raf-1 in vivo.

  5. Substitution of the transmembrane domain of Vpu in simian-human immunodeficiency virus (SHIV{sub KU1bMC33}) with that of M2 of influenza A results in a virus that is sensitive to inhibitors of the M2 ion channel and is pathogenic for pig-tailed macaques

    SciTech Connect

    Hout, David R.; Gomez, Melissa L.; Pacyniak, Erik; Gomez, Lisa M.; Fegley, Barbara; Mulcahy, Ellyn R.; Hill, M. Sarah; Culley, Nathan; Pinson, David M.; Nothnick, Warren; Powers, Michael F.; Wong, Scott W.; Stephens, Edward B. . E-mail: estephen@kumc.edu

    2006-01-20

    the parental SHIV{sub KU-1bMC33} virus, two pig-tailed macaques were inoculated and followed for up to 8 months. Both pig-tailed macaques developed severe CD4{sup +} T cell loss within 1 month of inoculation, high viral loads, and histological lesions consistent with lymphoid depletion similar to the parental SHIV{sub KU-1bMC33}. Taken together, these results indicate for the first time that the TM domain of the Vpu protein can be functionally substituted with the TM of M2 of influenza A virus, and shows that compounds that target the TM domain of Vpu protein of HIV-1 could serve as novel anti-HIV-1 drugs.

  6. Modelling Cometary Sodium Tails

    NASA Astrophysics Data System (ADS)

    Birkett, K. S.; Jones, G. H.; Coates, A. J.

    2013-12-01

    Neutral sodium is readily observed in cometary spectra and can be seen to form its own distinct tail at high activity comets. Solar radiation pressure accelerates the sodium atoms antisunward and, as strong sodium absorption lines are present in the solar spectrum, the magnitude of this force is dependent upon the Doppler shift of the incident solar radiation. Therefore the heliocentric velocity of the sodium atom directly determines its acceleration. This can produce unique effects, such as a stagnation region. Sodium is relatively easy to detect and so can potentially be used to trace mechanisms in the coma that are otherwise difficult to observe. The source of neutral sodium in the tail currently remains unknown. We have therefore developed a new, three dimensional Monte-Carlo model of neutral cometary sodium in order to facilitate testing of different source production functions. It includes weightings due to neutral sodium lifetime, variation of cometary sodium emission due to Fraunhofer absorption lines and solar flux variation with heliocentric distance. The Swings and Greenstein effects, which can have particularly dramatic effects in near-Sun comets, are also considered comprehensively. Preliminary results from this model are presented, focusing on a comparison of predictions of the neutral sodium tail of Comet C/2012 S1 (ISON) with initial observations.

  7. Molecular modelling and experimental studies of mutation and cell-adhesion sites in the fibronectin type III and whey acidic protein domains of human anosmin-1.

    PubMed Central

    Robertson, A; MacColl, G S; Nash, J A; Boehm, M K; Perkins, S J; Bouloux, P M

    2001-01-01

    Anosmin-1, the gene product of the KAL gene, is implicated in the pathogenesis of X-linked Kallmann's syndrome. Anosmin-1 protein expression is restricted to the basement membrane and interstitial matrix of tissues affected in this syndrome during development. The anosmin-1 sequence indicates an N-terminal cysteine-rich domain, a whey acidic protein (WAP) domain, four fibronectin type III (FnIII) domains and a C-terminal histidine-rich region, and shows similarity with cell-adhesion molecules, such as neural cell-adhesion molecule, TAG-1 and L1. We investigated the structural and functional significance of three loss-of-function missense mutations of anosmin-1 using comparative modelling of the four FnIII and the WAP domains based on known NMR and crystal structures. Three missense mutation-encoded amino acid substitutions, N267K, E514K and F517L, were mapped to structurally defined positions on the GFCC' beta-sheet face of the first and third FnIII domains. Electrostatic maps demonstrated large basic surfaces containing clusters of conserved predicted heparan sulphate-binding residues adjacent to these mutation sites. To examine these modelling results anosmin-1 was expressed in insect cells. The incorporation of the three mutations into recombinant anosmin-1 had no effect on its secretion. The removal of two dibasic motifs that may constitute potential physiological cleavage sites for anosmin-1 had no effect on cleavage. Peptides based on the anosmin-1 sequences R254--K285 and P504--K527 were then synthesized in order to assess the effect of the three mutations on cellular adhesion, using cell lines that represented potential functional targets of anosmin-1. Peptides (10 microg/ml) incorporating the N267K and E514K substitutions promoted enhanced adhesion to 13.S.1.24 rat olfactory epithelial cells and canine MDCK1 kidney epithelial cells (P<0.01) compared with the wild-type peptides. This result was attributed to the introduction of a lysine residue adjacent to

  8. A tale of tails: How histone tails mediate chromatin compaction in different salt and linker histone environments

    PubMed Central

    Arya, Gaurav; Schlick, Tamar

    2009-01-01

    To elucidate the role of the histone tails in chromatin compaction and in higher-order folding of chromatin under physiological conditions, we extend a mesoscale model of chromatin [Arya, Zhang, and Schlick, Biophys. J. 91, 133 (2006); Arya and Schlick, Proc. Natl. Acad. Sci. USA 103, 16236 (2006)] to account for divalent cations (Mg2+) and linker histones. Configurations of 24-nucleosome oligonucleosomes in different salt environments and in the presence and absence of linker histones are sampled by a mixture of local and global Monte Carlo methods. Analyses of the resulting ensembles reveals a dynamic synergism between the histone tails, linker histones, and physiological ions in forming compact higher-order structures of chromatin. In the presence of monovalent salt alone, oligonucleosomes remain relatively unfolded and the histone tails do not mediate many internucleosomal interactions. Upon the addition of linker histones and divalent cations, the oligonucleosomes undergo a significant compaction triggered by: a dramatic increase in the internucleosomal interactions mediated by the histone tails; formation of a rigid linker DNA “stem” around the linker histones' C-terminal domains; and reducion in the electrostatic repulsion between linker DNAs via sharp bending in some linker DNAs caused by the divalent cations. Among all histone tails, the H4 tails mediate the most internucleosomal interactions, consistent with experimental observations, followed by the H3, H2A, and H2B tails in decreasing order. Apart from mediating internucleosomal interactions, the H3 tails also contribute to chromatin compaction by attaching to the entering and exiting linker DNA to screen electrotatic repulsion among the linker DNAs. This tendency of the H3 tails to attach to linker DNA, however, decreases significantly upon the addition of linker histones due to competition effects. The H2A and H2B tails do not mediate significant internucleosomal interactions but are important for

  9. Complete cDNA and derived amino acid sequence of human factor V.

    PubMed Central

    Jenny, R J; Pittman, D D; Toole, J J; Kriz, R W; Aldape, R A; Hewick, R M; Kaufman, R J; Mann, K G

    1987-01-01

    cDNA clones encoding human factor V have been isolated from an oligo(dT)-primed human fetal liver cDNA library prepared with vector Charon 21A. The cDNA sequence of factor V from three overlapping clones includes a 6672-base-pair (bp) coding region, a 90-bp 5' untranslated region, and a 163-bp 3' untranslated region within which is a poly(A) tail. The deduced amino acid sequence consists of 2224 amino acids inclusive of a 28-amino acid leader peptide. Direct comparison with human factor VIII reveals considerable homology between proteins in amino acid sequence and domain structure: a triplicated A domain and duplicated C domain show approximately equal to 40% identity with the corresponding domains in factor VIII. As in factor VIII, the A domains of factor V share approximately 40% amino acid-sequence homology with the three highly conserved domains in ceruloplasmin. The B domain of factor V contains 35 tandem and approximately 9 additional semiconserved repeats of nine amino acids of the form Asp-Leu-Ser-Gln-Thr-Thr/Asn-Leu-Ser-Pro and 2 additional semiconserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues. Images PMID:3110773

  10. Complete cDNA and derived amino acid sequence of human factor V

    SciTech Connect

    Jenny, R.J.; Pittman, D.D.; Toole, J.J.; Kriz, R.W.; Aldape, R.A.; Hewick, R.M.; Kaufman, R.J.; Mann, K.G.

    1987-07-01

    cDNA clones encoding human factor V have been isolated from an oligo(dT)-primed human fetal liver cDNA library prepared with vector Charon 21A. The cDNA sequence of factor V from three overlapping clones includes a 6672-base-pair (bp) coding region, a 90-bp 5' untranslated region, and a 163-bp 3' untranslated region within which is a poly(A)tail. The deduced amino acid sequence consists of 2224 amino acids inclusive of a 28-amino acid leader peptide. Direct comparison with human factor VIII reveals considerable homology between proteins in amino acid sequence and domain structure: a triplicated A domain and duplicated C domain show approx. 40% identity with the corresponding domains in factor VIII. As in factor VIII, the A domains of factor V share approx. 40% amino acid-sequence homology with the three highly conserved domains in ceruloplasmin. The B domain of factor V contains 35 tandem and approx. 9 additional semiconserved repeats of nine amino acids of the form Asp-Leu-Ser-Gln-Thr-Thr/Asn-Leu-Ser-Pro and 2 additional semiconserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues.

  11. NF-Y binding is required for transactivation of neuronal aromatic L-amino acid decarboxylase gene promoter by the POU-domain protein Brn-2.

    PubMed

    Dugast, C; Weber, M J

    2001-04-18

    We have previously characterized binding sites for the NF-Y transcription factor (-71/-52) and Brn-2 POU-domain protein (-92/-71) in the neuronal promoter of the human aromatic L-amino acid decarboxylase gene [Mol. Brain Res. 56 (1998) 227]. We have now explored the functional role of these binding sites in transfected SK-N-BE neuroblastoma cells. Mutations of the NF-Y site that abolish binding depressed expression of a luciferase reporter gene up to 25-fold. The overexpression of a dominant negative mutant of NF-YA subunit depressed expression by 60%. Promoter activity was increased by the overexpression of Brn-2. Mutations or deletion of the binding site of Brn-2 did not suppress transcriptional activation by overexpressed Brn-2, while promoters defective in NF-Y binding were not transactivated by Brn-2. A GST-pulldown experiment showed that recombinant human Brn-2 protein weakly interacts with recombinant NF-Y outside of DNA. Cooperative binding of recombinant NF-Y and GST--Brn-2 proteins on the neuronal promoter was evidenced by an electrophoretic mobility shift assay. The POU-domain of Brn-2 was sufficient for such interaction. The results thus suggest that the activation of the neuronal promoter of the aromatic L-amino acid decarboxylase gene requires a direct interaction between the ubiquitous NF-Y factor and a cell-specific POU-domain protein. The NF-Y, but not the Brn-2 binding site, is essential for the recruitment of the NF-Y/Brn-2 complex on the promoter. PMID:11311976

  12. Human ERCC5 cDNA-cosmid complementation for excision repair and bipartite amino acid domains conserved with RAD proteins of saccharomyces cerevisiae and schizosaccharomyces pombe

    SciTech Connect

    MacInnes, M.A.; Dickson, J.A.; Hernandez, R.R.; Lin, G.Y.; Park, M.S.; Schauer, S.; Reynolds, R.J.; Strniste, G.F. ); Learmonth, D. ); Mudgett, J.S. ); Yu, J.Y. )

    1993-10-01

    Several human genes related to DNA excision repair (ER) have been isolated via ER cross-species complementation (ERCC) of UV-sensitive CHO cells. The authors have now isolated and characterized cDNAs for the human ERCC5 gene that complement CHO UV135 cells. The ERCC5 mRNA size is about 4.6 kb. Their available cDNA clones are partial length, and no single clone was active for UV135 complementation. When cDNAs were mixed pairwise with a cosmid clone containing an overlapping 5[prime]-end segment of the ERCC5 gene, DNA transfer produced UV-resistant colonies with 60 to 95% correction of UV resistance relative to either a genomic ERCC5 DNA transformant or the CHO AA8 progenitor cells. cDNA-cosmid transformants regained intermediate levels (20 to 45%) of ER-dependent reactivation of a UV-damaged pSVCATgpt reporter plasmid. Their evidence strongly implicates an in situ recombination mechanism in cDNA-cosmid complementation for ER. The complete deduced amino acid sequence of ERCC5 was reconstructed for several cDNA clones encoding a predicted protein of 1,186 amino acids. The ERCC5 protein has extensive sequence similarities, in bipartite domains A and B, to products of RAD repair genes of two yeast, Saccharomyces cerevisiae RAD2 and Schizosaccharomyces pombe rad13. Sequence, structural, and functional data taken together indicate that ERCC5 and its relatives are probable functional homologs. A second locus represented by S. cerevisiae YKL510 and S. pombe rad2 genes is structurally distinct from the ERCC5 locus but retains vestigial A and B domain similarities. Their analyses suggest that ERCC5 is a nuclear-localized protein with one or more highly conserved helix-loop-helix segments within domains A and B. 69 refs., 6 figs., 1 tab.

  13. Identification of the plakoglobin-binding domain in desmoglein and its role in plaque assembly and intermediate filament anchorage

    PubMed Central

    1994-01-01

    The carboxyterminal cytoplasmic portions (tails) of desmosomal cadherins of both the desmoglein (Dsg) and desmocollin type are integral components of the desmosomal plaque and are involved in desmosome assembly and the anchorage of intermediate-sized filaments. When additional Dsg tails were introduced by cDNA transfection into cultured human epithelial cells, in the form of chimeras with the aminoterminal membrane insertion domain of rat connexin32 (Co32), the resulting stably transfected cells showed a dominant-negative defect specific for desmosomal junctions: despite the continual presence of all desmosomal proteins, the endogenous desmosomes disappeared and the formation of Co32-Dsg chimeric gap junctions was inhibited. Using cell transfection in combination with immunoprecipitation techniques, we have examined a series of deletion mutants of the Dsg1 tail in Co32-Dsg chimeras. We show that upon removal of the last 262 amino acids the truncated Dsg tail still effects the binding of plakoglobin but not of detectable amounts of any catenin and induces the dominant-negative phenotype. However, further truncation or excision of the next 41 amino acids, which correspond to the highly conserved carboxyterminus of the C-domain in other cadherins, abolishes plakoglobin binding and allows desmosomes to reform. Therefore, we conclude that this short segment provides a plakoglobin-binding site and is important for plaque assembly and the specific anchorage of either actin filaments in adherens junctions or IFs in desmosomes. PMID:7929560

  14. Staphylococcal superantigen-like protein 10 (SSL10) inhibits blood coagulation by binding to prothrombin and factor Xa via their γ-carboxyglutamic acid (Gla) domain.

    PubMed

    Itoh, Saotomo; Yokoyama, Ryosuke; Kamoshida, Go; Fujiwara, Toshinobu; Okada, Hiromi; Takii, Takemasa; Tsuji, Tsutomu; Fujii, Satoshi; Hashizume, Hideki; Onozaki, Kikuo

    2013-07-26

    The staphylococcal superantigen-like protein (SSL) family is composed of 14 exoproteins sharing structural similarity with superantigens but no superantigenic activity. Target proteins of four SSLs have been identified to be involved in host immune responses. However, the counterparts of other SSLs have been functionally uncharacterized. In this study, we have identified porcine plasma prothrombin as SSL10-binding protein by affinity purification using SSL10-conjugated Sepharose. The resin recovered the prodomain of prothrombin (fragment 1 + 2) as well as factor Xa in pull-down analysis. The equilibrium dissociation constant between SSL10 and prothrombin was 1.36 × 10(-7) M in surface plasmon resonance analysis. On the other hand, the resin failed to recover γ-carboxyglutamic acid (Gla) domain-less coagulation factors and prothrombin from warfarin-treated mice, suggesting that the Gla domain of the coagulation factors is essential for the interaction. SSL10 prolonged plasma clotting induced by the addition of Ca(2+) and factor Xa. SSL10 did not affect the protease activity of thrombin but inhibited the generation of thrombin activity in recalcified plasma. S. aureus produces coagulase that non-enzymatically activates prothrombin. SSL10 attenuated clotting induced by coagulase, but the inhibitory effect was weaker than that on physiological clotting, and SSL10 did not inhibit protease activity of staphylothrombin, the complex of prothrombin with coagulase. These results indicate that SSL10 inhibits blood coagulation by interfering with activation of coagulation cascade via binding to the Gla domain of coagulation factor but not by directly inhibiting thrombin activity. This is the first finding that the bacterial protein inhibits blood coagulation via targeting the Gla domain of coagulation factors.

  15. Speciation And Colloid Transport of Arsenic From Mine Tailings

    SciTech Connect

    Slowey, A.J.; Johnson, S.B.; Newville, M.; Brown, G.E., Jr.

    2007-07-13

    In addition to affecting biogeochemical transformations, the speciation of As also influences its transport from tailings at inoperative mines. The speciation of As in tailings from the Sulfur Bank Mercury Mine site in Clear Lake, California (USA) (a hot-spring Hg deposit) and particles mobilized from these tailings have been examined during laboratory-column experiments. Solutions containing two common, plant-derived organic acids (oxalic and citric acid) were pumped at 13 pore volumes d{sup -1} through 25 by 500 mm columns of calcined Hg ore, analogous to the pedogenesis of tailings. Chemical analysis of column effluent indicated that all of the As mobilized was particulate (1.5 mg, or 6% of the total As in the column through 255 pore volumes of leaching). Arsenic speciation was evaluated using X-ray absorption spectroscopy (XAS), indicating the dominance of arsenate [As(V)] sorbed to poorly crystalline Fe(III)-(hydr)oxides and coprecipitated with jarosite [KFe{sub 3}(SO{sub 4}, AsO{sub 4}){sub 2}(OH){sub 6}] with no detectable primary or secondary minerals in the tailings and mobilized particles. Sequential chemical extractions (SCE) of <45 {micro}m mine tailings fractions also suggest that As occurs adsorbed to Fe (hydr)oxides (35%) and coprecipitated within poorly crystalline phases (45%). In addition, SCEs suggest that As is associated with 1 N acid-soluble phases such as carbonate minerals (20%) and within crystalline Fe-(hydr)oxides (10%). The finding that As is transported from these mine tailings dominantly as As(V) adsorbed to Fe (hydr)oxides or coprecipitated within hydroxysulfates such as jarosite suggests that As release from soils and sediments contaminated with tailings will be controlled by either organic acid-promoted dissolution or reductive dissolution of host phases.

  16. The human ubiquitin-52 amino acid fusion protein gene shares several structural features with mammalian ribosomal protein genes.

    PubMed Central

    Baker, R T; Board, P G

    1991-01-01

    Complementary DNA clones encoding ubiquitin fused to a 52 amino acid tail protein were isolated from human placental and adrenal gland cDNA libraries. The deduced human 52 amino acid tail protein is very similar to the homologous protein from other species, including the conservation of the putative metal-binding, nucleic acid-binding domain observed in these proteins. Northern blot analysis with a tail-specific probe indicated that the previously identified UbA mRNA species most likely represents comigrating transcripts of the 52 amino acid tail (UbA52) and 80 amino acid tail (UbA80) ubiquitin fusion genes. The UbA52 gene was isolated from a human genomic library and consists of five exons distributed over 3400 base pairs. One intron is in the 5' non-coding region, two interrupt the single ubiquitin coding unit, and the fourth intron is within the tail coding region. Several members of the Alu family of repetitive DNA are associated with the gene. The UbA52 promoter has several features in common with mammalian ribosomal protein genes, including its location in a CpG-rich island, initiation of transcription within a polypyrimidine tract, the lack of a consensus TATA motif, and the presence of Sp1 binding sites, observations that are consistent with the recent identification of the ubiquitin-free tail proteins as ribosomal proteins. Thus, in spite of its unusual feature of being translationally fused to ubiquitin, the 52 amino acid tail ribosomal protein is expressed from a structurally typical ribosomal protein gene. Images PMID:1850507

  17. Amino acids 16-275 of minute virus of mice NS1 include a domain that specifically binds (ACCA)2-3-containing DNA.

    PubMed

    Mouw, M; Pintel, D J

    1998-11-10

    GST-NS1 purified from Escherichia coli and insect cells binds double-strand DNA in an (ACCA)2-3-dependent fashion under similar ionic conditions, independent of the presence of anti-NS1 antisera or exogenously supplied ATP and interacts with single-strand DNA and RNA in a sequence-independent manner. An amino-terminal domain (amino acids 1-275) of NS1 [GST-NS1(1-275)], representing 41% of the full-length NS1 molecule, includes a domain that binds double-strand DNA in a sequence-specific manner at levels comparable to full-length GST-NS1, as well as single-strand DNA and RNA in a sequence-independent manner. The deletion of 15 additional amino-terminal amino acids yielded a molecule [GST-NS1(1-275)] that maintained (ACCA)2-3-specific double-strand DNA binding; however, this molecule was more sensitive to increasing ionic conditions than full-length GST-NS1 and GST-NS1(1-275) and could not be demonstrated to bind single-strand nucleic acids. A quantitative filter binding assay showed that E. coli- and baculovirus-expressed GST-NS1 and E. coli GST-NS1(1-275) specifically bound double-strand DNA with similar equilibrium kinetics [as measured by their apparent equilibrium DNA binding constants (KD)], whereas GST-NS1(16-275) bound 4- to 8-fold less well.

  18. Mutational analysis of the Src SH3 domain: the same residues of the ligand binding surface are important for intra- and intermolecular interactions.

    PubMed Central

    Erpel, T; Superti-Furga, G; Courtneidge, S A

    1995-01-01

    The protein tyrosine kinase c-Src is negatively regulated by phosphorylation of Tyr527 in its C-terminal tail. The repressed state is achieved through intramolecular interactions involving the phosphorylated tail, the Src homology 2 (SH2) domain and the SH3 domain. Both the SH2 and SH3 domains have also been shown to mediate the intermolecular interaction of Src with several proteins. To test which amino acids of the Src SH3 domain are important for these interactions, and whether the intra- and intermolecular associations involve the same residues, we carried out a detailed mutational analysis of the presumptive interaction surface. All mutations of conserved hydrophobic residues had an effect on both inter- and intramolecular interactions of the Src SH3 domain, although not all amino acids were equally important. Chimeric molecules in which the Src SH3 domain was replaced with those of spectrin or Lck showed derepressed kinase activity, whereas a chimera containing the Fyn SH3 domain was fully regulated. Since spectrin and Lck SH3 domains share the conserved hydrophobic residues characteristic of SH3 domains, other amino acids must be important for specificity. Mutational analysis of non- or semi-conserved residues in the RT and n-Src loops showed that some of these were also involved in inter- and intramolecular interactions. Stable transfection of selected SH3 domain mutants into NIH-3T3 cells showed that despite elevated levels of phosphotyrosine, the cells were morphologically normal, indicating that the SH3 domain was required for efficient transformation of NIH-3T3 cells by Src. Images PMID:7534229

  19. Floods from tailings dam failures.

    PubMed

    Rico, M; Benito, G; Díez-Herrero, A

    2008-06-15

    This paper compiles the available information on historic tailings dam failures with the purpose to establish simple correlations between tailings ponds geometric parameters (e.g., dam height, tailings volume) and the hydraulic characteristics of floods resulting from released tailings. Following the collapse of a mining waste dam, only a part of tailings and polluted water stored at the dam is released, and this outflow volume is difficult to estimate prior the incident. In this study, tailings' volume stored at the time of failure was shown to have a good correlation (r2=0.86) with the tailings outflow volume, and the volume of spilled tailings was correlated with its run-out distance (r2=0.57). An envelope curve was drawn encompassing the majority of data points indicating the potential maximum downstream distance affected by a tailings' spill. The application of the described regression equations for prediction purposes needs to be treated with caution and with support of on-site measurement and observations. However, they may provide a universal baseline approximation on tailing outflow characteristics (even if detailed dam information is unavailable), which is of a great importance for risk analysis purposes.

  20. Influence of tailed-current on UXO prospecting

    NASA Astrophysics Data System (ADS)

    Zhang, Linlin; Zhang, Shuang; Chen, Shudong; Fu, Haoyang

    2015-08-01

    Time-domain electromagnetic method used in unexploded ordnance (UXO) detection has always faced the problem of the losing of early-time response due to tailed-current. In this article, the response of UXO like targets with different tailed-current are calculated and measured, and the influence of tailed-current on UXO prospecting is talked. The targets include a sphere, an iron pipe and a shell, and the tailed-current is set with switch-off time varies from 0μs to 230μs. According to magnetic surface modes(MSM), the step response of a compact steel target exhibits an early algebraic regime wherein the response transitions from t-1/2 to t-3/2 decay, followed by a late regime characterized by an exponentially decay. In fact, the transmitter current cannot be turned off immediately, especially for system with multiturn coil and large current. The switch-off process is decided by system parameters such as coil induction, coil resister, damping resister and maximum voltage across the coil. The response of the targets will be distorted dramatically by the tailed-current. The targets responses of tailed-current with different switch-off time are calculated through a convolution algorithm and measured with a specially designed system. The results show that the responses of UXO like targets are influenced by the tailed-current in two ways. Firstly, the primary response of the tailed-current will lead to signal saturation in the early times. Secondly, the off-time responses of UXO like targets are distorted by the tailed-current. All the influences will affect the system ability on detecting and discriminating the UXO like targets. An extra-fast switch-off system and deconvolution strategies are good advices to solve the problems.

  1. Cry1Aa binding to the cadherin receptor does not require conserved amino acid sequences in the domain II loops

    PubMed Central

    Fujii, Yuki; Tanaka, Shiho; Otsuki, Manami; Hoshino, Yasushi; Morimoto, Chinatsu; Kotani, Takuya; Harashima, Yuko; Endo, Haruka; Yoshizawa, Yasutaka; Sato, Ryoichi

    2012-01-01

    Characterizing the binding mechanism of Bt (Bacillus thuringiensis) Cry toxin to the cadherin receptor is indispensable to understanding the specific insecticidal activity of this toxin. To this end, we constructed 30 loop mutants by randomly inserting four serial amino acids covering all four receptor binding loops (loops α8, 1, 2 and 3) and analysed their binding affinities for Bombyx mori cadherin receptors via Biacore. High binding affinities were confirmed for all 30 mutants containing loop sequences that differed from those of wild-type. Insecticidal activities were confirmed in at least one mutant from loops 1, 2 and 3, suggesting that there is no critical amino acid sequence for the binding of the four loops to BtR175. When two mutations at different loops were integrated into one molecule, no reduction in binding affinity was observed compared with wild-type sequences. Based on these results, we discussed the binding mechanism of Cry toxin to cadherin protein. PMID:23145814

  2. The geomagnetic tail

    SciTech Connect

    Birn, J. )

    1991-01-01

    A review is presented of the plasma sheet and lobe regions of the magnetotail, focusing principally on large-scale processes or microprocesses with some large-scale effects. Consideration is given to quiet and average structures, not necessarily related to activity phases, with quasi-steady convection aspects, and with the characteristics of dynamic phases including acceleration mechanisms and single particle aspects. Attention is given to various activity models, average and quiet time properties, properties and effects of magnetospheric convection, dynamics of the magnetotail, and the near tail, substorm current wedge.

  3. Complete amino acid sequence of the variable domains of two human IgM anti-gamma globulins (Lay/Pom) with shared idiotypic specificities.

    PubMed

    Capra, J D; Klapper, D G

    1976-01-01

    On the basis of extensive shared idiotypic specificities, two human IgM anti-gamma-globulins (Lay/Pom) were selected for complete amino acid sequence analysis of their variable domains. Previous studies on the variable regions of the heavy chains of these proteins had shown but eight amino acid differences, only one of which was within a complementarity-determining hypervariable region. The complete amino acid sequence of the variable regions of the light chains of these two proteins is the subject of this report. Protein Lay is a typical VchiI protein with only five 'framework' differences when compared with protein Roy. Protein Pom is best classified as a VchiII, but in the 'framework' there are 16 differences between it and protein Ti. Although there are extensive differences in the first hypervariable region, the second and third light-chain hypervariable regions have an identical sequence. The finding of two identical light-chain and two identical heavy-chain hypervariable regions in these two proteins, which were selected on the basis of their combining specificities and their idiotypic cross-reactions, strongly implicates hypervariable regions in the constitution of the idiotypic determinants and the antibody combining site. Additionally, the finding of identical hypervariable regions in light chains of different V-region subgroups fulfills a prediction of the gene-interaction concept of antibody variability. PMID:824717

  4. Structural and functional characterization of a complex between the acidic transactivation domain of EBNA2 and the Tfb1/p62 subunit of TFIIH.

    PubMed

    Chabot, Philippe R; Raiola, Luca; Lussier-Price, Mathieu; Morse, Thomas; Arseneault, Genevieve; Archambault, Jacques; Omichinski, James G

    2014-03-01

    Infection with the Epstein-Barr virus (EBV) can lead to a number of human diseases including Hodgkin's and Burkitt's lymphomas. The development of these EBV-linked diseases is associated with the presence of nine viral latent proteins, including the nuclear antigen 2 (EBNA2). The EBNA2 protein plays a crucial role in EBV infection through its ability to activate transcription of both host and viral genes. As part of this function, EBNA2 associates with several host transcriptional regulatory proteins, including the Tfb1/p62 (yeast/human) subunit of the general transcription factor IIH (TFIIH) and the histone acetyltransferase CBP(CREB-binding protein)/p300, through interactions with its C-terminal transactivation domain (TAD). In this manuscript, we examine the interaction of the acidic TAD of EBNA2 (residues 431-487) with the Tfb1/p62 subunit of TFIIH and CBP/p300 using nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimeter (ITC) and transactivation studies in yeast. NMR studies show that the TAD of EBNA2 binds to the pleckstrin homology (PH) domain of Tfb1 (Tfb1PH) and that residues 448-471 (EBNA2₄₄₈₋₄₇₁) are necessary and sufficient for this interaction. NMR structural characterization of a Tfb1PH-EBNA2₄₄₈₋₄₇₁ complex demonstrates that the intrinsically disordered TAD of EBNA2 forms a 9-residue α-helix in complex with Tfb1PH. Within this helix, three hydrophobic amino acids (Trp458, Ile461 and Phe462) make a series of important interactions with Tfb1PH and their importance is validated in ITC and transactivation studies using mutants of EBNA2. In addition, NMR studies indicate that the same region of EBNA2 is also required for binding to the KIX domain of CBP/p300. This study provides an atomic level description of interactions involving the TAD of EBNA2 with target host proteins. In addition, comparison of the Tfb1PH-EBNA2₄₄₈₋₄₇₁ complex with structures of the TAD of p53 and VP16 bound to Tfb1

  5. Structural and Functional Characterization of a Complex between the Acidic Transactivation Domain of EBNA2 and the Tfb1/p62 Subunit of TFIIH

    PubMed Central

    Lussier-Price, Mathieu; Morse, Thomas; Arseneault, Genevieve; Archambault, Jacques; Omichinski, James G.

    2014-01-01

    Infection with the Epstein-Barr virus (EBV) can lead to a number of human diseases including Hodgkin's and Burkitt's lymphomas. The development of these EBV-linked diseases is associated with the presence of nine viral latent proteins, including the nuclear antigen 2 (EBNA2). The EBNA2 protein plays a crucial role in EBV infection through its ability to activate transcription of both host and viral genes. As part of this function, EBNA2 associates with several host transcriptional regulatory proteins, including the Tfb1/p62 (yeast/human) subunit of the general transcription factor IIH (TFIIH) and the histone acetyltransferase CBP(CREB-binding protein)/p300, through interactions with its C-terminal transactivation domain (TAD). In this manuscript, we examine the interaction of the acidic TAD of EBNA2 (residues 431–487) with the Tfb1/p62 subunit of TFIIH and CBP/p300 using nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimeter (ITC) and transactivation studies in yeast. NMR studies show that the TAD of EBNA2 binds to the pleckstrin homology (PH) domain of Tfb1 (Tfb1PH) and that residues 448–471 (EBNA2448–471) are necessary and sufficient for this interaction. NMR structural characterization of a Tfb1PH-EBNA2448–471 complex demonstrates that the intrinsically disordered TAD of EBNA2 forms a 9-residue α-helix in complex with Tfb1PH. Within this helix, three hydrophobic amino acids (Trp458, Ile461 and Phe462) make a series of important interactions with Tfb1PH and their importance is validated in ITC and transactivation studies using mutants of EBNA2. In addition, NMR studies indicate that the same region of EBNA2 is also required for binding to the KIX domain of CBP/p300. This study provides an atomic level description of interactions involving the TAD of EBNA2 with target host proteins. In addition, comparison of the Tfb1PH-EBNA2448–471 complex with structures of the TAD of p53 and VP16 bound to Tfb1PH highlights the versatility of

  6. Structural and functional characterization of a complex between the acidic transactivation domain of EBNA2 and the Tfb1/p62 subunit of TFIIH.

    PubMed

    Chabot, Philippe R; Raiola, Luca; Lussier-Price, Mathieu; Morse, Thomas; Arseneault, Genevieve; Archambault, Jacques; Omichinski, James G

    2014-03-01

    Infection with the Epstein-Barr virus (EBV) can lead to a number of human diseases including Hodgkin's and Burkitt's lymphomas. The development of these EBV-linked diseases is associated with the presence of nine viral latent proteins, including the nuclear antigen 2 (EBNA2). The EBNA2 protein plays a crucial role in EBV infection through its ability to activate transcription of both host and viral genes. As part of this function, EBNA2 associates with several host transcriptional regulatory proteins, including the Tfb1/p62 (yeast/human) subunit of the general transcription factor IIH (TFIIH) and the histone acetyltransferase CBP(CREB-binding protein)/p300, through interactions with its C-terminal transactivation domain (TAD). In this manuscript, we examine the interaction of the acidic TAD of EBNA2 (residues 431-487) with the Tfb1/p62 subunit of TFIIH and CBP/p300 using nuclear magnetic resonance (NMR) spectroscopy, isothermal titration calorimeter (ITC) and transactivation studies in yeast. NMR studies show that the TAD of EBNA2 binds to the pleckstrin homology (PH) domain of Tfb1 (Tfb1PH) and that residues 448-471 (EBNA2₄₄₈₋₄₇₁) are necessary and sufficient for this interaction. NMR structural characterization of a Tfb1PH-EBNA2₄₄₈₋₄₇₁ complex demonstrates that the intrinsically disordered TAD of EBNA2 forms a 9-residue α-helix in complex with Tfb1PH. Within this helix, three hydrophobic amino acids (Trp458, Ile461 and Phe462) make a series of important interactions with Tfb1PH and their importance is validated in ITC and transactivation studies using mutants of EBNA2. In addition, NMR studies indicate that the same region of EBNA2 is also required for binding to the KIX domain of CBP/p300. This study provides an atomic level description of interactions involving the TAD of EBNA2 with target host proteins. In addition, comparison of the Tfb1PH-EBNA2₄₄₈₋₄₇₁ complex with structures of the TAD of p53 and VP16 bound to Tfb1

  7. Crystallographic Insights into the Autocatalytic Assembly Mechanism of a Bacteriophage Tail Spike

    SciTech Connect

    Xiang, Ye; Leiman, Petr G.; Li, Long; Grimes, Shelley; Anderson, Dwight L.; Rossmann, Michael G.

    2010-02-03

    The tailed bacteriophage phi29 has 12 'appendages' (gene product 12, gp12) attached to its neck region that participate in host cell recognition and entry. In the cell, monomeric gp12 undergoes proteolytic processing that releases the C-terminal domain during assembly into trimers. We report here crystal structures of the protein before and after catalytic processing and show that the C-terminal domain of gp12 is an 'autochaperone' that aids trimerization. We also show that autocleavage of the C-terminal domain is a posttrimerization event that is followed by a unique ATP-dependent release. The posttranslationally modified N-terminal part has three domains that function to attach the appendages to the phage, digest the cell wall teichoic acids, and bind irreversibly to the host, respectively. Structural and sequence comparisons suggest that some eukaryotic and bacterial viruses as well as bacterial adhesins might have a similar maturation mechanism as is performed by phi29 gp12 for Bacillus subtilis.

  8. Kinesin's light chains inhibit the head- and microtubule-binding activity of its tail.

    PubMed

    Wong, Yao Liang; Rice, Sarah E

    2010-06-29

    Kinesin-1 is a microtubule-based motor comprising two heavy chains (KHCs) and two light chains (KLCs). Motor activity is precisely regulated to avoid futile ATP consumption and to ensure proper intracellular localization of kinesin-1 and its cargoes. The KHC tail inhibits ATPase activity by interacting with the enzymatic KHC heads, and the tail also binds microtubules. Here, we present a role for the KLCs in regulating both the head- and microtubule-binding activities of the kinesin-1 tail. We show that KLCs reduce the affinity of the head-tail interaction over tenfold and concomitantly repress the tail's regulatory activity. We also show that KLCs inhibit tail-microtubule binding by a separate mechanism. Inhibition of head-tail binding requires steric and electrostatic factors. Inhibition of tail-microtubule binding is largely electrostatic, pH dependent, and mediated partly by a highly negatively charged linker region between the KHC-interacting and cargo-binding domains of the KLCs. Our data support a model wherein KLCs promote activation of kinesin-1 for cargo transport by simultaneously suppressing tail-head and tail-microtubule interactions. KLC-mediated inhibition of tail-microtubule binding may also influence diffusional movement of kinesin-1 on microtubules, and kinesin-1's role in microtubule transport/sliding. PMID:20547877

  9. Susceptibility to virus-cell fusion at the plasma membrane is reduced through expression of HIV gp41 cytoplasmic domains.

    PubMed

    Malinowsky, Katharina; Luksza, Julia; Dittmar, Matthias T

    2008-06-20

    The cytoplasmic tail of the HIV transmembrane protein plays an important role in viral infection. In this study we analyzed the role of retroviral cytoplasmic tails in modulating the cytoskeleton and interfering with virus-cell fusion. HeLaP4 cells expressing different HIV cytoplasmic tail constructs showed reduced acetylated tubulin levels whereas the cytoplasmic tail of MLV did not alter microtubule stability indicating a unique function for the lentiviral cytoplasmic tail. The effect on tubulin is mediated through the membrane proximal region of the HIV cytoplasmic tail and was independent of membrane localization. Site-directed mutagenesis identified three motifs in the HIV-2 cytoplasmic tail required to effect the reduction in acetylated tubulin. Both the YxxPhi domain and amino acids 21 to 45 of the HIV-2 cytoplasmic tail need to be present to change the level of acetylated tubulin in transfected cells. T-cells stably expressing one HIV-2 cytoplasmic tail derived construct showed also a reduction in acetylated tubulin thus confirming the importance of this effect not only for HeLaP4 and 293T cells. Challenge experiments using transiently transfected HeLaP4 cells and T cells stably expressing an HIV cytoplasmic tail construct revealed both reduced virus-cell fusion and replication of HIV-1(NL4.3) compared to control cells. In the virus-cell fusion assay only virions pseudotyped with either HIV or MLV envelopes showed reduced fusion efficiency, whereas VSV-G pseudotyped virions where not affected by the expression of HIV derived cytoplasmic tail constructs, indicating that fusion at the plasma but not endosomal membrane is affected. Overexpression of human histone-deacetylase 6 (HDAC6) and constitutively active RhoA resulted in a reduction of acetylated tubulin and reduced virus-cell fusion as significant as that observed following expression of HIV cytoplasmic tail constructs. Inhibition of HDAC6 showed a strong increase in acetylated tubulin and increase of

  10. Susceptibility to virus-cell fusion at the plasma membrane is reduced through expression of HIV gp41 cytoplasmic domains

    SciTech Connect

    Malinowsky, Katharina; Luksza, Julia; Dittmar, Matthias T.

    2008-06-20

    The cytoplasmic tail of the HIV transmembrane protein plays an important role in viral infection. In this study we analyzed the role of retroviral cytoplasmic tails in modulating the cytoskeleton and interfering with virus-cell fusion. HeLaP4 cells expressing different HIV cytoplasmic tail constructs showed reduced acetylated tubulin levels whereas the cytoplasmic tail of MLV did not alter microtubule stability indicating a unique function for the lentiviral cytoplasmic tail. The effect on tubulin is mediated through the membrane proximal region of the HIV cytoplasmic tail and was independent of membrane localization. Site-directed mutagenesis identified three motifs in the HIV-2 cytoplasmic tail required to effect the reduction in acetylated tubulin. Both the Yxx{phi} domain and amino acids 21 to 45 of the HIV-2 cytoplasmic tail need to be present to change the level of acetylated tubulin in transfected cells. T-cells stably expressing one HIV-2 cytoplasmic tail derived construct showed also a reduction in acetylated tubulin thus confirming the importance of this effect not only for HeLaP4 and 293T cells. Challenge experiments using transiently transfected HeLaP4 cells and T cells stably expressing an HIV cytoplasmic tail construct revealed both reduced virus-cell fusion and replication of HIV-1{sub NL4.3} compared to control cells. In the virus-cell fusion assay only virions pseudotyped with either HIV or MLV envelopes showed reduced fusion efficiency, whereas VSV-G pseudotyped virions where not affected by the expression of HIV derived cytoplasmic tail constructs, indicating that fusion at the plasma but not endosomal membrane is affected. Overexpression of human histone-deacetylase 6 (HDAC6) and constitutively active RhoA resulted in a reduction of acetylated tubulin and reduced virus-cell fusion as significant as that observed following expression of HIV cytoplasmic tail constructs. Inhibition of HDAC6 showed a strong increase in acetylated tubulin and

  11. The multi-domain protein Np95 connects DNA methylation and histone modification.

    PubMed

    Rottach, Andrea; Frauer, Carina; Pichler, Garwin; Bonapace, Ian Marc; Spada, Fabio; Leonhardt, Heinrich

    2010-04-01

    DNA methylation and histone modifications play a central role in the epigenetic regulation of gene expression and cell differentiation. Recently, Np95 (also known as UHRF1 or ICBP90) has been found to interact with Dnmt1 and to bind hemimethylated DNA, indicating together with genetic studies a central role in the maintenance of DNA methylation. Using in vitro binding assays we observed a weak preference of Np95 and its SRA (SET- and Ring-associated) domain for hemimethylated CpG sites. However, the binding kinetics of Np95 in living cells was not affected by the complete loss of genomic methylation. Investigating further links with heterochromatin, we could show that Np95 preferentially binds histone H3 N-terminal tails with trimethylated (H3K9me3) but not acetylated lysine 9 via a tandem Tudor domain. This domain contains three highly conserved aromatic amino acids that form an aromatic cage similar to the one binding H3K9me3 in the chromodomain of HP1ss. Mutations targeting the aromatic cage of the Np95 tandem Tudor domain (Y188A and Y191A) abolished specific H3 histone tail binding. These multiple interactions of the multi-domain protein Np95 with hemimethylated DNA and repressive histone marks as well as with DNA and histone methyltransferases integrate the two major epigenetic silencing pathways. PMID:20026581

  12. Escherichia coli methionyl-tRNA formyltransferase: role of amino acids conserved in the linker region and in the C-terminal domain on the specific recognition of the initiator tRNA.

    PubMed

    Gite, S; Li, Y; Ramesh, V; RajBhandary, U L

    2000-03-01

    The formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is important for the initiation of protein synthesis in eubacteria. We are studying the molecular mechanisms of recognition of the initiator tRNA by Escherichia coli MTF. MTF from eubacteria contains an approximately 100-amino acid C-terminal extension that is not found in the E. coli glycinamide ribonucleotide formyltransferase, which, like MTF, use N(10)-formyltetrahydrofolate as a formyl group donor. This C-terminal extension, which forms a distinct structural domain, is attached to the N-terminal domain through a linker region. Here, we describe the effect of (i) substitution mutations on some nineteen basic, aromatic and other conserved amino acids in the linker region and in the C-terminal domain of MTF and (ii) deletion mutations from the C-terminus on enzyme activity. We show that the positive charge on two of the lysine residues in the linker region leading to the C-terminal domain are important for enzyme activity. Mutation of some of the basic amino acids in the C-terminal domain to alanine has mostly small effects on the kinetic parameters, whereas mutation to glutamic acid has large effects. However, the deletion of 18, 20, or 80 amino acids from the C-terminus has very large effects on enzyme activity. Overall, our results support the notion that the basic amino acid residues in the C-terminal domain provide a positively charged channel that is used for the nonspecific binding of tRNA, whereas some of the amino acids in the linker region play an important role in activity of MTF.

  13. Long tail kinetics in biophysics?

    PubMed Central

    Nagle, J F

    1992-01-01

    Long tail kinetics describe a variety of data from complex, disordered materials that cannot be described by conventional kinetics. It is suggested that the kinetics of diffusive motion in complex biological media, such as cytoplasm or biomembranes, might also have long tails. The effects of long tail kinetics are investigated for two standard biophysical measurements, fluorescence recovery after photobleaching (FRAP), and dynamic light scattering (DLS). It is shown that long tail kinetic data would yield significantly distorted and misleading results when analyzed assuming conventional kinetics. PMID:1420883

  14. Dopamine uptake and cocaine binding mechanisms: the involvement of charged amino acids from the transmembrane domains of the human dopamine transporter.

    PubMed

    Dar, Dalit E; Metzger, Thomas G; Vandenbergh, David J; Uhl, George R

    2006-05-24

    The wild type human dopamine transporter (DAT) and five DAT mutants were transfected into COS-7 cells and their ability to uptake dopamine or to bind cocaine was examine three days later. In each mutant, a single charged amino acid, located in areas that initial hydrophobic analysis had indicated were DAT transmembrane domains was substituted by alanine. Mutants used in this study were lysines 257 and 525 (termed K257A and K525A), arginines 283 and 521 (termed R283A and R521A), and glutamate 491 (termed E491A). Dopamine affinity was significantly enhanced in the K257A and R283A mutants, and the IC(50) for displacement of the radioactive cocaine analog 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane (CFT) by cocaine was significantly elevated in the E491A mutant. All mutants displayed a reduction or complete loss of the maximal velocity (V(m)) of dopamine transport. PMID:16674939

  15. Biochemical and virological analysis of the 18-residue C-terminal tail of HIV-1 integrase

    PubMed Central

    Dar, Mohd J; Monel, Blandine; Krishnan, Lavanya; Shun, Ming-Chieh; Di Nunzio, Francesca; Helland, Dag E; Engelman, Alan

    2009-01-01

    Background The 18 residue tail abutting the SH3 fold that comprises the heart of the C-terminal domain is the only part of HIV-1 integrase yet to be visualized by structural biology. To ascertain the role of the tail region in integrase function and HIV-1 replication, a set of deletion mutants that successively lacked three amino acids was constructed and analyzed in a variety of biochemical and virus infection assays. HIV-1/2 chimers, which harbored the analogous 23-mer HIV-2 tail in place of the HIV-1 sequence, were also studied. Because integrase mutations can affect steps in the replication cycle other than integration, defective mutant viruses were tested for integrase protein content and reverse transcription in addition to integration. The F185K core domain mutation, which increases integrase protein solubility, was furthermore analyzed in a subset of mutants. Results Purified proteins were assessed for in vitro levels of 3' processing and DNA strand transfer activities whereas HIV-1 infectivity was measured using luciferase reporter viruses. Deletions lacking up to 9 amino acids (1-285, 1-282, and 1-279) displayed near wild-type activities in vitro and during infection. Further deletion yielded two viruses, HIV-11-276 and HIV-11-273, that displayed approximately two and 5-fold infectivity defects, respectively, due to reduced integrase function. Deletion mutant HIV-11-270 and the HIV-1/2 chimera were non-infectious and displayed approximately 3 to 4-fold reverse transcription in addition to severe integration defects. Removal of four additional residues, which encompassed the C-terminal β strand of the SH3 fold, further compromised integrase incorporation into virions and reverse transcription. Conclusion HIV-11-270, HIV-11-266, and the HIV-1/2 chimera were typed as class II mutant viruses due to their pleiotropic replication defects. We speculate that residues 271-273 might play a role in mediating the known integrase-reverse transcriptase interaction, as

  16. The cysteine-rich region and the whey acidic protein domain are essential for anosmin-1 biological functions.

    PubMed

    Esteban, Pedro F; Murcia-Belmonte, Verónica; García-González, Diego; de Castro, Fernando

    2013-03-01

    The protein anosmin-1, coded by the KAL1 gene responsible for the X-linked form of Kallmann syndrome (KS), exerts its biological effects mainly through the interaction with and signal modulation of fibroblast growth factor receptor 1 (FGFR1). We have previously shown the interaction of the third fibronectin-like type 3 (FnIII) domain and the N-terminal region of anosmin-1 with FGFR1. Here, we demonstrate that missense mutations reported in patients with KS, C172R and N267K did not alter or substantially reduce, respectively, the binding to FGFR1. These substitutions annulled the chemoattraction of the full-length protein over subventricular zone (SVZ) neuronal precursors (NPs), but they did not annul it in the N-terminal-truncated protein (A1Nt). We also show that although not essential for binding to FGFR1, the cysteine-rich (CR) region is necessary for anosmin-1 function and that FnIII.3 cannot substitute for FnIII.1 function. Truncated proteins recapitulating nonsense mutations found in KS patients did not show the chemotropic effect on SVZ NPs, suggesting that the presence behind FnIII.1 of any part of anosmin-1 produces an unstable protein incapable of action. We also identify the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway as necessary for the chemotropic effect exerted by FGF2 and anosmin-1 on rat SVZ NPs.

  17. Germ plasm localisation of the HELICc of Vasa in Drosophila: analysis of domain sufficiency and amino acids critical for localisation

    PubMed Central

    Wang, Szu-Chieh; Hsu, Hao-Jen; Lin, Gee-way; Wang, Ting-Fang; Chang, Chun-che; Lin, Ming-Der

    2015-01-01

    Formation of the germ plasm drives germline specification in Drosophila and some other insects such as aphids. Identification of the DEAD-box protein Vasa (Vas) as a conserved germline marker in flies and aphids suggests that they share common components for assembling the germ plasm. However, to which extent the assembly order is conserved and the correlation between functions and sequences of Vas remain unclear. Ectopic expression of the pea aphid Vas (ApVas1) in Drosophila did not drive its localisation to the germ plasm, but ApVas1 with a replaced C-terminal domain (HELICc) of Drosophila Vas (DmVas) became germ-plasm restricted. We found that HELICc itself, through the interaction with Oskar (Osk), was sufficient for germ-plasm localisation. Similarly, HELICc of the grasshopper Vas could be recruited to the germ plasm in Drosophila. Nonetheless, germ-plasm localisation was not seen in the Drosophila oocytes expressing HELICcs of Vas orthologues from aphids, crickets, and mice. We further identified that glutamine (Gln) 527 within HELICc of DmVas was critical for localisation, and its corresponding residue could also be detected in grasshopper Vas yet missing in the other three species. This suggests that Gln527 is a direct target of Osk or critical to the maintenance of HELICc conformation. PMID:26419889

  18. Germ plasm localisation of the HELICc of Vasa in Drosophila: analysis of domain sufficiency and amino acids critical for localisation

    NASA Astrophysics Data System (ADS)

    Wang, Szu-Chieh; Hsu, Hao-Jen; Lin, Gee-Way; Wang, Ting-Fang; Chang, Chun-Che; Lin, Ming-Der

    2015-09-01

    Formation of the germ plasm drives germline specification in Drosophila and some other insects such as aphids. Identification of the DEAD-box protein Vasa (Vas) as a conserved germline marker in flies and aphids suggests that they share common components for assembling the germ plasm. However, to which extent the assembly order is conserved and the correlation between functions and sequences of Vas remain unclear. Ectopic expression of the pea aphid Vas (ApVas1) in Drosophila did not drive its localisation to the germ plasm, but ApVas1 with a replaced C-terminal domain (HELICc) of Drosophila Vas (DmVas) became germ-plasm restricted. We found that HELICc itself, through the interaction with Oskar (Osk), was sufficient for germ-plasm localisation. Similarly, HELICc of the grasshopper Vas could be recruited to the germ plasm in Drosophila. Nonetheless, germ-plasm localisation was not seen in the Drosophila oocytes expressing HELICcs of Vas orthologues from aphids, crickets, and mice. We further identified that glutamine (Gln) 527 within HELICc of DmVas was critical for localisation, and its corresponding residue could also be detected in grasshopper Vas yet missing in the other three species. This suggests that Gln527 is a direct target of Osk or critical to the maintenance of HELICc conformation.

  19. Germ plasm localisation of the HELICc of Vasa in Drosophila: analysis of domain sufficiency and amino acids critical for localisation.

    PubMed

    Wang, Szu-Chieh; Hsu, Hao-Jen; Lin, Gee-way; Wang, Ting-Fang; Chang, Chun-che; Lin, Ming-Der

    2015-09-30

    Formation of the germ plasm drives germline specification in Drosophila and some other insects such as aphids. Identification of the DEAD-box protein Vasa (Vas) as a conserved germline marker in flies and aphids suggests that they share common components for assembling the germ plasm. However, to which extent the assembly order is conserved and the correlation between functions and sequences of Vas remain unclear. Ectopic expression of the pea aphid Vas (ApVas1) in Drosophila did not drive its localisation to the germ plasm, but ApVas1 with a replaced C-terminal domain (HELICc) of Drosophila Vas (DmVas) became germ-plasm restricted. We found that HELICc itself, through the interaction with Oskar (Osk), was sufficient for germ-plasm localisation. Similarly, HELICc of the grasshopper Vas could be recruited to the germ plasm in Drosophila. Nonetheless, germ-plasm localisation was not seen in the Drosophila oocytes expressing HELICcs of Vas orthologues from aphids, crickets, and mice. We further identified that glutamine (Gln) 527 within HELICc of DmVas was critical for localisation, and its corresponding residue could also be detected in grasshopper Vas yet missing in the other three species. This suggests that Gln527 is a direct target of Osk or critical to the maintenance of HELICc conformation.

  20. Two Proteases, Trypsin Domain-containing 1 (Tysnd1) and Peroxisomal Lon Protease (PsLon), Cooperatively Regulate Fatty Acid β-Oxidation in Peroxisomal Matrix*

    PubMed Central

    Okumoto, Kanji; Kametani, Yukari; Fujiki, Yukio

    2011-01-01

    The molecular mechanisms underlying protein turnover and enzyme regulation in the peroxisomal matrix remain largely unknown. Trypsin domain-containing 1 (Tysnd1) and peroxisomal Lon protease (PsLon) are newly identified peroxisomal matrix proteins that harbor both a serine protease-like domain and a peroxisome-targeting signal 1 (PTS1) sequence. Tysnd1 processes several PTS1-containing proteins and cleaves N-terminal presequences from PTS2-containing protein precursors. Here we report that knockdown of Tysnd1, but not PsLon, resulted in accumulation of endogenous β-oxidation enzymes in their premature form. The protease activity of Tysnd1 was inactivated by intermolecular self-conversion of the 60-kDa form to 15- and 45-kDa chains, which were preferentially degraded by PsLon. Peroxisomal β-oxidation of a very long fatty acid was significantly decreased by knockdown of Tysnd1 and partially lowered by PsLon knockdown. Taken together, these data suggest that Tysnd1 is a key regulator of the peroxisomal β-oxidation pathway via proteolytic processing of β-oxidation enzymes. The proteolytic activity of oligomeric Tysnd1 is in turn controlled by self-cleavage of Tysnd1 and degradation of Tysnd1 cleavage products by PsLon. PMID:22002062

  1. Identification of a Lipoteichoic Acid Glycosyltransferase Enzyme Reveals that GW-Domain-Containing Proteins Can Be Retained in the Cell Wall of Listeria monocytogenes in the Absence of Lipoteichoic Acid or Its Modifications

    PubMed Central

    Percy, Matthew G.; Karinou, Eleni; Webb, Alexander J.

    2016-01-01

    ABSTRACT Listeria monocytogenes is a foodborne Gram-positive bacterial pathogen, and many of its virulence factors are either secreted proteins or proteins covalently or noncovalently attached to the cell wall. Previous work has indicated that noncovalently attached proteins with GW (glycine-tryptophan) domains are retained in the cell wall by binding to the cell wall polymer lipoteichoic acid (LTA). LTA is a glycerol phosphate polymer, which is modified in L. monocytogenes with galactose and d-alanine residues. We identified Lmo0933 as the cytoplasmic glycosyltransferase required for the LTA glycosylation process and renamed the protein GtlA, for glycosyltransferase LTA A. Using L. monocytogenes mutants lacking galactose or d-alanine modifications or the complete LTA polymer, we show that GW domain proteins are retained within the cell wall, indicating that other cell wall polymers are involved in the retention of GW domain proteins. Further experiments revealed peptidoglycan as the binding receptor as a purified GW domain fusion protein can bind to L. monocytogenes cells lacking wall teichoic acid (WTA) as well as purified peptidoglycan derived from a wild-type or WTA-negative strain. With this, we not only identify the first enzyme involved in the LTA glycosylation process, but we also provide new insight into the binding mechanism of noncovalently attached cell wall proteins. IMPORTANCE Over the past 20 years, a large number of bacterial genome sequences have become available. Computational approaches are used for the genome annotation and identification of genes and encoded proteins. However, the function of many proteins is still unknown and often cannot be predicted bioinformatically. Here, we show that the previously uncharacterized Listeria monocytogenes gene lmo0933 likely codes for a glycosyltransferase required for the decoration of the cell wall polymer lipoteichoic acid (LTA) with galactose residues. Using L. monocytogenes mutants lacking LTA

  2. The multivalent PDZ domain-containing protein CIPP is a partner of acid-sensing ion channel 3 in sensory neurons.

    PubMed

    Anzai, Naohiko; Deval, Emmanuel; Schaefer, Lionel; Friend, Valerie; Lazdunski, Michel; Lingueglia, Eric

    2002-05-10

    Acid-sensing ion channels (ASICs) are cationic channels activated by extracellular pH. They are present in the brain, where they are thought to participate in signal transduction associated with local pH variations, and in sensory neurons, where they have been involved in pain perception associated with tissue acidosis and in mechanoperception. The ASIC3 subunit is mainly expressed in dorsal root ganglion neurons. Its expression is associated with a rapidly inactivating current followed by a slowly activating sustained current thought to be required for the tonic sensation of pain caused by acids. We report here the interaction of this channel subunit with the multivalent PDZ (PSD-95 Drosophila discs-large protein, Zonula occludens protein 1) domain-containing protein CIPP. This interaction requires the C-terminal region of ASIC3 and the fourth PDZ domain of CIPP. Co-expression of CIPP and ASIC3 in COS cells increases the maximal ASIC3 peak current density by a factor of 5 and slightly shifts the pH(0.5) for activation from pH 6.2 to pH 6.4. CIPP mRNA is found at a significant level in the same dorsal root ganglion neuronal cell population that expresses the ASIC3 subunit, i.e. mainly in the small nociceptive neurons. CIPP is thus a scaffolding protein that could both enhance the surface expression of ASIC3 and bring together ASIC3 and functionally related proteins in the membrane of sensory neurons.

  3. Distinct Amino Acids in the C-Linker Domain of the Arabidopsis K+ Channel KAT2 Determine Its Subcellular Localization and Activity at the Plasma Membrane1[W

    PubMed Central

    Nieves-Cordones, Manuel; Chavanieu, Alain; Jeanguenin, Linda; Alcon, Carine; Szponarski, Wojciech; Estaran, Sebastien; Chérel, Isabelle; Zimmermann, Sabine; Sentenac, Hervé; Gaillard, Isabelle

    2014-01-01

    Shaker K+ channels form the major K+ conductance of the plasma membrane in plants. They are composed of four subunits arranged around a central ion-conducting pore. The intracellular carboxy-terminal region of each subunit contains several regulatory elements, including a C-linker region and a cyclic nucleotide-binding domain (CNBD). The C-linker is the first domain present downstream of the sixth transmembrane segment and connects the CNBD to the transmembrane core. With the aim of identifying the role of the C-linker in the Shaker channel properties, we performed subdomain swapping between the C-linker of two Arabidopsis (Arabidopsis thaliana) Shaker subunits, K+ channel in Arabidopsis thaliana2 (KAT2) and Arabidopsis thaliana K+ rectifying channel1 (AtKC1). These two subunits contribute to K+ transport in planta by forming heteromeric channels with other Shaker subunits. However, they display contrasting behavior when expressed in tobacco mesophyll protoplasts: KAT2 forms homotetrameric channels active at the plasma membrane, whereas AtKC1 is retained in the endoplasmic reticulum when expressed alone. The resulting chimeric/mutated constructs were analyzed for subcellular localization and functionally characterized. We identified two contiguous amino acids, valine-381 and serine-382, located in the C-linker carboxy-terminal end, which prevent KAT2 surface expression when mutated into the equivalent residues from AtKC1. Moreover, we demonstrated that the nine-amino acid stretch 312TVRAASEFA320 that composes the first C-linker α-helix located just below the pore is a crucial determinant of KAT2 channel activity. A KAT2 C-linker/CNBD three-dimensional model, based on animal HCN (for Hyperpolarization-activated, cyclic nucleotide-gated K+) channels as structure templates, has been built and used to discuss the role of the C-linker in plant Shaker inward channel structure and function. PMID:24406792

  4. A three amino acid deletion in the transmembrane domain of the nicotinic acetylcholine receptor α6 subunit confers high-level resistance to spinosad in Plutella xylostella

    PubMed Central

    Wang, Jing; Wang, Xingliang; Lansdell, Stuart J.; Zhang, Jianheng; Millar, Neil S.; Wu, Yidong

    2016-01-01

    Spinosad is a macrocyclic lactone insecticide that acts primarily at the nicotinic acetylcholine receptors (nAChRs) of target insects. Here we describe evidence that high levels of resistance to spinosad in the diamondback moth (Plutella xylostella) are associated with a three amino acid (3-aa) deletion in the fourth transmembrane domain (TM4) of the nAChR α6 subunit (Pxα6). Following laboratory selection with spinosad, the SZ-SpinR strain of P. xylostella exhibited 940-fold resistance to spinosad. In addition, the selected insect population had 1060-fold cross-resistance to spinetoram but, in contrast, no cross-resistance to abamectin was observed. Genetic analysis indicates that spinosad resistance in SZ-SpinR is inherited as a recessive and autosomal trait, and that the 3-aa deletion (IIA) in TM4 of Pxα6 is tightly linked to spinosad resistance. Because of well-established difficulties in functional expression of cloned insect nAChRs, the analogous resistance-associated deletion mutation was introduced into a prototype nAChR (the cloned human α7 subunit). Two-electrode voltage-clamp recording with wild-type and mutated nAChRs expressed in Xenopus laevis oocytes indicated that the mutation causes a complete loss of agonist activation. In addition, radioligand binding studies indicated that the 3-aa deletion resulted in significantly lower-affinity binding of the extracellular neurotransmitter-binding site. These findings are consistent with the 3-amino acid (IIA) deletion within the transmembrane domain of Pxα6 being responsible for target-site resistance to spinosad in the SZ-SpinR strain of P. xylostella. PMID:26855198

  5. Characterization of a bacterial community in an abandoned semiarid lead-zinc mine tailing site.

    PubMed

    Mendez, Monica O; Neilson, Julia W; Maier, Raina M

    2008-06-01

    Bacterial diversity in mine tailing microbial communities has not been thoroughly investigated despite the correlations that have been observed between the relative microbial diversity and the success of revegetation efforts at tailing sites. This study employed phylogenetic analyses of 16S rRNA genes to compare the bacterial communities present in highly disturbed, extremely (pH 2.7) and moderately (pH 5.7) acidic lead-zinc mine tailing samples from a semiarid environment with those from a vegetated off-site (OS) control sample (pH 8). Phylotype richness in these communities decreased from 42 in the OS control to 24 in the moderately acidic samples and 8 in the extremely acidic tailing samples. The clones in the extremely acidic tailing sample were most closely related to acidophiles, none of which were detected in the OS control sample. The comparison generated by this study between the bacteria present in extremely acidic tailing and that in moderately acidic tailing communities with those in an OS control soil provides a reference point from which to evaluate the successful restoration of mine tailing disposal sites by phytostabilization.

  6. 3. VIEW OF WEST TAILING DAM, LARGE TANK, AND TAILING, ...

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

    3. VIEW OF WEST TAILING DAM, LARGE TANK, AND TAILING, LOOKING NORTHEAST. A SIX-FOOT SCALE IS LOCATED AGAINST WALL ON LEFT. PURPOSE OF TANK IS UNKNOWN, BUT APPEARS TO HAVE FALLEN FROM ITS ORIGINAL LOCATION AT THE MILL SITE, UP AND TO THE RIGHT OF THIS VIEW. - Skidoo Mine, Park Route 38 (Skidoo Road), Death Valley Junction, Inyo County, CA

  7. Increased Crystalline Cellulose Activity via Combinations of Amino Acid Changes in the Family 9 Catalytic Domain and Family 3c Cellulose Binding Module of Thermobifida fusca Cel9A ▿

    PubMed Central

    Li, Yongchao; Irwin, Diana C.; Wilson, David B.

    2010-01-01

    Amino acid modifications of the Thermobifida fusca Cel9A-68 catalytic domain or carbohydrate binding module 3c (CBM3c) were combined to create enzymes with changed amino acids in both domains. Bacterial crystalline cellulose (BC) and swollen cellulose (SWC) assays of the expressed and purified enzymes showed that three combinations resulted in 150% and 200% increased activity, respectively, and also increased synergistic activity with other cellulases. Several other combinations resulted in drastically lowered activity, giving insight into the need for a balance between the binding in the catalytic cleft on either side of the cleavage site, as well as coordination between binding affinity for the catalytic domain and CBM3c. The same combinations of amino acid variants in the whole enzyme, Cel9A-90, did not increase BC or SWC activity but did have higher filter paper (FP) activity at 12% digestion. PMID:20173060

  8. Retinoblastoma-binding protein 1 has an interdigitated double Tudor domain with DNA binding activity.

    PubMed

    Gong, Weibin; Wang, Jinfeng; Perrett, Sarah; Feng, Yingang

    2014-02-21

    Retinoblastoma-binding protein 1 (RBBP1) is a tumor and leukemia suppressor that binds both methylated histone tails and DNA. Our previous studies indicated that RBBP1 possesses a Tudor domain, which cannot bind histone marks. In order to clarify the function of the Tudor domain, the solution structure of the RBBP1 Tudor domain was determined by NMR and is presented here. Although the proteins are unrelated, the RBBP1 Tudor domain forms an interdigitated double Tudor structure similar to the Tudor domain of JMJD2A, which is an epigenetic mark reader. This indicates the functional diversity of Tudor domains. The RBBP1 Tudor domain structure has a significant area of positively charged surface, which reveals a capability of the RBBP1 Tudor domain to bind nucleic acids. NMR titration and isothermal titration calorimetry experiments indicate that the RBBP1 Tudor domain binds both double- and single-stranded DNA with an affinity of 10-100 μM; no apparent DNA sequence specificity was detected. The DNA binding mode and key interaction residues were analyzed in detail based on a model structure of the Tudor domain-dsDNA complex, built by HADDOCK docking using the NMR data. Electrostatic interactions mediate the binding of the Tudor domain with DNA, which is consistent with NMR experiments performed at high salt concentration. The DNA-binding residues are conserved in Tudor domains of the RBBP1 protein family, resulting in conservation of the DNA-binding function in the RBBP1 Tudor domains. Our results provide further insights into the structure and function of RBBP1.

  9. Power-law cross-correlations estimation under heavy tails

    NASA Astrophysics Data System (ADS)

    Kristoufek, Ladislav

    2016-11-01

    We examine the performance of six estimators of the power-law cross-correlations-the detrended cross-correlation analysis, the detrending moving-average cross-correlation analysis, the height cross-correlation analysis, the averaged periodogram estimator, the cross-periodogram estimator and the local cross-Whittle estimator-under heavy-tailed distributions. The selection of estimators allows to separate these into the time and frequency domain estimators. By varying the characteristic exponent of the α-stable distributions which controls the tails behavior, we report several interesting findings. First, the frequency domain estimators are practically unaffected by heavy tails bias-wise. Second, the time domain estimators are upward biased for heavy tails but they have lower estimator variance than the other group for short series. Third, specific estimators are more appropriate depending on distributional properties and length of the analyzed series. In addition, we provide a discussion of implications of these results for empirical applications as well as theoretical explanations.

  10. ALS-Causing Mutations Significantly Perturb the Self-Assembly and Interaction with Nucleic Acid of the Intrinsically Disordered Prion-Like Domain of TDP-43

    PubMed Central

    Lim, Liangzhong; Wei, Yuanyuan; Lu, Yimei; Song, Jianxing

    2016-01-01

    TAR-DNA-binding protein-43 (TDP-43) C-terminus encodes a prion-like domain widely presented in RNA-binding proteins, which functions to form dynamic oligomers and also, amazingly, hosts most amyotrophic lateral sclerosis (ALS)-causing mutations. Here, as facilitated by our previous discovery, by circular dichroism (CD), fluorescence and nuclear magnetic resonance (NMR) spectroscopy, we have successfully determined conformations, dynamics, and self-associations of the full-length prion-like domains of the wild type and three ALS-causing mutants (A315E, Q331K, and M337V) in both aqueous solutions and membrane environments. The study decodes the following: (1) The TDP-43 prion-like domain is intrinsically disordered only with some nascent secondary structures in aqueous solutions, but owns the capacity to assemble into dynamic oligomers rich in β-sheet structures. By contrast, despite having highly similar conformations, three mutants gained the ability to form amyloid oligomers. The wild type and three mutants all formed amyloid fibrils after incubation as imaged by electron microscopy. (2) The interaction with nucleic acid enhances the self-assembly for the wild type but triggers quick aggregation for three mutants. (3) A membrane-interacting subdomain has been identified over residues Met311-Gln343 indispensable for TDP-43 neurotoxicity, which transforms into a well-folded Ω-loop-helix structure in membrane environments. Furthermore, despite having very similar membrane-embedded conformations, three mutants will undergo further self-association in the membrane environment. Our study implies that the TDP-43 prion-like domain appears to have an energy landscape, which allows the assembly of the wild-type sequence into dynamic oligomers only under very limited condition sets, and ALS-causing point mutations are sufficient to remodel it to more favor the amyloid formation or irreversible aggregation, thus supporting the emerging view that the pathologic aggregation

  11. Functional Role of Residues Corresponding to Helical Domain II (Amino Acids 35 to 46) of Human Immunodeficiency Virus Type 1 Vpr

    PubMed Central

    Singh, Satya P.; Tomkowicz, Brian; Lai, Derhsing; Cartas, Maria; Mahalingam, Sundarasamy; Kalyanaraman, Vaniambadi S.; Murali, Ramachandran; Srinivasan, Alagarsamy

    2000-01-01

    Vpr, encoded by the human immunodeficiency virus type 1 genome, contains 96 amino acids and is a multifunctional protein with features which include cell cycle arrest at G2, nuclear localization, participation in transport of the preintegration complex, cation channel activity, oligomerization, and interaction with cellular proteins, in addition to its incorporation into the virus particles. Recently, structural studies based on nuclear magnetic resonance and circular dichroism spectroscopy showed that Vpr contains a helix (HI)-turn-helix (HII) core at the amino terminus and an amphipathic helix (HIII) in the middle region. Though the importance of helical domains HI and HIII has been defined with respect to Vpr functions, the role of helical domain HII is not known. To address this issue, we constructed a series of mutants in which the HII domain was altered by deletion, insertion, and/or substitution mutagenesis. To enable the detection of Vpr, the sequence corresponding to the Flag epitope (DYKDDDDK) was added, in frame, to the Vpr coding sequences. Mutants, expressed through the in vitro transcription/translation system and in cells, showed an altered migration corresponding to deletions in Vpr. Substitution mutational analysis of residues in HII showed reduced stability for VprW38S-FL, VprL42G-FL, and VprH45W-FL. An assay involving cotransfection of NLΔVpr proviral DNA and a Vpr expression plasmid was employed to analyze the virion incorporation property of Vpr. Mutant Vpr containing deletions and specific substitutions (VprW38S-FL, VprL39G-FL, VprL42G-FL, VprG43P-FL, and VprI46G-FL) exhibited a negative virion incorporation phenotype. Further, mutant Vpr-FL containing deletions also failed to associate with wild-type Vpr, indicating a possible defect in the oligomerization feature of Vpr. Subcellular localization studies indicated that mutants VprΔ35-50-H-FL, VprR36W-FL, VprL39G-FL, and VprI46G-FL exhibited both cytoplasmic and nuclear localization, unlike

  12. Functional role of residues corresponding to helical domain II (amino acids 35 to 46) of human immunodeficiency virus type 1 Vpr.

    PubMed

    Singh, S P; Tomkowicz, B; Lai, D; Cartas, M; Mahalingam, S; Kalyanaraman, V S; Murali, R; Srinivasan, A

    2000-11-01

    Vpr, encoded by the human immunodeficiency virus type 1 genome, contains 96 amino acids and is a multifunctional protein with features which include cell cycle arrest at G(2), nuclear localization, participation in transport of the preintegration complex, cation channel activity, oligomerization, and interaction with cellular proteins, in addition to its incorporation into the virus particles. Recently, structural studies based on nuclear magnetic resonance and circular dichroism spectroscopy showed that Vpr contains a helix (HI)-turn-helix (HII) core at the amino terminus and an amphipathic helix (HIII) in the middle region. Though the importance of helical domains HI and HIII has been defined with respect to Vpr functions, the role of helical domain HII is not known. To address this issue, we constructed a series of mutants in which the HII domain was altered by deletion, insertion, and/or substitution mutagenesis. To enable the detection of Vpr, the sequence corresponding to the Flag epitope (DYKDDDDK) was added, in frame, to the Vpr coding sequences. Mutants, expressed through the in vitro transcription/translation system and in cells, showed an altered migration corresponding to deletions in Vpr. Substitution mutational analysis of residues in HII showed reduced stability for VprW38S-FL, VprL42G-FL, and VprH45W-FL. An assay involving cotransfection of NLDeltaVpr proviral DNA and a Vpr expression plasmid was employed to analyze the virion incorporation property of Vpr. Mutant Vpr containing deletions and specific substitutions (VprW38S-FL, VprL39G-FL, VprL42G-FL, VprG43P-FL, and VprI46G-FL) exhibited a negative virion incorporation phenotype. Further, mutant Vpr-FL containing deletions also failed to associate with wild-type Vpr, indicating a possible defect in the oligomerization feature of Vpr. Subcellular localization studies indicated that mutants VprDelta35-50-H-FL, VprR36W-FL, VprL39G-FL, and VprI46G-FL exhibited both cytoplasmic and nuclear localization

  13. Cysteine Substitution of Transmembrane Domain Amino Acids Alters the Ethanol Inhibition of GluN1/GluN2A N-Methyl-d-Aspartate Receptors

    PubMed Central

    Xu, Minfu; Smothers, C. Thetford

    2015-01-01

    N-Methyl-d-aspartate receptors (NMDARs) are inhibited by behaviorally relevant concentrations of ethanol, and residues within transmembrane (TM) domains of NMDARs, including TM3 GluN1 phenylalanine 639 (F639), regulate this sensitivity. In the present study, we used cysteine (C) mutagenesis to determine whether there are additional residues within nearby TM domains that regulate ethanol inhibition on NMDARs. GluN1(F639C)/GluN2A receptors were less inhibited by ethanol than wild-type receptors, and inhibition was restored to wild-type levels following treatment with ethanol-like methanethiosulfonate reagents. Molecular modeling identified six residues in the GluN1 TM1 domain (valine V566; serine S569) and the GluN2A TM4 domain (methionine, M817; V820, F821, and leucine, L824) that were in close vicinity to the TM3 F639 residue, and these were individually mutated to cysteine and tested for ethanol inhibition and receptor function. The F639C-induced decrease in ethanol inhibition was blunted by coexpression of GluN1 TM1 mutants V566C and S569C, and statistically significant interactions were observed for ethanol inhibition among V566C, F639C, and GluN2A TM4 mutants V820C and F821C and S569C, F639C, and GluN2A TM4 mutants F821C and L824C. Ethanol inhibition was also reduced when either GluN1 TM1 mutant V566C or S569C was combined with GluN2A V820C, suggesting a novel TM1:TM4 intrasubunit site of action for ethanol. Cysteines substituted at TM3 and TM4 sites previously suggested to interact with ethanol had less dramatic effects on ethanol inhibition. Overall, the results from these studies suggest that interactions among TM1, TM3, and TM4 amino acids in NMDARs are important determinants of ethanol action at these receptors. PMID:25635140

  14. 4-O-methylation of glucuronic acid in Arabidopsis glucuronoxylan is catalyzed by a domain of unknown function family 579 protein

    PubMed Central

    Urbanowicz, Breeanna R.; Peña, Maria J.; Ratnaparkhe, Supriya; Avci, Utku; Backe, Jason; Steet, Heather F.; Foston, Marcus; Li, Hongjia; O’Neill, Malcolm A.; Ragauskas, Arthur J.; Darvill, Alan G.; Wyman, Charles; Gilbert, Harry J.; York, William S.

    2012-01-01

    The hemicellulose 4-O-methyl glucuronoxylan is one of the principle components present in the secondary cell walls of eudicotyledonous plants. However, the biochemical mechanisms leading to the formation of this polysaccharide and the effects of modulating its structure on the physical properties of the cell wall are poorly understood. We have identified and functionally characterized an Arabidopsis glucuronoxylan methyltransferase (GXMT) that catalyzes 4-O-methylation of the glucuronic acid substituents of this polysaccharide. AtGXMT1, which was previously classified as a domain of unknown function (DUF) 579 protein, specifically transfers the methyl group from S-adenosyl-l-methionine to O-4 of α-d-glucopyranosyluronic acid residues that are linked to O-2 of the xylan backbone. Biochemical characterization of the recombinant enzyme indicates that GXMT1 is localized in the Golgi apparatus and requires Co2+ for optimal activity in vitro. Plants lacking GXMT1 synthesize glucuronoxylan in which the degree of 4-O-methylation is reduced by 75%. This result is correlated to a change in lignin monomer composition and an increase in glucuronoxylan release during hydrothermal treatment of secondary cell walls. We propose that the DUF579 proteins constitute a previously undescribed family of cation-dependent, polysaccharide-specific O-methyl-transferases. This knowledge provides new opportunities to selectively manipulate polysaccharide O-methylation and extends the portfolio of structural targets that can be modified either alone or in combination to modulate biopolymer interactions in the plant cell wall. PMID:22893684

  15. Amino Acid Residues in Transmembrane Domain 10 of Organic Anion Transporting Polypeptide 1B3 are Critical for Cholecystokinin Octapeptide Transport†

    PubMed Central

    Gui, Chunshan; Hagenbuch, Bruno

    2008-01-01

    Human organic anion transporting polypeptides (OATP) 1B1 and 1B3 are multi-specific transporters that mediate uptake of amphipathic organic compounds into hepatocytes. The two OATPs contain twelve transmembrane domains (TMs) and share 80% amino acid sequence identity. Besides common substrates with OATP1B1, OATP1B3 specifically transports cholecystokinin octapeptide (CCK-8). To determine which structural domains/residues are important for the substrate selectivity of OATP1B3, we constructed a series of chimeric proteins between OATP1B3 and 1B1, expressed them in HEK293 cells and determined uptake of CCK-8 along with surface expression of the proteins. Replacing TM10 in OATP1B3 with TM10 of OATP1B1 resulted in dramatically reduced CCK-8 transport, indicating that TM10 is crucial for recognition and/or translocation of CCK-8. Using site-directed mutagenesis, we identified three key residues within TM10, namely Y537, S545 and T550. When we replaced these residues by the corresponding amino acid residues found in OATP1B1, CCK-8 transport was similarly low as for the replacement of the whole TM10. Kinetic experiments showedthat the Km values for CCK-8 transport in the TM10-replacement and triple mutant were only 1.3 and 1.1 μM, respectively as compared to 16.3 μM for wild-type OATP1B3. Similarly, the Vmax values dropped from 495.5 pmol/normalized mg/min for wild-type OATP1B3 to 13.3 and 19.0 for the TM10-replacement and triple mutant, respectively. Molecular modeling indicated that two of the three identified residues might form hydrogen bonds with CCK-8. In conclusion, we have identified three amino acid residues (Y537, S545 and T550) in TM10 of OATP1B3 that are important for CCK-8 transport. PMID:18690707

  16. Identification of two Amino Acids in the C-terminal Domain of Mouse CRY2 Essential for PER2 Interaction

    PubMed Central

    2010-01-01

    Background Cryptochromes (CRYs) are a class of flavoprotein blue-light signaling receptors found in plants and animals, and they control plant development and the entrainment of circadian rhythms. They also act as integral parts of the central circadian oscillator in humans and other animals. In mammals, the CLOCK-BMAL1 heterodimer activates transcription of the Per and Cry genes as well as clock-regulated genes. The PER2 proteins interact with CRY and CKIε, and the resulting ternary complexes translocate into the nucleus, where they negatively regulate the transcription of Per and Cry core clock genes and other clock-regulated output genes. Recent studies have indicated that the extended C-termini of the mammalian CRYs, as compared to photolyase proteins, interact with PER proteins. Results We identified a region on mCRY2 (between residues 493 and 512) responsible for direct physical interaction with mPER2 by mammalian two-hybrid and co-immunoprecipitation assays. Moreover, using oligonucleotide-based degenerate PCR, we discovered that mutation of Arg-501 and Lys-503 of mCRY2 within this C-terminal region totally abolishes interaction with PER2. Conclusions Our results identify mCRY2 amino acid residues that interact with the mPER2 binding region and suggest the potential for rational drug design to inhibit CRYs for specific therapeutic approaches. PMID:20840750

  17. Amino acid 72 of mouse and human GDF9 mature domain is responsible for altered homodimer bioactivities but has subtle effects on GDF9:BMP15 heterodimer activities.

    PubMed

    Peng, Jia; Wigglesworth, Karen; Rangarajan, Adithya; Eppig, John J; Thompson, Thomas B; Matzuk, Martin M

    2014-12-01

    Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-secreted paralogs of the transforming growth factor beta (TGFbeta) superfamily. In mammals, these two growth factors play critical roles in folliculogenesis. As previously reported, an arginine in the pre-helix loop of GDF5 defines the high binding specificity to its type 1 receptor. Interestingly, bioactive mouse GDF9 and human BMP15 share the conserved arginine in the pre-helix loop, but their low-activity counterparts (mouse BMP15 and human GDF9) have a glycine or a proline instead. To address the question of whether the arginine residue defines the different activities of GDF9 and BMP15 homodimers and their heterodimers in human and mouse, we used site-directed mutagenesis to change the species-specific residues in human and mouse proteins, and examined their activities in our in vitro assays. Although amino acid 72 of mature GDF9 is responsible for altered homodimer bioactivities, neither the corresponding BMP15 amino acid 62 nor the intact pre-helix loop is indispensable for BMP15 homodimer activity. However, amino acid 72 in GDF9 only has only subtle effects on GDF9:BMP15 heterodimer activity. Based on previous studies and our recent findings, we provide hypothetical models to understand the molecular mechanism to define activities of the homodimeric and heterodimeric ligands. The arginine residue in the pre-helix loop of GDF9 homodimer may prevent the inhibition from its pro-domain or directly alter receptor binding, but this residue in GDF9 does not significantly affect the heterodimer activity, because of suggested conformational changes during heterodimer formation.

  18. Differences in activation of aryl hydrocarbon receptors of white sturgeon relative to lake sturgeon are predicted by identities of key amino acids in the ligand binding domain.

    PubMed

    Doering, Jon A; Farmahin, Reza; Wiseman, Steve; Beitel, Shawn C; Kennedy, Sean W; Giesy, John P; Hecker, Markus

    2015-04-01

    Dioxin-like compounds (DLCs) are pollutants of global environmental concern. DLCs elicit their adverse outcomes through activation of the aryl hydrocarbon receptor (AhR). However, there is limited understanding of the mechanisms that result in differences in sensitivity to DLCs among different species of fishes. Understanding these mechanisms is critical for protection of the diversity of fishes exposed to DLCs, including endangered species. This study investigated specific mechanisms that drive responses of two endangered fishes, white sturgeon (Acipenser transmontanus) and lake sturgeon (Acipenser fulvescens) to DLCs. It determined whether differences in sensitivity to activation of AhRs (AhR1 and AhR2) can be predicted based on identities of key amino acids in the ligand binding domain (LBD). White sturgeon were 3- to 30-fold more sensitive than lake sturgeon to exposure to 5 different DLCs based on activation of AhR2. There were no differences in sensitivity between white sturgeon and lake sturgeon based on activation of AhR1. Adverse outcomes as a result of exposure to DLCs have been shown to be mediated through activation of AhR2, but not AhR1, in all fishes studied to date. This indicates that white sturgeon are likely to have greater sensitivity in vivo relative to lake sturgeon. Homology modeling and in silico mutagenesis suggests that differences in sensitivity to activation of AhR2 result from differences in key amino acids at position 388 in the LBD of AhR2 of white sturgeon (Ala-388) and lake sturgeon (Thr-388). This indicates that identities of key amino acids in the LBD of AhR2 could be predictive of both in vitro activation by DLCs and in vivo sensitivity to DLCs in these, and potentially other, fishes.

  19. Characterizing Aciniform Silk Repetitive Domain Backbone Dynamics and Hydrodynamic Modularity.

    PubMed

    Tremblay, Marie-Laurence; Xu, Lingling; Sarker, Muzaddid; Liu, Xiang-Qin; Rainey, Jan K

    2016-01-01

    Spider aciniform (wrapping) silk is a remarkable fibrillar biomaterial with outstanding mechanical properties. It is a modular protein consisting, in Argiope trifasciata, of a core repetitive domain of 200 amino acid units (W units). In solution, the W units comprise a globular folded core, with five α-helices, and disordered tails that are linked to form a ~63-residue intrinsically disordered linker in concatemers. Herein, we present nuclear magnetic resonance (NMR) spectroscopy-based (15)N spin relaxation analysis, allowing characterization of backbone dynamics as a function of residue on the ps-ns timescale in the context of the single W unit (W₁) and the two unit concatemer (W₂). Unambiguous mapping of backbone dynamics throughout W₂ was made possible by segmental NMR active isotope-enrichment through split intein-mediated trans-splicing. Spectral density mapping for W₁ and W₂ reveals a striking disparity in dynamics between the folded core and the disordered linker and tail regions. These data are also consistent with rotational diffusion behaviour where each globular domain tumbles almost independently of its neighbour. At a localized level, helix 5 exhibits elevated high frequency dynamics relative to the proximal helix 4, supporting a model of fibrillogenesis where this helix unfolds as part of the transition to a mixed α-helix/β-sheet fibre. PMID:27517921

  20. Characterizing Aciniform Silk Repetitive Domain Backbone Dynamics and Hydrodynamic Modularity

    PubMed Central

    Tremblay, Marie-Laurence; Xu, Lingling; Sarker, Muzaddid; Liu, Xiang-Qin; Rainey, Jan K.

    2016-01-01

    Spider aciniform (wrapping) silk is a remarkable fibrillar biomaterial with outstanding mechanical properties. It is a modular protein consisting, in Argiope trifasciata, of a core repetitive domain of 200 amino acid units (W units). In solution, the W units comprise a globular folded core, with five α-helices, and disordered tails that are linked to form a ~63-residue intrinsically disordered linker in concatemers. Herein, we present nuclear magnetic resonance (NMR) spectroscopy-based 15N spin relaxation analysis, allowing characterization of backbone dynamics as a function of residue on the ps–ns timescale in the context of the single W unit (W1) and the two unit concatemer (W2). Unambiguous mapping of backbone dynamics throughout W2 was made possible by segmental NMR active isotope-enrichment through split intein-mediated trans-splicing. Spectral density mapping for W1 and W2 reveals a striking disparity in dynamics between the folded core and the disordered linker and tail regions. These data are also consistent with rotational diffusion behaviour where each globular domain tumbles almost independently of its neighbour. At a localized level, helix 5 exhibits elevated high frequency dynamics relative to the proximal helix 4, supporting a model of fibrillogenesis where this helix unfolds as part of the transition to a mixed α-helix/β-sheet fibre. PMID:27517921

  1. Negative polarity of phenyl-C{sub 61} butyric acid methyl ester adjacent to donor macromolecule domains

    SciTech Connect

    Alley, Olivia J.; Dawidczyk, Thomas J.; Hardigree, Josué F. Martínez; Katz, Howard E.; Wu, Meng-Yin; Johns, Gary L.; Markovic, Nina; Arnold, Michael S.

    2015-01-19

    Interfacial fields within organic photovoltaics influence the movement of free charge carriers, including exciton dissociation and recombination. Open circuit voltage (V{sub oc}) can also be dependent on the interfacial fields, in the event that they modulate the energy gap between donor HOMO and acceptor LUMO. A rise in the vacuum level of the acceptor will increase the gap and the V{sub oc}, which can be beneficial for device efficiency. Here, we measure the interfacial potential differences at donor-acceptor junctions using Scanning Kelvin Probe Microscopy, and quantify how much of the potential difference originates from physical contact between the donor and acceptor. We see a statistically significant and pervasive negative polarity on the phenyl-C{sub 61} butyric acid methyl ester (PCBM) side of PCBM/donor junctions, which should also be present at the complex interfaces in bulk heterojunctions. This potential difference may originate from molecular dipoles, interfacial interactions with donor materials, and/or equilibrium charge transfer due to the higher work function and electron affinity of PCBM. We show that the contact between PCBM and poly(3-hexylthiophene) doubles the interfacial potential difference, a statistically significant difference. Control experiments determined that this potential difference was not due to charges trapped in the underlying substrate. The direction of the observed potential difference would lead to increased V{sub oc}, but would also pose a barrier to electrons being injected into the PCBM and make recombination more favorable. Our method may allow unique information to be obtained in new donor-acceptor junctions.

  2. [Tail Plane Icing

    NASA Technical Reports Server (NTRS)

    1997-01-01

    The Aviation Safety Program initiated by NASA in 1997 has put greater emphasis in safety related research activities. Ice-contaminated-tailplane stall (ICTS) has been identified by the NASA Lewis Icing Technology Branch as an important activity for aircraft safety related research. The ICTS phenomenon is characterized as a sudden, often uncontrollable aircraft nose- down pitching moment, which occurs due to increased angle-of-attack of the horizontal tailplane resulting in tailplane stall. Typically, this phenomenon occurs when lowering the flaps during final approach while operating in or recently departing from icing conditions. Ice formation on the tailplane leading edge can reduce tailplane angle-of-attack range and cause flow separation resulting in a significant reduction or complete loss of aircraft pitch control. In 1993, the Federal Aviation Authority (FAA) and NASA embarked upon a four-year research program to address the problem of tailplane stall and to quantify the effect of tailplane ice accretion on aircraft performance and handling characteristics. The goals of this program, which was completed in March 1998, were to collect aerodynamic data for an aircraft tail with and without ice contamination and to develop analytical methods for predicting the effects of tailplane ice contamination. Extensive dry air and icing tunnel tests which resulted in a database of the aerodynamic effects associated with tailplane ice contamination. Although the FAA/NASA tailplane icing program generated some answers regarding ice-contaminated-tailplane stall (ICTS) phenomena, NASA researchers have found many open questions that warrant further investigation into ICTS. In addition, several aircraft manufacturers have expressed interest in a second research program to expand the database to other tail configurations and to develop experimental and computational methodologies for evaluating the ICTS phenomenon. In 1998, the icing branch at NASA Lewis initiated a second

  3. Wall Teichoic Acids Restrict Access of Bacteriophage Endolysin Ply118, Ply511, and PlyP40 Cell Wall Binding Domains to the Listeria monocytogenes Peptidoglycan

    PubMed Central

    Eugster, Marcel R.

    2012-01-01

    The C-terminal cell wall binding domains (CBDs) of phage endolysins direct the enzymes to their binding ligands on the bacterial cell wall with high affinity and specificity. The Listeria monocytogenes Ply118, Ply511, and PlyP40 endolysins feature related CBDs which recognize the directly cross-linked peptidoglycan backbone structure of Listeria. However, decoration with fluorescently labeled CBDs primarily occurs at the poles and septal regions of the rod-shaped cells. To elucidate the potential role of secondary cell wall-associated carbohydrates such as the abundant wall teichoic acid (WTA) on this phenomenon, we investigated CBD binding using L. monocytogenes serovar 1/2 and 4 cells deficient in WTA. Mutants were obtained by deletion of two redundant tagO homologues, whose products catalyze synthesis of the WTA linkage unit. While inactivation of either tagO1 (EGDe lmo0959) or tagO2 (EGDe lmo2519) alone did not affect WTA content, removal of both alleles following conditional complementation yielded WTA-deficient Listeria cells. Substitution of tagO from an isopropyl-β-d-thiogalactopyranoside-inducible single-copy integration vector restored the original phenotype. Although WTA-deficient cells are viable, they featured severe growth inhibition and an unusual coccoid morphology. In contrast to CBDs from other Listeria phage endolysins which directly utilize WTA as binding ligand, the data presented here show that WTAs are not required for attachment of CBD118, CBD511, and CBDP40. Instead, lack of the cell wall polymers enables unrestricted spatial access of CBDs to the cell wall surface, indicating that the abundant WTA can negatively regulate sidewall localization of the cell wall binding domains. PMID:23002226

  4. Identifying possible sites for antibody neutralization escape: Implications for unique functional properties of the C-terminal tail of Human Immunodeficiency Virus Type 1 gp41.

    PubMed

    Lu, Zhifeng; Huang, Yushen; Tan, Yue; Yu, Yang; Wang, Junyi; Chen, Ying-Hua

    2016-07-01

    A previous amino acid sequence analyses from our laboratory reported nine potential sites in gp41 glycoprotein of HIV-1 that may contribute to virus escape from antibody neutralization. Besides four sites found outside the membrane of HIV-1 virus, five located in the C-terminal tail of gp41 specifically in the lentivirus lytic peptides motifs (LLPs). To further study the bioinformatical results, the virus infectivity assay and the standard neutralization assay were conducted on conservatively mutated virus. Two sites in the LLP3 domain stood out with the ability to alter the resistance of HIV-1 virus to certain broadly neutralizing antibodies (bNAbs). While the glycoprotein incorporation on the viral membrane and the interaction of the LLP3 domain with the lipid membrane remained unaltered, the increase in neutralization resistance of the mutant virus was associated with the changes on Env conformation. Our findings demonstrate different sensibility of bNAbs to mutations in the C-terminal tail and indicate an unrecognized potential role for even minor sequence variation in the C-terminal tail in modulating the antigenicity of the ectodomain of HIV-1 envelope glycoprotein complex.

  5. Domain Engineering

    NASA Astrophysics Data System (ADS)

    Bjørner, Dines

    Before software can be designed we must know its requirements. Before requirements can be expressed we must understand the domain. So it follows, from our dogma, that we must first establish precise descriptions of domains; then, from such descriptions, “derive” at least domain and interface requirements; and from those and machine requirements design the software, or, more generally, the computing systems.

  6. Structural and Functional Studies of γ-Carboxyglutamic Acid Domains of Factor VIIa and Activated Protein C: Role of Magnesium at Physiological Calcium

    PubMed Central

    Vadivel, Kanagasabai; Agah, Sayeh; Messer, Amanda S.; Cascio, Duilio; Bajaj, Madhu S.; Krishnaswamy, Sriram; Esmon, Charles T; Padmanabhan, Kaillathe; Bajaj, S. Paul

    2014-01-01

    Crystal structures of factor (F) VIIa/soluble (s) tissue factor (TF), obtained under high Mg2+ (50 mM Mg2+/5 mM Ca2+), have three of seven Ca2+-sites in the γ-Carboxyglutamic Acid (Gla) domain replaced by Mg2+ at positions 1, 4 and 7. We now report structures under low Mg2+ (2.5 mM Mg2+/5 mM Ca2+) as well as under high Ca2+ (5 mM Mg2+/45 mM Ca2+). Under low Mg2+, four Ca2+ and three Mg2+ occupy the same positions as in high Mg2+ structures. Conversely, under low Mg2+, reexamination of the structure of Gla domain of activated Protein C (APC) complexed with sEPCR has position 4 occupied by Ca2+ and positions 1 and 7 by Mg2+. Nonetheless, in direct binding experiments, Mg2+ replaced three Ca2+-sites in the unliganded Protein C or APC. Further, the high Ca2+-condition was necessary to replace Mg4 in the FVIIa/sTF structure. In biological studies, Mg2+ enhanced phospholipid binding to FVIIa and APC at physiological Ca2+. Additionally, Mg2+ potentiated phospholipid-dependent activations of FIX and FX by FVIIa/TF, and inactivation of FVa by APC. Since APC and FVIIa bind to sEPCR involving similar interactions, we conclude that under the low Mg2+-condition, sEPCR binding to APC-Gla (or FVIIa-Gla) replaces Mg4 by Ca4 with an attendant conformational change in the Gla domain ω-loop. Moreover, since phospholipid and sEPCR bind to FVIIa or APC via the ω-loop, we predict that phospholipid-binding also induces the functional Ca4 conformation in this loop. Cumulatively, the data illustrate that Mg2+ and Ca2+ act in concert to promote coagulation and anticoagulation. PMID:23454357

  7. ORA47 (octadecanoid-responsive AP2/ERF-domain transcription factor 47) regulates jasmonic acid and abscisic acid biosynthesis and signaling through binding to a novel cis-element.

    PubMed

    Chen, Hsing-Yu; Hsieh, En-Jung; Cheng, Mei-Chun; Chen, Chien-Yu; Hwang, Shih-Ying; Lin, Tsan-Piao

    2016-07-01

    ORA47 (octadecanoid-responsive AP2/ERF-domain transcription factor 47) of Arabidopsis thaliana is an AP2/ERF domain transcription factor that regulates jasmonate (JA) biosynthesis and is induced by methyl JA treatment. The regulatory mechanism of ORA47 remains unclear. ORA47 is shown to bind to the cis-element (NC/GT)CGNCCA, which is referred to as the O-box, in the promoter of ABI2. We proposed that ORA47 acts as a connection between ABA INSENSITIVE1 (ABI1) and ABI2 and mediates an ABI1-ORA47-ABI2 positive feedback loop. PORA47:ORA47-GFP transgenic plants were used in a chromatin immunoprecipitation (ChIP) assay to show that ORA47 participates in the biosynthesis and/or signaling pathways of nine phytohormones. Specifically, many abscisic acid (ABA) and JA biosynthesis and signaling genes were direct targets of ORA47 under stress conditions. The JA content of the P35S:ORA47-GR lines was highly induced under wounding and moderately induced under water stress relative to that of the wild-type plants. The wounding treatment moderately increased ABA accumulation in the transgenic lines, whereas the water stress treatment repressed the ABA content. ORA47 is proposed to play a role in the biosynthesis of JA and ABA and in regulating the biosynthesis and/or signaling of a suite of phytohormone genes when plants are subjected to wounding and water stress. PMID:26974851

  8. Mercury's Dynamic Magnetic Tail

    NASA Technical Reports Server (NTRS)

    Slavin, James A.

    2010-01-01

    The Mariner 10 and MESSENGER flybys of Mercury have revealed a magnetosphere that is likely the most responsive to upstream interplanetary conditions of any in the solar system. The source of the great dynamic variability observed during these brief passages is due to Mercury's proximity to the Sun and the inverse proportionality between reconnection rate and solar wind Alfven Mach number. However, this planet's lack of an ionosphere and its small physical dimensions also contribute to Mercury's very brief Dungey cycle, approx. 2 min, which governs the time scale for internal plasma circulation. Current observations and understanding of the structure and dynamics of Mercury's magnetotail are summarized and discussed. Special emphasis will be placed upon such questions as: 1) How much access does the solar wind have to this small magnetosphere as a function of upstream conditions? 2) What roles do heavy planetary ions play? 3) Do Earth-like substorms take place at Mercury? 4) How does Mercury's tail respond to extreme solar wind events such coronal mass ejections? Prospects for progress due to advances in the global magnetohydrodynamic and hybrid simulation modeling and the measurements to be taken by MESSENGER after it enters Mercury orbit on March 18, 2011 will be discussed.

  9. [Amino acids 395-416 in DNA binding domain of STAT4 is involved in IL-12-induced nuclear import of STAT4].

    PubMed

    Huang, Yu-Mei; Wen, Ya-Ping; Li, Xuan-An; Yuan, Yuan; Luo, Qi-Zhi; Li, Ming

    2012-08-25

    The purpose of the present study is to explore the mechanism of IL-12-induced nuclear import of signal transducer and activator of transcription 4 (STAT4). Assayed by analyses of homology alignment of STATs, amino acids 395-416 in DNA binding domain was found to be a potential dimer-specific nuclear localization signal (dsNLS) of STAT4. Therefore, several plasmids were constructed. Wild-type STAT4 was inserted into the SalI and BamHI sites of pEGFP-C1 for the construction of plasmid pEGFP-STAT4. The DNA fragment of STAT4 with the deletion of amino acids 395-416 was amplified by RCR and introduced into the SalI and BamHI sites of pEGFP-C1 which was named pEGFP-STAT4-Del. Classic NLS DNA sequence of SV40 T antigen was inserted into the XhoI and HindIII sites of pEGFP-C1. This plasmid was named as pEGFP-NLS and used as a positive control. Plasmid pEGFP-NLS-STAT4-Del was constructed by inserting STAT4-Del into SalI and BamHI sites of pEGFP-NLS. These plasmids were transiently transfected into Caski cells, respectively. The results showed that, after these transfected cells were stimulated by IL-12, wild type STAT4 existed in the cytoplasm at 0 min, and was predominantly localized to the nucleus at 45 min, and distributed in both cytoplasm and nucleus at 60 min, suggesting that STAT4 translocates from cytoplasm into nucleus and finally re-entries into the cytoplasm during the stimulation of IL-12. However, deletion mutant of STAT4 was arrested in cytoplasm during the IL-12 stimulation. Leptomycin B, which specifically blocks protein export from nucleus into cytoplasm, was used to further demonstrate whether STAT4-Del is transferred into nucleus even with stimulation of IL-12. After the transfected cells were pre-treated by leptomycin B, the wild type STAT4 was mainly localized in nucleus after the IL-12 stimulation, suggesting that STAT4 was translocated from cytoplasm into nucleus by the stimulation of IL-12. On the other hand, the deletion mutant of STAT4 distributed

  10. The deletion of several amino acid stretches of Escherichia coli alpha-hemolysin (HlyA) suggests that the channel-forming domain contains beta-strands.

    PubMed

    Benz, Roland; Maier, Elke; Bauer, Susanne; Ludwig, Albrecht

    2014-01-01

    Escherichia coli α-hemolysin (HlyA) is a pore-forming protein of 110 kDa belonging to the family of RTX toxins. A hydrophobic region between the amino acid residues 238 and 410 in the N-terminal half of HlyA has previously been suggested to form hydrophobic and/or amphipathic α-helices and has been shown to be important for hemolytic activity and pore formation in biological and artificial membranes. The structure of the HlyA transmembrane channel is, however, largely unknown. For further investigation of the channel structure, we deleted in HlyA different stretches of amino acids that could form amphipathic β-strands according to secondary structure predictions (residues 71-110, 158-167, 180-203, and 264-286). These deletions resulted in HlyA mutants with strongly reduced hemolytic activity. Lipid bilayer measurements demonstrated that HlyAΔ71-110 and HlyAΔ264-286 formed channels with much smaller single-channel conductance than wildtype HlyA, whereas their channel-forming activity was virtually as high as that of the wildtype toxin. HlyAΔ158-167 and HlyAΔ180-203 were unable to form defined channels in lipid bilayers. Calculations based on the single-channel data indicated that the channels generated by HlyAΔ71-110 and HlyAΔ264-286 had a smaller size (diameter about 1.4 to 1.8 nm) than wildtype HlyA channels (diameter about 2.0 to 2.6 nm), suggesting that in these mutants part of the channel-forming domain was removed. Osmotic protection experiments with erythrocytes confirmed that HlyA, HlyAΔ71-110, and HlyAΔ264-286 form defined transmembrane pores and suggested channel diameters that largely agreed with those estimated from the single-channel data. Taken together, these results suggest that the channel-forming domain of HlyA might contain β-strands, possibly in addition to α-helical structures. PMID:25463653

  11. The deletion of several amino acid stretches of Escherichia coli alpha-hemolysin (HlyA) suggests that the channel-forming domain contains beta-strands.

    PubMed

    Benz, Roland; Maier, Elke; Bauer, Susanne; Ludwig, Albrecht

    2014-01-01

    Escherichia coli α-hemolysin (HlyA) is a pore-forming protein of 110 kDa belonging to the family of RTX toxins. A hydrophobic region between the amino acid residues 238 and 410 in the N-terminal half of HlyA has previously been suggested to form hydrophobic and/or amphipathic α-helices and has been shown to be important for hemolytic activity and pore formation in biological and artificial membranes. The structure of the HlyA transmembrane channel is, however, largely unknown. For further investigation of the channel structure, we deleted in HlyA different stretches of amino acids that could form amphipathic β-strands according to secondary structure predictions (residues 71-110, 158-167, 180-203, and 264-286). These deletions resulted in HlyA mutants with strongly reduced hemolytic activity. Lipid bilayer measurements demonstrated that HlyAΔ71-110 and HlyAΔ264-286 formed channels with much smaller single-channel conductance than wildtype HlyA, whereas their channel-forming activity was virtually as high as that of the wildtype toxin. HlyAΔ158-167 and HlyAΔ180-203 were unable to form defined channels in lipid bilayers. Calculations based on the single-channel data indicated that the channels generated by HlyAΔ71-110 and HlyAΔ264-286 had a smaller size (diameter about 1.4 to 1.8 nm) than wildtype HlyA channels (diameter about 2.0 to 2.6 nm), suggesting that in these mutants part of the channel-forming domain was removed. Osmotic protection experiments with erythrocytes confirmed that HlyA, HlyAΔ71-110, and HlyAΔ264-286 form defined transmembrane pores and suggested channel diameters that largely agreed with those estimated from the single-channel data. Taken together, these results suggest that the channel-forming domain of HlyA might contain β-strands, possibly in addition to α-helical structures.

  12. Native plant restoration of biosolids-amended copper mine tailings

    SciTech Connect

    Kramer, P.A.; Zabowski, D.; Everett, R.L.; Scherer, G.

    1998-12-31

    Copper mine tailings are difficult to revegetate due to nutrient deficiencies, high levels of acidity, and potential metal toxicities. An amendment of biosolids could ameliorate these harsh growing conditions through the addition of available nutrients, improvement of physical soil properties (e.g., increased water holding capacity), and possible lowering of toxic metal availability through complexation with organic matter. A study was conducted on mine tailings at Holden, WA to evaluate the effect of an amendment of biosolids on the survival and growth of five native plant species (Sitka alder, big leaf maple, fireweed, w. yarrow, and pearly everlasting). Plots were established in tailings, gravel over tailings (G/T), and biosolids plus gravel over tailings. Each of the native plant species, except maple, had their highest survival in the biosolids-amended plot with 3 species at 100% survival. The biosolids amendment was shown to improve the growth of all species except maple. Fireweed produced 62 times more biomass in the biosolids-amended plot compared to the unamended plot (G/T). Plant analysis revealed a dramatic increase in nutrient content with the amendment of biosolids. Biosolids improved the survival, growth, and nutritional status of native plant species on the copper mine tailings.

  13. Structure of the SANT domain from the Xenopus chromatin remodeling factor ISWI

    SciTech Connect

    Horton, John R.; Elgar, Stuart J.; Khan, Seema I.; Zhang, Xing; Wade, Paul A.; Cheng, Xiaodong

    2008-09-17

    The SANT (Swi3, Ada2, N-Cor, and TFIIIB) module was first described as a putative DNA-binding domain with strong similarity to the helix-turn-helix DNA binding domain of Myb-related proteins. The X-ray structure of the C-terminal one third portion of the ATPase ISWI of Drosophila melangoaster, containing both SANT and SLIDE (SANT-Like ISWI Domain), confirmed the overall helix-turn-helix structural architecture of SANT as well as SLIDE. However, the DNA-contacting residues in Myb are not conserved in SANT and the structurally corresponding residues in the ISWI SANT domain are acidic, and therefore incompatible with DNA interaction. Recent studies suggested that SANT domains might be a histone-tail-binding module, including the DNA binding SANT domain of c-Myb. Here they present the X-ray structure of Xenopus laevis ISWI SANT domain, derived from limited proteolysis of a C-terminal fragment of ISWI protein.

  14. A unique alkaline pH-regulated and fatty acid-activated tandem pore domain potassium channel (K2P) from a marine sponge

    PubMed Central

    Wells, Gregory D.; Tang, Qiong-Yao; Heler, Robert; Tompkins-MacDonald, Gabrielle J.; Pritchard, Erica N.; Leys, Sally P.; Logothetis, Diomedes E.; Boland, Linda M.

    2012-01-01

    SUMMARY A cDNA encoding a potassium channel of the two-pore domain family (K2P, KCNK) of leak channels was cloned from the marine sponge Amphimedon queenslandica. Phylogenetic analysis indicated that AquK2P cannot be placed into any of the established functional groups of mammalian K2P channels. We used the Xenopus oocyte expression system, a two-electrode voltage clamp and inside-out patch clamp electrophysiology to determine the physiological properties of AquK2P. In whole cells, non-inactivating, voltage-independent, outwardly rectifying K+ currents were generated by external application of micromolar concentrations of arachidonic acid (AA; EC50 ∼30 μmol l–1), when applied in an alkaline solution (≥pH 8.0). Prior activation of channels facilitated the pH-regulated, AA-dependent activation of AquK2P but external pH changes alone did not activate the channels. Unlike certain mammalian fatty-acid-activated K2P channels, the sponge K2P channel was not activated by temperature and was insensitive to osmotically induced membrane distortion. In inside-out patch recordings, alkalinization of the internal pH (pKa 8.18) activated the AquK2P channels independently of AA and also facilitated activation by internally applied AA. The gating of the sponge K2P channel suggests that voltage-independent outward rectification and sensitivity to pH and AA are ancient and fundamental properties of animal K2P channels. In addition, the membrane potential of some poriferan cells may be dynamically regulated by pH and AA. PMID:22723483

  15. A single amino acid substitution (R441A) in the receptor-binding domain of SARS coronavirus spike protein disrupts the antigenic structure and binding activity

    SciTech Connect

    He Yuxian . E-mail: yhe@nybloodcenter.org; Li Jingjing; Jiang Shibo

    2006-05-26

    The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) has two major functions: interacting with the receptor to mediate virus entry and inducing protective immunity. Coincidently, the receptor-binding domain (RBD, residues 318-510) of SAR-CoV S protein is a major antigenic site to induce neutralizing antibodies. Here, we used RBD-Fc, a fusion protein containing the RBD and human IgG1 Fc, as a model in the studies and found that a single amino acid substitution in the RBD (R441A) could abolish the immunogenicity of RBD to induce neutralizing antibodies in immunized mice and rabbits. With a panel of anti-RBD mAbs as probes, we observed that R441A substitution was able to disrupt the majority of neutralizing epitopes in the RBD, suggesting that this residue is critical for the antigenic structure responsible for inducing protective immune responses. We also demonstrated that the RBD-Fc bearing R441A mutation could not bind to soluble and cell-associated angiotensin-converting enzyme 2 (ACE2), the functional receptor for SARS-CoV and failed to block S protein-mediated pseudovirus entry, indicating that this point mutation also disrupted the receptor-binding motif (RBM) in the RBD. Taken together, these data provide direct evidence to show that a single amino acid residue at key position in the RBD can determine the major function of SARS-CoV S protein and imply for designing SARS vaccines and therapeutics.

  16. Two independent targeting signals in the cytoplasmic domain determine trans-Golgi network localization and endosomal trafficking of the proprotein convertase furin.

    PubMed Central

    Schäfer, W; Stroh, A; Berghöfer, S; Seiler, J; Vey, M; Kruse, M L; Kern, H F; Klenk, H D; Garten, W

    1995-01-01

    Furin, a subtilisin-like eukaryotic endoprotease, is responsible for proteolytic cleavage of cellular and viral proteins transported via the constitutive secretory pathway. Cleavage occurs at the C-terminus of basic amino acid sequences, such as R-X-K/R-R and R-X-X-R. Furin was found predominantly in the trans-Golgi network (TGN), but also in clathrin-coated vesicles dispatched from the TGN, on the plasma membrane as an integral membrane protein and in the medium as an anchorless enzyme. When furin was vectorially expressed in normal rat kidney (NRK) cells it accumulated in the TGN similarly to the endogenous glycoprotein TGN38, often used as a TGN marker protein. The signals determining TGN targeting of furin were investigated by mutational analysis of the cytoplasmic tail of furin and by using the hemagglutinin (HA) of fowl plague virus, a protein with cell surface destination, as a reporter molecule, in which membrane anchor and cytoplasmic tail were replaced by the respective domains of furin. The membrane-spanning domain of furin grafted to HA does not localize the chimeric molecule to the TGN, whereas the cytoplasmic domain does. Results obtained on furin mutants with substitutions and deletions of amino acids in the cytoplasmic tail indicate that wild-type furin is concentrated in the TGN by a mechanism involving two independent targeting signals, which consist of the acidic peptide CPSDSEEDEG783 and the tetrapeptide YKGL765. The acidic signal in the cytoplasmic domain of a HA-furin chimera is necessary and sufficient to localize the reporter molecule to the TGN, whereas YKGL is a determinant for targeting to the endosomes. The data support the concept that the acidic signal, which is the dominant one, retains furin in the TGN, whereas the YKGL motif acts as a retrieval signal for furin that has escaped to the cell surface. Images PMID:7781597

  17. Vegetation successfully prevents oxidization of sulfide minerals in mine tailings.

    PubMed

    Li, Yang; Sun, Qingye; Zhan, Jing; Yang, Yang; Wang, Dan

    2016-07-15

    The oxidization of metal sulfide in tailings causes acid mine drainage. However, it remains unclear whether vegetation prevents the oxidization of metal sulfides. The oxidization characteristics and microbial indices of the tailings in the presence of various plant species were investigated to explore the effects of vegetation on the oxidization of sulfide minerals in tailings. The pH, reducing sulfur, free iron oxides (Fed), chemical oxygen consumption (COC) and biological oxygen consumption (BOC) were measured. Key iron- and sulfur-oxidizing bacteria (Acidithiobacillus spp., Leptospirillum spp. and Thiobacillus spp.) were quantified using real-time PCR. The results indicate that vegetation growing on tailings can effectively prevent the oxidization of sulfide minerals in tailings. A higher pH and reducing-sulfur content and lower Fed were observed in the 0-30 cm depth interval in the presence of vegetation compared to bare tailings (BT). The COC gradually decreased with depth in all of the soil profiles; specifically, the COC rapidly decreased in the 10-20 cm interval in the presence of vegetation but gradually decreased in the BT profiles. Imperata cylindrica (IC) and Chrysopogon zizanoides (CZ) profiles contained the highest BOC in the 10-20 cm interval. The abundance of key iron- and sulfur-oxidizing bacteria in the vegetated tailings were significantly lower than in the BT; in particular, IC was associated with the lowest iron- and sulfur-oxidizing bacterial abundance. In conclusion, vegetation successfully prevented the oxidization of sulfide minerals in the tailings, and Imperata cylindrica is the most effective in reducing the number of iron- and sulfur-oxidizing bacteria and helped to prevent the oxidization of sulfide minerals in the long term.

  18. Plant Growth-Promoting Bacteria for Phytostabilization of Mine Tailings

    SciTech Connect

    Grandlic, C.J.; Mendez, M.O.; Chorover, J.; Machado, B.; Maier, R.M.

    2009-05-19

    Eolian dispersion of mine tailings in arid and semiarid environments is an emerging global issue for which economical remediation alternatives are needed. Phytostabilization, the revegetation of these sites with native plants, is one such alternative. Revegetation often requires the addition of bulky amendments such as compost which greatly increases cost. We report the use of plant growth-promoting bacteria (PGPB) to enhance the revegetation of mine tailings and minimize the need for compost amendment. Twenty promising PGPB isolates were used as seed inoculants in a series of greenhouse studies to examine revegetation of an extremely acidic, high metal content tailings sample previously shown to require 15% compost amendment for normal plant growth. Several isolates significantly enhanced growth of two native species, quailbush and buffalo grass, in tailings. In this study, PGPB/compost outcomes were plant specific; for quailbush, PGPB were most effective in combination with 10% compost addition while for buffalo grass, PGPB enhanced growth in the complete absence of compost. Results indicate that selected PGPB can improve plant establishment and reduce the need for compost amendment. Further, PGPB activities necessary for aiding plant growth in mine tailings likely include tolerance to acidic pH and metals.

  19. Characterization of Emergent Data Networks Among Long-Tail Data

    NASA Astrophysics Data System (ADS)

    Elag, Mostafa; Kumar, Praveen; Hedstrom, Margaret; Myers, James; Plale, Beth; Marini, Luigi; McDonald, Robert

    2014-05-01

    Data curation underpins data-driven scientific advancements. It manages the information flux across multiple users throughout data life cycle as well as increases data sustainability and reusability. The exponential growth in data production spanning across the Earth Science involving individual and small research groups, which is termed as log-tail data, increases the data-knowledge latency among related domains. It has become clear that an advanced framework-agnostic metadata and ontologies for long-tail data is required to increase their visibility to each other, and provide concise and meaningful descriptions that reveal their connectivity. Despite the advancement that has been achieved by various sophisticated data management models in different Earth Science disciplines, it is not always straightforward to derive relationships among long-tail data. Semantic data clustering algorithms and pre-defined logic rules that are oriented toward prediction of possible data relationships, is one method to address these challenges. Our work advances the connectivity of related long-tail data by introducing the design for an ontology-based knowledge management system. In this work, we present the system architecture, its components, and illustrate how it can be used to scrutinize the connectivity among datasets. To demonstrate the capabilities of this "data network" prototype, we implemented this approach within the Sustainable Environment Actionable Data (SEAD) environment, an open-source semantic content repository that provides a RDF database for long-tail data, and show how emergent relationships among datasets can be identified.

  20. The structure of comet tails

    NASA Technical Reports Server (NTRS)

    Brandt, J. C.; Niedner, M. B., Jr.

    1986-01-01

    Present models of the plasma tails of comets are described. The interaction of the solar wind with ions from the cometary atmosphere is discussed, and the phenomenon of magnetic reconnection observed in plasma tails is explained. The accomplishments of the ICE mission to the Comet Giacobini-Zinner are summarized, and the tasks and expected contributions from upcoming Soviet, European, and Japanese missions to Comet Halley are addressed.

  1. Tail phenomena. [of Halley's comet

    NASA Technical Reports Server (NTRS)

    Brandt, J. C.; Niedner, M. B., Jr.

    1985-01-01

    An overview of tail phenomena is presented based on worldwide submissions to the Large-Scale Phenomena Discipline Specialist Team of the International Halley Watch. Examples of tail phenomena and science are presented along with estimates of total expected yield from the Network. The archive of this material will clearly be very valuable for studying the solar-wind/comet interaction during the 1985-1986 apparition of Halley's Comet.

  2. Theseus Tail Being Unloaded

    NASA Technical Reports Server (NTRS)

    1996-01-01

    The tail of the Theseus prototype research aircraft is seen here being unloaded at NASA's Dryden Flight Research Center, Edwards, California, in May of 1996. The Theseus aircraft, built and operated by Aurora Flight Sciences Corporation, Manassas, Virginia, was a unique aircraft flown at NASA's Dryden Flight Research Center, Edwards, California, under a cooperative agreement between NASA and Aurora. Dryden hosted the Theseus program, providing hangar space and range safety for flight testing. Aurora Flight Sciences was responsible for the actual flight testing, vehicle flight safety, and operation of the aircraft. The Theseus remotely piloted aircraft flew its maiden flight on May 24, 1996, at Dryden. During its sixth flight on November 12, 1996, Theseus experienced an in-flight structural failure that resulted in the loss of the aircraft. As of the beginning of the year 2000, Aurora had not rebuilt the aircraft. Theseus was built for NASA under an innovative, $4.9 million fixed-price contract by Aurora Flight Sciences Corporation and its partners, West Virginia University, Morgantown, West Virginia, and Fairmont State College, Fairmont, West Virginia. The twin-engine, unpiloted vehicle had a 140-foot wingspan, and was constructed largely of composite materials. Powered by two 80-horsepower, turbocharged piston engines that drove twin 9-foot-diameter propellers, Theseus was designed to fly autonomously at high altitudes, with takeoff and landing under the active control of a ground-based pilot in a ground control station 'cockpit.' With the potential ability to carry 700 pounds of science instruments to altitudes above 60,000 feet for durations of greater than 24 hours, Theseus was intended to support research in areas such as stratospheric ozone depletion and the atmospheric effects of future high-speed civil transport aircraft engines. Instruments carried aboard Theseus also would be able to validate satellite-based global environmental change measurements

  3. FcLDP1, a Gene Encoding a Late Embryogenesis Abundant (LEA) Domain Protein, Responds to Brassinosteroids and Abscisic Acid during the Development of Fruits in Fragaria chiloensis.

    PubMed

    Espinoza, Analía; Contreras, Rodrigo; Zúñiga, Gustavo E; Herrera, Raúl; Moya-León, María Alejandra; Norambuena, Lorena; Handford, Michael

    2016-01-01

    White Chilean strawberries (Fragaria chiloensis) are non-climacteric fruits, with an exotic color and aroma. In order to discover genes involved in the development of these fruits, we identified a fragment of a gene encoding a late embryogenesis abundant domain protein, FcLDP1, that was expressed in early stages of fruit development, particularly in receptacles. Hormones play key roles in regulating the development of non-climacteric fruits. We show that the brassinosteroid content of the white strawberry varies during development. Additionally, FcLDP1 as well as the closest ortholog in the woodland strawberry, F. vesca (FvLDP1) possess multiple brassinosteroid, as well as abscisic acid (ABA) response motifs in the promoter region, consistent with the response of transiently expressed FcLDP1 promoter-GFP fusions to these hormones, and the rise in FcLDP1 transcript levels in white strawberry fruits treated with brassinosteroids or ABA. These findings suggest that both hormones regulate FcLDP1 expression during the development of white strawberries.

  4. FcLDP1, a Gene Encoding a Late Embryogenesis Abundant (LEA) Domain Protein, Responds to Brassinosteroids and Abscisic Acid during the Development of Fruits in Fragaria chiloensis.

    PubMed

    Espinoza, Analía; Contreras, Rodrigo; Zúñiga, Gustavo E; Herrera, Raúl; Moya-León, María Alejandra; Norambuena, Lorena; Handford, Michael

    2016-01-01

    White Chilean strawberries (Fragaria chiloensis) are non-climacteric fruits, with an exotic color and aroma. In order to discover genes involved in the development of these fruits, we identified a fragment of a gene encoding a late embryogenesis abundant domain protein, FcLDP1, that was expressed in early stages of fruit development, particularly in receptacles. Hormones play key roles in regulating the development of non-climacteric fruits. We show that the brassinosteroid content of the white strawberry varies during development. Additionally, FcLDP1 as well as the closest ortholog in the woodland strawberry, F. vesca (FvLDP1) possess multiple brassinosteroid, as well as abscisic acid (ABA) response motifs in the promoter region, consistent with the response of transiently expressed FcLDP1 promoter-GFP fusions to these hormones, and the rise in FcLDP1 transcript levels in white strawberry fruits treated with brassinosteroids or ABA. These findings suggest that both hormones regulate FcLDP1 expression during the development of white strawberries. PMID:27379111

  5. FcLDP1, a Gene Encoding a Late Embryogenesis Abundant (LEA) Domain Protein, Responds to Brassinosteroids and Abscisic Acid during the Development of Fruits in Fragaria chiloensis

    PubMed Central

    Espinoza, Analía; Contreras, Rodrigo; Zúñiga, Gustavo E.; Herrera, Raúl; Moya-León, María Alejandra; Norambuena, Lorena; Handford, Michael

    2016-01-01

    White Chilean strawberries (Fragaria chiloensis) are non-climacteric fruits, with an exotic color and aroma. In order to discover genes involved in the development of these fruits, we identified a fragment of a gene encoding a late embryogenesis abundant domain protein, FcLDP1, that was expressed in early stages of fruit development, particularly in receptacles. Hormones play key roles in regulating the development of non-climacteric fruits. We show that the brassinosteroid content of the white strawberry varies during development. Additionally, FcLDP1 as well as the closest ortholog in the woodland strawberry, F. vesca (FvLDP1) possess multiple brassinosteroid, as well as abscisic acid (ABA) response motifs in the promoter region, consistent with the response of transiently expressed FcLDP1 promoter-GFP fusions to these hormones, and the rise in FcLDP1 transcript levels in white strawberry fruits treated with brassinosteroids or ABA. These findings suggest that both hormones regulate FcLDP1 expression during the development of white strawberries. PMID:27379111

  6. Disease causing mutants of TDP-43 nucleic acid binding domains are resistant to aggregation and have increased stability and half-life

    PubMed Central

    Austin, James A.; Wright, Gareth S. A.; Watanabe, Seiji; Grossmann, J. Günter; Antonyuk, Svetlana V.; Yamanaka, Koji; Hasnain, S. Samar

    2014-01-01

    Over the last two decades many secrets of the age-related human neural proteinopathies have been revealed. A common feature of these diseases is abnormal, and possibly pathogenic, aggregation of specific proteins in the effected tissue often resulting from inherent or decreased structural stability. An archetype example of this is superoxide dismutase-1, the first genetic factor to be linked with amyotrophic lateral sclerosis (ALS). Mutant or posttranslationally modified TAR DNA binding protein-32 (TDP-43) is also strongly associated with ALS and an increasingly large number of other neurodegenerative diseases, including frontotemporal lobar degeneration (FTLD). Cytoplasmic mislocalization and elevated half-life is a characteristic of mutant TDP-43. Furthermore, patient age at the onset of disease symptoms shows a good inverse correlation with mutant TDP-43 half-life. Here we show that ALS and FTLD-associated TDP-43 mutations in the central nucleic acid binding domains lead to elevated half-life and this is commensurate with increased thermal stability and inhibition of aggregation. It is achieved without impact on secondary, tertiary, or quaternary structure. We propose that tighter structural cohesion contributes to reduced protein turnover, increasingly abnormal proteostasis and, ultimately, faster onset of disease symptoms. These results contrast our perception of neurodegenerative diseases as misfolded proteinopathies and delineate a novel path from the molecular characteristics of mutant TDP-43 to aberrant cellular effects and patient phenotype. PMID:24591609

  7. Surface-specific interaction of the extracellular domain of protein L1 with nitrilotriacetic acid-terminated self-assembled monolayers.

    PubMed

    Fick, Jörg; Wolfram, Tobias; Belz, Ferdinand; Roke, Sylvie

    2010-01-19

    We report a study on the interaction of the extracellular domain of trans-membrane proteins N-cadherin and L1 with nitrilotriacetic acid (NTA)-terminated self-assembled monolayers (SAMs) grown on silver and gold surfaces. Quartz crystal microbalance (QCM) and reflection absorption infrared spectroscopy (RAIRS) measurements reveal that upon addition of protein to an NTA-SAM there is a subsequent change in the mass and average chemical structure inside the films formed on the metal substrates. By using vibrational sum frequency generation (VSFG) spectroscopy and making a comparison to SAMs prepared with n-alkanethiols, we find that the formed NTA-SAMs are terminated by ethanol molecules from solution. The ethanol signature vanishes after the addition of L1, which indicates that the L1 proteins can interact specifically with the NTA complex. Although the RAIRS spectra display signatures in the amide and fingerprint regions, the VSFG spectra display only a weak feature at 866 cm(-1), which possibly indicates that some of the abundant phenyl rings in the complex are ordered. Although cell biology experiments suggest the directional complexation of L1, the VSFG experiments suggest that the alpha-helices and beta-sheets of L1 lack any preferential ordering.

  8. The central acidic domain of MDM2 is critical in inhibition of retinoblastoma-mediated suppression of E2F and cell growth.

    PubMed

    Sdek, Patima; Ying, Haoqiang; Zheng, Hongwu; Margulis, Alexander; Tang, Xiaoren; Tian, Kui; Xiao, Zhi-Xiong Jim

    2004-12-17

    Retinoblastoma (Rb) protein is a paradigm of tumor suppressors. Inactivation of Rb plays a critical role in the development of human malignancies. MDM2, an oncogene frequently found amplified and overexpressed in a variety of human tumors and cancers, directly interacts and inhibits the p53 tumor suppressor protein. In addition, MDM2 has been shown to stimulate E2F transactivation activity and promote S-phase entry independent of p53, yet the mechanism of which is still not fully understood. In this study, we demonstrate that MDM2 specifically binds to Rb C-pocket and that the central acidic domain of MDM2 is essential for Rb interaction. In addition, we show that overexpression of MDM2 reduces Rb-E2F complexes in vivo. Moreover, the ectopic expression of the wild type MDM2, but not mutant MDM2 defective in Rb interaction, stimulates E2F transactivation activity and inhibits Rb growth suppression function. Taken together, these results suggest that MDM2-mediated inhibition of Rb likely contributes to MDM2 oncogenic activity. PMID:15485814

  9. Insights into the phosphoregulation of beta-secretase sorting signal by the VHS domain of GGA1.

    PubMed

    Shiba, Tomoo; Kametaka, Satoshi; Kawasaki, Masato; Shibata, Masahiro; Waguri, Satoshi; Uchiyama, Yasuo; Wakatsuki, Soichi

    2004-06-01

    BACE (beta-site amyloid precursor protein cleaving enzyme, beta-secretase) is a type-I membrane protein which functions as an aspartic protease in the production of beta-amyloid peptide, a causative agent of Alzheimer's disease. Its cytoplasmic tail has a characteristic acidic-cluster dileucine motif recognized by the VHS domain of adaptor proteins, GGAs (Golgi-localizing, gamma-adaptin ear homology domain, ARF-interacting). Here we show that BACE is colocalized with GGAs in the trans-Golgi network and peripheral structures, and phosphorylation of a serine residue in the cytoplasmic tail enhances interaction with the VHS domain of GGA1 by about threefold. The X-ray crystal structure of the complex between the GGA1-VHS domain and the BACE C-terminal peptide illustrates a similar recognition mechanism as mannose 6-phosphate receptors except that a glutamine residue closes in to fill the gap created by the shorter BACE peptide. The serine and lysine of the BACE peptide point their side chains towards the solvent. However, phosphorylation of the serine affects the lysine side chain and the peptide backbone, resulting in one additional hydrogen bond and a stronger electrostatic interaction with the VHS domain, hence the reversible increase in affinity.

  10. l-Ala-γ-d-Glu-meso-diaminopimelic Acid (DAP) Interacts Directly with Leucine-rich Region Domain of Nucleotide-binding Oligomerization Domain 1, Increasing Phosphorylation Activity of Receptor-interacting Serine/Threonine-protein Kinase 2 and Its Interaction with Nucleotide-binding Oligomerization Domain 1*

    PubMed Central

    Laroui, Hamed; Yan, Yutao; Narui, Yoshie; Ingersoll, Sarah A.; Ayyadurai, Saravanan; Charania, Moiz A.; Zhou, Feimeng; Wang, Binghe; Salaita, Khalid; Sitaraman, Shanthi V.; Merlin, Didier

    2011-01-01

    The oligopeptide transporter PepT1 expressed in inflamed colonic epithelial cells transports small bacterial peptides, such as muramyl dipeptide (MDP) and l-Ala-γ-d-Glu-meso-diaminopimelic acid (Tri-DAP) into cells. The innate immune system uses various proteins to sense pathogen-associated molecular patterns. Nucleotide-binding oligomerization domain (NOD)-like receptors of which there are more than 20 related family members are present in the cytosol and recognize intracellular ligands. NOD proteins mediate NF-κB activation via receptor-interacting serine/threonine-protein kinase 2 (RICK or RIPK). The specific ligands for some NOD-like receptors have been identified. NOD type 1 (NOD1) is activated by peptides that contain a diaminophilic acid, such as the PepT1 substrate Tri-DAP. In other words, PepT1 transport activity plays an important role in controlling intracellular loading of ligands for NOD1 in turn determining the activation level of downstream inflammatory pathways. However, no direct interaction between Tri-DAP and NOD1 has been identified. In the present work, surface plasmon resonance and atomic force microscopy experiments showed direct binding between NOD1 and Tri-DAP with a Kd value of 34.5 μm. In contrast, no significant binding was evident between muramyl dipeptide and NOD1. Furthermore, leucine-rich region (LRR)-truncated NOD1 did not interact with Tri-DAP, indicating that Tri-DAP interacts with the LRR domain of NOD1. Next, we examined binding between RICK and NOD1 proteins and found that such binding was significant with a Kd value of 4.13 μm. However, NOD1/RICK binding was of higher affinity (Kd of 3.26 μm) when NOD1 was prebound to Tri-DAP. Furthermore, RICK phosphorylation activity was increased when NOD was prebound to Tri-DAP. In conclusion, we have shown that Tri-DAP interacts directly with the LRR domain of NOD1 and consequently increases RICK/NOD1 association and RICK phosphorylation activity. PMID:21757725

  11. Morphogenesis of the T4 tail and tail fibers

    PubMed Central

    2010-01-01

    Remarkable progress has been made during the past ten years in elucidating the structure of the bacteriophage T4 tail by a combination of three-dimensional image reconstruction from electron micrographs and X-ray crystallography of the components. Partial and complete structures of nine out of twenty tail structural proteins have been determined by X-ray crystallography and have been fitted into the 3D-reconstituted structure of the "extended" tail. The 3D structure of the "contracted" tail was also determined and interpreted in terms of component proteins. Given the pseudo-atomic tail structures both before and after contraction, it is now possible to understand the gross conformational change of the baseplate in terms of the change in the relative positions of the subunit proteins. These studies have explained how the conformational change of the baseplate and contraction of the tail are related to the tail's host cell recognition and membrane penetration function. On the other hand, the baseplate assembly process has been recently reexamined in detail in a precise system involving recombinant proteins (unlike the earlier studies with phage mutants). These experiments showed that the sequential association of the subunits of the baseplate wedge is based on the induced-fit upon association of each subunit. It was also found that, upon association of gp53 (gene product 53), the penultimate subunit of the wedge, six of the wedge intermediates spontaneously associate to form a baseplate-like structure in the absence of the central hub. Structure determination of the rest of the subunits and intermediate complexes and the assembly of the hub still require further study. PMID:21129200

  12. Three-dimensional reconstructions of the bacteriophage CUS-3 virion reveal a conserved coat protein I-domain but a distinct tailspike receptor-binding domain

    SciTech Connect

    Parent, Kristin N.; Tang, Jinghua; Cardone, Giovanni; Gilcrease, Eddie B.; Janssen, Mandy E.; Olson, Norman H.; Casjens, Sherwood R.; Baker, Timothy S.

    2014-09-15

    CUS-3 is a short-tailed, dsDNA bacteriophage that infects serotype K1 Escherichia coli. We report icosahedrally averaged and asymmetric, three-dimensional, cryo-electron microscopic reconstructions of the CUS-3 virion. Its coat protein structure adopts the “HK97-fold” shared by other tailed phages and is quite similar to that in phages P22 and Sf6 despite only weak amino acid sequence similarity. In addition, these coat proteins share a unique extra external domain (“I-domain”), suggesting that the group of P22-like phages has evolved over a very long time period without acquiring a new coat protein gene from another phage group. On the other hand, the morphology of the CUS-3 tailspike differs significantly from that of P22 or Sf6, but is similar to the tailspike of phage K1F, a member of the extremely distantly related T7 group of phages. We conclude that CUS-3 obtained its tailspike gene from a distantly related phage quite recently. - Highlights: • Asymmetric and symmetric three-dimensional reconstructions of phage CUS-3 are presented. • CUS-3 major capsid protein has a conserved I-domain, which is found in all three categories of “P22-like phage”. • CUS-3 has very different tailspike receptor binding domain from those of P22 and Sf6. • The CUS-3 tailspike likely was acquired by horizontal gene transfer.

  13. The inhibition of the GTPase activating protein-Ha-ras interaction by acidic lipids is due to physical association of the C-terminal domain of the GTPase activating protein with micellar structures.

    PubMed Central

    Serth, J; Lautwein, A; Frech, M; Wittinghofer, A; Pingoud, A

    1991-01-01

    The effects of fatty acids and phospholipids on the interaction of the full-length GTPase activating protein (GAP) as well as its isolated C-terminal domain and the Ha-ras proto-oncogene product p21 were studied by various methods, viz. GTPase activity measurements, fluorescence titrations and gel permeation chromatography. It is shown that all fatty acids and acidic phospholipids tested, provided the critical micellar concentration and the critical micellar temperature are reached, inhibit the GAP stimulated p21 GTPase activity. This is interpreted to mean that it is not the molecular structure of acidic lipid molecules per se but rather their physical state of aggregation which is responsible for the inhibitory effect of lipids on the GTPase activity. The relative inhibitory potency of various lipids was measured under defined conditions with mixed Triton X-100 micelles to follow the order: unsaturated fatty acids greater than saturated acids approximately phosphatidic acids greater than or equal to phosphatidylinositol phosphates much greater than phosphatidylinositol and phosphatidylserine. GTPase experiments with varying concentrations of p21 and constant concentrations of GAP and lipids indicate that the binding of GAP by the lipid micelles is responsible for the inhibition, a finding which was confirmed by fluorescence titrations and gel filtrations which show that the C-terminal domain of GAP is bound by lipid micelles. PMID:2026138

  14. Use of cemented paste backfill in arsenic-rich tailings

    NASA Astrophysics Data System (ADS)

    Hamberg, Roger; Maurice, Christian; Alakangas, Lena

    2015-04-01

    Gold is extracted by cyanide leaching from inclusions in arsenopyrite from a mine in the north of Sweden. The major ore mineral assemblage consists of pyrrhotite and arsenopyrite-loellingite. Effluents from the gold extraction were treated with Fe2(SO4)3, with the aim to form stable As-bearing Fe-precipitates (FEP). The use of the method called cemented paste backfill (CPB) is sometimes suggested for the management of tailings. In CPB, tailings are commonly mixed with low proportions (3 - 7 %) of cement and backfilled into underground excavated area. To reduce costs, amendments such as granulated blast furnace slag (GBFS), biofuel fly ash (BFA) and cement kiln dust (CKD) are used for partial replacement of cement in CPB due to their pozzolanic and alkaline properties. The objective for this study was to evaluate the leaching behaviour of As in CPB-mixtures with low proportions (1 - 3 %) of BFA and ordinary cement and unmodified tailings. The selection of CPB-recipies was made based on technical and economical criterias to adress the demands deriving from the mining operations. Speciation of the As in ore and tailings samples revealed that mining processes have dissolved the majority of the arsenopyrite in the ore, causing secondary As phases to co-precipitate with newly formed FEP:s. Tank leaching tests (TLT) and weathering cells (WCT) were used to compare leaching behaviour in a monolithic mass contra a crushed material. Quantification of the presumed benefit of CPB was made by calculation of the cumulative leaching of As. Results from the leaching tests (TLT and WCT) showed that the inclusion of As-rich tailings into a cementitious matrix increased leaching of As. This behaviour could partially be explained by an increase of pH. The addition of alkaline binder materials to tailings increased As leaching due to the relocation of desorbed As from FEPs into less acid-tolerant species such as Ca-arsenates and cementitious As-phases. Unmodified tailings generated an

  15. SUBAQUEOUS DISPOSAL OF MILL TAILINGS

    SciTech Connect

    Neeraj K. Mendiratta; Roe-Hoan Yoon; Paul Richardson

    1999-09-03

    A study of mill tailings and sulfide minerals was carried out in order to understand their behavior under subaqueous conditions. A series of electrochemical experiments, namely, cyclic voltammetry, electrochemical impedance spectroscopy and galvanic coupling tests were carried out in artificial seawater and in pH 6.8 buffer solutions with chloride and ferric salts. Two mill tailings samples, one from the Kensington Mine, Alaska, and the other from the Holden Mine, Washington, were studied along with pyrite, galena, chalcopyrite and copper-activated sphalerite. SEM analysis of mill tailings revealed absence of sulfide minerals from the Kensington Mine mill tailings, whereas the Holden Mine mill tailings contained approximately 8% pyrite and 1% sphalerite. In order to conduct electrochemical tests, carbon matrix composite (CMC) electrodes of mill tailings, pyrite and galena were prepared and their feasibility was established by conducting a series of cyclic voltammetry tests. The cyclic voltammetry experiments carried out in artificial seawater and pH 6.8 buffer with chloride salts showed that chloride ions play an important role in the redox processes of sulfide minerals. For pyrite and galena, peaks were observed for the formation of chloride complexes, whereas pitting behavior was observed for the CMC electrodes of the Kensington Mine mill tailings. The electrochemical impedance spectroscopy conducted in artificial seawater provided with the Nyquist plots of pyrite and galena. The Nyquist plots of pyrite and galena exhibited an inert range of potential indicating a slower rate of leaching of sulfide minerals in marine environments. The galvanic coupling experiments were carried out to study the oxidation of sulfide minerals in the absence of oxygen. It was shown that in the absence of oxygen, ferric (Fe3+) ions might oxidize the sulfide minerals, thereby releasing undesirable oxidation products in the marine environment. The source of Fe{sup 3{minus}} ions may be

  16. Niemann-Pick type C 1 function requires lumenal domain residues that mediate cholesterol-dependent NPC2 binding.

    PubMed

    Deffieu, Maika S; Pfeffer, Suzanne R

    2011-11-22

    Niemann-Pick type C1 (NPC1) protein is needed for cellular utilization of low-density lipoprotein-derived cholesterol that has been delivered to lysosomes. The protein has 13 transmembrane domains, three large lumenal domains, and a cytoplasmic tail. NPC1's lumenally oriented, N-terminal domain binds cholesterol and has been proposed to receive cholesterol from NPC2 protein as part of the process by which cholesterol is exported from lysosomes into the cytosol. Using surface plasmon resonance and affinity chromatography, we show here that the second lumenal domain of NPC1 binds directly to NPC2 protein. For these experiments, a soluble NPC1 lumenal domain 2 was engineered by replacing adjacent transmembrane domains with antiparallel coiled-coil sequences. Interaction of NPC2 with NPC1 lumenal domain 2 is only detected at acidic pH, conditions that are optimal for cholesterol binding to NPC2 and transfer to NPC1; the pH is also appropriate for the acidic environment where binding would take place. Binding to NPC1 domain 2 requires the presence of cholesterol on NPC2 protein, a finding that supports directional transfer of cholesterol from NPC2 onto NPC1's N-terminal domain. Finally, human disease-causing mutations in NPC1 domain 2 decrease NPC2 binding, suggesting that NPC2 binding is necessary for NPC1 function in humans. These data support a model in which NPC1 domain 2 holds NPC2 in position to facilitate directional cholesterol transfer from NPC2 onto NPC1 protein for export from lysosomes.

  17. Does climate have heavy tails?

    NASA Astrophysics Data System (ADS)

    Bermejo, Miguel; Mudelsee, Manfred

    2013-04-01

    When we speak about a distribution with heavy tails, we are referring to the probability of the existence of extreme values will be relatively large. Several heavy-tail models are constructed from Poisson processes, which are the most tractable models. Among such processes, one of the most important are the Lévy processes, which are those process with independent, stationary increments and stochastic continuity. If the random component of a climate process that generates the data exhibits a heavy-tail distribution, and if that fact is ignored by assuming a finite-variance distribution, then there would be serious consequences (in the form, e.g., of bias) for the analysis of extreme values. Yet, it appears that it is an open question to what extent and degree climate data exhibit heavy-tail phenomena. We present a study about the statistical inference in the presence of heavy-tail distribution. In particular, we explore (1) the estimation of tail index of the marginal distribution using several estimation techniques (e.g., Hill estimator, Pickands estimator) and (2) the power of hypothesis tests. The performance of the different methods are compared using artificial time-series by means of Monte Carlo experiments. We systematically apply the heavy tail inference to observed climate data, in particular we focus on time series data. We study several proxy and directly observed climate variables from the instrumental period, the Holocene and the Pleistocene. This work receives financial support from the European Commission (Marie Curie Initial Training Network LINC, No. 289447, within the 7th Framework Programme).

  18. Biogeochemistry of metalliferous mine tailings during phytostabilizatio

    NASA Astrophysics Data System (ADS)

    Chorover, J.; Root, R. A.; Hammond, C.; Wang, Y.; Maier, R. M.

    2015-12-01

    In the semi-arid southwest US, legacy mine tailings and the associated metal(loid) contaminants, are prone to wind dispersion and water erosion. Without remediation, tailings can remain barren for decades to centuries, providing a point source of toxic contamination. Successful mitigation of toxins (As, Pb) from fugitive dust is often limited to confinement and stabilization. Capping mine tailings with soil or gravel is an accepted, although expensive, strategy to reduce erosion. Revegetation via assisted direct planting (also known as phytostabilization) has the potential to be a cost-effective and self-sustaining alternative "green-technology" to expensive capping. The impact of phytostabilization, and requisite added organic carbon and irrigation on mechanisms of contaminant mobility is being investigated with concurrent highly-instrumented greenhouse mesocosms and in situ field studies using advanced microbiological tools and synchrotron x-ray based molecular probes. Composted treatments initially neutralized the near surface acid tailings (~2 to ~6.5). However, after 9 mo the mesocosms showed a gradual and eventual decrease back to pH 2. The exception was the root zone of Atriplex lentiformis, which buffered the acidic conditions for 12 months. Rhizosphere microbiota experienced a 5-log increase in the compost-amended compared to control greenhouse mesocosms. Weathering of the primary sulfidic mineral assemblage, indicated by the iron and sulfur speciation, was shown to control the mobility, speciation and bioavailability of both As and Pb via sequestration in (meta)stable neoformed jarosite phases as plumbojarosite and As(V) substituted for sulfate in hydronium jarosite, with important implications for human and environmental health risk management. We conclude that the disequilibrium imposed by phytostabilization results in an increase of heterotrophic biomass that is concurrent with a time series of geochemical transformations, which controls the species

  19. Importance of the short cytoplasmic domain of the feline immunodeficiency virus transmembrane glycoprotein for fusion activity and envelope glycoprotein incorporation into virions

    SciTech Connect

    Celma, Cristina C.P.; Paladino, Monica G.; Gonzalez, Silvia A.; Affranchino, Jose L.

    2007-09-30

    The mature form of the envelope (Env) glycoprotein of lentiviruses is a heterodimer composed of the surface (SU) and transmembrane (TM) subunits. Feline immunodeficiency virus (FIV) possesses a TM glycoprotein with a cytoplasmic tail of approximately 53 amino acids which is unusually short compared with that of the other lentiviral glycoproteins (more than 100 residues). To investigate the relevance of the FIV TM cytoplasmic domain to Env-mediated viral functions, we characterized the biological properties of a series of Env glycoproteins progressively shortened from the carboxyl terminus. All the mutant Env proteins were efficiently expressed in feline cells and processed into the SU and TM subunits. Deletion of 5 or 11 amino acids from the TM C-terminus did not significantly affect Env surface expression, fusogenic activity or Env incorporation into virions, whereas removal of 17 or 23 residues impaired Env-mediated cell-to-cell fusion. Further truncation of the FIV TM by 29 residues resulted in an Env glycoprotein that was poorly expressed at the cell surface, exhibited only 20% of the wild-type Env fusogenic capacity and was inefficiently incorporated into virions. Remarkably, deletion of the TM C-terminal 35 or 41 amino acids restored or even enhanced Env biological functions. Indeed, these mutant Env glycoproteins bearing cytoplasmic domains of 18 or 12 amino acids were found to be significantly more fusogenic than the wild-type Env and were efficiently incorporated into virions. Interestingly, truncation of the TM cytoplasmic domain to only 6 amino acids did not affect Env incorporation into virions but abrogated Env fusogenicity. Finally, removal of the entire TM cytoplasmic tail or deletion of as many as 6 amino acids into the membrane-spanning domain led to a complete loss of Env functions. Our results demonstrate that despite its relatively short length, the FIV TM cytoplasmic domain plays an important role in modulating Env-mediated viral functions.

  20. Mira's Tail There All Along

    NASA Technical Reports Server (NTRS)

    2007-01-01

    NASA's Galaxy Evolution Explorer discovered an exceptionally long comet-like tail of material trailing behind Mira -- a star that has been studied thoroughly for about 400 years. So, why had this tail gone unnoticed for so long? The answer is that nobody had scanned the extended region around Mira in ultraviolet light until now.

    As this composite demonstrates, the tail is only visible in ultraviolet light (top), and does not show up in visible light (bottom). Incidentally, Mira is much brighter in visible than ultraviolet light due to its low surface temperature of about 3,000 kelvin (about 5,000 degrees Fahrenheit).

    The Galaxy Evolution Explorer, one of NASA's Small Explorer class missions, is the first all-sky survey in ultraviolet light. It found Mira's tail by chance during a routine scan. Since the mission's launch more than four years ago, it has surveyed millions of galaxies and stars. Such vast collections of data often bring welcome surprises, such as Mira's unusual tail.

    The visible-light image is from the United Kingdom Schmidt Telescope in Australia, via the Digitized Sky Survey, a program affiliated with the Space Telescope Science Institute, Baltimore, Md.

  1. Examination of Lipopolysaccharide (O-Antigen) Populations of Thiobacillus ferrooxidans from Two Mine Tailings

    PubMed Central

    Southam, G.; Beveridge, T. J.

    1993-01-01

    Net acid-generating capacities of 39.74 kg of H2SO4 per ton (ca. 0.05 kg/kg) (pH 2.68) for the Lemoine copper mine tailings (closed ca. 8 years ago; located 40 km west of Chibougamau, Quebec, Canada) and 16.07 kg of H2SO4 per ton (ca. 0.02 kg/kg) (pH 3.01) for the Copper Rand tailings (in current use and 50 km distant [east] from those of Lemoine) demonstrate that these sulfide tailings can support populations of acidophilic thiobacilli. Oxidized regions in both tailings environments were readily visible, were extremely acidic (Lemoine, pH 2.36; Copper Rand, pH 3.07), and provided natural isolates for our study. A 10% (wt/vol) oxalic acid treatment, which solubilizes both ferric sulfate and ferric hydroxide precipitates (B. Ramsay, J. Ramsay, M. deTremblay, and C. Chavarie, Geomicrobiol. J. 6:171-177, 1988), enabled the recovery of intact bacterial cells from the tailings material and from liquid synthetic medium for lipopolysaccharide analysis. No viable cells could be cultured after this oxalic acid treatment. Sodium dodecyl sulfate-polyacrylamide gel electro-phoretic profiles of lipopolysaccharides extracted from the Lemoine tailings were complex, indicating a heterogeneous population of Thiobacillus ferrooxidans. Six T. ferrooxidans subspecies as identified by lipopolysaccharide analysis (i.e., lipopolysaccharide chemotypes) were eventually isolated from a total of 112 cultures from the Lemoine tailings. Using the same isolate and lipopolysaccharide typing techniques, we identified only a single lipopolysaccharide chemotype from 20 cultures of T. ferrooxidans isolated from the Copper Rand tailings. This homogeneity of lipopolysaccharide chemotype was much different from what was found for the older Lemoine tailings and may reflect a progressive lipopolysaccharide heterogeneity of Thiobacillus isolates as tailings leach and age. Images PMID:16348925

  2. Core domain mutant Y220C of p53 protein has a key role in copper homeostasis in case of free fatty acids overload.

    PubMed

    Arciello, Mario; Longo, Alessia; Viscomi, Carmela; Capo, Concetta; Angeloni, Antonio; Rossi, Luisa; Balsano, Clara

    2015-12-01

    Nonalcoholic fatty liver disease (NAFLD) is a pathology that includes a wide variety of clinical conditions ranging from simple steatosis to end-stage liver diseases. Despite the huge amount of researches, the molecular basis of NAFLD are still not fully understood. Recently, it was suggested a role for p53 in NAFLD pathogenesis. Among its targets there is Synthesis of Cytochrome c Oxidase 2 (SCO2), a copper chaperone, involved in both aerobic respiration and metal cellular excretion. Copper seems to play a role in NAFLD. It was demonstrated a low hepatic copper content in NAFLD patients, which correlates with metabolic syndrome parameters. Copper homeostasis deregulation, in fact, seems to be related to lipid metabolism alteration and insulin resistance. Here we provide evidence on the role of p53 in the modulation of copper homeostasis, in an experimental model of NAFLD. We used two different hepatoma cell lines, HepG2 and Huh 7.5.1, characterized by the presence of wt p53 and its Y220C mutant, respectively, treated with a free fatty acids (FFAs) solution. Interestingly, p53 activation correlated with the intracellular copper level maintenance. We demonstrated that, in hepatoma cell lines, core domain mutant Y220C of p53 affects the modulation of SCO2 and Copper transporter 1 (CTR1), influencing, in this way, intracellular copper homeostasis in presence of FFAs accumulation, and that the 220 residue of the protein is crucial for such control. The role of p53 we highlighted may have deep implications in clinical conditions where copper homeostasis is deregulated.

  3. Sequential processing of lysosomal acid phosphatase by a cytoplasmic thiol proteinase and a lysosomal aspartyl proteinase.

    PubMed Central

    Gottschalk, S; Waheed, A; Schmidt, B; Laidler, P; von Figura, K

    1989-01-01

    BHK cells expressing human lysosomal acid phosphatase (LAP) transport LAP to lysosomes as an integral membrane protein. In lysosomes LAP is released from the membrane by proteolytic processing, which involves at least two cleavages at the C terminus of LAP. The first cleavage is catalysed by a thiol proteinase at the outside of the lysosomal membrane and removes the bulk of the cytoplasmic tail of LAP. The second cleavage is catalysed by an aspartyl proteinase inside the lysosomes and releases the luminal part of LAP from the membrane-spanning domain. The first cleavage at the cytoplasmic side of the lysosomal membrane depends on acidification of lysosomes and the second cleavage inside the lysosomes depends on prior processing of the cytoplasmic tail. These results suggest that the cytoplasmic tail controls the conformation of the luminal portion of LAP and vice versa. Images PMID:2684640

  4. STAS Domain Structure and Function

    PubMed Central

    Sharma, Alok K.; Rigby, Alan C.; Alper, Seth L.

    2011-01-01

    Pendrin shares with nearly all SLC26/SulP anion transporters a carboxy-terminal cytoplasmic segment organized around a Sulfate Transporter and Anti-Sigma factor antagonist (STAS) domain. STAS domains of divergent amino acid sequence exhibit a conserved fold of 4 β strands interspersed among 5 α helices. The first STAS domain proteins studied were single-domain anti-sigma factor antagonists (anti-anti-σ). These anti-anti-σ indirectly stimulate bacterial RNA polymerase by inactivating inhibitory anti-σ kinases, liberating σ factors to direct specific transcription of target genes or operons. Some STAS domains are nucleotide-binding phosphoproteins or nucleotidases. Others are interaction/transduction modules within multidomain sensors of light, oxygen and other gasotransmitters, cyclic nucleotides, inositol phosphates, and G proteins. Additional multidomain STAS protein sequences suggest functions in sensing, metabolism, or transport of nutrients such as sugars, amino acids, lipids, anions, vitamins, or hydrocarbons. Still other multidomain STAS polypeptides include histidine and serine/threonine kinase domains and ligand-activated transcription factor domains. SulP/SLC26 STAS domains and adjacent sequences interact with other transporters, cytoskeletal scaffolds, and with enzymes metabolizing transported anion substrates, forming putative metabolons. STAS domains are central to membrane targeting of many SulP/SLC26 anion transporters, and STAS domain mutations are associated with at least three human recessive diseases. This review summarizes STAS domain structure and function. PMID:22116355

  5. Acidic phospholipids govern the enhanced activation of IgG-B cell receptor.

    PubMed

    Chen, Xiangjun; Pan, Weiling; Sui, Yinqiang; Li, Hua; Shi, Xiaoshan; Guo, Xingdong; Qi, Hai; Xu, Chenqi; Liu, Wanli

    2015-10-06

    B cells that express the isotype-switched IgG-B cell receptor (IgG-BCR) are one of the driving forces for antibody memory. To allow for a rapid memory IgG antibody response, IgG-BCR evolved into a highly effective signalling machine. Here, we report that the positively charged cytoplasmic domain of mIgG (mIgG-tail) specifically interacts with negatively charged acidic phospholipids. The key immunoglobulin tail tyrosine (ITT) in mIgG-tail is thus sequestered in the membrane hydrophobic core in quiescent B cells. Pre-disruption of such interaction leads to excessive recruitment of BCRs and inflated BCR signalling upon antigen stimulation, resulting in hyperproliferation of primary B cells. Physiologically, membrane-sequestered mIgG-tail can be released by antigen engagement or Ca(2+) mobilization in the initiation of B cell activation. Our studies suggest a novel regulatory mechanism for how dynamic association of mIgG-tail with acidic phospholipids governs the enhanced activation of IgG-BCR.

  6. Spectral induced polarization (SIP) response of mine tailings.

    PubMed

    Placencia-Gómez, Edmundo; Parviainen, Annika; Slater, Lee; Leveinen, Jussi

    2015-02-01

    Mine tailings impoundments are a source of leachates known as acid mine drainage (AMD) which can pose a contamination risk for surrounding surface and groundwater. Methodologies which can help management of this environmental issue are needed. We carried out a laboratory study of the spectral induced polarization (SIP) response of tailings from the Haveri Au-Cu mine, SW Finland. The primary objectives were, (1) to determine possible correlations between SIP parameters and textural properties associated with oxidative-weathering mechanisms, mineralogical composition and metallic content, and (2) to evaluate the effects of the pore water chemistry on SIP parameters associated with redox-inactive and redox-active electrolytes varying in molar concentration, conductivity and pH. The Haveri tailings exhibit well defined relaxation spectra between 100 and 10,000Hz. The relaxation magnitudes are governed by the in-situ oxidative-weathering conditions on sulphide mineral surfaces contained in the tailings, and decrease with the oxidation degree. The oxidation-driven textural variation in the tailings results in changes to the frequency peak of the phase angle, the imaginary conductivity and chargeability, when plotted versus the pore water conductivity. In contrast, the real and the formation electrical conductivity components show a single linear dependence on the pore water conductivity. The increase of the pore water conductivity (dominated by the increase of ions concentration in solution) along with a transition to acidic conditions shifts the polarization peak towards higher frequencies. These findings show the unique sensitivity of the SIP method to potentially discriminate AMD discharges from reactive oxidation zones in tailings, suggesting a significant advantage for monitoring threatened aquifers.

  7. Spectral induced polarization (SIP) response of mine tailings

    NASA Astrophysics Data System (ADS)

    Placencia-Gómez, Edmundo; Parviainen, Annika; Slater, Lee; Leveinen, Jussi

    2015-02-01

    Mine tailings impoundments are a source of leachates known as acid mine drainage (AMD) which can pose a contamination risk for surrounding surface and groundwater. Methodologies which can help management of this environmental issue are needed. We carried out a laboratory study of the spectral induced polarization (SIP) response of tailings from the Haveri Au-Cu mine, SW Finland. The primary objectives were, (1) to determine possible correlations between SIP parameters and textural properties associated with oxidative-weathering mechanisms, mineralogical composition and metallic content, and (2) to evaluate the effects of the pore water chemistry on SIP parameters associated with redox-inactive and redox-active electrolytes varying in molar concentration, conductivity and pH. The Haveri tailings exhibit well defined relaxation spectra between 100 and 10,000 Hz. The relaxation magnitudes are governed by the in-situ oxidative-weathering conditions on sulphide mineral surfaces contained in the tailings, and decrease with the oxidation degree. The oxidation-driven textural variation in the tailings results in changes to the frequency peak of the phase angle, the imaginary conductivity and chargeability, when plotted versus the pore water conductivity. In contrast, the real and the formation electrical conductivity components show a single linear dependence on the pore water conductivity. The increase of the pore water conductivity (dominated by the increase of ions concentration in solution) along with a transition to acidic conditions shifts the polarization peak towards higher frequencies. These findings show the unique sensitivity of the SIP method to potentially discriminate AMD discharges from reactive oxidation zones in tailings, suggesting a significant advantage for monitoring threatened aquifers.

  8. Regulation of enzyme localization by polymerization: polymer formation by the SAM domain of diacylglycerol kinase delta1.

    PubMed

    Harada, Bryan T; Knight, Mary Jane; Imai, Shin-Ichi; Qiao, Feng; Ramachander, Ranjini; Sawaya, Michael R; Gingery, Mari; Sakane, Fumio; Bowie, James U

    2008-03-01

    The diacylglycerol kinase (DGK) enzymes function as regulators of intracellular signaling by altering the levels of the second messengers, diacylglycerol and phosphatidic acid. The DGK delta and eta isozymes possess a common protein-protein interaction module known as a sterile alpha-motif (SAM) domain. In DGK delta, SAM domain self-association inhibits the translocation of DGK delta to the plasma membrane. Here we show that DGK delta SAM forms a polymer and map the polymeric interface by a genetic selection for soluble mutants. A crystal structure reveals that DGKSAM forms helical polymers through a head-to-tail interaction similar to other SAM domain polymers. Disrupting polymerization by polymer interface mutations constitutively localizes DGK delta to the plasma membrane. Thus, polymerization of DGK delta regulates the activity of the enzyme by sequestering DGK delta in an inactive cellular location. Regulation by dynamic polymerization is an emerging theme in signal transduction.

  9. Inhibition of Mycobacterium tuberculosis PknG by non-catalytic rubredoxin domain specific modification: reaction of an electrophilic nitro-fatty acid with the Fe-S center.

    PubMed

    Gil, Magdalena; Graña, Martín; Schopfer, Francisco J; Wagner, Tristan; Denicola, Ana; Freeman, Bruce A; Alzari, Pedro M; Batthyány, Carlos; Durán, Rosario

    2013-12-01

    PknG from Mycobacterium tuberculosis is a Ser/Thr protein kinase that regulates key metabolic processes within the bacterial cell as well as signaling pathways from the infected host cell. This multidomain protein has a conserved canonical kinase domain with N- and C-terminal flanking regions of unclear functional roles. The N-terminus harbors a rubredoxin-like domain (Rbx), a bacterial protein module characterized by an iron ion coordinated by four cysteine residues. Disruption of the Rbx-metal binding site by simultaneous mutations of all the key cysteine residues significantly impairs PknG activity. This encouraged us to evaluate the effect of a nitro-fatty acid (9- and 10-nitro-octadeca-9-cis-enoic acid; OA-NO2) on PknG activity. Fatty acid nitroalkenes are electrophilic species produced during inflammation and metabolism that react with nucleophilic residues of target proteins (i.e., Cys and His), modulating protein function and subcellular distribution in a reversible manner. Here, we show that OA-NO2 inhibits kinase activity by covalently adducting PknG remote from the catalytic domain. Mass spectrometry-based analysis established that cysteines located at Rbx are the specific targets of the nitroalkene. Cys-nitroalkylation is a Michael addition reaction typically reverted by thiols. However, the reversible OA-NO2-mediated nitroalkylation of the kinase results in an irreversible inhibition of PknG. Cys adduction by OA-NO2 induced iron release from the Rbx domain, revealing a new strategy for the specific inhibition of PknG. These results affirm the relevance of the Rbx domain as a target for PknG inhibition and support that electrophilic lipid reactions of Rbx-Cys may represent a new drug strategy for specific PknG inhibition.

  10. Phytostabilization of gold mine tailings, New Zealand. Part 1: Plant establishment in alkaline saline substrate.

    PubMed

    Mains, D; Craw, D; Rufaut, C G; Smith, C M S

    2006-01-01

    Tailings from the Macraes mine, southern New Zealand, are prone to wind erosion. Use of a vegetation cover for physical stabilization is one potential solution to this environmental problem. This study used field trials contained in lysimeters to 1), test the ability of different plant species to grow in un/amended tailings and 2), provide background information on the nutrient and chemical content of waters in tailings. Barley (Hordeum vulgare), blue lupin (Lupinus angustifolius), and rye corn (Secale cereale) were trialed, using Superphosphate fertilizer and sewage sludge as amendments. Rye corn grew well in fertilizer-amended tailings, but poorly in unamended tailings; barley growth was similar in amended and unamended tailings; blue lupins grew poorly overall The tailings had alkaline pH (7-8.5) and water rapidly (< 1 mo) interacted with the tailings to become strongly saline. Minor acid generation was neutralized by calcite, with associated release of calcium and carbonate ions. Leachate waters were supersaturated with respect to calcite and dolomite. Dissolved sodium concentrations were up to 1000 mg L(-1), but elevated Ca2+ calcium and Mg2+ ensured that sodicity was lower than plant-toxic levels. Rye corn is a potentially useful plant for rapid phytostabilization of tailings, with only minor phosphate amendment required.

  11. Microscale measurements of oxygen diffusion and consumption in subaqueous sulfide tailings

    NASA Astrophysics Data System (ADS)

    Elberling, Bo; Damgaard, Lars Riis

    2001-06-01

    The disposal of sulfide mine tailings in an environmentally sound, yet cost-effective, manner is an issue facing most metal mines. Subaqueous tailing disposal is considered an attractive option for disposal that limits oxygen (O 2) availability within sulfide mine tailings and controlling sulfide oxidation and the resultant acid mine drainage (AMD). Assuming that O 2 profiles represent steady-state conditions, we aim to evaluate the depth-dependent and temperature-dependent rates of O 2 consumption in saturated mine tailings. Measurements include microscale O 2 gradients and diffusivity profiles within columns representing undisturbed mine tailing profiles from an impoundment near Nanisivik Mine in northern Canada. Measurements were made across the diffusive boundary layer (DBL) above the tailing-water interface as well as in the tailings below. Laboratory measurements of O 2 profiles are compared to in situ profiles. From the laboratory results it is possible to evaluate the O 2 flux across the DBL and the depth-integrated O 2 uptake. The results are compared with the average sulfate production rate over 3 months. O 2 uptake in saturated tailings is discussed in relation to O 2 uptake measured in columns after free drainage. The methods applied provide consistent O 2 consumption rates as well as reliable predictions for controlling AMD by keeping tailings under water.

  12. Structure of the Receptor-Binding Protein of Bacteriophage Det7: a Podoviral Tail Spike in a Myovirus▿

    PubMed Central

    Walter, Monika; Fiedler, Christian; Grassl, Renate; Biebl, Manfred; Rachel, Reinhard; Hermo-Parrado, X. Lois; Llamas-Saiz, Antonio L.; Seckler, Robert; Miller, Stefan; van Raaij, Mark J.

    2008-01-01

    A new Salmonella enterica phage, Det7, was isolated from sewage and shown by electron microscopy to belong to the Myoviridae morphogroup of bacteriophages. Det7 contains a 75-kDa protein with 50% overall sequence identity to the tail spike endorhamnosidase of podovirus P22. Adsorption of myoviruses to their bacterial hosts is normally mediated by long and short tail fibers attached to a contractile tail, whereas podoviruses do not contain fibers but attach to host cells through stubby tail spikes attached to a very short, noncontractile tail. The amino-terminal 150 residues of the Det7 protein lack homology to the P22 tail spike and are probably responsible for binding to the base plate of the myoviral tail. Det7 tail spike lacking this putative particle-binding domain was purified from Escherichia coli, and well-diffracting crystals of the protein were obtained. The structure, determined by molecular replacement and refined at a 1.6-Å resolution, is very similar to that of bacteriophage P22 tail spike. Fluorescence titrations with an octasaccharide suggest Det7 tail spike to bind its receptor lipopolysaccharide somewhat less tightly than the P22 tail spike. The Det7 tail spike is even more resistant to thermal unfolding than the already exceptionally stable homologue from P22. Folding and assembly of both trimeric proteins are equally temperature sensitive and equally slow. Despite the close structural, biochemical, and sequence similarities between both proteins, the Det7 tail spike lacks both carboxy-terminal cysteines previously proposed to form a transient disulfide during P22 tail spike assembly. Our data suggest receptor-binding module exchange between podoviruses and myoviruses in the course of bacteriophage evolution. PMID:18077713

  13. Plant growth-promoting bacteria for phytostabilization of mine tailings.

    PubMed

    Grandlic, Christopher J; Mendez, Monica O; Chorover, Jon; Machado, Blenda; Maier, Raina M

    2008-03-15

    Eolian dispersion of mine tailings in arid and semiarid environments is an emerging global issue for which economical remediation alternatives are needed. Phytostabilization, the revegetation of these sites with native plants, is one such alternative. Revegetation often requires the addition of bulky amendments such as compost which greatly increases cost. We report the use of plant growth-promoting bacteria (PGPB) to enhance the revegetation of mine tailings and minimize the need for compost amendment. Twenty promising PGPB isolates were used as seed inoculants in a series of greenhouse studies to examine revegetation of an extremely acidic, high metal contenttailings sample previously shown to require 15% compost amendment for normal plant growth. Several isolates significantly enhanced growth of two native species, quailbush and buffalo grass, in tailings. In this study, PGPB/compost outcomes were plant specific; for quailbush, PGPB were most effective in combination with 10% compost addition while for buffalo grass, PGPB enhanced growth in the complete absence of compost. Results indicate that selected PGPB can improve plant establishment and reduce the need for compost amendment. Further, PGPB activities necessary for aiding plant growth in mine tailings likely include tolerance to acidic pH and metals.

  14. The C-terminal tail of protein kinase D2 and protein kinase D3 regulates their intracellular distribution

    SciTech Connect

    Papazyan, Romeo; Rozengurt, Enrique; Rey, Osvaldo . E-mail: orey@mednet.ucla.edu

    2006-04-14

    We generated a set of GFP-tagged chimeras between protein kinase D2 (PKD2) and protein kinase D3 (PKD3) to examine in live cells the contribution of their C-terminal region to their intracellular localization. We found that the catalytic domain of PKD2 and PKD3 can localize to the nucleus when expressed without other kinase domains. However, when the C-terminal tail of PKD2 was added to its catalytic domain, the nuclear localization of the resulting protein was inhibited. In contrast, the nuclear localization of the CD of PKD3 was not inhibited by its C-terminal tail. Furthermore, the exchange of the C-terminal tail of PKD2 and PKD3 in the full-length proteins was sufficient to exchange their intracellular localization. Collectively, these data demonstrate that the short C-terminal tail of these kinases plays a critical role in determining their cytoplasmic/nuclear localization.

  15. Identification of guanylate cyclases and related signaling proteins in sperm tail from sea stars by mass spectrometry.

    PubMed

    Nakachi, Mia; Matsumoto, Midori; Terry, Philip M; Cerny, Ronald L; Moriyama, Hideaki

    2008-01-01

    Marine invertebrates employ external fertilization to take the advantages of sexual reproduction as one of excellent survival strategies. To prevent mismatching, successful fertilization can be made only after going though strictly defined steps in the fertilization. In sea stars, the fertilization process starts with the chemotaxis of sperm followed by hyperactivation of sperm upon arriving onto the egg coat, and then sperm penetrate to the egg coat before achieving the fusion. To investigate whether the initiation of chemotaxis and the following signaling has species specificity, we conducted comparative studies in the protein level among sea stars, Asterias amurensis, A. forbesi, and Asterina pectinifera. Since transcription of messenger ribonucleic acid (mRNA) has been suppressed in gamete, the roles of sperm proteins during the fertilization cannot be investigated by examining the mRNA profile. Therefore, proteomics analysis by mass spectrometry was used in this study. In sea stars, upon receiving asteroidal sperm-activating peptide (asterosap), the receptor membrane-bound guanylate cyclases in the sperm tail trigger sperm chemotaxis. We confirmed the presence of membrane-bound guanylate cyclases in the three sea star species, and they all had the same structural domains including the extracellular domain, kinase-like domain, and guanylate cyclase domain. The majority of peptides recovered were from alpha-helices distributed on the solvent side of the protein. More peptides were recovered from the intracellular domains. The transmembrane domain has not been recovered. The functions of the receptors seemed to be conserved among the species. Furthermore, we identified proteins that may be involved in the guanylate cyclase-triggered signaling pathway.

  16. Formation of nNOS/PSD-95 PDZ dimer requires a preformed beta-finger structure from the nNOS PDZ domain.

    PubMed

    Tochio, H; Mok, Y K; Zhang, Q; Kan, H M; Bredt, D S; Zhang, M

    2000-10-27

    PDZ domains are modular protein units that play important roles in organizing signal transduction complexes. PDZ domains mediate interactions with both C-terminal peptide ligands and other PDZ domains. Here, we used PDZ domains from neuronal nitric oxide synthase (nNOS) and postsynaptic density protein-95 (PSD-95) to explore the mechanism for PDZ-dimer formation. The nNOS PDZ domain terminates with a approximately 30 residue amino acid beta-finger peptide that is shown to be required for nNOS/PSD-95 PDZ dimer formation. In addition, formation of the PDZ dimer requires this beta-finger peptide to be physically anchored to the main body of the canonical nNOS PDZ domain. A buried salt bridge between the beta-finger and the PDZ domain induces and stabilizes the beta-hairpin structure of the nNOS PDZ domain. In apo-nNOS, the beta-finger peptide is partially flexible and adopts a transient beta-strand like structure that is stabilized upon PDZ dimer formation. The flexibility of the NOS PDZ beta-finger is likely to play a critical role in supporting the formation of nNOS/PSD-95 complex. The experimental data also suggest that nNOS PDZ and the second PDZ domain of PSD-95 form a "head-to-tail" dimer similar to the nNOS/syntrophin complex characterized by X-ray crystallography.

  17. Biochemical Characterization of Mycobacterium smegmatis RnhC (MSMEG_4305), a Bifunctional Enzyme Composed of Autonomous N-Terminal Type I RNase H and C-Terminal Acid Phosphatase Domains

    PubMed Central

    Jacewicz, Agata

    2015-01-01

    ABSTRACT Mycobacterium smegmatis encodes several DNA repair polymerases that are adept at incorporating ribonucleotides, which raises questions about how ribonucleotides in DNA are sensed and removed. RNase H enzymes, of which M. smegmatis encodes four, are strong candidates for a surveillance role. Here, we interrogate the biochemical activity and nucleic acid substrate specificity of M. smegmatis RnhC, a bifunctional RNase H and acid phosphatase. We report that (i) the RnhC nuclease is stringently specific for RNA:DNA hybrid duplexes; (ii) RnhC does not selectively recognize and cleave DNA-RNA or RNA-DNA junctions in duplex nucleic acid; (iii) RnhC cannot incise an embedded monoribonucleotide or diribonucleotide in duplex DNA; (iv) RnhC can incise tracts of 4 or more ribonucleotides embedded in duplex DNA, leaving two or more residual ribonucleotides at the cleaved 3′-OH end and at least one or two ribonucleotides on the 5′-PO4 end; (v) the RNase H activity is inherent in an autonomous 140-amino-acid (aa) N-terminal domain of RnhC; and (vi) the C-terminal 211-aa domain of RnhC is an autonomous acid phosphatase. The cleavage specificity of RnhC is clearly distinct from that of Escherichia coli RNase H2, which selectively incises at an RNA-DNA junction. Thus, we classify RnhC as a type I RNase H. The properties of RnhC are consistent with a role in Okazaki fragment RNA primer removal or in surveillance of oligoribonucleotide tracts embedded in DNA but not in excision repair of single misincorporated ribonucleotides. IMPORTANCE RNase H enzymes help cleanse the genome of ribonucleotides that are present either as ribotracts (e.g., RNA primers) or as single ribonucleotides embedded in duplex DNA. Mycobacterium smegmatis encodes four RNase H proteins, including RnhC, which is characterized in this study. The nucleic acid substrate and cleavage site specificities of RnhC are consistent with a role in initiating the removal of ribotracts but not in single

  18. The ionospheres and plasma tails of comets

    NASA Technical Reports Server (NTRS)

    Mendis, D. A.; Ip, W.-H.

    1977-01-01

    The paper reviews the current state of knowledge about cometary plasma (type I) tails and ionospheres. Observational statistics for type I tails are examined along with spectroscopic observations of plasma tails, identified ion species in such tails, and the morphology of cometary plasma tails and ionospheres. Evidence for a strong interaction between comets and the solar wind is evaluated on the basis of observations of plasma-tail orientations, large accelerations of tail structures, and correlations between disturbances in type I tails and solar-wind or geomagnetic disturbances. The use of comets as solar-wind probes is discussed, the nature of comet-solar-wind interactions is investigated, and ionization sources for cometary gases are considered. Hydrodynamic models of comet-solar-wind interaction are summarized, and the structure and ion chemistry of cometary ionospheres are studied. Observations suggesting that significant magnetic fields are associated with comets are briefly reviewed and interpreted.

  19. Lobster Tail Ice Formation on Aerosurface

    NASA Technical Reports Server (NTRS)

    1999-01-01

    Glace Ice formation commonly refered to as 'Lobster Tail' by scientists and engineers, is caused to form on the leading edge of a aircraft tail section in the icing research tunnel at the NASA Glenn Research Center, Cleveland, Ohio.

  20. Expression of a borage desaturase cDNA containing an N-terminal cytochrome b5 domain results in the accumulation of high levels of delta6-desaturated fatty acids in transgenic tobacco.

    PubMed

    Sayanova, O; Smith, M A; Lapinskas, P; Stobart, A K; Dobson, G; Christie, W W; Shewry, P R; Napier, J A

    1997-04-15

    gamma-Linolenic acid (GLA; C18:3 delta(6,9,12)) is a component of the seed oils of evening primrose (Oenothera spp.), borage (Borago officinalis L.), and some other plants. It is widely used as a dietary supplement and for treatment of various medical conditions. GLA is synthesized by a delta6-fatty acid desaturase using linoleic acid (C18:2 delta(9,12)) as a substrate. To enable the production of GLA in conventional oilseeds, we have isolated a cDNA encoding the delta6-fatty acid desaturase from developing seeds of borage and confirmed its function by expression in transgenic tobacco plants. Analysis of leaf lipids from a transformed plant demonstrated the accumulation of GLA and octadecatetraenoic acid (C18:4 delta(6,9,12,15)) to levels of 13.2% and 9.6% of the total fatty acids, respectively. The borage delta6-fatty acid desaturase differs from other desaturase enzymes, characterized from higher plants previously, by the presence of an N-terminal domain related to cytochrome b5.

  1. Subcellular Partitioning of Protein Tyrosine Phosphatase 1B to the Endoplasmic Reticulum and Mitochondria Depends Sensitively on the Composition of Its Tail Anchor

    PubMed Central

    Fueller, Julia; Egorov, Mikhail V.; Walther, Kirstin A.; Sabet, Ola; Mallah, Jana; Grabenbauer, Markus; Kinkhabwala, Ali

    2015-01-01

    The canonical protein tyrosine phosphatase PTP1B is an important regulator of diverse cellular signaling networks. PTP1B has long been thought to exert its influence solely from its perch on the endoplasmic reticulum (ER); however, an additional subpopulation of PTP1B has recently been detected in mitochondria extracted from rat brain tissue. Here, we show that PTP1B’s mitochondrial localization is general (observed across diverse mammalian cell lines) and sensitively dependent on the transmembrane domain length, C-terminal charge and hydropathy of its short (≤35 amino acid) tail anchor. Our electron microscopy of specific DAB precipitation revealed that PTP1B localizes via its tail anchor to the outer mitochondrial membrane (OMM), with fluorescence lifetime imaging microscopy establishing that this OMM pool contributes to the previously reported cytoplasmic interaction of PTP1B with endocytosed epidermal growth factor receptor. We additionally examined the mechanism of PTP1B’s insertion into the ER membrane through heterologous expression of PTP1B’s tail anchor in wild-type yeast and yeast mutants of major conserved ER insertion pathways: In none of these yeast strains was ER targeting significantly impeded, providing in vivo support for the hypothesis of spontaneous membrane insertion (as previously demonstrated in vitro). Further functional elucidation of the newly recognized mitochondrial pool of PTP1B will likely be important for understanding its complex roles in cellular responses to external stimuli, cell proliferation and diseased states. PMID:26431424

  2. Systematic mutational analysis of the amino-terminal domain of the Listeria monocytogenes ActA protein reveals novel functions in actin-based motility.

    PubMed

    Lauer, P; Theriot, J A; Skoble, J; Welch, M D; Portnoy, D A

    2001-12-01

    The Listeria monocytogenes ActA protein acts as a scaffold to assemble and activate host cell actin cytoskeletal factors at the bacterial surface, resulting in directional actin polymerization and propulsion of the bacterium through the cytoplasm. We have constructed 20 clustered charged-to-alanine mutations in the NH2-terminal domain of ActA and replaced the endogenous actA gene with these molecular variants. These 20 clones were evaluated in several biological assays for phenotypes associated with particular amino acid changes. Additionally, each protein variant was purified and tested for stimulation of the Arp2/3 complex, and a subset was tested for actin monomer binding. These specific mutations refined the two regions involved in Arp2/3 activation and suggest that the actin-binding sequence of ActA spans 40 amino acids. We also identified a 'motility rate and cloud-to-tail transition' region in which nine contiguous mutations spanning amino acids 165-260 caused motility rate defects and changed the ratio of intracellular bacteria associated with actin clouds and comet tails without affecting Arp2/3 activation. Several unusual motility phenotypes were associated with amino acid changes in this region, including altered paths through the cytoplasm, discontinuous actin tails in host cells and the tendency to 'skid' or dramatically change direction while moving. These unusual phenotypes illustrate the complexity of ActA functions that control the actin-based motility of L. monocytogenes.

  3. Systematic mutational analysis of the amino-terminal domain of the Listeria monocytogenes ActA protein reveals novel functions in actin-based motility.

    PubMed

    Lauer, P; Theriot, J A; Skoble, J; Welch, M D; Portnoy, D A

    2001-12-01

    The Listeria monocytogenes ActA protein acts as a scaffold to assemble and activate host cell actin cytoskeletal factors at the bacterial surface, resulting in directional actin polymerization and propulsion of the bacterium through the cytoplasm. We have constructed 20 clustered charged-to-alanine mutations in the NH2-terminal domain of ActA and replaced the endogenous actA gene with these molecular variants. These 20 clones were evaluated in several biological assays for phenotypes associated with particular amino acid changes. Additionally, each protein variant was purified and tested for stimulation of the Arp2/3 complex, and a subset was tested for actin monomer binding. These specific mutations refined the two regions involved in Arp2/3 activation and suggest that the actin-binding sequence of ActA spans 40 amino acids. We also identified a 'motility rate and cloud-to-tail transition' region in which nine contiguous mutations spanning amino acids 165-260 caused motility rate defects and changed the ratio of intracellular bacteria associated with actin clouds and comet tails without affecting Arp2/3 activation. Several unusual motility phenotypes were associated with amino acid changes in this region, including altered paths through the cytoplasm, discontinuous actin tails in host cells and the tendency to 'skid' or dramatically change direction while moving. These unusual phenotypes illustrate the complexity of ActA functions that control the actin-based motility of L. monocytogenes. PMID:11886549

  4. Magnetic domain wall gratings for magnetization reversal tuning and confined dynamic mode localization.

    PubMed

    Trützschler, Julia; Sentosun, Kadir; Mozooni, Babak; Mattheis, Roland; McCord, Jeffrey

    2016-01-01

    High density magnetic domain wall gratings are imprinted in ferromagnetic-antiferromagnetic thin films by local ion irradiation by which alternating head-to-tail-to-head-to-tail and head-to-head-to-tail-to-tail spatially overlapping domain wall networks are formed. Unique magnetic domain processes result from the interaction of anchored domain walls. Non-linear magnetization response is introduced by the laterally distributed magnetic anisotropy phases. The locally varying magnetic charge distribution gives rise to localized and guided magnetization spin-wave modes directly constrained by the narrow domain wall cores. The exchange coupled multiphase material structure leads to unprecedented static and locally modified dynamic magnetic material properties. PMID:27487941

  5. Magnetic domain wall gratings for magnetization reversal tuning and confined dynamic mode localization

    PubMed Central

    Trützschler, Julia; Sentosun, Kadir; Mozooni, Babak; Mattheis, Roland; McCord, Jeffrey

    2016-01-01

    High density magnetic domain wall gratings are imprinted in ferromagnetic-antiferromagnetic thin films by local ion irradiation by which alternating head-to-tail-to-head-to-tail and head-to-head-to-tail-to-tail spatially overlapping domain wall networks are formed. Unique magnetic domain processes result from the interaction of anchored domain walls. Non-linear magnetization response is introduced by the laterally distributed magnetic anisotropy phases. The locally varying magnetic charge distribution gives rise to localized and guided magnetization spin-wave modes directly constrained by the narrow domain wall cores. The exchange coupled multiphase material structure leads to unprecedented static and locally modified dynamic magnetic material properties. PMID:27487941

  6. Mechanics of biomimetic systems propelled by actin comet tails

    NASA Astrophysics Data System (ADS)

    Kang, Hyeran; Tambe, Dhananjay; Shenoy, Vivek; Tang, Jay

    2009-03-01

    The motility of intracellular bacterial pathogens such as Listeria monocytogenes is driven by filamentous actin comet tails in a variety of trajectories. Here, we present the in vitro study on the actin-based movements using spherical beads of different sizes coated with VCA protein, a partial domain of N-Wasp, in platelet extracts. Long term two-dimensional trajectories of the spherical beads motility show characteristic difference than those observed for bacteria, which have both elongated shape and asymmetric expression of the polymerization inducing enzyme. The trajectories also vary sensitively with the bead size and shape. These results provide a useful test to our new analytical model including the rotation of the bead relative to the tail.

  7. A Cytoplasmic Dynein Tail Mutation Impairs Motor Processivity

    PubMed Central

    Ori-McKenney, Kassandra M.; Xu, Jing; Gross, Steven P.; Vallee, Richard B.

    2012-01-01

    Mutations in the tail of the cytoplasmic dynein molecule have been reported to cause neurodegenerative disease in mice. The mutant mouse strain Legs at Odd Angles (Loa) exhibits impaired retrograde axonal transport, but the molecular deficiencies in the mutant dynein molecule, and how they contribute to neurodegeneration, are unknown. To address these questions, we purified wild-type and mutant mouse dynein. Using biochemical, single molecule, and live cell imaging techniques, we find a strong inhibition of motor run-length in vitro and in vivo, and significantly altered motor domain coordination in the mutant dynein. These results suggest a potential role for the dynein tail in motor function, and provide the first direct evidence for a link between single-motor processivity and disease. PMID:21102439

  8. Ezrin self-association involves binding of an N-terminal domain to a normally masked C-terminal domain that includes the F-actin binding site.

    PubMed Central

    Gary, R; Bretscher, A

    1995-01-01

    Ezrin is a membrane-cytoskeletal linking protein that is concentrated in actin-rich surface structures. It is closely related to the microvillar proteins radixin and moesin and to the tumor suppressor merlin/schwannomin. Cell extracts contain ezrin dimers and ezrin-moesin heterodimers in addition to monomers. Truncated ezrin fusion proteins were assayed by blot overlay to determine which regions mediate self-association. Here we report that ezrin self-association occurs by head-to-tail joining of distinct N-terminal and C-terminal domains. It is likely that these domains, termed N- and C-ERMADs (ezrin-radixin-moesin association domain), are responsible for homotypic and heterotypic associations among ERM family members. The N-ERMAD of ezrin resided within amino acids 1-296; deletion of 10 additional residues resulted in loss of activity. The C-ERMAD was mapped to the last 107 amino acids of ezrin, residues 479-585. The two residues at the C-terminus were required for activity, and the region from 530-585 was insufficient. The C-ERMAD was masked in the native monomer. Exposure of this domain required unfolding ezrin with sodium dodecyl sulfate or expressing the domain as part of a truncated protein. Intermolecular association could not occur unless the C-ERMAD had been made accessible to its N-terminal partner. It can be inferred that dimerization in vivo requires an activation step that exposes this masked domain. The conformationally inaccessible C-terminal region included the F-actin binding site, suggesting that this activity is likewise regulated by masking. Images PMID:7579708

  9. Uranium mill tailings quarterly report, January-March 1982

    SciTech Connect

    Latkovich, J.M.

    1982-05-01

    Progress is reported on: radon barrier systems for uranium mill tailings; liner evaluation for uranium mill tailings; revegetation/rock cover for stabilization of inactive U-tailings sites; and application of long-term chemical biobarriers for uranium tailings.

  10. Hepatitis C virus core protein interacts with the cytoplasmic tail of lymphotoxin-beta receptor.

    PubMed Central

    Matsumoto, M; Hsieh, T Y; Zhu, N; VanArsdale, T; Hwang, S B; Jeng, K S; Gorbalenya, A E; Lo, S Y; Ou, J H; Ware, C F; Lai, M M

    1997-01-01

    Hepatitis C virus (HCV) core protein is a multifunctional protein. We examined whether it can interact with cellular proteins, thus contributing to viral pathogenesis. Using the HCV core protein as a bait to screen a human liver cDNA library in a yeast two-hybrid screening system, we have isolated several positive clones encoding cellular proteins that interact with the HCV core protein. Interestingly, more than half of these clones encode the cytoplasmic domain of lymphotoxin-beta receptor (LT betaR), which is a member of the tumor necrosis factor receptor family. Their binding was confirmed by in vitro glutathione S-transferase fusion protein binding assay and protein-protein blotting assay to be direct and specific. The binding sites were mapped within a 58-amino-acid region of the cytoplasmic tail of LT betaR. The binding site in the HCV core protein was localized within amino acid residues 36 to 91 from the N terminus, corresponding to the hydrophilic region of the protein. In mammalian cells, the core protein was found to be associated with the membrane-bound LT betaR. Since the LT betaR is involved in germinal center formation and developmental regulation of peripheral lymphoid organs, lymph node development, and apoptotic signaling, the binding of HCV core protein to LT betaR suggests the possibility that this viral protein has an immunomodulating function and may explain the mechanism of viral persistence and pathogenesis of HCV. PMID:8995654

  11. Effect of Apex Flap Deflection on Vertical Tail Buffeting

    NASA Technical Reports Server (NTRS)

    Massey, Steven J.; Kandil, Osama A.

    1998-01-01

    A computational study of the effect of vortex breakdown location on vertical tail buffeting is conducted. The position of the breakdown is modified by employing an apex flap deflected by an experimentally determined optimal angle. The delayed breakdown flow and buffeting response is then compared to the nominal undeflected case. This multidisciplinary problem is solved sequentially for the fluid flow, the elastic tail deformations and the grid displacements. The fluid flow is simulated by time accurately solving the unsteady, compressible, Reynolds-averaged Navier-Stokes equations using an implicit, upwind, flux-difference splitting finite volume scheme. The elastic vibrations of the tails are modeled by uncoupled bending and torsion beam equations. These equations are solved accurately in time using the Galerkin method and a five-stage Runge-Kutta-Verner scheme. The grid for the fluid dynamics calculations is continuously deformed using interpolation functions to disperse the displacements smoothly throughout the computational domain. An angle-of-attack of 35 deg.is chosen such that the wing primary-vortex cores experience vortex breakdown and the resulting turbulent wake flow impinges on tile vertical tails. The dimensions and material properties of the vertical tails are chosen such that the deflections are large enough to insure interaction with the flow, and the natural frequencies are high enough to facilitate a practical computational solution. Results are presented for a baseline uncontrolled buffeting case and a delayed breakdown case in which the apex flap has been deflected 15 deg. The flap was found to be very effective in delaying the breakdown, increasing the location from 50%c to 94%c, which resulted in a 6% increase in lift coefficient and pitching moment. However, the integrated buffet loads and tip responses were roughly equivalent for the two cases.

  12. Translocation of Mitochondrially Synthesized Cox2 Domains from the Matrix to the Intermembrane Space▿

    PubMed Central

    Fiumera, Heather L.; Broadley, Sarah A.; Fox, Thomas D.

    2007-01-01

    The N-terminal and C-terminal domains of mitochondrially synthesized cytochrome c oxidase subunit II, Cox2, are translocated through the inner membrane to the intermembrane space (IMS). We investigated the distinct mechanisms of N-tail and C-tail export by analysis of epitope-tagged Cox2 variants encoded in Saccharomyces cerevisiae mitochondrial DNA. Both the N and C termini of a truncated protein lacking the Cox2 C-terminal domain were translocated to the IMS via a pathway dependent upon the conserved translocase Oxa1. The topology of this Cox2 variant, accumulated at steady state, was largely but not completely unaffected in mutants lacking proteins required for export of the C-tail domain, Cox18 and Mss2. C-tail export was blocked by truncation of the last 40 residues from the C-tail domain, indicating that sequence and/or structural features of this domain are required for its translocation. Mss2, a peripheral protein bound to the inner surface of the inner membrane, coimmunoprecipitated with full-length newly synthesized Cox2, whose leader peptide had already been cleaved in the IMS. Our data suggest that the C-tail domain is recognized posttranslationally by a specialized translocation apparatus after the N-tail has been translocated by Oxa1. PMID:17452441

  13. What Makes a Tidal Tail?

    NASA Astrophysics Data System (ADS)

    Rodruck, Michael; Konstantopoulos, I.; Charlton, J. C.

    2014-01-01

    Galaxy interactions are famous for creating some of the most visually stunning scenes in astronomy, particularly in the cases of tidal tails. These chaotic regions are known to house breeding grounds for young stellar clusters, as shown through past imaging and spectroscopic studies, but the underlying material remains a mystery. While we know that gas is easily stripped from the parent galaxies, what about the stars? The presence of an older stellar population is crucial to dynamical simulations of tidal tails, but has not yet been confirmed by observation. We use the twin tidal tails of NGC3256 as a case study for determining the presence of an old, underlying stellar population. Newly acq