Science.gov

Sample records for acid sites involved

  1. Mechanism of dehydroxylation of naturally occurring high-silica zeolites involving the formation of Lewis acid sites

    SciTech Connect

    Kazanskii, V.B.

    1987-11-01

    Using low-temperature adsorbed dihydrogen and carbon monoxide as molecular probes, the dehydroxylation of the hydrogen forms of the zeolites Y, and ZSM-5 has been studied. The high stability of the high-silica zeolites to dealumination and their difference from faujasites has been established as being due not only to the strength of their Broensted acid sites but also to the nature of their Lewis acid sites. The chemical properties of the Lewis acid sites and their possible role in catalytic reactions are discussed.

  2. Aspartic Acid 397 in Subunit B of the Na+-pumping NADH:Quinone Oxidoreductase from Vibrio cholerae Forms Part of a Sodium-binding Site, Is Involved in Cation Selectivity, and Affects Cation-binding Site Cooperativity

    PubMed Central

    Shea, Michael E.; Juárez, Oscar; Cho, Jonathan; Barquera, Blanca

    2013-01-01

    The Na+-pumping NADH:quinone complex is found in Vibrio cholerae and other marine and pathogenic bacteria. NADH:ubiquinone oxidoreductase oxidizes NADH and reduces ubiquinone, using the free energy released by this reaction to pump sodium ions across the cell membrane. In a previous report, a conserved aspartic acid residue in the NqrB subunit at position 397, located in the cytosolic face of this protein, was proposed to be involved in the capture of sodium. Here, we studied the role of this residue through the characterization of mutant enzymes in which this aspartic acid was substituted by other residues that change charge and size, such as arginine, serine, lysine, glutamic acid, and cysteine. Our results indicate that NqrB-Asp-397 forms part of one of the at least two sodium-binding sites and that both size and charge at this position are critical for the function of the enzyme. Moreover, we demonstrate that this residue is involved in cation selectivity, has a critical role in the communication between sodium-binding sites, by promoting cooperativity, and controls the electron transfer step involved in sodium uptake (2Fe-2S → FMNC). PMID:24030824

  3. Transcriptome sequencing revealed the transcriptional organization at ribosome-mediated attenuation sites in Corynebacterium glutamicum and identified a novel attenuator involved in aromatic amino acid biosynthesis.

    PubMed

    Neshat, Armin; Mentz, Almut; Rückert, Christian; Kalinowski, Jörn

    2014-11-20

    The Gram-positive bacterium Corynebacterium glutamicum belongs to the order Corynebacteriales and is used as a producer of amino acids at industrial scales. Due to its economic importance, gene expression and particularly the regulation of amino acid biosynthesis has been investigated extensively. Applying the high-resolution technique of transcriptome sequencing (RNA-seq), recently a vast amount of data has been generated that was used to comprehensively analyze the C. glutamicum transcriptome. By analyzing RNA-seq data from a small RNA cDNA library of C. glutamicum, short transcripts in the known transcriptional attenuators sites of the trp operon, the ilvBNC operon and the leuA gene were verified. Furthermore, whole transcriptome RNA-seq data were used to elucidate the transcriptional organization of these three amino acid biosynthesis operons. In addition, we discovered and analyzed the novel attenuator aroR, located upstream of the aroF gene (cg1129). The DAHP synthase encoded by aroF catalyzes the first step in aromatic amino acid synthesis. The AroR leader peptide contains the amino acid sequence motif F-Y-F, indicating a regulatory effect by phenylalanine and tyrosine. Analysis by real-time RT-PCR suggests that the attenuator regulates the transcription of aroF in dependence of the cellular amount of tRNA loaded with phenylalanine when comparing a phenylalanine-auxotrophic C. glutamicum mutant fed with limiting and excess amounts of a phenylalanine-containing dipeptide. Additionally, the very interesting finding was made that all analyzed attenuators are leaderless transcripts. PMID:24910972

  4. Implementation of an ex situ stabilization technique at the Sand Springs superfund site to solidify and stabilize acid tar sludges involving a quick-lime based stabilization process and innovative equipment design

    SciTech Connect

    McManus, R.W.; Grajczak, P.; Wilcoxson, J.C.; Webster, S.D.

    1997-12-31

    An old refinery site was safely remediated a year before schedule and for 25% less than final engineering estimates for the stabilization remedy thanks to energetic project management and innovative design involving ex situ stabilization/solidification of acid tar sludges. A quicklime based process, Dispersion by Chemical Reaction (DCR{trademark}), was employed to solidify and stabilize (SS) over 103,000 cubic meters (135,000 cubic yards) of petroleum waste, mostly acidic tarry sludge. The SS process was selected over competing methods because it afforded minimal volume increase, could readily achieve Record of Decision (ROD) specified physical and chemical treatment goals, could be implemented with treatment equipment that minimized emissions, and could be performed with low reagent usage and at low cost. To ensure treatment goals were achieved and an accelerated schedule met, a custom designed and fabricated transportable treatment unit (TTU) was employed to implement the process. The treated material was visually soil-like in character, it was left in stockpiles for periods of time, and it was placed and compacted in the on site landfill using standard earth-moving equipment.

  5. Low resolution X-ray structure of γ-glutamyltranspeptidase from Bacillus licheniformis: opened active site cleft and a cluster of acid residues potentially involved in the recognition of a metal ion.

    PubMed

    Lin, Long-Liu; Chen, Yi-Yu; Chi, Meng-Chun; Merlino, Antonello

    2014-09-01

    γ-Glutamyltranspeptidases (γ-GTs) cleave the γ-glutamyl amide bond of glutathione and transfer the released γ-glutamyl group to water (hydrolysis) or acceptor amino acids (transpeptidation). These ubiquitous enzymes play a key role in the biosynthesis and degradation of glutathione, and in xenobiotic detoxification. Here we report the 3Å resolution crystal structure of Bacillus licheniformis γ-GT (BlGT) and that of its complex with l-Glu. X-ray structures confirm that BlGT belongs to the N-terminal nucleophilic hydrolase superfamily and reveal that the protein possesses an opened active site cleft similar to that reported for the homologous enzyme from Bacillus subtilis, but different from those observed for human γ-GT and for γ-GTs from other microorganisms. Data suggest that the binding of l-Glu induces a reordering of the C-terminal tail of BlGT large subunit and allow the identification of a cluster of acid residues that are potentially involved in the recognition of a metal ion. The role of these residues on the conformational stability of BlGT has been studied by characterizing the autoprocessing, enzymatic activity, chemical and thermal denaturation of four new Ala single mutants. The results show that replacement of Asp568 with an Ala affects both the autoprocessing and structural stability of the protein. PMID:24780583

  6. Mapping of the Tacaribe Arenavirus Z-Protein Binding Sites on the L Protein Identified both Amino Acids within the Putative Polymerase Domain and a Region at the N Terminus of L That Are Critically Involved in Binding▿

    PubMed Central

    Wilda, Maximiliano; Lopez, Nora; Casabona, Juan Cruz; Franze-Fernandez, Maria T.

    2008-01-01

    Tacaribe virus (TacV) is the prototype of the New World group of arenaviruses. The TacV genome encodes four proteins: the nucleoprotein (N), the glycoprotein precursor, the polymerase (L), and a RING finger protein (Z). Using a reverse genetics system, we demonstrated that TacV N and L are sufficient to drive transcription and replication mediated by TacV-like RNAs and that Z is a powerful inhibitor of these processes (Lopez et al., J. Virol. 65:12241-12251, 2001). More recently, we provided the first evidence of an interaction between Z and L and showed that Z's inhibitory activity was dependent on its ability to bind to L (Jácamo et al., J. Virol. 77:10383-10393, 2003). In the present study, we mapped the TacV Z-binding sites on the 2,210-amino-acid L polymerase. To that end, we performed deletion analysis and point mutations of L and studied the Z-L interaction by coimmunoprecipitation with specific sera. We found that the C-terminal region of L was not essential for the interaction and identified two noncontiguous regions that were critical for binding: one at the N-terminus of L between residues 156 and 292 and a second one in the polymerase domain (domain III). The importance of domain III in binding was revealed by substitutions in D1188 and H1189 within motif A and in each residue of the conserved SDD sequence (residues 1328, 1329, and 1330) within motif C. Our results showed that of the substituted residues, only H1189 and D1329 appeared to be critically involved in binding Z. PMID:18799569

  7. ANTHROPOGENIC EMISSIONS INVOLVED IN ACIDIC DEPOSITION PROCESSES

    EPA Science Inventory

    The paper describes the methodology that was used to develop the 1985 National Acid Precipitation Assessment Program (NAPAP) Emissions Inventory, including quality control procedures. and summarizes the inventory contents. evelopment of the 1985 inventory required detailed invest...

  8. The 7-amino-acid site in the proline-rich region of the N-terminal domain of p53 is involved in the interaction with FAK and is critical for p53 functioning.

    PubMed

    Golubovskaya, Vita M; Finch, Richard; Zheng, Min; Kurenova, Elena V; Cance, William G

    2008-04-01

    It is known that p53 alterations are commonly found in tumour cells. Another marker of tumorigenesis is FAK (focal adhesion kinase), a non-receptor kinase that is overexpressed in many types of tumours. Previously we determined that the N-terminal domain of FAK physically interacted with the N-terminal domain of p53. In the present study, using phage display, sitedirected mutagenesis, pulldown and immunoprecipitation assays we localized the site of FAK binding to a 7-amino-acid region(amino acids 65-71) in the N-terminal proline-rich domain of human p53. Mutation of the binding site in p53 reversed the suppressive effect of FAK on p53-mediated transactivation ofp21, BAX (Bcl-2-associated X protein) and Mdm2 (murine double minute 2) promoters. In addition, to functionally test this p53 site, we conjugated p53 peptides [wild-type (containing the wild-type binding site) and mutant (with a mutated 7-aminoacid binding site)] to a TAT peptide sequence to penetrate the cells, and demonstrated that the wild-type p53 peptide disrupted binding of FAK and p53 proteins and significantly inhibited cell viability of HCT116 p53+/+ cells compared with the control mutant peptide and HCT116 p53-/- cells. Furthermore, the TAT-p53 peptide decreased the viability of MCF-7 cells, whereas the mutant peptide did not cause this effect. Normal fibroblast p53+/+ and p53-/- MEF (murine embryonic fibroblast) cells and breast MCF10A cells were not sensitive to p53 peptide. Thus, for the first time, we have identified the binding site of the p53 andFAK interaction and have demonstrated that mutating this site and targeting the site with peptides affects p53 functioning and viability in the cells. PMID:18215142

  9. Emissions involved in acidic deposition processes: Methodology and results

    SciTech Connect

    Placet, M.

    1990-01-01

    Data on the emissions involved in atmospheric acid-base chemistry are crucial to the assessment of acidic deposition and its effects. Sulfur dioxide (SO{sub 2}), nitrogen oxides (NO{sub x}), and volatile organic compounds (VOCs) are the primary chemical compounds involved in acidic deposition processes. In addition, other emission species -- e.g., ammonia, alkaline dust particles, hydrogen chloride, and hydrogen fluoride -- are involved in atmospheric acid-base chemistry, either by contributing acidic constituents or by neutralizing acidic species. Several emissions data bases have been developed under the auspices of the National Acid Precipitation Program (NAPAP). In addition to those developed by NAPAP, emissions data bases and emissions trends estimates also have been developed by organizations such as the Electric Power Research Institute (EPRI) and the U.S. Environmental Protection Agency (EPA). This paper briefly describes and compares the methods used in developing these emissions data bases and presents an overview of their emissions estimates. A more detailed discussion of these topics can be found in the State-of-Science Report on emissions recently released by NAPAP and in the references cited in that report. 14 refs., 4 figs., 1 tab.

  10. Computational studies of Bronsted acid sites in zeolites

    SciTech Connect

    Curtiss, L.A.; Iton, L.E.; Zygmunt, S.A.

    1995-01-01

    The authors have performed high-level ab initio calculations using both Hartree-Fock (HF) and Moller-Plesset perturbation theory (MP2) to study the geometry and energetics of the adsorption complex involving H{sub 2}O and the Bronsted acid site in the zeolite H-ZSM-5. In these calculations, which use aluminosilicate cluster models for the zeolite framework with as many a 28 T atoms (T = Si, Al), we included geometry optimization in the local vicinity of the acid site at the HF/6-31G(d) level of theory, and have calculated corrections for zero-point energies, extensions for zero-point energies, extensions to higher basis sets, and the influence of electron correlation. Results for the adsorption energy and geometry of this complex are reported and compared with previous theoretical and experimental values.

  11. Coarse-Grained Model for Water Involving a Virtual Site.

    PubMed

    Deng, Mingsen; Shen, Hujun

    2016-02-01

    In this work, we propose a new coarse-grained (CG) model for water by combining the features of two popular CG water models (BMW and MARTINI models) as well as by adopting a topology similar to that of the TIP4P water model. In this CG model, a CG unit, representing four real water molecules, consists of a virtual site, two positively charged particles, and a van der Waals (vdW) interaction center. Distance constraint is applied to the bonds formed between the vdW interaction center and the positively charged particles. The virtual site, which carries a negative charge, is determined by the locations of the two positively charged particles and the vdW interaction center. For the new CG model of water, we coined the name "CAVS" (charge is attached to a virtual site) due to the involvment of the virtual site. After being tested in molecular dynamic (MD) simulations of bulk water at various time steps, under different temperatures and in different salt (NaCl) concentrations, the CAVS model offers encouraging predictions for some bulk properties of water (such as density, dielectric constant, etc.) when compared to experimental ones. PMID:26747089

  12. Splicing of many human genes involves sites embedded within introns

    PubMed Central

    Kelly, Steven; Georgomanolis, Theodore; Zirkel, Anne; Diermeier, Sarah; O'Reilly, Dawn; Murphy, Shona; Längst, Gernot; Cook, Peter R.; Papantonis, Argyris

    2015-01-01

    The conventional model for splicing involves excision of each intron in one piece; we demonstrate this inaccurately describes splicing in many human genes. First, after switching on transcription of SAMD4A, a gene with a 134 kb-long first intron, splicing joins the 3′ end of exon 1 to successive points within intron 1 well before the acceptor site at exon 2 is made. Second, genome-wide analysis shows that >60% of active genes yield products generated by such intermediate intron splicing. These products are present at ∼15% the levels of primary transcripts, are encoded by conserved sequences similar to those found at canonical acceptors, and marked by distinctive structural and epigenetic features. Finally, using targeted genome editing, we demonstrate that inhibiting the formation of these splicing intermediates affects efficient exon–exon splicing. These findings greatly expand the functional and regulatory complexity of the human transcriptome. PMID:25897131

  13. The acid tolerance response of Salmonella typhimurium involves transient synthesis of key acid shock proteins.

    PubMed Central

    Foster, J W

    1993-01-01

    Although Salmonella typhimurium prefers neutral-pH environments, it can adapt to survive conditions of severe low-pH stress (pH 3.3). The process, termed the acid tolerance response (ATR), includes two distinct stages. The first stage, called pre-acid shock, is induced at pH 5.8 and involves the production of an inducible pH homeostasis system functional at external pH values below 4.0. The second stage occurs following an acid shock shift to pH 4.5 or below and is called the post-acid shock stage. During this stage of the ATR, 43 acid shock proteins (ASPs) are synthesized. The present data reveal that several ASPs important for pH 3.3 acid tolerance are only transiently produced. Their disappearance after 30 to 40 min of pH 4.4 acid shock coincides with an inability to survive subsequent pH 3.3 acid challenge. Clearly, an essential feature of inducible acid tolerance is an ability to synthesize these key ASPs. The pre-acid shock stage, with its inducible pH homeostasis system, offers the cell an enhanced ability to synthesize ASPs following rapid shifts to conditions below pH 4.0, an external pH that normally prevents ASP synthesis. The data also address possible signals for ASP synthesis. The inducing signal for 22 ASPs appears to be internal acidification, while external pH serves to induce 13 others. Of the 14 transient ASPs, 10 are induced in response to changes in internal pH. Mutations in the fur (ferric uptake regulator) locus that produce an Atr- acid-sensitive phenotype also eliminate induction of six transiently induced ASPs. Images PMID:8458840

  14. Environmental technology demonstrations involving explosives contamination at the Volunteer Site

    SciTech Connect

    Walker, A.J.; Broder, M.F.; Jayne, E.A.

    1997-08-01

    Managed by the US Army Environmental Center, the Army`s test site at Volunteer Army Ammunition Plant encompasses a 300-acre area formerly used for batch production of TNT. Soil and groundwater contamination in the test area is well characterized. A network of monitoring wells and detailed information regarding the volume, location, and concentration of soil contamination is available to potential demonstrators. On-site field and laboratory support is provided by ICI Americas Incorporated, the facility`s operator. Four demonstrations have been conducted at the test site and several are scheduled for 1997. Preliminary findings from the four demonstrations discussed will be available sometime in 1997.

  15. Legal Issues Involving the Operation of a University Web Site.

    ERIC Educational Resources Information Center

    Bockanic, William N.; Hogan, Patrick T.

    1999-01-01

    Discusses legal liability issues related to university Web sites, including copyright, defamation, and appropriation of images. Makes recommendations regarding domain names, intellectual-property protection, content, and security. (SK)

  16. Identification of amino acids involved in histamine potentiation of GABA A receptors.

    PubMed

    Thiel, Ulrike; Platt, Sarah J; Wolf, Steffen; Hatt, Hanns; Gisselmann, Günter

    2015-01-01

    Histamine is a neurotransmitter involved in a number of physiological and neuronal functions. In mammals, such as humans, and rodents, the histaminergic neurons found in the tuberomamillary nucleus project widely throughout the central nervous system. Histamine acts as positive modulator of GABAA receptors (GABAARs) and, in high concentrations (10 mM), as negative modulator of the strychnine-sensitive glycine receptor. However, the exact molecular mechanisms by which histamine acts on GABAARs are unknown. In our study, we aimed to identify amino acids potentially involved in the modulatory effect of histamine on GABAARs. We expressed GABAARs with 12 different point mutations in Xenopus laevis oocytes and characterized the effect of histamine on GABA-induced currents using the two-electrode voltage clamp technique. Our data demonstrate that the amino acid residues β2(N265) and β2(M286), which are important for modulation by propofol, are not involved in the action of histamine. However, we found that histamine modulation is dependent on the amino acid residues α1(R120), β2(Y157), β2(D163), β3(V175), and β3(Q185). We showed that the amino acid residues β2(Y157) and β3(Q185) mediate the positive modulatory effect of histamine on GABA-induced currents, whereas α1(R120) and β2(D163) form a potential histamine interaction site in GABAARs. PMID:26074818

  17. Identification of amino acids involved in histamine potentiation of GABAA receptors

    PubMed Central

    Thiel, Ulrike; Platt, Sarah J.; Wolf, Steffen; Hatt, Hanns; Gisselmann, Günter

    2015-01-01

    Histamine is a neurotransmitter involved in a number of physiological and neuronal functions. In mammals, such as humans, and rodents, the histaminergic neurons found in the tuberomamillary nucleus project widely throughout the central nervous system. Histamine acts as positive modulator of GABAA receptors (GABAARs) and, in high concentrations (10 mM), as negative modulator of the strychnine-sensitive glycine receptor. However, the exact molecular mechanisms by which histamine acts on GABAARs are unknown. In our study, we aimed to identify amino acids potentially involved in the modulatory effect of histamine on GABAARs. We expressed GABAARs with 12 different point mutations in Xenopus laevis oocytes and characterized the effect of histamine on GABA-induced currents using the two-electrode voltage clamp technique. Our data demonstrate that the amino acid residues β2(N265) and β2(M286), which are important for modulation by propofol, are not involved in the action of histamine. However, we found that histamine modulation is dependent on the amino acid residues α1(R120), β2(Y157), β2(D163), β3(V175), and β3(Q185). We showed that the amino acid residues β2(Y157) and β3(Q185) mediate the positive modulatory effect of histamine on GABA-induced currents, whereas α1(R120) and β2(D163) form a potential histamine interaction site in GABAARs. PMID:26074818

  18. Adverse experiences with nitric acid at the Savannah River Site

    SciTech Connect

    Durant, W.S.; Craig, D.K.; Vitacco, M.J.; McCormick, J.A.

    1991-06-01

    Nitric acid is used routinely at the Savannah River Site (SRS) in many processes. However, the site has experienced a number of adverse situations in handling nitric acid. These have ranged from minor injuries to personnel to significant explosions. This document compiles many of these events and includes discussions of process upsets, fires, injuries, and toxic effects of nitric acid and its decomposition products. The purpose of the publication is to apprise those using the acid that it is a potentially dangerous material and can react in many ways as demonstrated by SRS experience. 10 refs.

  19. Determination of the acidic sites of purified single-walled carbon nanotubes by acid base titration

    NASA Astrophysics Data System (ADS)

    Hu, H.; Bhowmik, P.; Zhao, B.; Hamon, M. A.; Itkis, M. E.; Haddon, R. C.

    2001-09-01

    We report the measurement of the acidic sites in three different samples of commercially available full-length purified single-walled carbon nanotubes (SWNTs) - as obtained from CarboLex (CLI), Carbon Solutions (CSI) and Tubes@Rice (TAR) - by simple acid-base titration methods. Titration of the purified SWNTs with NaOH and NaHCO 3 solutions was used to determine the total percentage of acidic sites and carboxylic acid groups, respectively. The total percentage of acidic sites in full length purified SWNTs from TAR, CLI and CSI are about 1-3%.

  20. Dissecting Abscisic Acid Signaling Pathways Involved in Cuticle Formation.

    PubMed

    Cui, Fuqiang; Brosché, Mikael; Lehtonen, Mikko T; Amiryousefi, Ali; Xu, Enjun; Punkkinen, Matleena; Valkonen, Jari P T; Fujii, Hiroaki; Overmyer, Kirk

    2016-06-01

    The cuticle is the outer physical barrier of aerial plant surfaces and an important interaction point between plants and the environment. Many environmental stresses affect cuticle formation, yet the regulatory pathways involved remain undefined. We used a genetics and gene expression analysis in Arabidopsis thaliana to define an abscisic acid (ABA) signaling loop that positively regulates cuticle formation via the core ABA signaling pathway, including the PYR/PYL receptors, PP2C phosphatase, and SNF1-Related Protein Kinase (SnRK) 2.2/SnRK2.3/SnRK2.6. Downstream of the SnRK2 kinases, cuticle formation was not regulated by the ABA-responsive element-binding transcription factors but rather by DEWAX, MYB16, MYB94, and MYB96. Additionally, low air humidity increased cuticle formation independent of the core ABA pathway and cell death/reactive oxygen species signaling attenuated expression of cuticle-biosynthesis genes. In Physcomitrella patens, exogenous ABA suppressed expression of cuticle-related genes, whose Arabidopsis orthologs were ABA-induced. Hence, the mechanisms regulating cuticle formation are conserved but sophisticated in land plants. Signaling specifically related to cuticle deficiency was identified to play a major role in the adaptation of ABA signaling pathway mutants to increased humidity and in modulating their immunity to Botrytis cinerea in Arabidopsis. These results define a cuticle-specific downstream branch in the ABA signaling pathway that regulates responses to the external environment. PMID:27060495

  1. Permeability of membranes to amino acids and modified amino acids: mechanisms involved in translocation

    NASA Technical Reports Server (NTRS)

    Chakrabarti, A. C.; Deamer, D. W. (Principal Investigator); Miller, S. L. (Principal Investigator)

    1994-01-01

    The amino acid permeability of membranes is of interest because they are one of the key solutes involved in cell function. Membrane permeability coefficients (P) for amino acid classes, including neutral, polar, hydrophobic, and charged species, have been measured and compared using a variety of techniques. Decreasing lipid chain length increased permeability slightly (5-fold), while variations in pH had only minor effects on the permeability coefficients of the amino acids tested in liposomes. Increasing the membrane surface charge increased the permeability of amino acids of the opposite charge, while increasing the cholesterol content decreased membrane permeability. The permeability coefficients for most amino acids tested were surprisingly similar to those previously measured for monovalent cations such as sodium and potassium (approximately 10(-12)-10(-13) cm s-1). This observation suggests that the permeation rates for the neutral, polar and charged amino acids are controlled by bilayer fluctuations and transient defects, rather than partition coefficients and Born energy barriers. Hydrophobic amino acids were 10(2) more permeable than the hydrophilic forms, reflecting their increased partition coefficient values. External pH had dramatic effects on the permeation rates for the modified amino acid lysine methyl ester in response to transmembrane pH gradients. It was established that lysine methyl ester and other modified short peptides permeate rapidly (P = 10(-2) cm s-1) as neutral (deprotonated) molecules. It was also shown that charge distributions dramatically alter permeation rates for modified di-peptides. These results may relate to the movement of peptides through membranes during protein translocation and to the origin of cellular membrane transport on the early Earth.

  2. Fatty acid transport and activation and the expression patterns of genes involved in fatty acid trafficking.

    PubMed

    Sandoval, Angel; Fraisl, Peter; Arias-Barrau, Elsa; Dirusso, Concetta C; Singer, Diane; Sealls, Whitney; Black, Paul N

    2008-09-15

    These studies defined the expression patterns of genes involved in fatty acid transport, activation and trafficking using quantitative PCR (qPCR) and established the kinetic constants of fatty acid transport in an effort to define whether vectorial acylation represents a common mechanism in different cell types (3T3-L1 fibroblasts and adipocytes, Caco-2 and HepG2 cells and three endothelial cell lines (b-END3, HAEC, and HMEC)). As expected, fatty acid transport protein (FATP)1 and long-chain acyl CoA synthetase (Acsl)1 were the predominant isoforms expressed in adipocytes consistent with their roles in the transport and activation of exogenous fatty acids destined for storage in the form of triglycerides. In cells involved in fatty acid processing including Caco-2 (intestinal-like) and HepG2 (liver-like), FATP2 was the predominant isoform. The patterns of Acsl expression were distinct between these two cell types with Acsl3 and Acsl5 being predominant in Caco-2 cells and Acsl4 in HepG2 cells. In the endothelial lines, FATP1 and FATP4 were the most highly expressed isoforms; the expression patterns for the different Acsl isoforms were highly variable between the different endothelial cell lines. The transport of the fluorescent long-chain fatty acid C(1)-BODIPY-C(12) in 3T3-L1 fibroblasts and 3T3-L1 adipocytes followed typical Michaelis-Menten kinetics; the apparent efficiency (k(cat)/K(T)) of this process increases over 2-fold (2.1 x 10(6)-4.5 x 10(6)s(-1)M(-1)) upon adipocyte differentiation. The V(max) values for fatty acid transport in Caco-2 and HepG2 cells were essentially the same, yet the efficiency was 55% higher in Caco-2 cells (2.3 x 10(6)s(-1)M(-1) versus 1.5 x 10(6)s(-1)M(-1)). The kinetic parameters for fatty acid transport in three endothelial cell types demonstrated they were the least efficient cell types for this process giving V(max) values that were nearly 4-fold lower than those defined form 3T3-L1 adipocytes, Caco-2 cells and HepG2 cells. The

  3. Brønsted acid sites in gmelinite

    NASA Astrophysics Data System (ADS)

    Benco, Łubomír; Demuth, Thomas; Hafner, Jürgen; Hutschka, François

    1999-10-01

    The structural properties of purely siliceous and Al-substituted protonated gmelinite, a zeolite with a medium-sized unit cell, are investigated by means of first-principles local-density functional calculations. For acid sites introduced into an Al-free structure, optimized geometries are compared with experimental data. The substitution of a Si-O fragment by Al-O-H induces a pronounced local deformation of the structure. Four symmetrically distinct O sites (O1-O4) are classified, according to the pattern of the deformation, into two categories showing rather localized (sites 2 and 3), and mostly delocalized (sites 1 and 4) distortions. Relative stabilities of protonated structures are shown to depend on the initial geometry of the site. Larger Si-O-Si angles lead to a higher stability of the acid site. Two approaches, a static and a dynamical one, are used to derive OH stretching frequencies. Both of them prove a rather complex relation between the infrared (IR) frequency, the acidity, and the local environment of the Bro/nsted acid site. The lowest OH stretching frequency is assigned to the O2 site as distinguished in the dynamical calculations. The shift occurs due to attractive interactions of H to the framework oxygen atoms.

  4. Effects of Folic Acid on Secretases Involved in Aβ Deposition in APP/PS1 Mice.

    PubMed

    Tian, Tian; Bai, Dong; Li, Wen; Huang, Guo-Wei; Liu, Huan

    2016-01-01

    Alzheimer's disease (AD) is the most common type of dementia. Amyloid-β protein (Aβ) is identified as the core protein of neuritic plaques. Aβ is generated by the sequential cleavage of the amyloid precursor protein (APP) via the APP cleaving enzyme (α-secretase, or β-secretase) and γ-secretase. Previous studies indicated that folate deficiency elevated Aβ deposition in APP/PS1 mice, and this rise was prevented by folic acid. In the present study, we aimed to investigate whether folic acid could influence the generation of Aβ by regulating α-, β-, and γ-secretase. Herein, we demonstrated that folic acid reduced the deposition of Aβ42 in APP/PS1 mice brain by decreasing the mRNA and protein expressions of β-secretase [beta-site APP-cleaving enzyme 1 (BACE1)] and γ-secretase complex catalytic component-presenilin 1 (PS1)-in APP/PS1 mice brain. Meanwhile, folic acid increased the levels of ADAM9 and ADAM10, which are important α-secretases in ADAM (a disintegrin and metalloprotease) family. However, folic acid has no impact on the protein expression of nicastrin (Nct), another component of γ-secretase complex. Moreover, folic acid regulated the expression of miR-126-3p and miR-339-5p, which target ADAM9 and BACE1, respectively. Taken together, the effect of folic acid on Aβ deposition may relate to making APP metabolism through non-amyloidogenic pathway by decreasing β-secretase and increasing α-secretase. MicroRNA (miRNA) may involve in the regulation mechanism of folic acid on secretase expression. PMID:27618097

  5. The involvement of uric acid in the pathogenesis of preeclampsia.

    PubMed

    Masoura, Sophia; Makedou, Kali; Theodoridis, Theodoros; Kourtis, Anargyros; Zepiridis, Leonidas; Athanasiadis, Apostolos

    2015-01-01

    Preeclampsia is a common disorder of human pregnancy and a major cause of worldwide pregnancy-related maternal and neonatal morbidity and mortality. Oxidative stress, angiogenic imbalance, placental ischemia and an inflammatory response have been proposed to play role in the pathogenesis of the disease. Hyperuricemia is a key biochemical feature in preeclampsia with elevated levels of uric acid being diagnosed as early as the 10(th) week of gestation. Traditionally, elevated uric acid levels were considered a result of renal dysfunction known to exist in preeclampsia. The contribution of uric acid in the pathogenesis of preeclampsia is being recently further investigated. We, hereby, review the possible mechanisms by which uric acid contributes to the development of the disease and its complications both on mother and fetus. PMID:26022211

  6. SITE PROGRAM EVALUATION OF INNOVATIVE ACID MINE DRAINAGE TREATMENT TECHNOLOGIES AT THE LEVIATHAN MINE SITE, CA

    EPA Science Inventory

    The EPA SITE Program is conducting a detailed sampling and evaluation of several innovative acid mine drainage treatment technologies at the Leviathan Mine Superfund site in California. Technologies include BiPhasic Lime Treatment Plant, an alkaline lagoon, and an innovative bio...

  7. A Brassica napus Lipase Locates at the Membrane Contact Sites Involved in Chloroplast Development

    PubMed Central

    Tan, Xiaoli; Wang, Qiuye; Tian, Baoxia; Zhang, Henan; Lu, Daoli; Zhou, Jia

    2011-01-01

    Background Fatty acids synthesized in chloroplast are transported to endoplasmic reticulum (ER) for triacylglycerols (TAGs) resembling. The development of chloroplast also requires lipids trafficking from ER to chloroplast. The membrane contact sites (MCSs) between ER and chloroplast has been demonstrated to be involved for the trafficking of lipids and proteins. Lipids trafficking between ER and chloroplast is often accompanied by lipids interconversion. However, it is rarely known how lipids interconversion happens during their trafficking. Methodology/Principal Findings We cloned a lipase gene from Brassica napus L., designated as BnCLIP1. Green fluorescence protein (GFP)-tagged BnCLIP1 was shown to locate at the MCSs between ER and chloroplasts in tobacco leaves. Heterogeneous expression of BnCLIP1 in Saccharomyces cerevisiae (pep4) reduced the total amount of fatty acid. Gas chromatography-mass spectrometry (GC-MS) analysis revealed that the truncated BnCLIP1 had a substrate preference for C16:0 lipids in Saccharomyces cerevisiae (pep4). To probe the physiological function of BnCLIP1, two Brassica napus lines with different oil-content were introduced to investigate the transcript patterns of BnCLIP1 during seed development. Intriguingly, the transcript level of BnCLIP1 was found to be immediately up-regulated during the natural seed senescence of both lines; the transcription response of BnCLIP1 in the high oil-content seeds was faster than the lower ones, suggesting a potential role of BnCLIP1 in affecting seed oil synthesis via regulating chloroplast integrity. Further researches showed that chemical disruption of leaf chloroplast also activated the transcription of BnCLIP1. Conclusions/Significance The findings of this study show that BnCLIP1 encodes a lipase, localizes at the MCSs and involves in chloroplast development. PMID:22046373

  8. Luminescence and IR characterization of acid sites on alumina

    SciTech Connect

    Shen, Yan-Fei; Suib, S.L. ); Deeba, M.; Koermer, G.S. )

    1994-04-01

    Luminescence and infrared (IR) spectroscopies of pyridine and ammonia adsorption have been used to measure acidities of [gamma]-alumina. Neither luminescence nor IR spectra of pyridine adsorption show an Bronsted acidity on [gamma]-alumina pretreated to 400[degrees]C. However, luminescence emission data reveal four weak OH bands even when pretreatment is done at 600[degrees]C. Pyridine and subsequent water adsorption yield six luminescence emission bands. A red shift of the pyridine emission band is found when pretreatment or desorption temperature is increased. IR spectra of ammonia on alumina pretreated at 400[degrees]C show three deformation bands at 1452, 1465, and 1485 cm[sup [minus]1]. The first band is also observed together with a band at 1554 cm[sup [minus]1] even for pretreatment at 950[degrees]C, and it corresponds to NH[sup +][sub 4] formed from dissociative adsorption of ammonia, while the other two bands are assigned to ammonia adsorbed on Bronsted acid sites. These two bands disappear along with the appearance of a new band at 1429 cm[sup [minus]1], when deuterated alumina is pretreated at 400[degrees]C and subsequently subjected to ammonia. This new band at 1429 cm[sup [minus]1] is due to NH[sub 3]D[sup +] formed from ammonia adsorbed on acidic OD sites. Consequently, ammonia IR results demonstrate the existence of Bronsted acid sites on alumina pretreated at 400[degrees]C. 38 refs., 9 figs., 2 tabs.

  9. Dehydration of lactic acid to acrylic acid over lanthanum phosphate catalysts: the role of Lewis acid sites.

    PubMed

    Guo, Zhen; Theng, De Sheng; Tang, Karen Yuanting; Zhang, Lili; Huang, Lin; Borgna, Armando; Wang, Chuan

    2016-09-14

    Lanthanum phosphate (LaP) nano-rods were synthesized using n-butylamine as a shape-directing agent (SDA). The resulting catalysts were applied in the dehydration of lactic acid to acrylic acid. Aiming to understand the nature of the active sites, the chemical and physical properties of LaP materials were studied using a variety of characterization techniques. This study showed that the SDA not only affected the porosity of the LaP materials but also modified the acid-base properties. Clearly, the modification of the acid-base properties played a more critical role in determining the catalytic performance than porosity. An optimized catalytic performance was obtained on the LaP catalyst with a higher concentration of Lewis acid sites. Basic sites showed negative effects on the stability of the catalysts. Good stability was achieved when the catalyst was prepared using the appropriate SDA/La ratio. PMID:27514871

  10. Activity of capryloyl collagenic acid against bacteria involved in acne.

    PubMed

    Fourniat, J; Bourlioux, P

    1989-12-01

    Synopsis Capryloyl collagenic acid (Lipacide C8Co) has similar bacteriostatic activity in vitro to that of benzoyl peroxide towards the bacteria found in acne lesions (Staphylococcus aureus, Staphylococcus epidermidis and Propionibacterium acnes) (MIC between 1 and 4 mg ml(-1) for C8Co, and between 0.5 and 5 mg ml(-1) for benzoyl peroxide). The presence of Emulgine M8 did not affect the bacteriostatic activity of C8Co. A 4% w/v solution of C8Co (incorporating Emulgine M8) fulfilled the criteria for an antiseptic preparation as laid down by the French Pharmacopoeia (10th Edition), and had a spectrum 5 bactericidal activity according to the French Standard AFNOR NF T 72-151. The excellent cutaneous tolerance of capryloyl collagenic acid would indicate that an aqueous solution might be of value for topical treatment of the bacterial component of acne. Résumé Activité antibactérienne de l'acide capryloyl-collagénique vis à vis des bactéries impliquées dans l'etiologie de l'acné L'acide capryloyl-collagénique (Lipacide C8Co) et le peroxyde de benzoyle présentent une activité bactériostatique in-vitroéquivalente vis à vis des espèces bactériennes retrouvées au niveau des lésions acnéiques (Staphylococcus aureus, S. epidermidis et Propionibacterium acnes) (CMI comprise entre 1 et 4 mg ml(-1) pour le lipoaminoacide, et 0,5 et 5 mg ml(-1) pour le peroxyde de benzoyle). La mise en solution aqueuse de l'acide capryloyl-collagénique en présence d'Emulgine M8 ne modifie pas son activité bactériostatique. Une telle solution, à 4% m/V d'acide capryloyl-collagénique et 5% m/V d'Emulgine M8, satisfait à l'essai d'activité des préparations antiseptiques décrit à la Pharmacopée Française (Xème Ed.) (concentration minimale antiseptique: 10% v/V, pour un temps de contact de 5 min à 32 degrees C entre les germes tests et la solution diluée en eau distillée), et posséde une activité bactéricide antiseptique spectre 5 conforme à la norme AFNOR NF T

  11. Functional characterization of amyrin synthase involved in ursolic acid biosynthesis in Catharanthus roseus leaf epidermis.

    PubMed

    Yu, Fang; Thamm, Antje M K; Reed, Darwin; Villa-Ruano, Nemesio; Quesada, Alfonso Lara; Gloria, Edmundo Lozoya; Covello, Patrick; De Luca, Vincenzo

    2013-07-01

    Catharanthus roseus accumulates high levels of the pentacyclic triterpene, ursolic acid, as a component of its wax exudate on the leaf surface. Bioinformatic analyses of transcripts derived from the leaf epidermis provide evidence for the specialized role of this tissue in the biosynthesis of ursolic acid. Cloning and functional expression in yeast of a triterpene synthase derived from this tissue showed it to be predominantly an α-amyrin synthase (CrAS), since the α-amyrin to β-amyrin reaction products accumulated in a 5:1 ratio. Expression analysis of CrAS showed that triterpene biosynthesis occurs predominantly in the youngest leaf tissues and in the earliest stages of seedling development. Further studies using laser capture microdissection to harvest RNA from epidermis, mesophyll, idioblasts, laticifers and vasculature of leaves showed the leaf epidermis to be the preferred sites of CrAS expression and provide conclusive evidence for the involvement of this tissue in the biosynthesis of ursolic acid in C. roseus. PMID:22652241

  12. Site contamination health risk assessment case study involving tenant relocation from a former gasworks site.

    PubMed

    Turczynowicz, Len; Fitzgerald, D James; Nitschke, Monika; Mangas, Sam; McLean, Angela

    2007-10-01

    An Adelaide suburban public-housing residential site with 16 apartments was investigated after complaints of odor in some yard areas. A distinct 0.5-m layer of dark, odorous (tarry), contaminant material, which in some areas had been covered with plastic sheeting, was subsequently found beneath the topsoil across most of the site. This material appeared to extend beneath the apartments. Analysis indicated high levels of cyanide and polycyclic aromatic hydrocarbons (PAHs), consistent with gasworks waste. Historical investigation revealed that the site was originally owned by a gas company and that a large gasometer (gas-storage tank) existed in one corner of the site. This finding of significant soil contamination precipitated a decision by the health and housing authorities to notify tenants immediately and to plan for their relocation. In addition to tending to the consequent personal disruption and logistical difficulties this posed, a detailed risk assessment process was developed. Urine samples were collected before and after relocation and analyzed for 1-hydroxypyrene (1-OHP), a biomarker for polycyclic aromatic hydrocarbon (PAH) exposure. In addition, samples of tap water, indoor and outdoor air, indoor and ceiling dust, carpets, and soil from tillage areas were analyzed for PAHs. Data indicated a low health risk associated with tenancy on the site. This report presents details of the health risk assessment process undertaken and discusses vindicative reasons for tenant relocation. PMID:17763082

  13. Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

    SciTech Connect

    Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya; Apicella, Michael A.; Ramaswamy, S.

    2014-07-01

    Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which often takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.

  14. Electrophysiological evidence for acidic, basic, and neutral amino acid olfactory receptor sites in the catfish.

    PubMed

    Caprio, J; Byrd, R P

    1984-09-01

    Electrophysiological experiments indicate that olfactory receptors of the channel catfish, Ictalurus punctatus, contain different receptor sites for the acidic (A), basic (B), and neutral amino acids; further, at least two partially interacting neutral sites exist, one for the hydrophilic neutral amino acids containing short side chains (SCN), and the second for the hydrophobic amino acids containing long side chains (LCN). The extent of cross-adaptation was determined by comparing the electro-olfactogram (EOG) responses to 20 "test" amino acids during continuous bathing of the olfactory mucosa with water only (control) to those during each of the eight "adapting" amino acid regimes. Both the adapting and test amino acids were adjusted in concentrations to provide approximately equal response magnitudes in the unadapted state. Under all eight adapting regimes, the test EOG responses were reduced from those obtained in the unadapted state, but substantial quantitative differences resulted, depending upon the molecular structure of the adapting stimulus. Analyses of the patterns of EOG responses to the test stimuli identified and characterized the respective "transduction processes," a term used to describe membrane events initiated by a particular subset of amino acid stimuli that are intricately linked to the origin of the olfactory receptor potential. Only when the stimulus compounds interact with different transduction processes are the stimuli assumed to bind to different membrane "sites." Four relatively independent L-alpha-amino acid transduction processes (and thus at least four binding sites) identified in this report include: (a) the A process for aspartic and glutamic acids; (b) the B process for arginine and lysine; (c) the SCN process for glycine, alanine, serine, glutamine, and possibly cysteine; (d) the LCN process for methionine, ethionine, valine, norvaline, leucine, norleucine, glutamic acid-gamma-methyl ester, histidine, phenylalanine, and also

  15. A novel colonic anastomosis technique involving fixed polyglycolic acid mesh

    PubMed Central

    Aysan, Erhan; Bektas, Hasan; Ersoz, Feyzullah; Sari, Serkan; Kaygusuz, Arslan

    2010-01-01

    Background: Polyglycolic acid mesh (PAM) reinforcement of colonic anastomoses were evaluated. Methods: Twenty female albino rabbits were divided into two groups. Each rabbit underwent segmental colonic resection with single-layer anastomosis. In one group of rabbits, PAM of length equal to the circumference of the anastomosis was applied. Rabbits were sacrificed on postoperative day 10 and peritoneal adhesions, anastomosis burst pressure, and anastomosis histopathological characteristics were evaluated. Results: The average burst pressure for the control and PAM groups was 149±15.95 mmHgand 224±124.5 mmHg, respectively (p=0.578). All control anastomoses burst, whereas only five (50%) PAM anastomoses burst (p<0.03). There was no anastomotic leakage in the control group, whereas three PAM group anastomoses leaked (p=0.210). The collagen fiber density and amount of neovascularization were lower in the PAM than the control group (p=0.001 and p=0.002, respectively). The average peritoneal adhesion value was 1.6±0.51 in the control group and 2.9±0.31 in the PAM group (p<0.0001). Conclusion: The new fixed PAM-reinforced anastomosis technique resulted in an increased risk of anastomosis leakage and peritoneal adhesion, but also higher in non-burst anastomoses. PMID:21072268

  16. Mechanism involved in enhancement of osteoblast differentiation by hyaluronic acid

    SciTech Connect

    Kawano, Michinao; Ariyoshi, Wataru; Iwanaga, Kenjiro; Okinaga, Toshinori; Habu, Manabu; Yoshioka, Izumi; Tominaga, Kazuhiro; Nishihara, Tatsuji

    2011-02-25

    Research highlights: {yields} In this study was to investigate the effects of HA on osteoblast differentiation induced by BMP-2. {yields} MG63 cells were incubated with BMP-2 and HA for various time periods. {yields} Phosphorylation of Smad 1/5/8, p38, and ERK proteins was determined by western blot analysis. To elucidate the nuclear translocation of phosphorylated Smad 1/5/8, stimulated cells were subjected to immunofluorescence microscopy. {yields} HA enhanced BMP-2 induces osteoblastic differentiation in MG63 cells via down-regulation of BMP-2 antagonists and ERK phosphorylation. -- Abstract: Objectives: Bone morphogenetic protein-2 (BMP-2) is expected to be utilized to fill bone defects and promote healing of fractures. However, it is unable to generate an adequate clinical response for use in bone regeneration. Recently, it was reported that glycosaminoglycans, including heparin, heparan sulfate, keratan sulfate, dermatan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, and hyaluronic acid (HA), regulate BMP-2 activity, though the mechanism by which HA regulates osteogenic activities has not been fully elucidated. The aim of this study was to investigate the effects of HA on osteoblast differentiation induced by BMP-2. Materials and methods: Monolayer cultures of osteoblastic lineage MG63 cells were incubated with BMP-2 and HA for various time periods. To determine osteoblastic differentiation, alkaline phosphatase (ALP) activity in the cell lysates was quantified. Phosphorylation of Smad 1/5/8, p38, and ERK proteins was determined by Western blot analysis. To elucidate the nuclear translocation of phosphorylated Smad 1/5/8, stimulated cells were subjected to immunofluorescence microscopy. To further elucidate the role of HA in enhancement of BMP-2-induced Smad signaling, mRNA expressions of the BMP-2 receptor antagonists noggin and follistatin were detected using real-time RT-PCR. Results: BMP-2-induced ALP activation, Smad 1/5/8 phosphorylation, and

  17. Receptor sites involved in posttranslational transport of apocytochrome c into mitochondria: specificity, affinity, and number of sites.

    PubMed Central

    Hennig, B; Koehler, H; Neupert, W

    1983-01-01

    Assembly of cytochrome c involves a series of steps: synthesis of apocytochrome c on free ribosomes, specific binding of apocytochrome c to the mitochondrial surface, transfer across the outer membrane, covalent addition of protoheme, refolding of the polypeptide chain, and association of holocytochrome c with its functional sites at the inner membrane. The binding step of apocytochrome c to Neurospora crassa mitochondria was studied by inhibiting the subsequent transfer steps with the heme analogue deuterohemin. The binding sites are highly specific for mitochondrial apocytochromes c. Bound labeled Neurospora apocytochrome c was competitively displaced by unlabeled apocytochrome c from various species. These exhibited different abilities for displacement. Apocytochrome c from Paracoccus denitrificans, the amino-terminal (heme-binding) fragment of Neurospora apocytochrome c, and Neurospora holocytochrome c did not recognize the binding sites. Polylysine did not interfere with apocytochrome c binding. Apocytochrome c is reversibly bound. The binding sites are present in limited number. High-affinity binding sites were present at about 90 pmol/mg of mitochondrial protein. They displayed an association constant of 2.2 X 10(7) M-1. Apocytochrome c was imported into mitochondria and converted to holocytochrome c directly from the binding sites when inhibition by deuterohemin was relieved. We conclude that the apocytochrome c binding sites on mitochondria represent receptors that function in the recognition and import of this precursor by mitochondria. PMID:6308663

  18. Inorganic Nitrogen Cycling in an Extreme Acid Mine Drainage Site

    NASA Astrophysics Data System (ADS)

    Kalnejais, L. H.; Smith, R. L.; Nordstrom, D. K.; Banfield, J. F.

    2006-12-01

    Weathering of the massive sulfide ore body at Iron Mountain, northern California has generated sulfuric acid solutions with pH values ranging from 0.5 to 1, temperatures up to 50°C and high concentrations of toxic metals. Communities of microorganisms catalyze the oxidation of iron and sulfur that generates this extreme environment. The nitrogen requirements of these organisms and the nitrogen cycling within these waters are not understood. By adapting the chemiluminescence detection method of Baeseman (2004) we have constrained the stability of nitrate and nitrite species in acidic, high ferrous iron solutions and have measured a time series of the nitrate concentrations at sites within Iron Mountain. The half-life of nitrite is less than an hour due to reactions with ferrous ions, while nitrate is found at concentrations of up to 10 μM within the mine. By coupling this information with geochemical and microbial community information at each site together with culture enrichment studies using various nitrogen sources we hope to gain insight into the pathways of nitrogen utilization in this extreme environment. References Baeseman, J.L. (2004) Denitrification in acid-impacted mountain stream sediments. Ph.D. Dissertation, University of Colorado, Department of Civil, Environmental, and Architectural Engineering.

  19. Metaproteomics and ultrastructure characterization of Komagataeibacter spp. involved in high-acid spirit vinegar production.

    PubMed

    Andrés-Barrao, Cristina; Saad, Maged M; Cabello Ferrete, Elena; Bravo, Daniel; Chappuis, Marie-Luise; Ortega Pérez, Ruben; Junier, Pilar; Perret, Xavier; Barja, François

    2016-05-01

    Acetic acid bacteria (AAB) are widespread microorganisms in nature, extensively used in food industry to transform alcohols and sugar alcohols into their corresponding organic acids. Specialized strains are used in the production of vinegar through the oxidative transformation of ethanol into acetic acid. The main AAB involved in the production of high-acid vinegars using the submerged fermentation method belong to the genus Komagataeibacter, characterized by their higher ADH stability and activity, and higher acetic acid resistance (15-20%), compared to other AAB. In this work, the bacteria involved in the production of high-acid spirit vinegar through a spontaneous acetic acid fermentation process was studied. The analysis using a culture-independent approach revealed a homogeneous bacterial population involved in the process, identified as Komagataeibacter spp. Differentially expressed proteins during acetic acid fermentation were investigated by using 2D-DIGE and mass spectrometry. Most of these proteins were functionally related to stress response, the TCA cycle and different metabolic processes. In addition, scanning and transmission electron microscopy and specific staining of polysaccharide SDS-PAGE gels confirmed that Komagataeibacter spp. lacked the characteristic polysaccharide layer surrounding the outer membrane that has been previously reported to have an important role in acetic acid resistance in the genus Acetobacter. PMID:26742622

  20. The putative Cationic Amino Acid Transporter 9 is targeted to vesicles and may be involved in plant amino acid homeostasis

    PubMed Central

    Yang, Huaiyu; Stierhof, York-Dieter; Ludewig, Uwe

    2015-01-01

    Amino acids are major primary metabolites. Their uptake, translocation, compartmentation, and re-mobilization require a diverse set of cellular transporters. Here, the broadly expressed gene product of CATIONIC AMINO ACID TRANSPORTER 9 (CAT9) was identified as mainly localized to vesicular membranes that are involved in vacuolar trafficking, including those of the trans-Golgi network. In order to probe whether and how these compartments are involved in amino acid homeostasis, a loss-of-function cat9-1 mutant and ectopic over-expressor plants were isolated. Under restricted nitrogen supply in soil, cat9-1 showed a chlorotic phenotype, which was reversed in the over-expressors. The total soluble amino acid pools were affected in the mutants, but this was only significant under poor nitrogen supply. Upon nitrogen starvation, the soluble amino acid leaf pools were lower in the over-expressor, compared with cat9-1. Over-expression generally affected total soluble amino acid concentrations, slightly delayed development, and finally improved the survival upon severe nitrogen starvation. The results potentially identify a novel function of vesicular amino acid transport mediated by CAT9 in the cellular nitrogen-dependent amino acid homeostasis. PMID:25883600

  1. The putative Cationic Amino Acid Transporter 9 is targeted to vesicles and may be involved in plant amino acid homeostasis.

    PubMed

    Yang, Huaiyu; Stierhof, York-Dieter; Ludewig, Uwe

    2015-01-01

    Amino acids are major primary metabolites. Their uptake, translocation, compartmentation, and re-mobilization require a diverse set of cellular transporters. Here, the broadly expressed gene product of CATIONIC AMINO ACID TRANSPORTER 9 (CAT9) was identified as mainly localized to vesicular membranes that are involved in vacuolar trafficking, including those of the trans-Golgi network. In order to probe whether and how these compartments are involved in amino acid homeostasis, a loss-of-function cat9-1 mutant and ectopic over-expressor plants were isolated. Under restricted nitrogen supply in soil, cat9-1 showed a chlorotic phenotype, which was reversed in the over-expressors. The total soluble amino acid pools were affected in the mutants, but this was only significant under poor nitrogen supply. Upon nitrogen starvation, the soluble amino acid leaf pools were lower in the over-expressor, compared with cat9-1. Over-expression generally affected total soluble amino acid concentrations, slightly delayed development, and finally improved the survival upon severe nitrogen starvation. The results potentially identify a novel function of vesicular amino acid transport mediated by CAT9 in the cellular nitrogen-dependent amino acid homeostasis. PMID:25883600

  2. Decreasing Risky Behavior on Social Network Sites: The Impact of Parental Involvement in Secondary Education Interventions.

    PubMed

    Vanderhoven, Ellen; Schellens, Tammy; Valcke, Martin

    2016-06-01

    Teenagers face significant risks when using increasingly popular social network sites. Prevention and intervention efforts to raise awareness about these risks and to change risky behavior (so-called "e-safety" interventions) are essential for the wellbeing of these minors. However, several studies have revealed that while school interventions often affect awareness, they have only a limited impact on pupils' unsafe behavior. Utilizing the Theory of Planned Behavior and theories about parental involvement, we hypothesized that involving parents in an e-safety intervention would positively influence pupils' intentions and behavior. In a quasi-experimental study with pre- and post-test measures involving 207 pupils in secondary education, we compared the impact of an intervention without parental involvement with one that included active parental involvement by means of a homework task. We found that whereas parental involvement was not necessary to improve the intervention's impact on risk awareness, it did change intentions to engage in certain unsafe behavior, such as posting personal and sexual information on the profile page of a social network site, and in reducing existing problematic behavior. This beneficial impact was particularly evident for boys. These findings suggest that developing prevention campaigns with active parental involvement is well worth the effort. Researchers and developers should therefore focus on other efficient strategies to involve parents. PMID:26821548

  3. Public perceptions of a radioactively contaminated site: concerns, remediation preferences, and desired involvement.

    PubMed Central

    Feldman, D L; Hanahan, R A

    1996-01-01

    A public attitudes survey was conducted in neighborhoods adjacent to a radioactively contaminated site whose remediation is now under the auspices of the U.S. Department of Energy's Formerly Utilized Sites Remedial Action Program (FUSRAP). The survey's purpose was to ascertain levels of actual and desired public involvement in the remediation process; to identify health, environmental, economic, and future land-use concerns associated with the site; and to solicit remediation strategy preferences. Surface water and groundwater contamination, desire for public involvement, and potential health risks were found to be the most highly ranked site concerns. Preferred remediation strategies included treatment of contaminated soil and excavation with off-site disposal. Among on-site remediation strategies, only institutional controls that leave the site undisturbed and do not require additional excavation of materials were viewed favorably. Cost of remediation appeared to influence remediation strategy preference; however, no strategy was viewed as a panacea. Respondents were also concerned with protecting future generations, better assessment of risks to health and the environment, and avoiding generation of additional contaminated materials. PMID:9118878

  4. Identification and transcriptional profiling of Pseudomonas putida genes involved in furoic acid metabolism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Furfural (2-furaldehyde) is a furan formed by dehydration of pentose sugars. Pseudomonas putida Fu1 metabolizes furfural through a pathway involving conversion to 2-oxoglutarate, via 2-furoic acid and Coenzyme A intermediates. To identify genes involved in furan metabolism, two P. putida transposo...

  5. A novel omega3-fatty acid desaturase involved in the biosynthesis of eicosapentaenoic acid.

    PubMed Central

    Pereira, Suzette L; Huang, Yung-Sheng; Bobik, Emil G; Kinney, Anthony J; Stecca, Kevin L; Packer, Jeremy C L; Mukerji, Pradip

    2004-01-01

    Long-chain n-3 PUFAs (polyunsaturated fatty acids) such as EPA (eicosapentaenoic acid; 20:5 n-3) have important therapeutic and nutritional benefits in humans. In plants, cyanobacteria and nematodes, omega3-desaturases catalyse the formation of these n-3 fatty acids from n-6 fatty acid precursors. Here we describe the isolation and characterization of a gene ( sdd17 ) derived from an EPA-rich fungus, Saprolegnia diclina, that encodes a novel omega3-desaturase. This gene was isolated by PCR amplification of an S. diclina cDNA library using oligonucleotide primers corresponding to conserved regions of known omega3-desaturases. Expression of this gene in Saccharomyces cerevisiae, in the presence of various fatty acid substrates, revealed that the recombinant protein could exclusively desaturate 20-carbon n-6 fatty acid substrates with a distinct preference for ARA (arachidonic acid; 20:4 n-6), converting it into EPA. This activity differs from that of the known omega3-desaturases from any organism. Plant and cyanobacterial omega3-desaturases exclusively desaturate 18-carbon n-6 PUFAs, and a Caenorhabditis elegans omega3-desaturase preferentially desaturated 18-carbon PUFAs over 20-carbon substrates, and could not convert ARA into EPA when expressed in yeast. The sdd17 -encoded desaturase was also functional in transgenic somatic soya bean embryos, resulting in the production of EPA from exogenously supplied ARA, thus demonstrating its potential for use in the production of EPA in transgenic oilseed crops. PMID:14651475

  6. Probing the Binding Site of Bile Acids in TGR5.

    PubMed

    Macchiarulo, Antonio; Gioiello, Antimo; Thomas, Charles; Pols, Thijs W H; Nuti, Roberto; Ferrari, Cristina; Giacchè, Nicola; De Franco, Francesca; Pruzanski, Mark; Auwerx, Johan; Schoonjans, Kristina; Pellicciari, Roberto

    2013-12-12

    TGR5 is a G-protein-coupled receptor (GPCR) mediating cellular responses to bile acids (BAs). Although some efforts have been devoted to generate homology models of TGR5 and draw structure-activity relationships of BAs, none of these studies has hitherto described how BAs bind to TGR5. Here, we present an integrated computational, chemical, and biological approach that has been instrumental to determine the binding mode of BAs to TGR5. As a result, key residues have been identified that are involved in mediating the binding of BAs to the receptor. Collectively, these results provide new hints to design potent and selective TGR5 agonists. PMID:24900622

  7. Nature of the sites involved in the process of cesium desorption from vermiculite.

    PubMed

    Dzene, Liva; Tertre, Emmanuel; Hubert, Fabien; Ferrage, Eric

    2015-10-01

    Three particle size fractions of sodium-saturated vermiculite (10-20, 1-2 and 0.1-0.2 μm), differing only in their ratios of external-to-total sorption sites, were used to probe the nature of the sites involved in desorption of cesium ions. The sorption was investigated for initial aqueous concentrations of cesium ranging from 5.6×10(-4) to 1.3×10(-2) mol/L, and the cesium desorption was probed by exchange with ammonium ions. The results showed that (1) the amounts of desorbed cesium were strongly dependent on the particle size for a given initial aqueous cesium concentration and (2) the amounts of desorbed cations (Na(+) and Cs(+)) strongly decreased with increasing initial cesium aqueous concentration, irrespective of the particle size investigated. Quantitative analysis of these results suggested that cesium ions sorbed on external (edge+basal) sorption sites can be desorbed by ammonium ions. As a contrast, most of cesium ions sorbed on interlayer sites remain fixed due to the collapse of the structure under aqueous conditions. This study provides important information, such as the nature of the sites involved in the exchange process, when the thermodynamic formalism is considered to describe the ion-exchange process involving cesium and high-charge swelling clay minerals in polluted soil environments. PMID:26073847

  8. Lipid Classes and Fatty Acids in Ophryotrocha cyclops, a Dorvilleid from Newfoundland Aquaculture Sites

    PubMed Central

    Salvo, Flora; Dufour, Suzanne C.; Hamoutene, Dounia; Parrish, Christopher C.

    2015-01-01

    A new opportunistic annelid (Ophryotrocha cyclops) discovered on benthic substrates underneath finfish aquaculture sites in Newfoundland (NL) may be involved in the remediation of organic wastes. At those aquaculture sites, bacterial mats and O. cyclops often coexist and are used as indicators of organic enrichment. Little is known on the trophic strategies used by these annelids, including whether they might consume bacteria or other aquaculture-derived wastes. We studied the lipid and fatty acid composition of the annelids and their potential food sources (degraded flocculent organic matter, fresh fish pellets and bacterial mats) to investigate feeding relationships in these habitats and compared the lipid and fatty acid composition of annelids before and after starvation. Fish pellets were rich in lipids, mainly terrestrially derived C18 fatty acids (18:1ω9, 18:2ω6, 18:3ω3), while bacterial samples were mainly composed of ω7 fatty acids, and flocculent matter appeared to be a mixture of fresh and degrading fish pellets, feces and bacteria. Ophryotrocha cyclops did not appear to store excessive amounts of lipids (13%) but showed a high concentration of ω3 and ω6 fatty acids, as well as a high proportion of the main fatty acids contained in fresh fish pellets and bacterial mats. The dorvilleids and all potential food sources differed significantly in their lipid and fatty acid composition. Interestingly, while all food sources contained low proportions of 20:5ω3 and 20:2ω6, the annelids showed high concentrations of these two fatty acids, along with 20:4ω6. A starvation period of 13 days did not result in a major decrease in total lipid content; however, microscopic observations revealed that very few visible lipid droplets remained in the gut epithelium after three months of starvation. Ophryotrocha cyclops appears well adapted to extreme environments and may rely on lipid-rich organic matter for survival and dispersal in cold environments. PMID:26308719

  9. Identification of highly conserved residues involved in inhibition of HIV-1 RNase H function by Diketo acid derivatives.

    PubMed

    Corona, Angela; Di Leva, Francesco Saverio; Thierry, Sylvain; Pescatori, Luca; Cuzzucoli Crucitti, Giuliana; Subra, Frederic; Delelis, Olivier; Esposito, Francesca; Rigogliuso, Giuseppe; Costi, Roberta; Cosconati, Sandro; Novellino, Ettore; Di Santo, Roberto; Tramontano, Enzo

    2014-10-01

    HIV-1 reverse transcriptase (RT)-associated RNase H activity is an essential function in viral genome retrotranscription. RNase H is a promising drug target for which no inhibitor is available for therapy. Diketo acid (DKA) derivatives are active site Mg(2+)-binding inhibitors of both HIV-1 RNase H and integrase (IN) activities. To investigate the DKA binding site of RNase H and the mechanism of action, six couples of ester and acid DKAs, derived from 6-[1-(4-fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid ethyl ester (RDS1643), were synthesized and tested on both RNase H and IN functions. Most of the ester derivatives showed selectivity for HIV-1 RNase H versus IN, while acids inhibited both functions. Molecular modeling and site-directed mutagenesis studies on the RNase H domain demonstrated different binding poses for ester and acid DKAs and proved that DKAs interact with residues (R448, N474, Q475, Y501, and R557) involved not in the catalytic motif but in highly conserved portions of the RNase H primer grip motif. The ester derivative RDS1759 selectively inhibited RNase H activity and viral replication in the low micromolar range, making contacts with residues Q475, N474, and Y501. Quantitative PCR studies and fluorescence-activated cell sorting (FACS) analyses showed that RDS1759 selectively inhibited reverse transcription in cell-based assays. Overall, we provide the first demonstration that RNase H inhibition by DKAs is due not only to their chelating properties but also to specific interactions with highly conserved amino acid residues in the RNase H domain, leading to effective targeting of HIV retrotranscription in cells and hence offering important insights for the rational design of RNase H inhibitors. PMID:25092689

  10. Nerve cell death induced in vivo by kainic acid and quinolinic acid does not involve apoptosis.

    PubMed

    Ignatowicz, E; Vezzani, A M; Rizzi, M; D'Incalci, M

    1991-11-01

    We investigated whether in vivo excitotoxicity was mediated by a mechanism of programmed cell death called apoptosis. Neurotoxic doses of kainic acid (1.2 nmol) and quinolinic acid (120 nmol) were unilaterally injected in the dorsal hippocampus of anesthetized rats. Eight or 16 h later the animals were killed and DNA was extracted from the injected hippocampi. DNA from mouse thymocytes exposed to methylprednisolone (10(-5) M for 6 h at 37 degrees C) was used as a positive control of apoptotic cells. No typical 'ladder' of DNA fragments (multimers of approximately 200 Kb) which characterizes apoptosis was seen in hippocampal cells after toxic doses of kainic or quinolinic acid, as assessed by agarose gel electrophoresis. This suggests that hippocampal nerve cell death induced in vivo by the excitotoxins is not mediated by apoptosis. PMID:1839770

  11. TaOPR2 encodes a 12-oxo-phytodienoic acid reductase involved in the biosynthesis of jasmonic acid in wheat (Triticum aestivum L.).

    PubMed

    Wang, Yukun; Yuan, Guoliang; Yuan, Shaohua; Duan, Wenjing; Wang, Peng; Bai, Jianfang; Zhang, Fengting; Gao, Shiqing; Zhang, Liping; Zhao, Changping

    2016-01-29

    The 12-oxo-phytodienoic acid reductases (OPRs) are involved in the various processes of growth and development in plants, and classified into the OPRⅠ and OPRⅡ subgroups. In higher plants, only OPRⅡ subgroup genes take part in the biosynthesis of endogenous jasmonic acid. In this study, we isolated a novel OPRⅡ subgroup gene named TaOPR2 (GeneBank accession: KM216389) from the thermo-sensitive genic male sterile (TGMS) wheat cultivar BS366. TaOPR2 was predicted to encode a protein with 390 amino acids. The encoded protein contained the typical oxidored_FMN domain, the C-terminus peroxisomal-targeting signal peptide, and conserved FMN-binding sites. TaOPR2 was mapped to wheat chromosome 7B and located on peroxisome. Protein evolution analysis revealed that TaOPR2 belongs to the OPRⅡ subgroup and shares a high degree of identity with other higher plant OPR proteins. The quantitative real-time PCR results indicated that the expression of TaOPR2 is inhibited by abscisic acid (ABA), salicylic acid (SA), gibberellic acid (GA3), low temperatures and high salinity. In contrast, the expression of TaOPR2 can be induced by wounding, drought and methyl jasmonate (MeJA). Furthermore, the transcription level of TaOPR2 increased after infection with Puccinia striiformis f. sp. tritici and Puccinia recondite f. sp. tritici. TaOPR2 has NADPH-dependent oxidoreductase activity. In addition, the constitutive expression of TaOPR2 can rescue the male sterility phenotype of Arabidopsis mutant opr3. These results suggest that TaOPR2 is involved in the biosynthesis of jasmonic acid (JA) in wheat. PMID:26778003

  12. Possible involvement of lipoic acid in binding protein-dependent transport systems in Escherichia coli.

    PubMed

    Richarme, G

    1985-04-01

    We describe the properties of the binding protein dependent-transport of ribose, galactose, and maltose and of the lactose permease, and the phosphoenolpyruvate-glucose phosphotransferase transport systems in a strain of Escherichia coli which is deficient in the synthesis of lipoic acid, a cofactor involved in alpha-keto acid dehydrogenation. Such a strain can grow in the absence of lipoic acid in minimal medium supplemented with acetate and succinate. Although the lactose permease and the phosphoenolypyruvate-glucose phosphotransferase are not affected by lipoic acid deprivation, the binding protein-dependent transports are reduced by 70% in conditions of lipoic acid deprivation when compared with their activity in conditions of lipoic acid supply. The remaining transport is not affected by arsenate but is inhibited by the uncoupler carbonylcyanide-m-chlorophenylhydrazone; however the lipoic acid-dependent transport is completely inhibited by arsenate and only weakly inhibited by carbonylcyanide-m-chlorophenylhydrazone. The known inhibitor of alpha-keto acid dehydrogenases, 5-methoxyindole-2-carboxylic acid, completely inhibits all binding protein-dependent transports whether in conditions of lipoic supply or deprivation; the results suggest a possible relation between binding protein-dependent transport and alpha-keto acid dehydrogenases and shed light on the inhibition of these transports by arsenicals and uncouplers. PMID:3920206

  13. Specific Initiation Site for Simian Virus 40 Deoxyribonucleic Acid Replication

    PubMed Central

    Thoren, Marilyn M.; Sebring, Edwin D.; Salzman, Norman P.

    1972-01-01

    Replicating simian virus 40 (SV40) deoxyribonucleic acid (DNA) molecules have been isolated under conditions in which the newly synthesized DNA is uniformly labeled with 3H-thymidine. These newly synthesized strands are released from the replicative intermediate molecules by alkaline treatment, and it has been possible to isolate single-stranded SV40 DNA which varies in size from 157,000 daltons (from molecules that are 10% replicated) to 1,360,000 daltons (85% replicated). The rates of duplex formation of newly synthesized DNA have been used to relate their genetic complexity to the extent of DNA replication. As DNA replication proceeds, the time required to effect 50% renaturation of the newly synthesized DNA increases at a proportional rate. The data establish that DNA replication is not initiated at random, but rather that there is a single specific initiation site for DNA replication. PMID:4342054

  14. Involvement of phylogenetically conserved acidic amino acid residues in catalysis by an oxidative DNA damage enzyme formamidopyrimidine glycosylase.

    PubMed

    Lavrukhin, O V; Lloyd, R S

    2000-12-12

    Formamidopyrimidine glycosylase (Fpg) is an important bacterial base excision repair enzyme, which initiates removal of damaged purines such as the highly mutagenic 8-oxoguanine. Similar to other glycosylase/AP lyases, catalysis by Fpg is known to proceed by a nucleophilic attack by an amino group (the secondary amine of its N-terminal proline) on C1' of the deoxyribose sugar at a damaged base, which results in the departure of the base from the DNA and removal of the sugar ring by beta/delta-elimination. However, in contrast to other enzymes in this class, in which acidic amino acids have been shown to be essential for glycosyl and phosphodiester bond scission, the catalytically essential acidic residues have not been documented for Fpg. Multiple sequence alignments of conserved acidic residues in all known bacterial Fpg-like proteins revealed six conserved glutamic and aspartic acid residues. Site-directed mutagenesis was used to change glutamic and aspartic acid residues to glutamines and asparagines, respectively. While the Asp to Asn mutants had no effect on the incision activity on 8-oxoguanine-containing DNA, several of the substitutions at glutamates reduced Fpg activity on the 8-oxoguanosine DNA, with the E3Q and E174Q mutants being essentially devoid of activity. The AP lyase activity of all of the glutamic acid mutants was slightly reduced as compared to the wild-type enzyme. Sodium borohydride trapping of wild-type Fpg and its E3Q and E174Q mutants on 8-oxoguanosine or AP site containing DNA correlated with the relative activity of the mutants on either of these substrates. PMID:11106507

  15. Protein binding sites involved in the assembly of the KplE1 prophage intasome.

    PubMed

    Panis, Gaël; Duverger, Yohann; Champ, Stéphanie; Ansaldi, Mireille

    2010-08-15

    The organization of the recombination regions of the KplE1 prophage in Escherichia coli K12 differs from that observed in the lambda prophage. Indeed, the binding sites characterized for the IntS integrase, the TorI recombination directionality factor (RDF) and the integration host factor (IHF) vary in number, spacing and orientation on the attL and attR regions. In this paper, we performed site-directed mutagenesis of the recombination sites to decipher if all sites are essential for the site-specific recombination reaction and how the KplE1 intasome is assembled. We also show that TorI and IntS form oligomers that are stabilized in the presence of their target DNA. Moreover, we found that IHF is the only nucleoid associated protein (NAP) involved in KplE1 recombination, although it is dispensable. This is consistent with the presence of only one functional IHF site on attR and none on attL. PMID:20494389

  16. Serum uric acid may not be involved in the development of preeclampsia.

    PubMed

    Chen, Q; Lau, S; Tong, M; Wei, J; Shen, F; Zhao, J; Zhao, M

    2016-02-01

    Higher serum levels of uric acid are associated with preeclampsia and may signal an early change in preeclampsia. However there is less evidence suggesting there is a meaningful association between uric acid and the development of preeclampsia. A total of 877 women with preeclampsia at presentation and 580 normotensive pregnancies were retrospectively recruited from January 2009 to May 2014. In addition, 5556 pregnant women were also prospectively recruited from September 2012 to December 2013. Retrospective serum levels of uric acid were obtained from women with preeclampsia at the time of presentation (n=877), and serum levels of uric acid in the first, second and third trimester were prospectively collected in women who later developed preeclampsia (n=78), as well as those who did not (n=5478). The serum levels of uric acid were significantly increased in women with preeclampsia at presentation from retrospective samples and this increase correlated with the time of onset and the severity of preeclampsia. However, in prospective samples, serum levels of uric acid were not increased in the first and second trimesters in women who later developed preeclampsia compared with those who did not. The serum level of uric acid in the first and second trimesters in women who developed preeclampsia was not different. Our results demonstrate that the serum levels of uric acid were only increased after the presentation of clinical symptoms of preeclampsia. Therefore, it is likely that uric acid is not involved in the development of preeclampsia and cannot be an early prediction biomarker of this disease. PMID:25994995

  17. Possible intermolecular interaction between quinolones and biphenylacetic acid inhibits gamma-aminobutyric acid receptor sites.

    PubMed

    Akahane, K; Kimura, Y; Tsutomi, Y; Hayakawa, I

    1994-10-01

    The combination of some new quinolone antibacterial agents with 4-biphenylacetic acid (BPAA), a metabolite of fenbufen, is known to specifically induce functional blockade of the gamma-aminobutyric acid (GABA) receptors. The mechanisms of these drug interactions were further examined. Scatchard analysis of [3H]muscimol binding to rat brain plasma membranes in the presence of enoxacin and BPAA revealed that a significant decrease in the number of muscimol binding sites was produced without affecting the affinity of binding to the receptors. In the presence of norfloxacin, BPAA inhibited muscimol binding the most potently of the six BPAA-related compounds tested. Fenbufen and 9,10-dihydro-gamma-oxo-2-phenanthrenebutyric acid also inhibited the binding, and 4-biphenylcarboxylic acid and methyl 4-biphenylacetate inhibited it slightly, but 3-benzoylpropionic acid exhibited no competitive inhibition. Accordingly, hybrid molecules of norfloxacin and BPAA were synthesized for stereochemical analysis of these drug interactions. A hybrid with a -CONH(CH2)3- chain between norfloxacin and BPAA (flexible structure) inhibited muscimol binding, and intracisternal injection of this hybrid caused clonic convulsions in mice more potently than the combination of norfloxacin and BPAA did. In contrast, a hybrid linked by -CONH- (stretched structure) showed almost no such inhibitory effect. 1H NMR analysis indicated the presence of intramolecular attraction at the quinoline ring of the hybrid exhibiting the antagonistic activity. These results suggest the possibility that quinolones and BPAA interact with the GABA receptor at nearby sites and that the binding affinity of quinolones to the GABA receptors is largely enhanced by the intermolecular interaction with BPAA. PMID:7840564

  18. Possible intermolecular interaction between quinolones and biphenylacetic acid inhibits gamma-aminobutyric acid receptor sites.

    PubMed Central

    Akahane, K; Kimura, Y; Tsutomi, Y; Hayakawa, I

    1994-01-01

    The combination of some new quinolone antibacterial agents with 4-biphenylacetic acid (BPAA), a metabolite of fenbufen, is known to specifically induce functional blockade of the gamma-aminobutyric acid (GABA) receptors. The mechanisms of these drug interactions were further examined. Scatchard analysis of [3H]muscimol binding to rat brain plasma membranes in the presence of enoxacin and BPAA revealed that a significant decrease in the number of muscimol binding sites was produced without affecting the affinity of binding to the receptors. In the presence of norfloxacin, BPAA inhibited muscimol binding the most potently of the six BPAA-related compounds tested. Fenbufen and 9,10-dihydro-gamma-oxo-2-phenanthrenebutyric acid also inhibited the binding, and 4-biphenylcarboxylic acid and methyl 4-biphenylacetate inhibited it slightly, but 3-benzoylpropionic acid exhibited no competitive inhibition. Accordingly, hybrid molecules of norfloxacin and BPAA were synthesized for stereochemical analysis of these drug interactions. A hybrid with a -CONH(CH2)3- chain between norfloxacin and BPAA (flexible structure) inhibited muscimol binding, and intracisternal injection of this hybrid caused clonic convulsions in mice more potently than the combination of norfloxacin and BPAA did. In contrast, a hybrid linked by -CONH- (stretched structure) showed almost no such inhibitory effect. 1H NMR analysis indicated the presence of intramolecular attraction at the quinoline ring of the hybrid exhibiting the antagonistic activity. These results suggest the possibility that quinolones and BPAA interact with the GABA receptor at nearby sites and that the binding affinity of quinolones to the GABA receptors is largely enhanced by the intermolecular interaction with BPAA. PMID:7840564

  19. Stakeholder involvement and public outreach at two Department of Energy sites

    SciTech Connect

    Gray, R.H.

    1996-06-01

    Outreach efforts at two U.S. Department of Energy sites (i.e., the Hanford Site in southeastern Washington and Pantex Plant in the Texas panhandle) have involved a broad spectrum of communications media, including technical presentations and articles, information brochures and fact sheets, video productions; interactive exhibits; presentations at civic and other public meetings; and proactive interactions with the news media, regulators and concerned citizens. In addition, representatives of local communities operate offsite environmental monitoring stations and Native Americans are involved in studying cultural resources, fisheries, and other issues at Hanford, and a program to obtain environmental samples from neighbors property is underway at Pantex. All major environmental programs (e.g., the reconstruction of past radiological doses to offsite human populations at Hanford) are conducted with open public participation, the Pantex Plant has opened an Information office in Amarillo, Texas, and both sites now have Citizen`s Advisory Boards. This presentation describes Hanford and Pantex public outreach and involvement efforts, our successes and failures, and the lessons learned.

  20. Site-specific incorporation of keto amino acids into functional G protein-coupled receptors using unnatural amino acid mutagenesis.

    PubMed

    Ye, Shixin; Köhrer, Caroline; Huber, Thomas; Kazmi, Manija; Sachdev, Pallavi; Yan, Elsa C Y; Bhagat, Aditi; RajBhandary, Uttam L; Sakmar, Thomas P

    2008-01-18

    G protein-coupled receptors (GPCRs) are ubiquitous heptahelical transmembrane proteins involved in a wide variety of signaling pathways. The work described here on application of unnatural amino acid mutagenesis to two GPCRs, the chemokine receptor CCR5 (a major co-receptor for the human immunodeficiency virus) and rhodopsin (the visual photoreceptor), adds a new dimension to studies of GPCRs. We incorporated the unnatural amino acids p-acetyl-L-phenylalanine (Acp) and p-benzoyl-L-phenylalanine (Bzp) into CCR5 at high efficiency in mammalian cells to produce functional receptors harboring reactive keto groups at three specific positions. We obtained functional mutant CCR5, at levels up to approximately 50% of wild type as judged by immunoblotting, cell surface expression, and ligand-dependent calcium flux. Rhodopsin containing Acp at three different sites was also purified in high yield (0.5-2 microg/10(7) cells) and reacted with fluorescein hydrazide in vitro to produce fluorescently labeled rhodopsin. The incorporation of reactive keto groups such as Acp or Bzp into GPCRs allows their reaction with different reagents to introduce a variety of spectroscopic and other probes. Bzp also provides the possibility of photo-cross-linking to identify precise sites of protein-protein interactions, including GPCR binding to G proteins and arrestins, and for understanding the molecular basis of ligand recognition by chemokine receptors. PMID:17993461

  1. Jasmonic acid involves in grape fruit ripening and resistant against Botrytis cinerea.

    PubMed

    Jia, Haifeng; Zhang, Cheng; Pervaiz, Tariq; Zhao, Pengcheng; Liu, Zhongjie; Wang, Baoju; Wang, Chen; Zhang, Lin; Fang, Jinggui; Qian, Jianpu

    2016-01-01

    Fruit ripening is a complex process that is regulated by a signal network. Whereas the regulatory mechanism of abscisic acid has been studied extensively in non-climacteric fruit, little is know about other signaling pathways involved in this process. In this study, we performed that plant hormone jasmonic acid plays an important role in grape fruit coloring and softening by increasing the transcription levels of several ripening-related genes, such as the color-related genes PAL1, DFR, CHI, F3H, GST, CHS, and UFGT; softening-related genes PG, PL, PE, Cell, EG1, and XTH1; and aroma-related genes Ecar, QR, and EGS. Lastly, the fruit anthocyanin, phenol, aroma, and cell wall materials were changed. Jasmonic acid positively regulated its biosynthesis pathway genes LOS, AOS, and 12-oxophytodienoate reductase (OPR) and signal pathway genes COI1 and JMT. RNA interference of grape jasmonic acid pathway gene VvAOS in strawberry fruit appeared fruit un-coloring phenotypes; exogenous jasmonic acid rescued this phenotypes. On the contrary, overexpression of grape jasmonic acid receptor VvCOI1 in the strawberry fruit accelerated the fruit-ripening process and induced some plant defense-related gene expression level. Furthermore, jasmonic acid treatment or strong jasmonic acid signal pathway in strawberry fruit make the fruit resistance against Botrytis cinerea. PMID:26498957

  2. Regulation of the expression of key genes involved in HDL metabolism by unsaturated fatty acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of this study was to determine the effects, and possible mechanisms of action, of unsaturated fatty acids on the expression of genes involved in HDL metabolism in HepG2 cells. The mRNA concentration of target genes was assessed by real time PCR. Protein concentrations were determined by wes...

  3. Integrated environmental site characterization involving geochemistry, geophysics, and geology: A shortcut to remediation

    SciTech Connect

    Viellenave, J.; Slatten, M.; Church, G.; Anderson, M.

    1996-11-01

    Environmental site characterization processes have evolved from simple drill-and-sample routines into more sophisticated evaluations of increasingly complex problems involving a variety of contaminants. Strategic integration of several geoscience tools into a more holistic approach benefits the site owner/operator by developing a synoptic perspective of the site at the earliest possible time, allowing for more selective and focused use of the expensive and invasive technologies. The ultimate effect is a better site characterization, including attention to difficult PRP issues, lower liability, fewer risks of bypassing potentially hazardous contaminant accumulations, and a result that is more targeted to environmental and human health risks. An integrated site investigation system requires good geology and hydrology, but is properly augmented by use of modem and sophisticated geochemical and geophysical tools. Establishing characterization objectives is critical in deciding what geoscience tool(s) to deploy in any given situation. For each tool, critical criteria are identified that will enable the user to best decide which to use for what purposes.

  4. Involvement of apurinic sites in the synergistic action of alkylating and intercalating drugs in Escherichia coli.

    PubMed

    Malvy, C; Safraoui, H; Bloch, E; Bertrand, J R

    1988-03-01

    The toxicity of the intercalating compounds 9-aminoellipticine (9AE) and isopropyl-oxazolopyridocarbazole (Ipr-OPC) were studied. The inhibitory effect of non-toxic doses of 9AE, which incises DNA at apurinic (AP) sites, or Ipr-OPC, which does not cleave DNA at AP sites, with non-toxic doses of the alkylating agent dimethylsulphate (DMS) on the growth of Escherichia coli strain AB1157, is additive. The same result has been observed with an exonuclease III mutant which has only 10% of the AP endonuclease activity. However, 9AE or Ipr-OPC display a synergistic toxic effect with a DMS concentration which allows 20% of E. coli AB1157 survival. This synergy is increased for 9AE in the AP endonuclease mutant when compared to the wild-type strain. Under identical conditions 9AE and Ipr-OPC have no synergistic effect on a mutant deficient in the enzymes which generate AP sites. Therefore AP sites are involved in the synergistic toxicity of DMS and the studied intercalating agents. However, the precise role of the interaction of intercalating agents with AP sites, either without cleavage (type 1 compounds) or with cleavage (type 2 compounds), in the observed effect remains an open question. PMID:3284541

  5. Site-Specific Mapping of Sialic Acid Linkage Isomers by Ion Mobility Spectrometry.

    PubMed

    Guttman, Miklos; Lee, Kelly K

    2016-05-17

    Detailed structural elucidation of protein glycosylation is a tedious process often involving several techniques. Glycomics and glycoproteomics approaches with mass spectrometry offer a rapid platform for glycan profiling but are limited by the inability to resolve isobaric species such as linkage and positional isomers. Recently, ion mobility spectrometry (IMS) has been shown to effectively resolve isobaric oligosaccharides, but the utility of IMS to obtain glycan structural information on a site-specific level with proteomic analyses has yet to be investigated. Here, we report that the addition of IMS to conventional glycoproteomics platforms adds additional information regarding glycan structure and is particularly useful for differentiation of sialic acid linkage isomers on both N- and O-linked glycopeptides. With further development IMS may hold the potential for rapid and complete structural elucidation of glycan chains at a site-specific level. PMID:27089023

  6. Identification and Functions of Amino Acid Residues in PotB and PotC Involved in Spermidine Uptake Activity*

    PubMed Central

    Higashi, Kyohei; Sakamaki, Yoshiharu; Herai, Emiko; Demizu, Risa; Uemura, Takeshi; Saroj, Sunil D.; Zenda, Risa; Terui, Yusuke; Nishimura, Kazuhiro; Toida, Toshihiko; Kashiwagi, Keiko; Igarashi, Kazuei

    2010-01-01

    Amino acid residues on PotB and PotC involved in spermidine uptake were identified by random and site-directed mutagenesis. It was found that Trp8, Tyr43, Trp100, Leu110, and Tyr261 in PotB and Trp46, Asp108, Glu169, Ser196, Asp198, and Asp199 in PotC were strongly involved in spermidine uptake and that Tyr160, Glu172, and Leu274 in PotB and Tyr19, Tyr88, Tyr148, Glu160, Leu195, and Tyr211 in PotC were moderately involved in spermidine uptake. Among 11 amino acid residues that were strongly involved in spermidine uptake, Trp8 in PotB was important for insertion of PotB and PotC into membranes. Tyr43, Trp100, and Leu110 in PotB and Trp46, Asp108, Ser196, and Asp198 in PotC were found to be involved in the interaction with PotD. Leu110 and Tyr261 in PotB and Asp108, Asp198, and Asp199 in PotC were involved in the recognition of spermidine, and Trp100 and Tyr261 in PotB and Asp108, Glu169, and Asp198 in PotC were involved in ATPase activity of PotA. Accordingly, Trp100 in PotB was involved in both PotD recognition and ATPase activity, Leu110 in PotB was involved in both PotD and spermidine recognition, and Tyr261 in PotB was involved in both spermidine recognition and ATPase activity. Asp108 and Asp198 in PotC were involved in PotD and spermidine recognition as well as ATPase activity. These results suggest that spermidine passage from PotD to the cytoplasm is coupled to the ATPase activity of PotA through a structural change of PotA by its ATPase activity. PMID:20937813

  7. Glutathione is involved in physiological response of Candida utilis to acid stress.

    PubMed

    Wang, Da-Hui; Zhang, Jun-Li; Dong, Ying-Ying; Wei, Gong-Yuan; Qi, Bin

    2015-12-01

    Candida utilis often encounters an acid stress environment when hexose and pentose are metabolized to produce acidic bio-based materials. In order to reveal the physiological role of glutathione (GSH) in the response of cells of this industrial yeast to acid stress, an efficient GSH-producing strain of C. utilis CCTCC M 209298 and its mutants deficient in GSH biosynthesis, C. utilis Δgsh1 and Δgsh2, were used in this study. A long-term mild acid challenge (pH 3.5 for 6 h) and a short-term severe acid challenge (pH 1.5 for 2 h) were conducted at 18 h during batch culture of the yeast to generate acid stress conditions. Differences in the physiological performances among the three strains under acid stress were analyzed in terms of GSH biosynthesis and distribution; intracellular pH; activities of γ-glutamylcysteine synthetase, catalase, and superoxide dismutase; intracellular ATP level; and ATP/ADP ratio. The intracellular GSH content of the yeast was found to be correlated with changes in physiological data, and a higher intracellular GSH content led to greater relief of cells to the acid stress, suggesting that GSH may be involved in protecting C. utilis against acid stress. Results presented in this manuscript not only increase our understanding of the impact of GSH on the physiology of C. utilis but also help us to comprehend the mechanism underlying the response to acid stress of eukaryotic microorganisms. PMID:26346268

  8. Comparative studies on the inhibitory activities of selected benzoic acid derivatives against secretory phospholipase A2, a key enzyme involved in the inflammatory pathway.

    PubMed

    Dileep, K V; Remya, C; Cerezo, J; Fassihi, A; Pérez-Sánchez, H; Sadasivan, C

    2015-07-01

    Inflammation is considered to be a key factor in major diseases like cancer, Alzheimer's disease, Parkinson's disease, etc. For the past few decades, pharmaceutical companies have explored new effective medications against inflammation. As a part of their detailed studies, many drug targets and drugs have been introduced against inflammation. In the present study, the inhibiting capacities of selected benzoic acid derivatives like gallic acid, vannilic acid, syringic acid and protocatechuic acid against secretory phospholipase A2 (sPLA2), a major enzyme involved in the inflammatory pathway, have been investigated. The detailed in vitro, biophysical and in silico studies carried out on these benzoic acid derivatives revealed that all the selected compounds have a uniform mode of binding in the active site of sPLA2 and are inhibitory in micromolar concentrations. The study also focuses on the non-selective inhibitory activity of an NSAID, aspirin, against sPLA2. PMID:25927625

  9. A Sialic Acid Binding Site in a Human Picornavirus

    PubMed Central

    Frank, Martin; Hähnlein-Schick, Irmgard; Ekström, Jens-Ola; Arnberg, Niklas; Stehle, Thilo

    2014-01-01

    The picornaviruses coxsackievirus A24 variant (CVA24v) and enterovirus 70 (EV70) cause continued outbreaks and pandemics of acute hemorrhagic conjunctivitis (AHC), a highly contagious eye disease against which neither vaccines nor antiviral drugs are currently available. Moreover, these viruses can cause symptoms in the cornea, upper respiratory tract, and neurological impairments such as acute flaccid paralysis. EV70 and CVA24v are both known to use 5-N-acetylneuraminic acid (Neu5Ac) for cell attachment, thus providing a putative link between the glycan receptor specificity and cell tropism and disease. We report the structures of an intact human picornavirus in complex with a range of glycans terminating in Neu5Ac. We determined the structure of the CVA24v to 1.40 Å resolution, screened different glycans bearing Neu5Ac for CVA24v binding, and structurally characterized interactions with candidate glycan receptors. Biochemical studies verified the relevance of the binding site and demonstrated a preference of CVA24v for α2,6-linked glycans. This preference can be rationalized by molecular dynamics simulations that show that α2,6-linked glycans can establish more contacts with the viral capsid. Our results form an excellent platform for the design of antiviral compounds to prevent AHC. PMID:25329320

  10. Age- and diabetes-related nonenzymatic crosslinks in collagen fibrils: candidate amino acids involved in Advanced Glycation End-products.

    PubMed

    Gautieri, Alfonso; Redaelli, Alberto; Buehler, Markus J; Vesentini, Simone

    2014-02-01

    Ageing and diabetes share a common deleterious phenomenon, the formation of Advanced Glycation Endproducts (AGEs), which accumulate predominantly in collagen due to its low turnover. Though the general picture of glycation has been identified, the detailed knowledge of which collagen amino acids are involved in AGEs is still missing. In this work we use an atomistic model of a collagen fibril to pinpoint, for the first time, the precise location of amino acids involved in the most relevant AGE, glucosepane. The results show that there are 14 specific lysine-arginine pairs that, due to their relative position and configuration, are likely to form glucosepane. We find that several residues involved in AGE crosslinks are within key collagen domains, such as binding sites for integrins, proteoglycans and collagenase, hence providing molecular-level explanations of previous experimental results showing decreased collagen affinity for key molecules. Altogether, these findings reveal the molecular mechanism by which glycation affects the biological properties of collagen tissues, which in turn contribute to age- and diabetes-related pathological states. PMID:24060753

  11. Epidermal Permeability Barrier Recovery Is Delayed in Vitiligo-Involved Sites

    PubMed Central

    Liu, J.; Man, W.Y.; Lv, C.Z.; Song, S.P.; Shi, Y.J.; Elias, P.M.; Man, M.Q.

    2010-01-01

    Background/Objectives Prior studies have demonstrated that both the skin surface pH and epidermal permeability barrier function vary with skin pigmentation types. Although melanin deficiency is the main feature of vitiligo, alterations in cutaneous biophysical properties in vitiligo have not yet been well defined. In the present study, stratum corneum (SC) hydration, the skin surface pH and epidermal permeability barrier function in vitiligo were evaluated. Methods A total of 30 volunteers with vitiligo comprising 19 males and 11 females aged 13–51 years (mean age: 27.91 ± 2.06 years) were enrolled in this study. The skin surface pH, SC hydration, melanin/erythema index and transepidermal water loss (TEWL) were measured by respective probes connected to a Courage-Khazaka MPA5. SC integrity was determined by measuring the TEWL following each D-Squame application. The barrier recovery rate was assessed at 5 h following barrier disruption by repeated tape stripping. Results In addition to SC hydration, both melanin and erythema index were significantly lower in vitiligo lesions than in contralateral, nonlesional sites, while no difference in skin surface pH between vitiligo-involved and uninvolved areas was observed. In addition, neither the basal TEWL nor SC integrity in the involved areas differed significantly from that in the uninvolved areas. However, barrier recovery in vitiligo-involved sites was significantly delayed in comparison with uninvolved sites (40.83 ± 5.39% vs. 58.30 ± 4.71%; t = 2.441; p < 0.02). Conclusion Barrier recovery following tape stripping of the SC is delayed in vitiligo. Therefore, improvement in epidermal permeability barrier function may be an important unrecognized factor to be considered in treating patients with vitiligo. PMID:20185976

  12. Design of a Brønsted acid with two different acidic sites: synthesis and application of aryl phosphinic acid-phosphoric acid as a Brønsted acid catalyst.

    PubMed

    Momiyama, N; Narumi, T; Terada, M

    2015-12-11

    A Brønsted acid with two different acidic sites, aryl phosphinic acid-phosphoric acid, has been synthesized. Its catalytic performance was assessed in the hetero-Diels-Alder reaction of aldehyde hydrates with Danishefsky's diene, achieving high reaction efficiency. PMID:26445921

  13. GroEL1: a dedicated chaperone involved in mycolic acid biosynthesis during biofilm formation in mycobacteria.

    PubMed

    Ojha, Anil; Anand, Mridula; Bhatt, Apoorva; Kremer, Laurent; Jacobs, William R; Hatfull, Graham F

    2005-12-01

    Mycobacteria are unusual in encoding two GroEL paralogs, GroEL1 and GroEL2. GroEL2 is essential--presumably providing the housekeeping chaperone functions--while groEL1 is nonessential, contains the attB site for phage Bxb1 integration, and encodes a putative chaperone with unusual structural features. Inactivation of the Mycobacterium smegmatis groEL1 gene by phage Bxb1 integration allows normal planktonic growth but prevents the formation of mature biofilms. GroEL1 modulates synthesis of mycolates--long-chain fatty acid components of the mycobacterial cell wall--specifically during biofilm formation and physically associates with KasA, a key component of the type II Fatty Acid Synthase involved in mycolic acid synthesis. Biofilm formation is associated with elevated synthesis of short-chain (C56-C68) fatty acids, and strains with altered mycolate profiles--including an InhA mutant resistant to the antituberculosis drug isoniazid and a strain overexpressing KasA--are defective in biofilm formation. PMID:16325580

  14. Highly selective Lewis acid sites in desilicated MFI zeolites for dihydroxyacetone isomerization to lactic acid.

    PubMed

    Dapsens, Pierre Y; Mondelli, Cecilia; Pérez-Ramírez, Javier

    2013-05-01

    Desilication of commercial MFI-type (ZSM-5) zeolites in solutions of alkali metal hydroxides is demonstrated to generate highly selective heterogeneous catalysts for the aqueous-phase isomerization of biobased dihydroxyacetone (DHA) to lactic acid (LA). The best hierarchical ZSM-5 sample attains a LA selectivity exceeding 90 %, which is comparable to that of the state-of-the-art catalyst (i.e., the Sn-beta zeolite); this optimized hierarchical catalyst is recyclable over three runs. The Lewis acid sites, which are created through desilication along with the introduction of mesoporosity, are shown to play a crucial role in the formation of the desired product; these cannot be achieved by using other post-synthetic methods, such as steaming or impregnation of aluminum species. Desilication of other metallosilicates, such as Ga-MFI, also leads to high LA selectivity. In the presence of a soluble aluminum source, such as aluminum nitrate, alkaline-assisted alumination can introduce these unique Lewis acid centers in all-silica MFI zeolites. These findings highlight the potential of zeolites in the field of biomass-to-chemical conversion, and expand the applicability of desilication for the generation of selective catalytic centers. PMID:23554234

  15. Characterization of the ArsRS regulon of Helicobacter pylori, involved in acid adaptation.

    PubMed

    Pflock, Michael; Finsterer, Nadja; Joseph, Biju; Mollenkopf, Hans; Meyer, Thomas F; Beier, Dagmar

    2006-05-01

    The human gastric pathogen Helicobacter pylori is extremely well adapted to the highly acidic conditions encountered in the stomach. The pronounced acid resistance of H. pylori relies mainly on the ammonia-producing enzyme urease; however, urease-independent mechanisms are likely to contribute to acid adaptation. Acid-responsive gene regulation is mediated at least in part by the ArsRS two-component system consisting of the essential OmpR-like response regulator ArsR and the nonessential cognate histidine kinase ArsS, whose autophosphorylation is triggered in response to low pH. In this study, by global transcriptional profiling of an ArsS-deficient H. pylori mutant grown at pH 5.0, we define the ArsR approximately P-dependent regulon consisting of 109 genes, including the urease gene cluster, the genes encoding the aliphatic amidases AmiE and AmiF, and the rocF gene encoding arginase. We show that ArsR approximately P controls the acid-induced transcription of amiE and amiF by binding to extended regions located upstream of the -10 box of the respective promoters. In contrast, transcription of rocF is repressed by ArsR approximately P at neutral, acidic, and mildly alkaline pH via high-affinity binding of the response regulator to a site overlapping the promoter of the rocF gene. PMID:16672598

  16. CHARACTERIZATION OF INDIVIDUAL CHEMICAL REACTIONS CONSUMING ACID DURING NUCLEAR WASTE PROCESSING AT THE SAVANNAH RIVER SITE - 136B

    SciTech Connect

    Koopman, D.; Pickenheim, B.; Lambert, D.; Newell, J.; Stone, M.

    2009-09-02

    Conversion of legacy radioactive high-level waste at the Savannah River Site into a stable glass waste form involves a chemical pretreatment process to prepare the waste for vitrification. Waste slurry is treated with nitric and formic acids to achieve certain goals. The total quantity of acid added to a batch of waste slurry is constrained by the catalytic activity of trace noble metal fission products in the waste that can convert formic acid into hydrogen gas at many hundreds of times the radiolytic hydrogen generation rate. A large block of experimental process simulations were performed to characterize the chemical reactions that consume acid prior to hydrogen generation. The analysis led to a new equation for predicting the quantity of acid required to process a given volume of waste slurry.

  17. Direct photoaffinity labeling of cellular retinoic acid-binding protein I (CRABP-I) with all-trans-retinoic acid: identification of amino acids in the ligand binding site.

    PubMed

    Chen, G; Radominska-Pandya, A

    2000-10-17

    Cellular retinoic acid-binding proteins I and II (CRABP-I and -II, respectively) are transport proteins for all-trans-retinoic acid (RA), an active metabolite of vitamin A (retinol), and have been reported to be directly involved in the metabolism of RA. In this study, direct photoaffinity labeling with [11,12-(3)H]RA was used to identify amino acids comprising the ligand binding site of CRABP-I. Photoaffinity labeling of CRABP-I with [(3)H]RA was light- and concentration-dependent and was protected by unlabeled RA and various retinoids, indicating that the labeling was directed to the RA-binding site. Photolabeled CRABP-I was hydrolyzed with endoproteinase Lys-C to yield radioactive peptides, which were separated by reversed-phase HPLC for analysis by Edman degradation peptide sequencing. This method identified five modified amino acids from five separate HPLC fractions: Trp7, Lys20, Arg29, Lys38, and Trp109. All five amino acids are located within one side of the "barrel" structure in the area indicated by the reported crystal structure as the ligand binding site. This is the first direct identification of specific amino acids in the RA-binding site of CRABPs by photoaffinity labeling. These results provide significant information about the ligand binding site of the CRABP-I molecule in solution. PMID:11027136

  18. Public/stakeholder involvement at two Department of Energy sites: Case studies

    SciTech Connect

    Gray, R.H.

    1995-12-31

    Efforts to communicate the results of environmental studies and involve the public in environmental decisions have increased nationwide. Outreach efforts at two US Department of Energy sites (i.e., the Hanford Site in southeastern Washington State and the Pantex Plant in the Texas Panhandle) have used a broad spectrum of communications media, including technical articles (open literature and symposium publications, annual and topical reports); information brochures and fact sheets; video productions; interactive exhibits, presentations at scientific, technical, civic, and other public meetings; and proactive interactions with the news media and with local, state, federal, and other agencies. In addition, representatives of local communities now operate offsite environmental monitoring stations and Native Americans are involved in studying cultural resources, fisheries, and other issues at Hanford and a program to obtain environmental samples from neighbor`s property is underway at the Pantex Plant. All major environmental programs, such as the multi-year effort to reconstruct past radiological doses to offsite human populations at Hanford, are now conducted with open public participation.

  19. 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.

  20. High-affinity binding sites involved in the import of porin into mitochondria.

    PubMed Central

    Pfaller, R; Neupert, W

    1987-01-01

    The specific recognition by mitochondria of the precursor of porin and the insertion into the outer membrane were studied with a radiolabeled water-soluble form of porin derived from the mature protein. High-affinity binding sites had a number of 5-10 pmol/mg mitochondrial protein and a ka of 1-5 X 10(8) M-1. Binding was abolished after trypsin pretreatment of mitochondria indicating that binding sites were of protein-aceous nature. Specifically bound porin could be extracted at alkaline pH but not by high salt and was protected against low concentrations of proteinase K. It could be chased to a highly protease resistant form corresponding to mature porin. High-affinity binding sites could be extracted from mitochondria with detergent and reconstituted in asolectin-ergosterol liposomes. Water-soluble porin competed for the specific binding and import of the precursor of the ADP/ATP carrier, an inner membrane protein. We suggest that (i) binding of precursors to proteinaceous receptors serves as an initial step for recognition, (ii) the receptor for porin may also be involved in the import of precursors of inner membrane proteins, and (iii) interaction with the receptor triggers partial insertion of the precursor into the outer membrane. Images Fig. 4. PMID:2960520

  1. Involved site radiation therapy for the treatment of early-stage Hodgkin lymphoma in adolescents and young adults

    PubMed Central

    Portlock, Carol S

    2016-01-01

    Radiation therapy technology has permitted the development of new treatment planning techniques. Involved field, involved node, and involved site radiotherapy fields are discussed and compared. Indications for and implications of combined modality therapy are examined, particularly as pertinent to the adolescent and young adult population. PMID:26767184

  2. Detection of genes involved in fatty acid elongation and desaturation in thraustochytrid marine eukaryotes.

    PubMed

    Nagano, Naoki; Sakaguchi, Keishi; Taoka, Yousuke; Okita, Yuji; Honda, Daiske; Ito, Makoto; Hayashi, Masahiro

    2011-01-01

    Heterotrophic marine protists known as thraustochytrids can synthesize polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA). The biosynthetic pathways of PUFAs in thraustochytrids are poorly understood, however. In this study, we attempted to reveal the enzymes involved in DHA synthesis in thraustochytrids. Nine thraustochytrid strains representing 3 genera (Aurantiochytrium, Schizochytrium, and Thraustochytrium) were used for PCR-based detection of the genes encoding Δ5-elongase and Δ4-desaturase and for fatty acid analysis. The degenerate primers were designed to amplify the Δ5-elongase and Δ4-desaturase genes, and the partial sequences of the enzymes were obtained from the genera Thraustochytrium and Schizochytrium. These fragments were identical to those of known Δ5-elongase and Δ4-desaturase. Neither Δ5-elongase nor Δ4-desaturase was detected in the strains belonging to the genus Aurantiochytrium, however, suggesting that this group likely synthesizes DHA not via the elongation/desaturation pathway but via an alternate pathway such as the polyketide synthase pathway. The fatty acid profiles of thraustochytrids were consistent with the presence of genes involved in PUFA biosynthesis in thraustochytrid genera. Thus, our findings suggest that two biosynthetic pathways for PUFAs exist in these organisms. PMID:21852747

  3. Nitrous Acid at Concordia (Inland Site) and Dumont d'Urville (Costal Site), East Antarctica

    NASA Astrophysics Data System (ADS)

    Kerbrat, M.; Legrand, M.; Preunkert, S.; Gallée, H.; Kleffman, J.

    2012-04-01

    One of the most recent important finding made in Antarctica after the discovery of the appearance of the Antarctic ozone hole in the early 80's was the discovery of a very oxidizing canopy over the South Pole region in relation with unexpected high levels of NO. There is a strong need however to extend investigations of the oxidation capacity of the lower atmosphere at the scale of the whole Antarctic continent, and in particular, over East Antarctica. That motivated the OPALE (Oxidant Production over Antarctic Land and its Export) project. Indeed the limited data gained by using aircraft sampling during ANTCI 2003 suggest that over the East Antarctic plateau even higher NO emissions persist. Among several not yet resolved questions related to the high level of oxidants over Antarctica is the role of nitrous acid (HONO). During the austral summer 2010/2011 the levels of nitrous acid (HONO) were for the first time investigated at Concordia (75°06'S, 123°33'E) and Dumont D'Urville (66°40'S, 140°01'E), two stations located in East Antarctica. Also for the first time in Antarctica, HONO was measured by deploying a long path absorption photometer (LOPAP). At Concordia, from the end of December 2010 to mid January 2011 HONO mixing ratios at 1 m above the snow surface ranged between 5 and 60 pptv. Diurnal cycles were observed with levels peaking in the morning (06:30 to 07:30) and the evening (19:00 to 20:00). At Dumont d'Urville, background mixing ratios close to 2 pptv were observed in February 2011. No clear diurnal cycles were observed at that site but several events of air masses export from inland Antarctica were encountered with enhanced HONO levels reaching 10 pptv at night. These first HONO data gained in East Antarctica are discussed in terms of sources and sinks along with synoptic weather conditions.

  4. CD36 is involved in oleic acid detection by the murine olfactory system

    PubMed Central

    Oberland, Sonja; Ackels, Tobias; Gaab, Stefanie; Pelz, Thomas; Spehr, Jennifer; Spehr, Marc; Neuhaus, Eva M.

    2015-01-01

    Olfactory signals influence food intake in a variety of species. To maximize the chances of finding a source of calories, an animal’s preference for fatty foods and triglycerides already becomes apparent during olfactory food search behavior. However, the molecular identity of both receptors and ligands mediating olfactory-dependent fatty acid recognition are, so far, undescribed. We here describe that a subset of olfactory sensory neurons expresses the fatty acid receptor CD36 and demonstrate a receptor-like localization of CD36 in olfactory cilia by STED microscopy. CD36-positive olfactory neurons share olfaction-specific transduction elements and project to numerous glomeruli in the ventral olfactory bulb. In accordance with the described roles of CD36 as fatty acid receptor or co-receptor in other sensory systems, the number of olfactory neurons responding to oleic acid, a major milk component, in Ca2+ imaging experiments is drastically reduced in young CD36 knock-out mice. Strikingly, we also observe marked age-dependent changes in CD36 localization, which is prominently present in the ciliary compartment only during the suckling period. Our results support the involvement of CD36 in fatty acid detection by the mammalian olfactory system. PMID:26441537

  5. Changes in actin dynamics are involved in salicylic acid signaling pathway.

    PubMed

    Matoušková, Jindřiška; Janda, Martin; Fišer, Radovan; Sašek, Vladimír; Kocourková, Daniela; Burketová, Lenka; Dušková, Jiřina; Martinec, Jan; Valentová, Olga

    2014-06-01

    Changes in actin cytoskeleton dynamics are one of the crucial players in many physiological as well as non-physiological processes in plant cells. Positioning of actin filament arrays is necessary for successful establishment of primary lines of defense toward pathogen attack, depolymerization leads very often to the enhanced susceptibility to the invading pathogen. On the other hand it was also shown that the disruption of actin cytoskeleton leads to the induction of defense response leading to the expression of PATHOGENESIS RELATED proteins (PR). In this study we show that pharmacological actin depolymerization leads to the specific induction of genes in salicylic acid pathway but not that involved in jasmonic acid signaling. Life imaging of leafs of Arabidopsis thaliana with GFP-tagged fimbrin (GFP-fABD2) treated with 1 mM salicylic acid revealed rapid disruption of actin filaments resembling the pattern viewed after treatment with 200 nM latrunculin B. The effect of salicylic acid on actin filament fragmentation was prevented by exogenous addition of phosphatidic acid, which binds to the capping protein and thus promotes actin polymerization. The quantitative evaluation of actin filament dynamics is also presented. PMID:24767113

  6. Mutagenesis identifies the critical amino acid residues of human endonuclease G involved in catalysis, magnesium coordination, and substrate specificity

    PubMed Central

    Wu, Shih-Lu; Li, Chia-Cheng; Chen, Jaw-Chyun; Chen, Yi-Jin; Lin, Ching-Ting; Ho, Tin-Yun; Hsiang, Chien-Yun

    2009-01-01

    Background Endonuclease G (EndoG), a member of DNA/RNA nonspecific ββα-Me-finger nucleases, is involved in apoptosis and normal cellular proliferation. In this study, we analyzed the critical amino acid residues of EndoG and proposed the catalytic mechanism of EndoG. Methods To identify the critical amino acid residues of human EndoG, we replaced the conserved histidine, asparagine, and arginine residues with alanine. The catalytic efficacies of Escherichia coli-expressed EndoG variants were further analyzed by kinetic studies. Results Diethyl pyrocarbonate modification assay revealed that histidine residues were involved in EndoG activity. His-141, Asn-163, and Asn-172 in the H-N-H motif of EndoG were critical for catalysis and substrate specificity. H141A mutant required a higher magnesium concentration to achieve its activity, suggesting the unique role of His-141 in both catalysis and magnesium coordination. Furthermore, an additional catalytic residue (Asn-251) and an additional metal ion binding site (Glu-271) of human EndoG were identified. Conclusion Based on the mutational analysis and homology modeling, we proposed that human EndoG shared a similar catalytic mechanism with nuclease A from Anabaena. PMID:19272175

  7. Contrasting microbial functional genes in two distinct saline-alkali and slightly acidic oil-contaminated sites.

    PubMed

    Liang, Yuting; Zhao, Huihui; Zhang, Xu; Zhou, Jizhong; Li, Guanghe

    2014-07-15

    To compare the functional gene structure and diversity of microbial communities in saline-alkali and slightly acidic oil-contaminated sites, 40 soil samples were collected from two typical oil exploration sites in North and South China and analyzed with a comprehensive functional gene array (GeoChip 3.0). The overall microbial pattern was significantly different between the two sites, and a more divergent pattern was observed in slightly acidic soils. Response ratio was calculated to compare the microbial functional genes involved in organic contaminant degradation and carbon, nitrogen, phosphorus, and sulfur cycling. The results indicated a significantly low abundance of most genes involved in organic contaminant degradation and in the cycling of nitrogen and phosphorus in saline-alkali soils. By contrast, most carbon degradation genes and all carbon fixation genes had similar abundance at both sites. Based on the relationship between the environmental variables and microbial functional structure, pH was the major factor influencing the microbial distribution pattern in the two sites. This study demonstrated that microbial functional diversity and heterogeneity in oil-contaminated environments can vary significantly in relation to local environmental conditions. The limitation of nitrogen and phosphorus and the low degradation capacity of organic contaminant should be carefully considered, particularly in most oil-exploration sites with saline-alkali soils. PMID:24784752

  8. The heparan and heparin metabolism pathway is involved in regulation of fatty acid composition.

    PubMed

    Jiang, Zhihua; Michal, Jennifer J; Wu, Xiao-Lin; Pan, Zengxiang; MacNeil, Michael D

    2011-01-01

    Six genes involved in the heparan sulfate and heparin metabolism pathway, DSEL (dermatan sulfate epimerase-like), EXTL1 (exostoses (multiple)-like 1), HS6ST1 (heparan sulfate 6-O-sulfotransferase 1), HS6ST3 (heparan sulfate 6-O-sulfotransferase 3), NDST3 (N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 3), and SULT1A1 (sulfotransferase family, cytosolic, 1A, phenol-preferring, member 1), were investigated for their associations with muscle lipid composition using cattle as a model organism. Nineteen single nucleotide polymorphisms (SNPs)/multiple nucleotide length polymorphisms (MNLPs) were identified in five of these six genes. Six of these mutations were then genotyped on 246 Wagyu x Limousin F(2) animals, which were measured for 5 carcass, 6 eating quality and 8 fatty acid composition traits. Association analysis revealed that DSEL, EXTL1 and HS6ST1 significantly affected two stearoyl-CoA desaturase activity indices, the amount of conjugated linoleic acid (CLA), and the relative amount of saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) in skeletal muscle (P<0.05). In particular, HS6ST1 joined our previously reported SCD1 and UQCRC1 genes to form a three gene network for one of the stearoyl-CoA desaturase activity indices. These results provide evidence that genes involved in heparan sulfate and heparin metabolism are also involved in regulation of lipid metabolism in bovine muscle. Whether the SNPs affected heparan sulfate proteoglycan structure is unknown and warrants further investigation. PMID:21647334

  9. Mineral phosphate solubilization by Streptomyces sp. CTM396 involves the excretion of gluconic acid and is stimulated by humic acids.

    PubMed

    Farhat, Mounira Ben; Boukhris, Ines; Chouayekh, Hichem

    2015-03-01

    The actinomycetes isolates (128) which were taken from agricultural soil samples and collected near a rock phosphate processing unit were screened for mineral phosphate-solubilizing (MPS) ability. A significant MPS activity was observed for 30 isolates on various phosphate sources when grown in the National Botanical Research Institute's phosphate broth. CTM396 and CTM397 strains which showed the highest MPS abilities were identified by 16S rDNA sequencing as members of the genus Streptomyces. Their MPS activity was proved to be concomitant with a drop in pH due to the secretion of gluconic acid (GA). This was correlated with the simultaneous detection by PCR of genes gdh [encoding the glucose dehydrogenase (GDH) responsible for GA production from glucose] and pqq (involved in biosynthesis of the pyrroloquinoline quinone cofactor of GDH), as well as the highlighting of GHD enzyme activity, for the first time in a Streptomyces sp. strain producing GA. Furthermore, the 0.05% of humic acids proved to have a stimulatory effect on the growth and the ability of CTM396 to solubilize Gafsa rock phosphate. According to this study, it is possible to use humic acids and Gafsa rock phosphate in association with spores of ad hoc Streptomyces strains as natural and efficient amendments to improve plant growth with no need of costly and pollutant transformation of Gafsa rock phosphate. PMID:25743071

  10. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    2011-12-06

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  11. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    2011-03-22

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  12. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    2008-10-07

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  13. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    2012-02-14

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  14. Site specific incorporation of keto amino acids into proteins

    DOEpatents

    Schultz, Peter G.; Wang, Lei

    2009-04-28

    Compositions and methods of producing components of protein biosynthetic machinery that include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, and orthogonal pairs of tRNAs/synthetases, which incorporate keto amino acids into proteins are provided. Methods for identifying these orthogonal pairs are also provided along with methods of producing proteins with keto amino acids using these orthogonal pairs.

  15. Neuronal adaptation involves rapid expansion of the action potential initiation site.

    PubMed

    Scott, Ricardo S; Henneberger, Christian; Padmashri, Ragunathan; Anders, Stefanie; Jensen, Thomas P; Rusakov, Dmitri A

    2014-01-01

    Action potential (AP) generation is the key to information-processing in the brain. Although APs are normally initiated in the axonal initial segment, developmental adaptation or prolonged network activity may alter the initiation site geometry thus affecting cell excitability. Here we find that hippocampal dentate granule cells adapt their spiking threshold to the kinetics of the ongoing dendrosomatic excitatory input by expanding the AP-initiation area away from the soma while also decelerating local axonal spikes. Dual-patch soma-axon recordings combined with axonal Na(+) and Ca(2+) imaging and biophysical modelling show that the underlying mechanism involves distance-dependent inactivation of axonal Na(+) channels due to somatic depolarization propagating into the axon. Thus, the ensuing changes in the AP-initiation zone and local AP propagation could provide activity-dependent control of cell excitability and spiking on a relatively rapid timescale. PMID:24851940

  16. MNGIE with lack of skeletal muscle involvement and a novel TP splice site mutation.

    PubMed

    Szigeti, K; Wong, L-J C; Perng, C-L; Saifi, G M; Eldin, K; Adesina, A M; Cass, D L; Hirano, M; Lupski, J R; Scaglia, F

    2004-02-01

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive multisystem disorder caused by thymidine phosphorylase (TP) deficiency, resulting in severe gastrointestinal dysmotility and skeletal muscle abnormalities. A patient is reported with a classical MNGIE clinical presentation but without skeletal muscle involvement at morphological, enzymatic, or mitochondrial DNA level, though gastrointestinal myopathy was present. MNGIE was diagnosed by markedly raised plasma thymidine and reduced thymidine phosphorylase activity. Molecular genetic analysis showed a homozygous novel splice site mutation in TP. On immunohistochemical studies there was marked TP expression in the CNS, in contrast to what has been observed in rodents. It is important to examine the most significantly affected tissue and to measure TP activity and plasma thymidine in order to arrive at an accurate diagnosis in this condition. PMID:14757860

  17. Neuronal adaptation involves rapid expansion of the action potential initiation site

    PubMed Central

    Scott, Ricardo S.; Henneberger, Christian; Padmashri, Ragunathan; Anders, Stefanie; Jensen, Thomas P.; Rusakov, Dmitri A.

    2014-01-01

    Action potential (AP) generation is the key to information-processing in the brain. Although APs are normally initiated in the axonal initial segment, developmental adaptation or prolonged network activity may alter the initiation site geometry thus affecting cell excitability. Here we find that hippocampal dentate granule cells adapt their spiking threshold to the kinetics of the ongoing dendrosomatic excitatory input by expanding the AP-initiation area away from the soma while also decelerating local axonal spikes. Dual-patch soma–axon recordings combined with axonal Na+ and Ca2+ imaging and biophysical modelling show that the underlying mechanism involves distance-dependent inactivation of axonal Na+ channels due to somatic depolarization propagating into the axon. Thus, the ensuing changes in the AP-initiation zone and local AP propagation could provide activity-dependent control of cell excitability and spiking on a relatively rapid timescale. PMID:24851940

  18. Locating high-affinity fatty acid-binding sites on albumin by x-ray crystallography and NMR spectroscopy

    PubMed Central

    Simard, J. R.; Zunszain, P. A.; Ha, C.-E.; Yang, J. S.; Bhagavan, N. V.; Petitpas, I.; Curry, S.; Hamilton, J. A.

    2005-01-01

    Human serum albumin (HSA) is a versatile transport protein for endogenous compounds and drugs. To evaluate physiologically relevant interactions between ligands for the protein, it is necessary to determine the locations and relative affinities of different ligands for their binding site(s). We present a site-specific investigation of the relative affinities of binding sites on HSA for fatty acids (FA), the primary physiological ligand for the protein. Titration of HSA with [13C]carboxyl-labeled FA was used initially to identify three NMR chemical shifts that are associated with high-affinity binding pockets on the protein. To correlate these peaks with FA-binding sites identified from the crystal structures of FA–HSA complexes, HSA mutants were engineered with substitutions of amino acids involved in coordination of the bound FA carboxyl. Titration of [13C]palmitate into solutions of HSA mutants for either FA site four (R410A/Y411A) or site five (K525A) within domain III of HSA each revealed loss of a specific NMR peak that was present in spectra of wild-type protein. Because these peaks are among the first three to be observed on titration of HSA with palmitate, sites four and five represent two of the three high-affinity long-chain FA-binding sites on HSA. These assignments were confirmed by titration of [13C]palmitate into recombinant domain III of HSA, which contains only sites four and five. These results establish a protocol for direct probing of the relative affinities of FA-binding sites, one that may be extended to examine competition between FA and other ligands for specific binding sites. PMID:16330771

  19. Locating high-affinity fatty acid-binding sites on albumin by x-ray crystallography and NMR spectroscopy.

    PubMed

    Simard, J R; Zunszain, P A; Ha, C-E; Yang, J S; Bhagavan, N V; Petitpas, I; Curry, S; Hamilton, J A

    2005-12-13

    Human serum albumin (HSA) is a versatile transport protein for endogenous compounds and drugs. To evaluate physiologically relevant interactions between ligands for the protein, it is necessary to determine the locations and relative affinities of different ligands for their binding site(s). We present a site-specific investigation of the relative affinities of binding sites on HSA for fatty acids (FA), the primary physiological ligand for the protein. Titration of HSA with [(13)C]carboxyl-labeled FA was used initially to identify three NMR chemical shifts that are associated with high-affinity binding pockets on the protein. To correlate these peaks with FA-binding sites identified from the crystal structures of FA-HSA complexes, HSA mutants were engineered with substitutions of amino acids involved in coordination of the bound FA carboxyl. Titration of [(13)C]palmitate into solutions of HSA mutants for either FA site four (R410A/Y411A) or site five (K525A) within domain III of HSA each revealed loss of a specific NMR peak that was present in spectra of wild-type protein. Because these peaks are among the first three to be observed on titration of HSA with palmitate, sites four and five represent two of the three high-affinity long-chain FA-binding sites on HSA. These assignments were confirmed by titration of [(13)C]palmitate into recombinant domain III of HSA, which contains only sites four and five. These results establish a protocol for direct probing of the relative affinities of FA-binding sites, one that may be extended to examine competition between FA and other ligands for specific binding sites. PMID:16330771

  20. Hypericum perforatum hydroxyalkylpyrone synthase involved in sporopollenin biosynthesis--phylogeny, site-directed mutagenesis, and expression in nonanther tissues.

    PubMed

    Jepson, Christina; Karppinen, Katja; Daku, Rhys M; Sterenberg, Brian T; Suh, Dae-Yeon

    2014-09-01

    Anther-specific chalcone synthase-like enzyme (ASCL), an ancient plant type III polyketide synthase, is involved in the biosynthesis of sporopollenin, the stable biopolymer found in the exine layer of the wall of a spore or pollen grain. The gene encoding polyketide synthase 1 from Hypericum perforatum (HpPKS1) was previously shown to be expressed mainly in young flower buds, but also in leaves and other tissues at lower levels. Angiosperm ASCLs, identified by sequence and phylogenetic analyses, are divided into two sister clades, the Ala-clade and the Val-clade, and HpPKS1 belongs to the Ala-clade. Recombinant HpPKS1 produced triketide and, to a lesser extent, tetraketide alkylpyrones from medium-chain (C6) to very long-chain (C24) fatty acyl-CoA substrates. Like other ASCLs, HpPKS1 also preferred hydroxyl fatty acyl-CoA esters over the analogous unsubstituted fatty acyl-CoA esters. To study the structural basis of the substrate preference, mutants of Ala200 and Ala215 at the putative active site and Arg202 and Asp211 at the modeled acyl-binding tunnel were constructed. The A200T/A215Q mutant accepted decanoyl-CoA, a poor substrate for the wild-type enzyme, possibly because of active site constriction by bulkier substitutions. The substrate preference of the A215V and A200T/A215Q mutants shifted toward nonhydroxylated, medium-chain to long-chain fatty acyl-CoA substrates. The R202L/D211V double mutant was selective for acyl-CoA with chain lengths of C16-C18, and showed a diminished preference for the hydroxylated acyl-CoA substrates. Transient upregulation by abscisic acid and downregulation by jasmonic acid and wounding suggested that HpPKS1, and possibly other Ala-clade ASCLs, may be involved in the biosynthesis of minor cell wall components in nonanther tissues. PMID:25040801

  1. Interaction of metal ions with acid sites of biosorbents peat moss and Vaucheria and model substances alginic and humic acids

    SciTech Connect

    Crist, R.H.; Martin, J.R.; Crist, D.R.

    1999-07-01

    The interaction between added metal ions and acid sites of two biosorbents, peat moss and the alga Vaucheria, was studied. Results were interpreted in terms of two model substances, alginic acid, a copolymer of guluronic and mannuronic acids present in marine algae, and humic acid in peat moss. For peat moss and Vaucheria at pH 4--6, two protons were displaced per Cd sorbed, after correction for sorbed metals also displaced by the heavy metal. The frequent neglect of exchange of heavy metals for metals either sorbed on the native material or added for pH adjustment leads to erroneous conclusions about proton displacement stoichiometry. Proton displacement constants K{sub ex}{sup H} decreased logarithmically with pH and had similar slopes for alginic acid and biosorbents. This pH effect was interpreted as an electrostatic effect of increasing anionic charge making proton removal less favorable. The maximum number of exchangeable acid sites (capacity C{sub H}) decreased with pH for alginic acid but increased with pH for biosorbents. Consistent with titration behavior, this difference was explained in terms of more weak acid sites in the biosorbents.

  2. Mitochondrial Dysfunction Is Involved in the Toxic Activity of Boric Acid against Saprolegnia

    PubMed Central

    Ali, Shimaa E.; Thoen, Even; Evensen, Øystein; Wiik-Nielsen, Jannicke; Gamil, Amr A. A.; Skaar, Ida

    2014-01-01

    There has been a significant increase in the incidence of Saprolegnia infections over the past decades, especially after the banning of malachite green. Very often these infections are associated with high economic losses in salmonid farms and hatcheries. The use of boric acid to control the disease has been investigated recently both under in vitro and in vivo conditions, however its possible mode of action against fish pathogenic Saprolegnia is not known. In this study, we have explored the transformation in Saprolegnia spores/hyphae after exposure to boric acid (1 g/L) over a period 4–24 h post treatment. Using transmission electron microscopy (TEM), early changes in Saprolegnia spores were detected. Mitochondrial degeneration was the most obvious sign observed following 4 h treatment in about 20% of randomly selected spores. We also investigated the effect of the treatment on nuclear division, mitochondrial activity and function using confocal laser scanning microscopy (CLSM). Fluorescence microscopy was also used to test the effect of treatment on mitochondrial membrane potential and formation of reactive oxygen species. Additionally, the viability and proliferation of treated spores that correlated to mitochondrial enzymatic activity were tested using an MTS assay. All obtained data pointed towards changes in the mitochondrial structure, membrane potential and enzymatic activity following treatment. We have found that boric acid has no effect on the integrity of membranes of Saprolegnia spores at concentrations tested. It is therefore likely that mitochondrial dysfunction is involved in the toxic activity of boric acid against Saprolegnia spp. PMID:25354209

  3. Expression analysis for genes involved in arachidonic acid biosynthesis in Mortierella alpina CBS 754.68

    PubMed Central

    Samadlouie, Hamid-Reza; Hamidi-Esfahani, Zohreh; Alavi, Seyed-Mehdi; Varastegani, Boshra

    2014-01-01

    The time courses for production of fungal biomass, lipid, phenolic and arachidonic acid (ARA) as well as expression of the genes involved in biosynthesis of ARA and lipid were examined in Mortierella alpina CBS 754.68. A significant increase in the arachidonic acid content in lipids that coincided with reduced levels of lipid was obtained. Reduced gene expression occurred presumably due to the steady reduction of carbon and nitrogen resources. However, these energy resources were inefficiently compensated by the breakdown of the accumulated lipids that in turn, induced up-regulated expression of the candidate genes. The results further indicated that the expression of the GLELO encoding gene is a rate-limiting step in the biosynthesis of ARA in the early growth phase. PMID:25242926

  4. Composite active site of chondroitin lyase ABC accepting both epimers of uronic acid

    SciTech Connect

    Shaya, D.; Hahn, Bum-Soo; Bjerkan, Tonje Marita; Kim, Wan Seok; Park, Nam Young; Sim, Joon-Soo; Kim, Yeong-Shik; Cygler, M.

    2008-03-19

    Enzymes have evolved as catalysts with high degrees of stereospecificity. When both enantiomers are biologically important, enzymes with two different folds usually catalyze reactions with the individual enantiomers. In rare cases a single enzyme can process both enantiomers efficiently, but no molecular basis for such catalysis has been established. The family of bacterial chondroitin lyases ABC comprises such enzymes. They can degrade both chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans at the nonreducing end of either glucuronic acid (CS) or its epimer iduronic acid (DS) by a {beta}-elimination mechanism, which commences with the removal of the C-5 proton from the uronic acid. Two other structural folds evolved to perform these reactions in an epimer-specific fashion: ({alpha}/{alpha}){sub 5} for CS (chondroitin lyases AC) and {beta}-helix for DS (chondroitin lyases B); their catalytic mechanisms have been established at the molecular level. The structure of chondroitinase ABC from Proteus vulgaris showed surprising similarity to chondroitinase AC, including the presence of a Tyr-His-Glu-Arg catalytic tetrad, which provided a possible mechanism for CS degradation but not for DS degradation. We determined the structure of a distantly related Bacteroides thetaiotaomicron chondroitinase ABC to identify additional structurally conserved residues potentially involved in catalysis. We found a conserved cluster located {approx}12 {angstrom} from the catalytic tetrad. We demonstrate that a histidine in this cluster is essential for catalysis of DS but not CS. The enzyme utilizes a single substrate-binding site while having two partially overlapping active sites catalyzing the respective reactions. The spatial separation of the two sets of residues suggests a substrate-induced conformational change that brings all catalytically essential residues close together.

  5. Myeloid ecotropic viral integration site 1 (MEIS) 1 involvement in embryonic implantation

    PubMed Central

    Xu, Bei; Geerts, Dirk; Qian, Kun; Zhang, Hanwang; Zhu, Guijin

    2008-01-01

    BACKGROUND The HOXA10 homeobox gene controls embryonic uterine development and adult endometrial receptivity. The three-amino-acid loop extension (TALE) family homeobox genes like myeloid ecotropic viral integration site 1 (MEIS) provide enhanced target gene activation and specificity in HOX-regulated cellular processes by acting as HOX cofactors. METHODS AND RESULTS Analysis of an Affymetrix data set in the public domain showed high expression of MEIS1 in human endometrium. MEIS1 expression was confirmed during the human menstrual cycle by RT–PCR and in situ hybridization and was increased during the secretory compared with proliferative phase of the cycle (P = 0.0001), the time of implantation. To assess the importance of maternal Meis1 expression in a mouse model, the uteri of Day 2 pregnant mice were injected with Meis1 over-expression or small interfering RNA (siRNA) constructs. Blocking Meis1 expression by siRNA before implantation significantly reduced average implantation rates (P = 0.00001). Increased or decreased Meis1 expression significantly increased or decreased the expression of integrin β3, a transcriptional target of HOXA10 and an important factor in early embryo-endometrium interactions (P = 0.006). Manipulating Meis1 expression before implantation also dramatically affected the number of pinopodes, uterine endometrial epithelial projections that develop at the time of endometrial receptivity. CONCLUSIONS The results suggest that in mouse, meis1 contributes to regulating endometrial development during the menstrual cycle and establishing the conditions necessary for implantation. PMID:18408019

  6. Adsorption and characterization of MCPA on DDTMA- and raw-montmorillonite: Surface sites involved.

    PubMed

    Santiago, Cintia C; Fernández, Mariela A; Torres Sánchez, Rosa M

    2016-01-01

    The 4-chloro-2-methylphenoxy acid (MCPA) is an herbicide widely used in agriculture, which generates a great concern about contamination of surface water and serious consequences for human health and the environment. In this work, the adsorption of MCPA on an Argentine montmorillonite (MMT) and its organo-montmorillonite product (OMMT) with different dodecyl trimethyl ammonium loading was investigated. MCPA adsorption on OMMT increases at least 3 times, with respect to the amount determined for MMT. X-ray diffraction and zeta potential analyses indicated the inner (interlayer) and outer surface participate as adsorption sites. Changes in surface electric charge and also interlayer expansion suggest that dimethyl amine (MCPA counterion) was also surface-adsorbed. The larger aggregates of OMMT, without and with MCPA, obtained compared to those of MMT samples, generate an improvement in the coagulation efficiency. This property, particularly after MCPA retention, allows an easier separation of the solids from the solution and enables a simple technological process application. PMID:26786275

  7. PTH1 Receptor Is Involved in Mediating Cellular Response to Long-Chain Polyunsaturated Fatty Acids

    PubMed Central

    Chachisvilis, Mirianas

    2012-01-01

    The molecular pathways by which long chain polyunsaturated fatty acids (LCPUFA) influence skeletal health remain elusive. Both LCPUFA and parathyroid hormone type 1 receptor (PTH1R) are known to be involved in bone metabolism while any direct link between the two is yet to be established. Here we report that LCPUFA are capable of direct, PTH1R dependent activation of extracellular ligand-regulated kinases (ERK). From a wide range of fatty acids studied, varying in chain length, saturation, and position of double bonds, eicosapentaenoic (EPA) and docosahexaenoic fatty acids (DHA) caused the highest ERK phosphorylation. Moreover, EPA potentiated the effect of parathyroid hormone (PTH(1–34)) in a superagonistic manner. EPA or DHA dependent ERK phosphorylation was inhibited by the PTH1R antagonist and by knockdown of PTH1R. Inhibition of PTH1R downstream signaling molecules, protein kinases A (PKA) and C (PKC), reduced EPA and DHA dependent ERK phosphorylation indicating that fatty acids predominantly activate G-protein pathway and not the β-arrestin pathway. Using picosecond time-resolved fluorescence microscopy and a genetically engineered PTH1R sensor (PTH-CC), we detected conformational responses to EPA similar to those caused by PTH(1–34). PTH1R antagonist blocked the EPA induced conformational response of the PTH-CC. Competitive binding studies using fluorescence anisotropy technique showed that EPA and DHA competitively bind to and alter the affinity of PTH1 receptor to PTH(1–34) leading to a superagonistic response. Finally, we showed that EPA stimulates protein kinase B (Akt) phosphorylation in a PTH1R-dependent manner and affects the osteoblast survival pathway, by inhibiting glucocorticoid-induced cell death. Our findings demonstrate for the first time that LCPUFAs, EPA and DHA, can activate PTH1R receptor at nanomolar concentrations and consequently provide a putative molecular mechanism for the action of fatty acids in bone. PMID:23300710

  8. Hydrogen Peroxide Is Involved in Salicylic Acid-Elicited Rosmarinic Acid Production in Salvia miltiorrhiza Cell Cultures

    PubMed Central

    Hao, Wenfang; Zhang, Jingyi; Hu, Gege; Yao, Yaqin; Dong, Juane

    2014-01-01

    Salicylic acid (SA) is an elicitor to induce the biosynthesis of secondary metabolites in plant cells. Hydrogen peroxide (H2O2) plays an important role as a key signaling molecule in response to various stimuli and is involved in the accumulation of secondary metabolites. However, the relationship between them is unclear and their synergetic functions on accumulation of secondary metabolites are unknown. In this paper, the roles of SA and H2O2 in rosmarinic acid (RA) production in Salvia miltiorrhiza cell cultures were investigated. The results showed that SA significantly enhanced H2O2 production, phenylalanine ammonia-lyase (PAL) activity, and RA accumulation. Exogenous H2O2 could also promote PAL activity and enhance RA production. If H2O2 production was inhibited by NADPH oxidase inhibitor (IMD) or scavenged by quencher (DMTU), RA accumulation would be blocked. These results indicated that H2O2 is secondary messenger for signal transduction, which can be induced by SA, significantly and promotes RA accumulation. PMID:24995364

  9. Ginseng Gintonin Activates the Human Cardiac Delayed Rectifier K+ Channel: Involvement of Ca2+/Calmodulin Binding Sites

    PubMed Central

    Choi, Sun-Hye; Lee, Byung-Hwan; Kim, Hyeon-Joong; Jung, Seok-Won; Kim, Hyun-Sook; Shin, Ho-Chul; Lee, Jun-Hee; Kim, Hyoung-Chun; Rhim, Hyewhon; Hwang, Sung-Hee; Ha, Tal soo; Kim, Hyun-Ji; Cho, Hana; Nah, Seung-Yeol

    2014-01-01

    Gintonin, a novel, ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand, elicits [Ca2+]i transients in neuronal and non-neuronal cells via pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. The slowly activating delayed rectifier K+ (IKs) channel is a cardiac K+ channel composed of KCNQ1 and KCNE1 subunits. The C terminus of the KCNQ1 channel protein has two calmodulin-binding sites that are involved in regulating IKs channels. In this study, we investigated the molecular mechanisms of gintonin-mediated activation of human IKs channel activity by expressing human IKs channels in Xenopus oocytes. We found that gintonin enhances IKs channel currents in concentration- and voltage-dependent manners. The EC50 for the IKs channel was 0.05 ± 0.01 μg/ml. Gintonin-mediated activation of the IKs channels was blocked by an LPA1/3 receptor antagonist, an active phospholipase C inhibitor, an IP3 receptor antagonist, and the calcium chelator BAPTA. Gintonin-mediated activation of both the IKs channel was also blocked by the calmodulin (CaM) blocker calmidazolium. Mutations in the KCNQ1 [Ca2+]i/CaM-binding IQ motif sites (S373P, W392R, or R539W)blocked the action of gintonin on IKs channel. However, gintonin had no effect on hERG K+ channel activity. These results show that gintonin-mediated enhancement of IKs channel currents is achieved through binding of the [Ca2+]i/CaM complex to the C terminus of KCNQ1 subunit. PMID:25234465

  10. Analysis of the Proteolytic Processing of ABCA3: Identification of Cleavage Site and Involved Proteases

    PubMed Central

    Hofmann, Nicole; Galetskiy, Dmitry; Rauch, Daniela; Wittmann, Thomas; Marquardt, Andreas; Griese, Matthias; Zarbock, Ralf

    2016-01-01

    Rationale ABCA3 is a lipid transporter in the limiting membrane of lamellar bodies in alveolar type II cells. Mutations in the ABCA3 gene cause respiratory distress syndrome in new-borns and childhood interstitial lung disease. ABCA3 is N-terminally cleaved by an as yet unknown protease, a process believed to regulate ABCA3 activity. Methods The exact site where ABCA3 is cleaved was localized using mass spectrometry (MS). Proteases involved in ABCA3 processing were identified using small molecule inhibitors and siRNA mediated gene knockdown. Results were verified by in vitro digestion of a synthetic peptide substrate mimicking ABCA3’s cleavage region, followed by MS analysis. Results We found that cleavage of ABCA3 occurs after Lys174 which is located in the proteins’ first luminal loop. Inhibition of cathepsin L and, to a lesser extent, cathepsin B resulted in attenuation of ABCA3 cleavage. Both enzymes showed activity against the ABCA3 peptide in vitro with cathepsin L being more active. Conclusion We show here that, like some other proteins of the lysosomal membrane, ABCA3 is a substrate of cathepsin L. Therefore, cathepsin L may represent a potential target to therapeutically influence ABCA3 activity in ABCA3-associated lung disease. PMID:27031696

  11. Mutations at sites involved in Suc1 binding inactivate Cdc2.

    PubMed Central

    Ducommun, B; Brambilla, P; Draetta, G

    1991-01-01

    suc1+ encodes an essential cell cycle regulator of the fission yeast Schizosaccharomyces pombe. Its product, a 13-kDa protein, interacts with the Cdc2 protein kinase. Both positive and negative effects on cell cycle progression have been attributed to Suc1. To date, the exact mechanisms and the physiological role of the interaction between Suc1 and Cdc2 remain unclear. Here we have studied the molecular basis of this association. We show that Cdc2 can bind Suc1 or its mammalian homolog directly in the absence of any additional protein component. Using an alanine scanning mutagenesis method, we analyzed the interaction between Cdc2 and Suc1. We show that the integrity of several domains on the Cdc2 protein, including sites directly involved in catalytic activity, is required for binding to Suc1. Furthermore, Cdc2 mutant proteins unable to bind Suc1 (but able to bind cyclins) are nonfunctional when overexpressed in S. pombe, indicating that a specific interaction with Suc1 is required for Cdc2 function. Images PMID:1944283

  12. Interatomic Coulombic Decay Effects in Theoretical DNA Recombination Systems Involving Protein Interaction Sites

    NASA Astrophysics Data System (ADS)

    Vargas, E. L.; Rivas, D. A.; Duot, A. C.; Hovey, R. T.; Andrianarijaona, V. M.

    2015-03-01

    DNA replication is the basis for all biological reproduction. A strand of DNA will ``unzip'' and bind with a complimentary strand, creating two identical strands. In this study, we are considering how this process is affected by Interatomic Coulombic Decay (ICD), specifically how ICD affects the individual coding proteins' ability to hold together. ICD mainly deals with how the electron returns to its original state after excitation and how this affects its immediate atomic environment, sometimes affecting the connectivity between interaction sites on proteins involved in the DNA coding process. Biological heredity is fundamentally controlled by DNA and its replication therefore it affects every living thing. The small nature of the proteins (within the range of nanometers) makes it a good candidate for research of this scale. Understanding how ICD affects DNA molecules can give us invaluable insight into the human genetic code and the processes behind cell mutations that can lead to cancer. Authors wish to give special thanks to Pacific Union College Student Senate in Angwin, California, for their financial support.

  13. Overlapping sites for constitutive and induced DNA binding factors involved in interferon-stimulated transcription.

    PubMed Central

    Dale, T C; Rosen, J M; Guille, M J; Lewin, A R; Porter, A G; Kerr, I M; Stark, G R

    1989-01-01

    A 14 bp interferon (IFN)-stimulated response element (ISRE) from 6-16, a human gene regulated by alpha-IFN, confers IFN inducibility on a heterologous thymidine kinase promoter. A 39 bp double-stranded oligonucleotide corresponding to a 5' region of 6-16 which includes the ISRE competes for factors required for gene expression by alpha-IFN in transfected cells and a single base change (A-11 to C) within the ISRE (GGGAAAATGAAACT) abolishes this competition. Band-shift assays performed with whole-cell extracts and the 39 bp oligonucleotide reveal specific complexes formed by rapidly induced and constitutive factors, both of which fail to bind to the A-11 to C oligonucleotide. A detailed footprinting analysis reveals that these two types of factors bind to overlapping sites within the ISRE, but in very different ways. These data were used to design oligonucleotides which decreased the formation of the inducible complex without affecting the constitutive one. Changes at the 5' margin of the ISRE and upstream of it markedly decrease formation of the induced but not the constitutive complex and also abolish the ability of the 39 bp sequence to function as an inducible enhancer with the thymidine kinase promoter. Thus, induction of 6-16 transcription in IFN-treated cells is likely to be stimulated by binding of the induced factor to the ISRE and upstream sequences, while the subsequent suppression of transcription may involve competition for the ISRE by the other class of factors. Images PMID:2721502

  14. Structural and electronic features of a Broensted acid site in H-ZSM-5

    SciTech Connect

    Cook, S.J.; Chakraborty, A.K.; Bell, A.T.; Theodorou, D.N. Univ. of California, Berkeley )

    1993-06-24

    The authors report the results of local density functional theory calculations on a Broensted acid site of the zeolite H-ZSM-5. They have investigated the structural and electronic properties of the site. Comparison is made between their results and existing experimental data. It is shown that structural relaxation around an acid site must be performed to obtain accurate energetics for substitution of aluminum into the zeolite framework. The effects of cluster termination are studied by comparing results obtained for both isolated clusters and clusters embedded in a Madelung field generated by the zeolite framework. The properties of the electron density distribution in the region around an acidic proton indicate that the acidic moiety may be characterized as a rather soft acid. 47 refs., 5 figs., 4 tabs.

  15. Evidence that Chemical Chaperone 4-Phenylbutyric Acid Binds to Human Serum Albumin at Fatty Acid Binding Sites

    PubMed Central

    James, Joel; Shihabudeen, Mohamed Sham; Kulshrestha, Shweta; Goel, Varun; Thirumurugan, Kavitha

    2015-01-01

    Endoplasmic reticulum stress elicits unfolded protein response to counteract the accumulating unfolded protein load inside a cell. The chemical chaperone, 4-Phenylbutyric acid (4-PBA) is a FDA approved drug that alleviates endoplasmic reticulum stress by assisting protein folding. It is found efficacious to augment pathological conditions like type 2 diabetes, obesity and neurodegeneration. This study explores the binding nature of 4-PBA with human serum albumin (HSA) through spectroscopic and molecular dynamics approaches, and the results show that 4-PBA has high binding specificity to Sudlow Site II (Fatty acid binding site 3, subdomain IIIA). Ligand displacement studies, RMSD stabilization profiles and MM-PBSA binding free energy calculation confirm the same. The binding constant as calculated from fluorescence spectroscopic studies was found to be kPBA = 2.69 x 105 M-1. Like long chain fatty acids, 4-PBA induces conformational changes on HSA as shown by circular dichroism, and it elicits stable binding at Sudlow Site II (fatty acid binding site 3) by forming strong hydrogen bonding and a salt bridge between domain II and III of HSA. This minimizes the fluctuation of HSA backbone as shown by limited conformational space occupancy in the principal component analysis. The overall hydrophobicity of W214 pocket (located at subdomain IIA), increases upon occupancy of 4-PBA at any FA site. Descriptors of this pocket formed by residues from other subdomains largely play a role in compensating the dynamic movement of W214. PMID:26181488

  16. Induction of liver alpha-1 acid glycoprotein gene expression involves both positive and negative transcription factors.

    PubMed Central

    Lee, Y M; Tsai, W H; Lai, M Y; Chen, D S; Lee, S C

    1993-01-01

    Expression of the alpha-1 acid glycoprotein (AGP) gene is liver specific and acute phase responsive. Within the 180-bp region of the AGP promoter, at least five cis elements have been found to interact with trans-acting factors. Four of these elements (A, C, D, and E) interacted with AGP/EBP, a liver-enriched transcription factor, as shown by footprinting analysis and by an anti-AGP/EBP antibody-induced supershift in a gel retardation assay. Modification of these sites by site-directed mutagenesis coupled with transfection analysis indicated that AGP/EBP binding to all of these sites resulted in positive regulation of the promoter. Dose-response data suggest that AGP/EBP binding to these sites results in the cooperative activation of the promoter. In contrast, functional assays showed that element B is a negative regulatory element; this element is recognized by heat-stable DNA-binding factors which are found in many cells and tissues. The regulation of these binding proteins was studied in rat liver treated with lipopolysaccharide (LPS), which induced an acute-phase reaction. We found that LPS treatment resulted in a two- to threefold increase in AGP/EBP activity and a severalfold decrease in the activity of factors that bind to element B in the liver. These results indicate that expression of the AGP gene can be regulated by both positive and negative factors and that the modulation of these factors can account for the LPS induction of the AGP gene. Images PMID:8417341

  17. Cinnamic Acid Is Partially Involved in Propolis Immunomodulatory Action on Human Monocytes

    PubMed Central

    Conti, Bruno José; Búfalo, Michelle Cristiane; Golim, Marjorie de Assis; Sforcin, José Maurício

    2013-01-01

    Propolis is a beehive product used in traditional medicine due to its biological properties. It shows a complex chemical composition including phenolics, such as cinnamic acid (Ci). The mechanisms of action of propolis have been the subject of research recently; however, the involvement of Ci on propolis activity was not investigated on immune cells. Ci effects were evaluated on human monocytes, assessing the expression of Toll-like receptors (TLRs), HLA-DR, and CD80. Cytokine production (TNF-α and IL-10) and the fungicidal activity of monocytes were evaluated as well. Data showed that Ci downregulated TLR-2, HLA-DR, and CD80 and upregulated TLR-4 expression by human monocytes. High concentrations of Ci inhibited both TNF-α and IL-10 production, whereas the same concentrations induced a higher fungicidal activity against Candida albicans. TNF-α and IL-10 production was decreased by blocking TLR-4, while the fungicidal activity of monocytes was not affected by blocking TLRs. These results suggest that Ci modulated antigen receptors, cytokine production, and the fungicidal activity of human monocytes depending on concentration, and TLR-4 may be involved in its mechanism of action. Ci seemed to be partially involved in propolis activities. PMID:23762102

  18. Cinnamic Acid is partially involved in propolis immunomodulatory action on human monocytes.

    PubMed

    Conti, Bruno José; Búfalo, Michelle Cristiane; Golim, Marjorie de Assis; Bankova, Vassya; Sforcin, José Maurício

    2013-01-01

    Propolis is a beehive product used in traditional medicine due to its biological properties. It shows a complex chemical composition including phenolics, such as cinnamic acid (Ci). The mechanisms of action of propolis have been the subject of research recently; however, the involvement of Ci on propolis activity was not investigated on immune cells. Ci effects were evaluated on human monocytes, assessing the expression of Toll-like receptors (TLRs), HLA-DR, and CD80. Cytokine production (TNF- α and IL-10) and the fungicidal activity of monocytes were evaluated as well. Data showed that Ci downregulated TLR-2, HLA-DR, and CD80 and upregulated TLR-4 expression by human monocytes. High concentrations of Ci inhibited both TNF- α and IL-10 production, whereas the same concentrations induced a higher fungicidal activity against Candida albicans. TNF- α and IL-10 production was decreased by blocking TLR-4, while the fungicidal activity of monocytes was not affected by blocking TLRs. These results suggest that Ci modulated antigen receptors, cytokine production, and the fungicidal activity of human monocytes depending on concentration, and TLR-4 may be involved in its mechanism of action. Ci seemed to be partially involved in propolis activities. PMID:23762102

  19. Organic acids in cloud water and rainwater at a mountain site in acid rain areas of South China.

    PubMed

    Sun, Xiao; Wang, Yan; Li, Haiyan; Yang, Xueqiao; Sun, Lei; Wang, Xinfeng; Wang, Tao; Wang, Wenxing

    2016-05-01

    To investigate the chemical characteristics of organic acids and to identify their source, cloud water and rainwater samples were collected at Mount Lu, a mountain site located in the acid rain-affected area of south China, from August to September of 2011 and March to May of 2012. The volume-weighted mean (VWM) concentration of organic acids in cloud water was 38.42 μeq/L, ranging from 7.45 to 111.46 μeq/L, contributing to 2.50 % of acidity. In rainwater samples, organic acid concentrations varied from 12.39 to 68.97 μeq/L (VWM of 33.39 μeq/L). Organic acids contributed significant acidity to rainwater, with a value of 17.66 %. Formic acid, acetic acid, and oxalic acid were the most common organic acids in both cloud water and rainwater. Organic acids had an obviously higher concentration in summer than in spring in cloud water, whereas there was much less discrimination in rainwater between the two seasons. The contribution of organic acids to acidity was lower during summer than during spring in both cloud water (2.20 % in summer vs 2.83 % in spring) and rainwater (12.24 % in summer vs 19.89 % in spring). The formic-to-acetic acid ratio (F/A) showed that organic acids were dominated by primary emissions in 71.31 % of the cloud water samples and whole rainwater samples. Positive matrix factorization (PMF) analysis determined four factors as the sources of organic acids in cloud water, including biogenic emissions (61.8 %), anthropogenic emissions (15.28 %), marine emissions (15.07 %) and soil emissions (7.85 %). The findings from this study imply an indispensable role of organic acids in wet deposition, but organic acids may have a limited capacity to increase ecological risks in local environments. PMID:26841776

  20. Jasmonic Acid, Abscisic Acid, and Salicylic Acid Are Involved in the Phytoalexin Responses of Rice to Fusarium fujikuroi, a High Gibberellin Producer Pathogen.

    PubMed

    Siciliano, Ilenia; Amaral Carneiro, Greice; Spadaro, Davide; Garibaldi, Angelo; Gullino, Maria Lodovica

    2015-09-23

    Fusarium fujikuroi, the causal agent of bakanae disease, is the main seedborne pathogen on rice. To understand the basis of rice resistance, a quantitative method to simultaneously detect phytohormones and phytoalexins was developed by using HPLC-MS/MS. With this method dynamic profiles and possible interactions of defense-related phytohormones and phytoalexins were investigated on two rice cultivars, inoculated or not with F. fujikuroi. In the resistant cultivar Selenio, the presence of pathogen induced high production of phytoalexins, mainly sakuranetin, and symptoms of bakanae were not observed. On the contrary, in the susceptible genotype Dorella, the pathogen induced the production of gibberellin and abscisic acid and inhibited jasmonic acid production, phytoalexins were very low, and bakanae symptoms were observed. The results suggested that a wide range of secondary metabolites are involved in plant defense against pathogens and phytoalexin synthesis could be an important factor for rice resistance against bakanae disease. PMID:26323788

  1. Involvement of I2-imidazoline binding sites in positive and negative morphine analgesia modulatory effects.

    PubMed

    Gentili, Francesco; Cardinaletti, Claudia; Carrieri, Antonio; Ghelfi, Francesca; Mattioli, Laura; Perfumi, Marina; Vesprini, Cristian; Pigini, Maria

    2006-12-28

    Some studies, suggesting the involvement of I(2)-imidazoline binding sites (I(2)-IBS) in morphine analgesia modulation, prompted us to examine on mice antinociceptive assays the effect produced by 1 (phenyzoline), that in view of its high I(2)-IBS affinity and high I(2)-IBS selectivity with regard to I(1)-IBS, alpha(2)-adrenoreceptors and mu-opioid receptors might be considered the first interesting I(2)-IBS ligand. The study was also applied to its ortho phenyl derivative 2 (diphenyzoline), designed and prepared in order to produce a possible modification of the biological profile of 1. Diphenyzoline (2) retains a significant I(2)-IBS selectivity with regard to I(1)-IBS, alpha(2)-adrenoreceptors and mu-opioid receptors. Moreover, by the functional assays 1 and 2 proved inactive at all alpha(2)-adrenoreceptors subtypes up to 10(-3) M. As expected, phenyzoline and diphenyzoline, which are structurally related, highlighted an interesting "positive" or "negative", respectively, morphine analgesia modulatory effect. In fact, 1 (s.c. 10 mg/kg) enhanced morphine analgesia (60% and 40% in mouse tail-flick and mouse hot-plate, respectively), while 2 (s.c. 10 mg/kg) decreased it (-41% and -20%, respectively). The ability to decrease morphine analgesia had never been observed before in I(2)-IBS ligands. These effects were not affected by i.p. treatment of animals with yohimbine (a selective alpha(2)-adrenoreceptor antagonist, 0.625 mg/kg) or efaroxan (an I(1)-IBS/alpha(2)-adrenoreceptor antagonist, 1.0 mg/kg). In contrast, they were completely reversed by i.p. treatment of animals with idazoxan (an I(2)-IBS/alpha(2)-adrenoreceptor antagonist, 2 mg/kg). Moreover, compound 2, in mouse tail-flick test, was able to potentiate by 23% the naloxone-induced decrease of morphine analgesia. Therefore, the results of this study indicate the crucial involvement of I(2)-IBS in the morphine analgesia modulatory effects of 1 and 2. PMID:17081513

  2. Three charged amino acids in extracellular loop 1 are involved in maintaining the outer pore architecture of CFTR

    PubMed Central

    Cui, Guiying; Rahman, Kazi S.; Infield, Daniel T.; Kuang, Christopher; Prince, Chengyu Z.

    2014-01-01

    The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) bears six extracellular loops (ECL1–6); ECL1 is the site of several mutations associated with CF. Mutation R117H has been reported to reduce current amplitude, whereas D110H, E116K, and R117C/L/P may impair channel stability. We hypothesized that these amino acids might not be directly involved in ion conduction and permeation but may contribute to stabilizing the outer vestibule architecture in CFTR. We used cRNA injected oocytes combined with electrophysiological techniques to test this hypothesis. Mutants bearing cysteine at these sites were not functionally modified by extracellular MTS reagents and were blocked by GlyH-101 similarly to WT-CFTR. These results suggest that these three residues do not contribute directly to permeation in CFTR. In contrast, mutants D110R-, E116R-, and R117A-CFTR exhibited instability of the open state and significantly shortened burst duration compared with WT-CFTR and failed to be locked into the open state by AMP-PNP (adenosine 5′-(β,γ-imido) triphosphate); charge-retaining mutants showed mainly the full open state with comparably longer open burst duration. These interactions suggest that these ECL1 residues might be involved in maintaining the outer pore architecture of CFTR. A CFTR homology model suggested that E116 interacts with R104 in both the closed and open states, D110 interacts with K892 in the fully closed state, and R117 interacts with E1126 in the open state. These interactions were confirmed experimentally. The results suggest that D110, E116, and R117 may contribute to stabilizing the architecture of the outer pore of CFTR by interactions with other charged residues. PMID:25024266

  3. Three charged amino acids in extracellular loop 1 are involved in maintaining the outer pore architecture of CFTR.

    PubMed

    Cui, Guiying; Rahman, Kazi S; Infield, Daniel T; Kuang, Christopher; Prince, Chengyu Z; McCarty, Nael A

    2014-08-01

    The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) bears six extracellular loops (ECL1-6); ECL1 is the site of several mutations associated with CF. Mutation R117H has been reported to reduce current amplitude, whereas D110H, E116K, and R117C/L/P may impair channel stability. We hypothesized that these amino acids might not be directly involved in ion conduction and permeation but may contribute to stabilizing the outer vestibule architecture in CFTR. We used cRNA injected oocytes combined with electrophysiological techniques to test this hypothesis. Mutants bearing cysteine at these sites were not functionally modified by extracellular MTS reagents and were blocked by GlyH-101 similarly to WT-CFTR. These results suggest that these three residues do not contribute directly to permeation in CFTR. In contrast, mutants D110R-, E116R-, and R117A-CFTR exhibited instability of the open state and significantly shortened burst duration compared with WT-CFTR and failed to be locked into the open state by AMP-PNP (adenosine 5'-(β,γ-imido) triphosphate); charge-retaining mutants showed mainly the full open state with comparably longer open burst duration. These interactions suggest that these ECL1 residues might be involved in maintaining the outer pore architecture of CFTR. A CFTR homology model suggested that E116 interacts with R104 in both the closed and open states, D110 interacts with K892 in the fully closed state, and R117 interacts with E1126 in the open state. These interactions were confirmed experimentally. The results suggest that D110, E116, and R117 may contribute to stabilizing the architecture of the outer pore of CFTR by interactions with other charged residues. PMID:25024266

  4. Structural and Functional Characterization of BaiA, An Enzyme Involved in Secondary Bile Acid Synthesis in Human Gut Microbe

    PubMed Central

    Bhowmik, Shiva; Jones, David H.; Chiu, Hsien-Po; Park, In-Hee; Chiu, Hsiu-Ju; Axelrod, Herbert L.; Farr, Carol L.; Tien, Henry J.; Agarwalla, Sanjay; Lesley, Scott A.

    2014-01-01

    Despite significant influence of secondary bile acids on human health and disease, limited structural and biochemical information is available for the key gut microbial enzymes catalyzing its synthesis. Herein, we report apo- and co-factor bound crystal structures of BaiA2, a short chain dehydrogenase/reductase from Clostridium scindens VPI 12708 that represent the first protein structure of this pathway. The structures elucidated the basis of co-factor specificity and mechanism of proton relay. A conformational restriction involving Glu42 located in the co-factor binding site seems crucial in determining co-factor specificity. Limited flexibility of Glu42 results in imminent steric and electrostatic hindrance with 2′-phosphate group of NADP(H). Consistent with crystal structures, steady-state kinetic characterization performed with both BaiA2 and BaiA1, a close homolog with 92% sequence identity, revealed specificity constant (kcat/KM) of NADP+ at least an order of magnitude lower than NAD+. Substitution of Glu42 with Ala improved specificity towards NADP+ by 10- fold compared to wild type. The co-factor bound structure uncovered a novel nicotinamide-hydroxyl ion (NAD+-OH−) adduct contraposing previously reported adducts. The OH− of the adduct in BaiA2 is distal to C4 atom of nicotinamide and proximal to 2′-hydroxyl group of the ribose moiety. Moreover, it is located at intermediary distances between terminal functional groups of active site residues Tyr157 (2.7 Å) and Lys161 (4.5 Å). Based on these observations we propose an involvement of NAD+-OH− adduct in proton relay instead of hydride transfer as noted for previous adducts. PMID:23836456

  5. 1-FFT amino acids involved in high DP inulin accumulation in Viguiera discolor

    PubMed Central

    De Sadeleer, Emerik; Vergauwen, Rudy; Struyf, Tom; Le Roy, Katrien; Van den Ende, Wim

    2015-01-01

    Fructans are important vacuolar reserve carbohydrates with drought, cold, ROS and general abiotic stress mediating properties. They occur in 15% of all flowering plants and are believed to display health benefits as a prebiotic and dietary fiber. Fructans are synthesized by specific fructosyltransferases and classified based on the linkage type between fructosyl units. Inulins, one of these fructan types with β(2-1) linkages, are elongated by fructan:fructan 1-fructosyltransferases (1-FFT) using a fructosyl unit from a donor inulin to elongate the acceptor inulin molecule. The sequence identity of the 1-FFT of Viguiera discolor (Vd) and Helianthus tuberosus (Ht) is 91% although these enzymes produce distinct fructans. The Vd 1-FFT produces high degree of polymerization (DP) inulins by preferring the elongation of long chain inulins, in contrast to the Ht 1-FFT which prefers small molecules (DP3 or 4) as acceptor. Since higher DP inulins have interesting properties for industrial, food and medical applications, we report here on the influence of two amino acids on the high DP inulin production capacity of the Vd 1-FFT. Introducing the M19F and H308T mutations in the active site of the Vd 1-FFT greatly reduces its capacity to produce high DP inulin molecules. Both amino acids can be considered important to this capacity, although the double mutation had a much higher impact than the single mutations. PMID:26322058

  6. 1-FFT amino acids involved in high DP inulin accumulation in Viguiera discolor.

    PubMed

    De Sadeleer, Emerik; Vergauwen, Rudy; Struyf, Tom; Le Roy, Katrien; Van den Ende, Wim

    2015-01-01

    Fructans are important vacuolar reserve carbohydrates with drought, cold, ROS and general abiotic stress mediating properties. They occur in 15% of all flowering plants and are believed to display health benefits as a prebiotic and dietary fiber. Fructans are synthesized by specific fructosyltransferases and classified based on the linkage type between fructosyl units. Inulins, one of these fructan types with β(2-1) linkages, are elongated by fructan:fructan 1-fructosyltransferases (1-FFT) using a fructosyl unit from a donor inulin to elongate the acceptor inulin molecule. The sequence identity of the 1-FFT of Viguiera discolor (Vd) and Helianthus tuberosus (Ht) is 91% although these enzymes produce distinct fructans. The Vd 1-FFT produces high degree of polymerization (DP) inulins by preferring the elongation of long chain inulins, in contrast to the Ht 1-FFT which prefers small molecules (DP3 or 4) as acceptor. Since higher DP inulins have interesting properties for industrial, food and medical applications, we report here on the influence of two amino acids on the high DP inulin production capacity of the Vd 1-FFT. Introducing the M19F and H308T mutations in the active site of the Vd 1-FFT greatly reduces its capacity to produce high DP inulin molecules. Both amino acids can be considered important to this capacity, although the double mutation had a much higher impact than the single mutations. PMID:26322058

  7. Study of the possible mechanisms involved in the mucosal immune system activation by lactic acid bacteria.

    PubMed

    Perdigón, G; Vintiñi, E; Alvarez, S; Medina, M; Medici, M

    1999-06-01

    The induction of a mucosal immune response is not easy due to the development of oral tolerance, but under some conditions, bacteria can activate this immune system. Antigens administered orally can interact with M cells of Peyer's patches or bind to the epithelial cells. We have demonstrated that certain lactic acid bacteria are able to induce specific secretory immunity, and others will enhance the gut inflammatory immune response. The aim of this work was to establish the reason for these different behaviors and to define possible mechanisms involved in the interaction of lactic acid bacteria at the intestinal level. We studied IgA+ and IgM+ B cells comparatively in bronchus and intestine and CD4+ T cells and IgA anti-lactic acid bacteria antibodies in the intestinal fluid, induced by oral administration of Lactobacillus casei, Lb. delbrueckii ssp. bulgaricus, Lb. acidophilus, Lb. plantarum, Lb. rhamnosus, Lactococcus lactis, and Streptococcus salivarius ssp. thermophilus. The increase in the IgA+ B cells in the bronchus means that these lactic acid bacteria were able to induce the IgA cycle by interaction with M cells from Peyer's patches or intestinal epithelial cells. The IgM+ cells increased when the stimulus did not induce the switch from IgM+ to IgA+. The increase in the CD4+ cells suggests interaction of Peyer's patches and enhancement of the B- and T-cell migration. The anti-lactic acid bacteria antibody is related to the processing and presentation of the microorganisms to the immune cells. We demonstrated that Lb. casei and Lb. plantarum were able to interact with Peyer's patch cells and showed an increase in IgA-, CD4+ cells, and antibodies specific for the stimulating strain. Lactobacillus acidophilus induced gut mucosal activation by interaction with the epithelial cells without increase in the immune cells associated with the bronchus. Although Lb. rhamnosus and Strep. salivarius ssp. thermophilus interact with epithelial cells, they also induced

  8. Improving Evolutionary Models for Mitochondrial Protein Data with Site-Class Specific Amino Acid Exchangeability Matrices

    PubMed Central

    Dunn, Katherine A.; Jiang, Wenyi; Field, Christopher; Bielawski, Joseph P.

    2013-01-01

    Adequate modeling of mitochondrial sequence evolution is an essential component of mitochondrial phylogenomics (comparative mitogenomics). There is wide recognition within the field that lineage-specific aspects of mitochondrial evolution should be accommodated through lineage-specific amino-acid exchangeability matrices (e.g., mtMam for mammalian data). However, such a matrix must be applied to all sites and this implies that all sites are subject to the same, or largely similar, evolutionary constraints. This assumption is unjustified. Indeed, substantial differences are expected to arise from three-dimensional structures that impose different physiochemical environments on individual amino acid residues. The objectives of this paper are (1) to investigate the extent to which amino acid evolution varies among sites of mitochondrial proteins, and (2) to assess the potential benefits of explicitly modeling such variability. To achieve this, we developed a novel method for partitioning sites based on amino acid physiochemical properties. We apply this method to two datasets derived from complete mitochondrial genomes of mammals and fish, and use maximum likelihood to estimate amino acid exchangeabilities for the different groups of sites. Using this approach we identified large groups of sites evolving under unique physiochemical constraints. Estimates of amino acid exchangeabilities differed significantly among such groups. Moreover, we found that joint estimates of amino acid exchangeabilities do not adequately represent the natural variability in evolutionary processes among sites of mitochondrial proteins. Significant improvements in likelihood are obtained when the new matrices are employed. We also find that maximum likelihood estimates of branch lengths can be strongly impacted. We provide sets of matrices suitable for groups of sites subject to similar physiochemical constraints, and discuss how they might be used to analyze real data. We also discuss how

  9. Chemiluminescence involving acidic and ambient ion light emitters. The chemiluminescence of the 9-acridinepercarboxylate anion

    SciTech Connect

    White, E.H.; Roswell, D.F.; Dupont, A.C.; Wilson, A.A.

    1987-08-19

    The reaction of phenyl 9-acridinecarboxylate with an excess of peroxide ion in THF/water (67/33 mol %) leads to the emission of either bright yellow-green light or bright blue light, depending on the reaction conditions. The blue emission is favored by high concentrations of hydrogen peroxide and water, for example. 9-Acridinepercarboxylic acid is a common intermediate in the reactions. The light emitter responsible for the blue chemiluminescence is acridone, whereas that responsible for the yellow-green chemiluminescence is the anion of acridone. The effects of base concentration and solvent composition on the relative proportions of these two emitters have produced evidence that, contrary to the expectation of simple theory, a dioxetanone is not an intermediate in the reaction. Other cases where chemiluminescence may involve percarboxylate and peroxide ions are discussed.

  10. Involvement of omega-3 fatty acids in emotional responses and hyperactive symptoms.

    PubMed

    Lavialle, Monique; Denis, Isabelle; Guesnet, Philippe; Vancassel, Sylvie

    2010-10-01

    Biochemical evidence suggests a role for n-3 polyunsaturated fatty acids (n-3 PUFAs) in the regulation of behavioral disturbances. A number of studies have revealed an association between reduced n-3 PUFA levels and attention-deficit hyperactivity disorder or depression. Here, we summarize the main findings regarding the association between n-3 PUFA and hyperactive and emotional disorders, and discuss potential mechanisms of action. Because the basal ganglia are involved in the control of locomotion and emotion, we examined published data regarding the role of n-3 PUFA in dopamine (DA) regulation in the basal ganglia. These results are discussed in the light of recent data from our laboratory suggesting an association between the drop in melatonin in the pineal gland and the increase in DA in the striatum and nucleus accumbens of n-3 PUFA-deprived rodents. PMID:20434321

  11. Interstitial telomeric sequences in human chromosomes cluster with common fragile sites, mutagen sensitive sites, viral integration sites, cancer breakpoints, proto-oncogenes and breakpoints involved in primate evolution

    SciTech Connect

    Adekunle, S.S.A.; Wyandt, H.; Mark, H.F.L.

    1994-09-01

    Recently we mapped the telomeric repeat sequences to 111 interstitial sites in the human genome and to sites of gaps and breaks induced by aphidicolin and sister chromatid exchange sites detected by BrdU. Many of these sites correspond to conserved fragile sites in man, gorilla and chimpazee, to sites of conserved sister chromatid exchange in the mammalian X chromosome, to mutagenic sensitive sites, mapped locations of proto-oncogenes, breakpoints implicated in primate evolution and to breakpoints indicated as the sole anomaly in neoplasia. This observation prompted us to investigate if the interstitial telomeric sites cluster with these sites. An extensive literature search was carried out to find all the available published sites mentioned above. For comparison, we also carried out a statistical analysis of the clustering of the sites of the telomeric repeats with the gene locations where only nucleotide mutations have been observed as the only chromosomal abnormality. Our results indicate that the telomeric repeats cluster most with fragile sites, mutagenic sensitive sites and breakpoints implicated in primate evolution and least with cancer breakpoints, mapped locations of proto-oncogenes and other genes with nucleotide mutations.

  12. Transport and signaling via the amino acid binding site of the yeast Gap1 amino acid transceptor.

    PubMed

    Van Zeebroeck, Griet; Bonini, Beatriz Monge; Versele, Matthias; Thevelein, Johan M

    2009-01-01

    Transporter-related nutrient sensors, called transceptors, mediate nutrient activation of signaling pathways through the plasma membrane. The mechanism of action of transporting and nontransporting transceptors is unknown. We have screened 319 amino acid analogs to identify compounds that act on Gap1, a transporting amino acid transceptor in yeast that triggers activation of the protein kinase A pathway. We identified competitive and noncompetitive inhibitors of transport, either with or without agonist action for signaling, including nontransported agonists. Using substituted cysteine accessibility method (SCAM) analysis, we identified Ser388 and Val389 as being exposed into the amino acid binding site, and we show that agonist action for signaling uses the same binding site as used for transport. Our results provide the first insight, to our knowledge, into the mechanism of action of transceptors. They indicate that signaling requires a ligand-induced specific conformational change that may be part of but does not require the complete transport cycle. PMID:19060912

  13. Na+ Inhibits the Epithelial Na+ Channel by Binding to a Site in an Extracellular Acidic Cleft*

    PubMed Central

    Kashlan, Ossama B.; Blobner, Brandon M.; Zuzek, Zachary; Tolino, Michael; Kleyman, Thomas R.

    2015-01-01

    The epithelial Na+ channel (ENaC) has a key role in the regulation of extracellular fluid volume and blood pressure. ENaC belongs to a family of ion channels that sense the external environment. These channels have large extracellular regions that are thought to interact with environmental cues, such as Na+, Cl−, protons, proteases, and shear stress, which modulate gating behavior. We sought to determine the molecular mechanism by which ENaC senses high external Na+ concentrations, resulting in an inhibition of channel activity. Both our structural model of an ENaC α subunit and the resolved structure of an acid-sensing ion channel (ASIC1) have conserved acidic pockets in the periphery of the extracellular region of the channel. We hypothesized that these acidic pockets host inhibitory allosteric Na+ binding sites. Through site-directed mutagenesis targeting the acidic pocket, we modified the inhibitory response to external Na+. Mutations at selected sites altered the cation inhibitory preference to favor Li+ or K+ rather than Na+. Channel activity was reduced in response to restraining movement within this region by cross-linking structures across the acidic pocket. Our results suggest that residues within the acidic pocket form an allosteric effector binding site for Na+. Our study supports the hypothesis that an acidic cleft is a key ligand binding locus for ENaC and perhaps other members of the ENaC/degenerin family. PMID:25389295

  14. ABC transporter AtABCG25 is involved in abscisic acid transport and responses

    PubMed Central

    Kuromori, Takashi; Miyaji, Takaaki; Yabuuchi, Hikaru; Shimizu, Hidetada; Sugimoto, Eriko; Kamiya, Asako; Moriyama, Yoshinori; Shinozaki, Kazuo

    2010-01-01

    Abscisic acid (ABA) is one of the most important phytohormones involved in abiotic stress responses, seed maturation, germination, and senescence. ABA is predominantly produced in vascular tissues and exerts hormonal responses in various cells, including guard cells. Although ABA responses require extrusion of ABA from ABA-producing cells in an intercellular ABA signaling pathway, the transport mechanisms of ABA through the plasma membrane remain unknown. Here we isolated an ATP-binding cassette (ABC) transporter gene, AtABCG25, from Arabidopsis by genetically screening for ABA sensitivity. AtABCG25 was expressed mainly in vascular tissues. The fluorescent protein-fused AtABCG25 was localized at the plasma membrane in plant cells. In membrane vesicles derived from AtABCG25-expressing insect cells, AtABCG25 exhibited ATP-dependent ABA transport. The AtABCG25-overexpressing plants showed higher leaf temperatures, implying an influence on stomatal regulation. These results strongly suggest that AtABCG25 is an exporter of ABA and is involved in the intercellular ABA signaling pathway. The presence of the ABA transport mechanism sheds light on the active control of multicellular ABA responses to environmental stresses among plant cells. PMID:20133881

  15. The vasorelaxant effect of gallic acid involves endothelium-dependent and -independent mechanisms.

    PubMed

    de Oliveira, Lais Moraes; de Oliveira, Thiago Sardinha; da Costa, Rafael Menezes; de Souza Gil, Eric; Costa, Elson Alves; Passaglia, Rita de Cassia Aleixo Tostes; Filgueira, Fernando Paranaíba; Ghedini, Paulo César

    2016-06-01

    The mechanisms of action involved in the vasorelaxant effect of gallic acid (GA) were examined in the isolated rat thoracic aorta. GA exerted a relaxant effect in the highest concentrations (0.4-10mM) in both endothelium-intact and endothelium-denuded aortic rings. Pre-incubation with L-NAME, ODQ, calmidazolium, TEA, 4-aminopyridine, and barium chloride significantly reduced the pEC50 values. Moreover, this effect was not modified by indomethacin, wortmannin, PP2, glibenclamide, or paxillin. Pre-incubation of GA (1, 3, and 10mM) in a Ca(2+)-free Krebs solution attenuated CaCl2-induced contractions and blocked BAY K8644-induced vascular contractions, but it did not inhibit a contraction induced by the release of Ca(2+) from the sarcoplasmatic reticulum stores. In addition, a Western blot analysis showed that GA induces phosphorylation of eNOS in rat thoracic aorta. These results suggest that GA induces relaxation in rat aortic rings through an endothelium-dependent pathway, resulting in eNOS phosphorylation and opening potassium channels. Additionally, the relaxant effect by an endothelium-independent pathway involves the blockade of the Ca(2+) influx via L-type Ca(2+) channels. PMID:26643780

  16. Turnover of binding sites for transcription factors involved in early Drosophila development.

    PubMed

    Costas, Javier; Casares, Fernando; Vieira, Jorge

    2003-05-22

    Despite the importance of cis-regulatory regions in evolution, little is know about their evolutionary dynamics. In this report, we analyze the process of evolution of binding sites for transcription factors using as a model a well characterized system, the Drosophila early developmental enhancers. We compare the sequences of eight enhancer regions for early developmental genes between Drosophila melanogaster and other two species, Drosophila virilis and Drosophila pseudoobscura, searching for the presence/absence of 104 biochemically verified binding sites from D. melanogaster. We also modeled the binding specificity of each binding site by the use of well-defined positional weight matrices (PWMs). The comparisons showed that turnover of binding sites seems to fit a molecular clock, at an approximate rate of 0.94% of gain/loss of binding sites per million years. This intense turnover affects both high and low affinity binding sites at the same extent. Furthermore, the subset of overlapping binding sites is also subjected to this high turnover. Conserved binding sites seem to be constrained to maintain not only location but also the exact sequence at each particular position. Finally, we detected a significant decrease in mean PWM scores for the D. virilis binding sites in the case of Hunchback. Possible explanations for this fact are discussed. PMID:12801649

  17. Site specific incorporation of heavy atom-containing unnatural amino acids into proteins for structure determination

    DOEpatents

    Xie, Jianming; Wang, Lei; Wu, Ning; Schultz, Peter G.

    2008-07-15

    Translation systems and other compositions including orthogonal aminoacyl tRNA-synthetases that preferentially charge an orthogonal tRNA with an iodinated or brominated amino acid are provided. Nucleic acids encoding such synthetases are also described, as are methods and kits for producing proteins including heavy atom-containing amino acids, e.g., brominated or iodinated amino acids. Methods of determining the structure of a protein, e.g., a protein into which a heavy atom has been site-specifically incorporated through use of an orthogonal tRNA/aminoacyl tRNA-synthetase pair, are also described.

  18. Identification of genes and pathways involved in the synthesis of Mead acid (20:3n-9), an indicator of essential fatty acid deficiency.

    PubMed

    Ichi, Ikuyo; Kono, Nozomu; Arita, Yuka; Haga, Shizuka; Arisawa, Kotoko; Yamano, Misato; Nagase, Mana; Fujiwara, Yoko; Arai, Hiroyuki

    2014-01-01

    In mammals, 5,8,11-eicosatrienoic acid (Mead acid, 20:3n-9) is synthesized from oleic acid during a state of essential fatty acid deficiency (EFAD). Mead acid is thought to be produced by the same enzymes that synthesize arachidonic acid and eicosapentaenoic acid, but the genes and the pathways involved in the conversion of oleic acid to Mead acid have not been fully elucidated. The levels of polyunsaturated fatty acids in cultured cells are generally very low compared to those in mammalian tissues. In this study, we found that cultured cells, such as NIH3T3 and Hepa1-6 cells, have significant levels of Mead acid, indicating that cells in culture are in an EFAD state under normal culture conditions. We then examined the effect of siRNA-mediated knockdown of fatty acid desaturases and elongases on the level of Mead acid, and found that knockdown of Elovl5, Fads1, or Fads2 decreased the level of Mead acid. This and the measured levels of possible intermediate products for the synthesis of Mead acid such as 18:2n-9, 20:1n-9 and 20:2n-9 in the knocked down cells indicate two pathways for the synthesis of Mead acid: pathway 1) 18:1n-9→(Fads2)→18:2n-9→(Elovl5)→20:2n-9→(Fads1)→20:3n-9 and pathway 2) 18:1n-9→(Elovl5)→20:1n-9→(Fads2)→20:2n-9→(Fads1)→20:3n-9. PMID:24184513

  19. Involvement of two classes of binding sites in the interactions of cyclophilin B with peripheral blood T-lymphocytes.

    PubMed Central

    Denys, A; Allain, F; Carpentier, M; Spik, G

    1998-01-01

    Cyclophilin B (CyPB) is a cyclosporin A (CsA)-binding protein, mainly associated with the secretory pathway, and is released in biological fluids. We recently reported that CyPB specifically binds to T-lymphocytes and promotes enhanced incorporation of CsA. The interactions with cellular binding sites involved, at least in part, the specific N-terminal extension of the protein. In this study, we intended to specify further the nature of the CyPB-binding sites on peripheral blood T-lymphocytes. We first provide evidence that the CyPB binding to heparin-Sepharose is prevented by soluble sulphated glycosaminoglycans (GAG), raising the interesting possibility that such interactions may occur on the T-cell surface. We then characterized CyPB binding to T-cell surface GAG and found that these interactions involved the N-terminal extension of CyPB, but not its conserved CsA-binding domain. In addition, we determined the presence of a second CyPB binding site, which we termed a type I site, in contrast with type II for GAG interactions. The two binding sites exhibit a similar affinity but the expression of the type I site was 3-fold lower. The conclusion that CyPB binding to the type I site is distinct from the interactions with GAG was based on the findings that it was (1) resistant to NaCl wash and GAG-degrading enzyme treatments, (2) reduced in the presence of CsA or cyclophilin C, and (3) unmodified in the presence of either the N-terminal peptide of CyPB or protamine. Finally, we showed that the type I binding sites were involved in an endocytosis process, supporting the hypothesis that they may correspond to a functional receptor for CyPB. PMID:9841882

  20. Evolution of HLA class II molecules: Allelic and amino acid site variability across populations.

    PubMed Central

    Salamon, H; Klitz, W; Easteal, S; Gao, X; Erlich, H A; Fernandez-Viña, M; Trachtenberg, E A; McWeeney, S K; Nelson, M P; Thomson, G

    1999-01-01

    Analysis of the highly polymorphic beta1 domains of the HLA class II molecules encoded by the DRB1, DQB1, and DPB1 loci reveals contrasting levels of diversity at the allele and amino acid site levels. Statistics of allele frequency distributions, based on Watterson's homozygosity statistic F, reveal distinct evolutionary patterns for these loci in ethnically diverse samples (26 populations for DQB1 and DRB1 and 14 for DPB1). When examined over all populations, the DQB1 locus allelic variation exhibits striking balanced polymorphism (P < 10(-4)), DRB1 shows some evidence of balancing selection (P < 0.06), and while there is overall very little evidence for selection of DPB1 allele frequencies, there is a trend in the direction of balancing selection (P < 0.08). In contrast, at the amino acid level all three loci show strong evidence of balancing selection at some sites. Averaged over polymorphic amino acid sites, DQB1 and DPB1 show similar deviation from neutrality expectations, and both exhibit more balanced polymorphic amino acid sites than DRB1. Across ethnic groups, polymorphisms at many codons show evidence for balancing selection, yet data consistent with directional selection were observed at other codons. Both antigen-binding pocket- and non-pocket-forming amino acid sites show overall deviation from neutrality for all three loci. Only in the case of DRB1 was there a significant difference between pocket- and non-pocket-forming amino acid sites. Our findings indicate that balancing selection at the MHC occurs at the level of polymorphic amino acid residues, and that in many cases this selection is consistent across populations. PMID:10224269

  1. Student Involvement with the Regionally Important Geomorphological Site (RIGS) Scheme: An Opportunity to Learn Geomorphology and Gain Transferable Skills.

    ERIC Educational Resources Information Center

    McEwen, Lindsey

    1996-01-01

    Outlines student involvement with a conservation project that aims to develop a Regionally Important Geological/Geomorphological Site network (RIGS) at a county level in the United Kingdom. Emphasis is placed on identifying, describing, evaluating, and documenting land forms of educational, research, historical, and/or aesthetic value. (MJP)

  2. Correlated fragile site expression allows the identification of candidate fragile genes involved in immunity and associated with carcinogenesis

    PubMed Central

    Re, Angela; Cora, Davide; Puliti, Alda Maria; Caselle, Michele; Sbrana, Isabella

    2006-01-01

    Background Common fragile sites (cfs) are specific regions in the human genome that are particularly prone to genomic instability under conditions of replicative stress. Several investigations support the view that common fragile sites play a role in carcinogenesis. We discuss a genome-wide approach based on graph theory and Gene Ontology vocabulary for the functional characterization of common fragile sites and for the identification of genes that contribute to tumour cell biology. Results Common fragile sites were assembled in a network based on a simple measure of correlation among common fragile site patterns of expression. By applying robust measurements to capture in quantitative terms the non triviality of the network, we identified several topological features clearly indicating departure from the Erdos-Renyi random graph model. The most important outcome was the presence of an unexpected large connected component far below the percolation threshold. Most of the best characterized common fragile sites belonged to this connected component. By filtering this connected component with Gene Ontology, statistically significant shared functional features were detected. Common fragile sites were found to be enriched for genes associated to the immune response and to mechanisms involved in tumour progression such as extracellular space remodeling and angiogenesis. Moreover we showed how the internal organization of the graph in communities and even in very simple subgraphs can be a starting point for the identification of new factors of instability at common fragile sites. Conclusion We developed a computational method addressing the fundamental issue of studying the functional content of common fragile sites. Our analysis integrated two different approaches. First, data on common fragile site expression were analyzed in a complex networks framework. Second, outcomes of the network statistical description served as sources for the functional annotation of genes at

  3. Molecular evidence for an involvement of organic anion transporters (OATs) in aristolochic acid nephropathy.

    PubMed

    Bakhiya, Nadiya; Arlt, Volker M; Bahn, Andrew; Burckhardt, Gerhard; Phillips, David H; Glatt, Hansruedi

    2009-10-01

    Aristolochic acid (AA), present in Aristolochia species, is the major causative agent in the development of severe renal failure and urothelial cancers in patients with AA nephropathy. It may also be a cause of Balkan endemic nephropathy. Epithelial cells of the proximal tubule are the primary cellular target of AA. To study whether organic anion transporters (OATs) expressed in proximal tubule cells are involved in uptake of AA, we used human epithelial kidney (HEK293) cells stably expressing human (h) OAT1, OAT3 or OAT4. AA potently inhibited the uptake of characteristic substrates, p-aminohippurate for hOAT1 and estrone sulfate for hOAT3 and hOAT4. Aristolochic acid I (AAI), the more cytotoxic and genotoxic AA congener, exhibited high affinity for hOAT1 (K(i)=0.6 microM) as well as hOAT3 (K(i)=0.5 microM), and lower affinity for hOAT4 (K(i)=20.6 microM). Subsequently, AAI-DNA adduct formation (investigated by (32)P-postlabelling) was used as a measure of AAI uptake. Significantly higher levels of adducts occurred in hOAT-expressing cells than in control cells: this effect was abolished in the presence of the OAT inhibitor probenecid. In Xenopus laevis oocytes hOAT-mediated efflux of p-aminohippurate was trans-stimulated by extracellular AA, providing further molecular evidence for AA translocation by hOATs. Our study indicates that OATs can mediate the uptake of AA into proximal tubule cells and thereby participate in kidney cell damage by this toxin. PMID:19643159

  4. Identification of a Cyanobacterial RND-Type Efflux System Involved in Export of Free Fatty Acids.

    PubMed

    Kato, Akihiro; Takatani, Nobuyuki; Use, Kazuhide; Uesaka, Kazuma; Ikeda, Kazutaka; Chang, Yajun; Kojima, Kouji; Aichi, Makiko; Ihara, Kunio; Nakahigashi, Kenji; Maeda, Shin-Ichi; Omata, Tatsuo

    2015-12-01

    An RND (resistance-nodulation-division)-type transporter having the capacity to export free fatty acids (FFAs) was identified in the cyanobacterium Synechococcus elongatus strain PCC 7942 during characterization of a mutant strain engineered to produce FFAs. The basic strategy for construction of the FFA-producing mutant was a commonly used one, involving inactivation of the endogenous acyl-acyl carrier protein synthetase gene (aas) and introduction of a foreign thioesterase gene ('tesA), but a nitrate transport mutant NA3 was used as the parental strain to achieve slow, nitrate-limited growth in batch cultures. Also, a nitrogen-regulated promoter PnirA was used to drive 'tesA to maximize thioesterase expression during the nitrate-limited growth. The resulting mutant (dAS2T) was, however, incapable of growth under the conditions of nitrate limitation, presumably due to toxicity associated with FFA overproduction. Incubation of the mutant culture under the non-permissive conditions allowed for isolation of a pseudorevertant (dAS2T-pr1) capable of growth on nitrate. Genome sequence and gene expression analyses of this strain suggested that expression of an RND-type efflux system had rescued growth on nitrate. Targeted inactivation of the RND-type transporter genes in the wild-type strain resulted in loss of tolerance to exogenously added FFAs including capric, lauric, myristic, oleic and linolenic acids. Overexpression of the genes in dAS2T, on the other hand, enhanced FFA excretion and cell growth in nitrate-containing medium, verifying that the genes encode an efflux pump for FFAs. These results demonstrate the importance of the efflux system in efficient FFA production using genetically engineered cyanobacteria. PMID:26468506

  5. Atmospheric geochemistry of formic and acetic acids at a mid-latitude temperate site

    NASA Technical Reports Server (NTRS)

    Talbot, R. W.; Beecher, K. M.; Harriss, R. C.; Cofer, R. W., III

    1988-01-01

    Tropospheric concentrations of formic and acetic acids in the gas, the aerosol, and the rainwater phases were determined in samples collected 1-2 m above ground level at an open field site in eastern Virginia. These acids were found to occur principally (98 percent or above) in the gas phase, with a marked annual seasonality, averaging 1890 ppt for formate and 1310 ppt for acetate during the growing season, as compared to 695 ppt and 700 ppt, respectively, over the nongrowing season. The data support the hypothesis that biogenic emissions from vegatation are important sources of atmospheric formic and acetic acid during the local growing season. The same time trends were observed for precipitation, although with less defined seasonality. The relative increase of the acetic acid/formic acid ratio during the nongrowing season points to the dominance of anthropogenic inputs of acetic acid from motor vehicles and biomass combustion in the wintertime.

  6. A model of compensatory molecular evolution involving multiple sites in RNA molecules.

    PubMed

    Kusumi, Junko; Ichinose, Motoshi; Takefu, Masasuke; Piskol, Robert; Stephan, Wolfgang; Iizuka, Masaru

    2016-01-01

    Consider two sites under compensatory fitness interaction, such as a Watson-Crick base pair in an RNA helix or two interacting residues in a protein. A mutation at any one of these two sites may reduce the fitness of an individual. However, fitness may be restored by the occurrence of a second mutation at the other site. Kimura modeled this process using a two-locus haploid fitness scheme with two alleles at each locus. He predicted that compensatory evolution following this model is very rare unless selection against the deleterious single mutations is weak and linkage between the interacting sites is tight. Here we investigate the question whether the rate of compensatory evolution increases if we take the context of the two directly interacting sites into account. By "context", we mean the effect of neighboring sites in an RNA helix. Interaction between the focal pair of sites under consideration and the context may lead to so-called indirect compensation. Thus, extending Kimura's classical model of compensatory evolution, we study the effects of both direct and indirect compensation on the rate of compensatory evolution. It is shown that the effects of indirect compensation are very strong. We find that recombination does not slow down the rate of compensatory evolution as predicted by the classical model. Instead, compensatory substitutions may be relatively frequent, even if linkage between the focal interacting sites is loose, selection against deleterious mutations is strong, and mutation rate is low. We compare our theoretical results with data on RNA secondary structures from vertebrate introns. PMID:26506471

  7. Quantification of MSCs involved in wound healing: use of SIS to transfer MSCs to wound site and quantification of MSCs involved in skin wound healing.

    PubMed

    Yeum, Chung Eun; Park, Eun Young; Lee, Seong-Beom; Chun, Heung-Jae; Chae, Gue-Tae

    2013-04-01

    Mesenchymal stem cells (MSCs) are known to be effective in wound healing, but not much has been reported on quantitative correlations between MSCs injected into the wound site and MSCs that actually participate in wound healing. This study traced MSCs participating in wound healing by using small intestinal submucosa (SIS) as a cell carrier, identified their moving path and calculated the number of MSCs involved in wound healing. First, MSCs were isolated from the nude mouse and 1 × 10(6) cells were seeded onto the centre of the SIS. MSC-seeded SIS complexes were injected onto full-thickness skin wounds made on the dorsum of nude mice. Tracing of MSC-seeded SIS complex transplanted to the wound site revealed that 27.6% of the MSCs were migrated to the wound site at the first attempt. Second, repeated injection of additional MSCs did not increase the number of MSCs participating in wound healing beyond a certain constant maximum amount. The number of MSCs present in the wound site remains constant in the range 2-3 × 10(5) from day 1 to day 10. The expression of skin regeneration-related growth factors was confirmed by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). MSCs participating in wound healing were found not only to suppress inflammation of the wound but also to increase the skin regeneration-related growth factors that enable the recovery of the skin. An optimal number of about 3 × 10(5) MSCs injected into the site was found to adapt themselves to the skin wound-healing process effectively. PMID:22278819

  8. Molecular modeling and simulation of FabG, an enzyme involved in the fatty acid pathway of Streptococcus pyogenes.

    PubMed

    Shafreen, Rajamohmed Beema; Pandian, Shunmugiah Karutha

    2013-09-01

    Streptococcus pyogenes (SP) is the major cause of pharyngitis accompanied by strep throat infections in humans. 3-keto acyl reductase (FabG), an important enzyme involved in the elongation cycle of the fatty acid pathway of S. pyogenes, is essential for synthesis of the cell-membrane, virulence factors and quorum sensing-related mechanisms. Targeting SPFabG may provide an important aid for the development of drugs against S. pyogenes. However, the absence of a crystal structure for FabG of S. pyogenes limits the development of structure-based drug designs. Hence, in the present study, a homology model of FabG was generated using the X-ray crystallographic structure of Aquifex aeolicus (PDB ID: 2PNF). The modeled structure was refined using energy minimization. Furthermore, active sites were predicted, and a large dataset of compounds was screened against SPFabG. The ligands were docked using the LigandFit module that is available from Discovery Studio version 2.5. From this list, 13 best hit ligands were chosen based on the docking score and binding energy. All of the 13 ligands were screened for Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) properties. From this, the two best descriptors, along with one descriptor that lay outside the ADMET plot, were selected for molecular dynamic (MD) simulation. In vitro testing of the ligands using biological assays further substantiated the efficacy of the ligands that were screened based on the in silico methods. PMID:23988477

  9. Secretion of a lysophospholipase D activity by adipocytes: involvement in lysophosphatidic acid synthesis

    PubMed Central

    Gesta, Stéphane; Simon, Marie-Françoise; Rey, Astrid; Sibrac, David; Girard, Alexia; Lafontan, Max; Valet, Philippe; Saulnier-Blache, Jean Sébastien

    2002-01-01

    The aim of the present work was to depict the metabolic pathways involved in extra-cellular production of lysophosphatidic acid (LPA) by adipocytes. LPA was followed by quantifying the accumulation of LPA in the incubation medium (conditioned medium: CM) of 3T3F442A adipocytes, or human adipose tissue explants, using a radioenzymatic assay. Surprisingly, after separation from the cells, the amount of LPA present in CM could significantly be increased by further incubation at 37°C. This suggested the presence of a LPA-synthesizing activity (LPA-SA) in CM. LPA-SA appeared as a soluble activity which was inhibited by divalent ion chelators: EDTA and phenanthrolin. The effect of EDTA was preferentially reverted by CoCl2, as described for a lysophospholipase D- (lyso-PLD) activity previously identified in rat plasma. LPA concentration could also be increased by treatment with a bacterial PLD, demonstrating the presence of PLD-sensitive LPA-precursors (mainly lysophosphatidylcholine) in adipocyte CM. LPA-SA could be increased by addition of exogenous lysophosphatidylcholine, lysophosphatidylglycerol, or lyso-platelet activating factor, demonstrating that LPA-SA resulted from the action of a lyso-PLD. LPA-SA was not inhibited, but rather activated, by primary alcohol (ethanol and 1-butanol), suggesting that adipocyte lyso-PLD was not a classical PLD. Finally, LPA-SA was found to be weaker in CM of undifferentiated adipocyte (preadipocytes) as compared to CM of differentiated adipocytes. In conclusion, our results reveal the existence of a secreted lyso-PLD activity regulated during adipocyte-differentiation and involved in extra-cellular production of synthesis of LPA by adipocytes. PMID:12032165

  10. The different interactions of Colletotrichum gloeosporioides with two strawberry varieties and the involvement of salicylic acid

    PubMed Central

    Zhang, Qing-Yu; Zhang, Li-Qing; Song, Li-Li; Duan, Ke; Li, Na; Wang, Yan-Xiu; Gao, Qing-Hua

    2016-01-01

    The disease symptoms recognized as ‘Anthracnose’ are caused by Colletotrichum spp. and lead to large-scale strawberry (Fragaria×ananassa Duchesne) losses worldwide in terms of both quality and production. Little is known regarding the mechanisms underlying the genetic variations in the strawberry–Colletotrichum spp. interaction. In this work, Colletotrichum gloeosporioides (C. gloeosporioides) infection was characterized in two varieties exhibiting different susceptibilities, and the involvement of salicylic acid (SA) was examined. Light microscopic observation showed that C. gloeosporioides conidia germinated earlier and faster on the leaf surface of the susceptible cultivar compared with the less-susceptible cultivar. Several PR genes were differentially expressed, with higher-amplitude changes observed in the less-susceptible cultivar. The less-susceptible cultivar contained a higher level of basal SA, and the SA levels increased rapidly upon infection, followed by a sharp decrease before the necrotrophic phase. External SA pretreatment reduced susceptibility and elevated the internal SA levels in both varieties, which were sharply reduced in the susceptible cultivar upon inoculation. The less-susceptible cultivar also displayed a more sensitive and marked increase in the transcripts of NB-LRR genes to C. gloeosporioides, and SA pretreatment differentially induced transcript accumulation in the two varieties during infection. Furthermore, SA directly inhibited the germination of C. gloeosporioides conidia; NB-LRR transcript accumulation in response to SA pretreatment was both dose- and cultivar-dependent. The results demonstrate that the less-susceptible cultivar showed reduced conidia germination. The contribution of SA might involve microbial isolate-specific sensitivity to SA, cultivar/tissue-specific SA homeostasis and signaling, and the sensitivity of R genes and the related defense network to SA and pathogens. PMID:27004126

  11. The different interactions of Colletotrichum gloeosporioides with two strawberry varieties and the involvement of salicylic acid.

    PubMed

    Zhang, Qing-Yu; Zhang, Li-Qing; Song, Li-Li; Duan, Ke; Li, Na; Wang, Yan-Xiu; Gao, Qing-Hua

    2016-01-01

    The disease symptoms recognized as 'Anthracnose' are caused by Colletotrichum spp. and lead to large-scale strawberry (Fragaria×ananassa Duchesne) losses worldwide in terms of both quality and production. Little is known regarding the mechanisms underlying the genetic variations in the strawberry-Colletotrichum spp. interaction. In this work, Colletotrichum gloeosporioides (C. gloeosporioides) infection was characterized in two varieties exhibiting different susceptibilities, and the involvement of salicylic acid (SA) was examined. Light microscopic observation showed that C. gloeosporioides conidia germinated earlier and faster on the leaf surface of the susceptible cultivar compared with the less-susceptible cultivar. Several PR genes were differentially expressed, with higher-amplitude changes observed in the less-susceptible cultivar. The less-susceptible cultivar contained a higher level of basal SA, and the SA levels increased rapidly upon infection, followed by a sharp decrease before the necrotrophic phase. External SA pretreatment reduced susceptibility and elevated the internal SA levels in both varieties, which were sharply reduced in the susceptible cultivar upon inoculation. The less-susceptible cultivar also displayed a more sensitive and marked increase in the transcripts of NB-LRR genes to C. gloeosporioides, and SA pretreatment differentially induced transcript accumulation in the two varieties during infection. Furthermore, SA directly inhibited the germination of C. gloeosporioides conidia; NB-LRR transcript accumulation in response to SA pretreatment was both dose- and cultivar-dependent. The results demonstrate that the less-susceptible cultivar showed reduced conidia germination. The contribution of SA might involve microbial isolate-specific sensitivity to SA, cultivar/tissue-specific SA homeostasis and signaling, and the sensitivity of R genes and the related defense network to SA and pathogens. PMID:27004126

  12. Lysyl oxidase-like 4 involvement in retinoic acid epithelial wound healing.

    PubMed

    Comptour, Aurélie; Rouzaire, Marion; Belville, Corinne; Bonnin, Nicolas; Daniel, Estelle; Chiambaretta, Frédéric; Blanchon, Loïc; Sapin, Vincent

    2016-01-01

    Vitamin A and its active forms (retinoic acids/RAs) are known to have pro-healing properties, but their mechanisms of action are still poorly understood. This work aimed to identify the cellular and molecular processes by which atRA (all-trans RA) improves wound healing, using an in vivo model of mouse corneal alkali burns and an in vitro cellular human corneal epithelial injury model. Regulation by atRA has been studied on most of the cellular events that occur in wound healing. We investigated the direct influence of atRA on a specific target gene known to be involved in the extracellular matrix (ECM) dynamics, one of the pathways contributing to epithelial repair. Our results demonstrate that atRA promotes corneal epithelial wound healing by acting preferentially on migration. The induction of lysyl oxidase-like 4 (LOXL4) expression by atRA in the corneal epithelium environment was established as essential in the mechanism of atRA-dependent wound healing. Our study describes for the first time a direct link between a retinoic-induced gene and protein, LOXL4, and its general clinical pro-healing properties in ECM dynamics. PMID:27597564

  13. Effect of citric acid, tetracycline, and doxycycline on instrumented periodontally involved root surfaces: A SEM study

    PubMed Central

    Chahal, Gurparkash Singh; Chhina, Kamalpreet; Chhabra, Vipin; Bhatnagar, Rakhi; Chahal, Amna

    2014-01-01

    Background: A surface smear layer consisting of organic and inorganic material is formed on the root surface following mechanical instrumentation and may inhibit the formation of new connective tissue attachment to the root surface. Modification of the tooth surface by root conditioning has resulted in improved connective tissue attachment and has advanced the goal of reconstructive periodontal treatment. Aim: The aim of this study was to compare the effects of citric acid, tetracycline, and doxycycline on the instrumented periodontally involved root surfaces in vitro using a scanning electron microscope. Settings and Design: A total of 45 dentin samples obtained from 15 extracted, scaled, and root planed teeth were divided into three groups. Materials and Methods: The root conditioning agents were applied with cotton pellets using the Passive burnishing technique for 5 minutes. The samples were then examined by the scanning electron microscope. Statistical Analysis Used: The statistical analysis was carried out using Statistical Package for Social Sciences (SPSS Inc., Chicago, IL, version 15.0 for Windows). For all quantitative variables means and standard deviations were calculated and compared. For more than two groups ANOVA was applied. For multiple comparisons post hoc tests with Bonferroni correction was used. Results: Upon statistical analysis the root conditioning agents used in this study were found to be effective in removing the smear layer, uncovering and widening the dentin tubules and unmasking the dentin collagen matrix. Conclusion: Tetracycline HCl was found to be the best root conditioner among the three agents used. PMID:24744541

  14. Lysyl oxidase-like 4 involvement in retinoic acid epithelial wound healing

    PubMed Central

    Comptour, Aurélie; Rouzaire, Marion; Belville, Corinne; Bonnin, Nicolas; Daniel, Estelle; Chiambaretta, Frédéric; Blanchon, Loïc; Sapin, Vincent

    2016-01-01

    Vitamin A and its active forms (retinoic acids/RAs) are known to have pro-healing properties, but their mechanisms of action are still poorly understood. This work aimed to identify the cellular and molecular processes by which atRA (all-trans RA) improves wound healing, using an in vivo model of mouse corneal alkali burns and an in vitro cellular human corneal epithelial injury model. Regulation by atRA has been studied on most of the cellular events that occur in wound healing. We investigated the direct influence of atRA on a specific target gene known to be involved in the extracellular matrix (ECM) dynamics, one of the pathways contributing to epithelial repair. Our results demonstrate that atRA promotes corneal epithelial wound healing by acting preferentially on migration. The induction of lysyl oxidase-like 4 (LOXL4) expression by atRA in the corneal epithelium environment was established as essential in the mechanism of atRA-dependent wound healing. Our study describes for the first time a direct link between a retinoic-induced gene and protein, LOXL4, and its general clinical pro-healing properties in ECM dynamics. PMID:27597564

  15. Are Phragmites australis enzymes involved in the degradation of the textile azo dye acid orange 7?

    PubMed

    Carias, Cátia C; Novais, Júlio M; Martins-Dias, Susete

    2008-01-01

    The role of antioxidant and detoxification enzymes of Phragmites australis, in the degradation of an azo dye, acid orange 7 (AO7), was studied. Activities of several enzymes involved in plant protection against stress were assayed through the activity characterization of superoxide dismutase (SOD), peroxidases (POD), catalase (CAT), ascorbate peroxidase (APOX), dehydroascorbate reductase (DHAR) and glutathione S-transferase (GST), obtained from P. australis crude extracts of leaves, stems and roots. A sub-surface vertical flow constructed wetland, planted with P. australis was used to test the plants response to the AO7 exposure at two different concentrations (130 and 700 mg l(-1)). An activity increase was detected for an AO7 concentration of 130 mg l(-1) for most enzymes studied (SOD, CAT and APOX), especially in leaves, suggesting a response of the reactive oxygen species scavenging enzymes to the chemical stress imposed. GST activity increase in this situation can also be interpreted as an activation of the detoxification pathway and subsequent AO7 conjugation. A totally different behaviour was observed for AO7 at 700 mg l(-1). An evident decrease in activity was observed for SOD, CAT, APOX and GST, probably due to enzymatic inhibition by AO7. Contrarily, DHAR activity augmented drastically in this situation. POD activity was not greatly affected during trial. Altogether these results suggest that P. australis effectively uses the ascorbate-glutathione pathway for the detoxification of AO7. PMID:17336060

  16. Involvement of endogenous salicylic acid in iron-deficiency responses in Arabidopsis.

    PubMed

    Shen, Chenjia; Yang, Yanjun; Liu, Kaidong; Zhang, Lei; Guo, Hong; Sun, Tao; Wang, Huizhong

    2016-07-01

    Several phytohormones have been demonstrated to be involved in iron (Fe) homeostasis. We took advantage of a salicylic acid (SA) biosynthesis defective mutant phytoalexin deficient 4 (pad4: T-DNA Salk_089936) to explore the possible effects of endogenous SA on the morphological and physiological responses to Fe deprivation. The morphological and physiological analysis was carried out between Col-0 and the pad4 mutant. Under an Fe-deficiency treatment, Col-0 showed more severe leaf chlorosis and root growth inhibition compared with the pad4 mutant. The soluble Fe concentrations were significantly higher in pad4 than in Col-0 under the Fe-deficiency treatment. Fe deficiency significantly induced SA accumulation in Col-0 and the loss-of-function of PAD4 blocked this process. The requirement of endogenous SA accumulation for Fe-deficiency responses was confirmed using a series of SA biosynthetic mutants and transgenic lines. Furthermore, a comparative RNA sequencing analysis of the whole seedling transcriptomes between Col-0 and the pad4 mutant was also performed. Based on the transcriptome data, the expression levels of many auxin- and ethylene-response genes were altered in pad4 compared with Col-0. Fe deficiency increases SA contents which elevates auxin and ethylene signalling, thereby activating Fe translocation via the bHLH38/39-mediated transcriptional regulation of downstream Fe genes. PMID:27208542

  17. Neuroinformatics analyses reveal GABAt and SSADH as major proteins involved in anticonvulsant activity of valproic acid.

    PubMed

    Piplani, Sakshi; Verma, Prabhakar Kumar; Kumar, Ajit

    2016-07-01

    The unequivocal hypotheses about anticonvulsant activity of valproic acid (VPA) have always been a basic hurdle in designing next generation neurotherapeutics, particularly the anti-epileptic drugs. The present study reports about a comprehensive in-silico investigation into qualitative and quantitative binding of VPA and corresponding natural ligands of four major enzymes involved in neurotransmissions, namely-GABA transaminase (GABAt), α-keto glutarate dehydrogenase (α-KGDH), Succinate Semialdehyde dehydrogenase (SSADH) and Glutamate Decarboxylase (GAD), respectively. The molecular docking analyses revealed that VPA inhibits GABAt and α-KGDH through allosteric while SSADH through competitive mode of binding. There is an observed elevation in binding of glutamate over GAD in the presence of VPA. The docking inhibition constant (Ki) of VPA to all the studied enzymatic receptors were observed to be well below the therapeutic concentration of VPA in blood, except for α-KGDH, thus favouring GABAergic over glutamatergic mode of anticonvulsant activity of VPA. The report is probably the first comprehensive in-silico molecular study about VPA action. PMID:27261619

  18. Dissolution of Simulated and Radioactive Savannah River Site High-Level Waste Sludges with Oxalic Acid & Citric Acid Solutions

    SciTech Connect

    STALLINGS, MARY

    2004-07-08

    This report presents findings from tests investigating the dissolution of simulated and radioactive Savannah River Site sludges with 4 per cent oxalic acid and mixtures of oxalic and citric acid previously recommended by a Russian team from the Khlopin Radium Institute and the Mining and Chemical Combine (MCC). Testing also included characterization of the simulated and radioactive waste sludges. Testing results showed the following: Dissolution of simulated HM and PUREX sludges with oxalic and citric acid mixtures at SRTC confirmed general trends reported previously by Russian testing. Unlike the previous Russian testing six sequential contacts of a mixture of oxalic acid citric acids at a 2:1 ratio (v/w) of acid to sludge did not produce complete dissolution of simulated HM and PUREX sludges. We observed that increased sludge dissolution occurred at a higher acid to sludge ratio, 50:1 (v/w), compared to the recommended ratio of 2:1 (v/w). We observed much lower dissolution of aluminum in a simulated HM sludge by sodium hydroxide leaching. We attribute the low aluminum dissolution in caustic to the high fraction of boehmite present in the simulated sludge. Dissolution of HLW sludges with 4 per cent oxalic acid and oxalic/citric acid followed general trends observed with simulated sludges. The limited testing suggests that a mixture of oxalic and citric acids is more efficient for dissolving HM and PUREX sludges and provides a more homogeneous dissolution of HM sludge than oxalic acid alone. Dissolution of HLW sludges in oxalic and oxalic/citric acid mixtures produced residual sludge solids that measured at higher neutron poison to equivalent 235U weight ratios than that in the untreated sludge solids. This finding suggests that residual solids do not present an increased nuclear criticality safety risk. Generally the neutron poison to equivalent 235U weight ratios of the acid solutions containing dissolved sludge components are lower than those in the untreated

  19. Evidence for a multigene family involved in bile acid 7-dehydroxylation in Eubacterium sp. strain VPI 12708.

    PubMed Central

    White, W B; Franklund, C V; Coleman, J P; Hylemon, P B

    1988-01-01

    Eubacterium sp. strain VPI 12708 is a human intestinal isolate which has an inducible bile acid 7-dehydroxylation activity. At least two cholic acid-induced polypeptides, with molecular masses of 27,000 and 45,000 daltons, respectively, coelute with bile acid 7-dehydroxylation activity. The 45,000-dalton polypeptide appears to be encoded by a cholic acid-induced mRNA species of greater than 6 kilobases, which suggests that the gene coding for this polypeptide is part of a larger operon. A gene has been cloned which flanks the gene encoding the 45,000-dalton polypeptide, in the upstream (5') direction. This gene appears to encode a second 27,000-dalton polypeptide. The gene bears striking homology at both the nucleotide (80%) and deduced amino acid sequence (89%) levels with the gene which encodes the 27,000-dalton polypeptide that has been shown previously to be involved in the bile acid 7-dehydroxylation reaction sequence. The implications of this homology and the possible function(s) of the two homologous genes in bile acid 7-dehydroxylation are discussed. Evidence is presented which suggests that the two homologous genes involved in bile acid 7-dehydroxylation may be part of a larger multigene family in Eubacterium sp. strain VPI 12708. Images PMID:3170477

  20. The Significance of Lewis Acid Sites for the Selective Catalytic Reduction of Nitric Oxide on Vanadium-Based Catalysts.

    PubMed

    Marberger, Adrian; Ferri, Davide; Elsener, Martin; Kröcher, Oliver

    2016-09-19

    The long debated reaction mechanisms of the selective catalytic reduction (SCR) of nitric oxide with ammonia (NH3 ) on vanadium-based catalysts rely on the involvement of Brønsted or Lewis acid sites. This issue has been clearly elucidated using a combination of transient perturbations of the catalyst environment with operando time-resolved spectroscopy to obtain unique molecular level insights. Nitric oxide reacts predominantly with NH3 coordinated to Lewis sites on vanadia on tungsta-titania (V2 O5 -WO3 -TiO2 ), while Brønsted sites are not involved in the catalytic cycle. The Lewis site is a mono-oxo vanadyl group that reduces only in the presence of both nitric oxide and NH3 . We were also able to verify the formation of the nitrosamide (NH2 NO) intermediate, which forms in tandem with vanadium reduction, and thus the entire mechanism of SCR. Our experimental approach, demonstrated in the specific case of SCR, promises to progress the understanding of chemical reactions of technological relevance. PMID:27553251

  1. Ethics Review for a Multi-Site Project Involving Tribal Nations in the Northern Plains.

    PubMed

    Angal, Jyoti; Petersen, Julie M; Tobacco, Deborah; Elliott, Amy J

    2016-04-01

    Increasingly, Tribal Nations are forming ethics review panels, which function separately from institutional review boards (IRBs). The emergence of strong community representation coincides with a widespread effort supported by the U.S. Department of Health & Human Services and other federal agencies to establish a single IRB for all multi-site research. This article underscores the value of a tribal ethics review board and describes the tribal oversight for the Safe Passage Study-a multi-site, community-based project in the Northern Plains. Our experience demonstrates the benefits of tribal ethics review and makes a strong argument for including tribal oversight in future regulatory guidance for multi-site, community-based research. PMID:26928897

  2. Gas phase acidity measurement of local acidic groups in multifunctional species: controlling the binding sites in hydroxycinnamic acids.

    PubMed

    Guerrero, Andres; Baer, Tomas; Chana, Antonio; González, Javier; Dávalos, Juan Z

    2013-07-01

    The applicability of the extended kinetic method (EKM) to determine the gas phase acidities (GA) of different deprotonable groups within the same molecule was tested by measuring the acidities of cinnamic, coumaric, and caffeic acids. These molecules differ not only in the number of acidic groups but in their nature, intramolecular distances, and calculated GAs. In order to determine independently the GA of groups within the same molecule using the EKM, it is necessary to selectively prepare pure forms of the hydrogen-bound heterodimer. In this work, the selectivity was achieved by the use of solvents of different vapor pressure (water and acetonitrile), as well as by variation of the drying temperature in the ESI source, which affected the production of heterodimers with different solvation energies and gas-phase dissociation energies. A particularly surprising finding is that the calculated solvation enthalpies of water and the aprotic acetonitrile are essentially identical, and that the different gas-phase products generated are apparently the result of their different vapor pressures, which affects the drying mechanism. This approach for the selective preparation of heterodimers, which is based on the energetics, appears to be quite general and should prove useful for other studies that require the selective production of heterodimers in ESI sources. The experimental results were supported by density functional theory (DFT) calculations of both gas-phase and solvated species. The experimental thermochemical parameters (deprotonation ΔG, ΔH, and ΔS) are in good agreement with the calculated values for the monofunctional cinnamic acid, as well as the multifunctional coumaric and caffeic acids. The measured GA for cinnamic acid is 334.5 ± 2.0 kcal/mol. The measured acidities for the COOH and OH groups of coumaric and caffeic acids are 332.7 ± 2.0, 318.7 ± 2.1, 332.2 ± 2.0, and 317.3 ± 2.2 kcal/mol, respectively. PMID:23799241

  3. Efficient solid acid catalyst containing Lewis and Brønsted Acid sites for the production of furfurals.

    PubMed

    Mazzotta, Michael G; Gupta, Dinesh; Saha, Basudeb; Patra, Astam K; Bhaumik, Asim; Abu-Omar, Mahdi M

    2014-08-01

    Self-assembled nanoparticulates of porous sulfonated carbonaceous TiO2 material that contain Brønsted and Lewis acidic sites were prepared by a one-pot synthesis method. The material was characterized by XRD, FTIR spectroscopy, NH3 temperature-programmed desorption, pyridine FTIR spectroscopy, field-emission scanning electron microscopy, high-resolution transmission electron microscopy, N2 -sorption, atomic absorbance spectroscopy, and inductively coupled plasma optical emission spectroscopy. The carbonaceous heterogeneous catalyst (Glu-TsOH-Ti) with a Brønsted-to-Lewis acid density ratio of 1.2 and more accessible acid sites was effective to produce 5-hydroxymethylfurfural and furfural from biomass-derived mono- and disaccharides and xylose in a biphasic solvent that comprised water and biorenewable methyltetrahydrofuran. The catalyst was recycled in four consecutive cycles with a total loss of only 3 % activity. Thus, Glu-TsOH-Ti, which contains isomerization and dehydration catalytic sites and is based on a cheap and biorenewable carbon support, is a sustainable catalyst for the production of furfurals, platform chemicals for biofuels and chemicals. PMID:24807741

  4. Identification of novel non-canonical RNA-binding sites in Gemin5 involved in internal initiation of translation

    PubMed Central

    Fernandez-Chamorro, Javier; Piñeiro, David; Gordon, James M. B.; Ramajo, Jorge; Francisco-Velilla, Rosario; Macias, Maria J.; Martinez-Salas, Encarnación

    2014-01-01

    Ribonucleic acid (RNA)-binding proteins are key players of gene expression control. We have shown that Gemin5 interacts with internal ribosome entry site (IRES) elements and modulates initiation of translation. However, little is known about the RNA-binding sites of this protein. Here we show that the C-terminal region of Gemin5 bears two non-canonical bipartite RNA-binding sites, encompassing amino acids 1297–1412 (RBS1) and 1383–1508 (RBS2). While RBS1 exhibits greater affinity for RNA than RBS2, it does not affect IRES-dependent translation in G5-depleted cells. In solution, the RBS1 three-dimensional structure behaves as an ensemble of flexible conformations rather than having a defined tertiary structure. However, expression of the polypeptide G51383–1508, bearing the low RNA-binding affinity RBS2, repressed IRES-dependent translation. A comparison of the RNA-binding capacity and translation control properties of constructs expressed in mammalian cells to that of the Gemin5 proteolysis products observed in infected cells reveals that non-repressive products accumulated during infection while the repressor polypeptide is not stable. Taken together, our results define the low affinity RNA-binding site as the minimal element of the protein being able to repress internal initiation of translation. PMID:24598255

  5. Modulation of a voltage-gated Na+ channel by sevoflurane involves multiple sites and distinct mechanisms

    PubMed Central

    Barber, Annika F.; Carnevale, Vincenzo; Klein, Michael L.; Eckenhoff, Roderic G.; Covarrubias, Manuel

    2014-01-01

    Halogenated inhaled general anesthetic agents modulate voltage-gated ion channels, but the underlying molecular mechanisms are not understood. Many general anesthetic agents regulate voltage-gated Na+ (NaV) channels, including the commonly used drug sevoflurane. Here, we investigated the putative binding sites and molecular mechanisms of sevoflurane action on the bacterial NaV channel NaChBac by using a combination of molecular dynamics simulation, electrophysiology, and kinetic analysis. Structural modeling revealed multiple sevoflurane interaction sites possibly associated with NaChBac modulation. Electrophysiologically, sevoflurane favors activation and inactivation at low concentrations (0.2 mM), and additionally accelerates current decay at high concentrations (2 mM). Explaining these observations, kinetic modeling suggests concurrent destabilization of closed states and low-affinity open channel block. We propose that the multiple effects of sevoflurane on NaChBac result from simultaneous interactions at multiple sites with distinct affinities. This multiple-site, multiple-mode hypothesis offers a framework to study the structural basis of general anesthetic action. PMID:24753583

  6. Effect of sun elevation upon remote sensing of ocean color over an acid waste dump site

    NASA Technical Reports Server (NTRS)

    Bressette, W. E.

    1978-01-01

    Photographic flights were made over an ocean acid waste dump site while dumping was in progress. The flights resulted in wide angle, broadband, spectral radiance film exposure data between the wavelengths of 500 to 900 nanometers for sun elevation angles ranging from 26 to 42 degrees. It is shown from densitometer data that the spectral signature of acid waste discharged into ocean water can be observed photographically, the influence of sun elevation upon remotely sensed apparent color can be normalized by using a single spectral band ratioing technique, and photographic quantification and mapping of acid waste through its suspended iron precipitate appears possible.

  7. Improvement of acetic acid tolerance of Saccharomyces cerevisiae using a zinc-finger-based artificial transcription factor and identification of novel genes involved in acetic acid tolerance.

    PubMed

    Ma, Cui; Wei, Xiaowen; Sun, Cuihuan; Zhang, Fei; Xu, Jianren; Zhao, Xinqing; Bai, Fengwu

    2015-03-01

    Acetic acid is present in cellulosic hydrolysate as a potent inhibitor, and the superior acetic acid tolerance of Saccharomyces cerevisiae ensures good cell viability and efficient ethanol production when cellulosic raw materials are used as substrates. In this study, a mutant strain of S. cerevisiae ATCC4126 (Sc4126-M01) with improved acetic acid tolerance was obtained through screening strains transformed with an artificial zinc finger protein transcription factor (ZFP-TF) library. Further analysis indicated that improved acetic acid tolerance was associated with improved catalase (CAT) activity. The ZFP coding sequence associated with the improved phenotype was identified, and real-time RT-PCR analysis revealed that three of the possible genes involved in the enhanced acetic acid tolerance regulated by this ZFP-TF, namely YFL040W, QDR3, and IKS1, showed decreased transcription levels in Sc4126-M01 in the presence of acetic acid, compared to those in the control strain. Sc4126-M01 mutants having QDR3 and IKS1 deletion (ΔQDR3 and ΔIKS1) exhibited higher acetic acid tolerance than the wild-type strain under acetic acid treatment. Glucose consumption rate and ethanol productivity in the presence of 5 g/L acetic acid were improved in the ΔQDR3 mutant compared to the wild-type strain. Our studies demonstrated that the synthetic ZFP-TF library can be used to improve acetic acid tolerance of S. cerevisiae and that the employment of an artificial transcription factor can facilitate the exploration of novel functional genes involved in stress tolerance of S. cerevisiae. PMID:25698512

  8. Nutritional and Hormonal Regulation of Citrate and Carnitine/Acylcarnitine Transporters: Two Mitochondrial Carriers Involved in Fatty Acid Metabolism.

    PubMed

    Giudetti, Anna M; Stanca, Eleonora; Siculella, Luisa; Gnoni, Gabriele V; Damiano, Fabrizio

    2016-01-01

    The transport of solutes across the inner mitochondrial membrane is catalyzed by a family of nuclear-encoded membrane-embedded proteins called mitochondrial carriers (MCs). The citrate carrier (CiC) and the carnitine/acylcarnitine transporter (CACT) are two members of the MCs family involved in fatty acid metabolism. By conveying acetyl-coenzyme A, in the form of citrate, from the mitochondria to the cytosol, CiC contributes to fatty acid and cholesterol synthesis; CACT allows fatty acid oxidation, transporting cytosolic fatty acids, in the form of acylcarnitines, into the mitochondrial matrix. Fatty acid synthesis and oxidation are inversely regulated so that when fatty acid synthesis is activated, the catabolism of fatty acids is turned-off. Malonyl-CoA, produced by acetyl-coenzyme A carboxylase, a key enzyme of cytosolic fatty acid synthesis, represents a regulator of both metabolic pathways. CiC and CACT activity and expression are regulated by different nutritional and hormonal conditions. Defects in the corresponding genes have been directly linked to various human diseases. This review will assess the current understanding of CiC and CACT regulation; underlining their roles in physio-pathological conditions. Emphasis will be placed on the molecular basis of the regulation of CiC and CACT associated with fatty acid metabolism. PMID:27231907

  9. Nutritional and Hormonal Regulation of Citrate and Carnitine/Acylcarnitine Transporters: Two Mitochondrial Carriers Involved in Fatty Acid Metabolism

    PubMed Central

    Giudetti, Anna M.; Stanca, Eleonora; Siculella, Luisa; Gnoni, Gabriele V.; Damiano, Fabrizio

    2016-01-01

    The transport of solutes across the inner mitochondrial membrane is catalyzed by a family of nuclear-encoded membrane-embedded proteins called mitochondrial carriers (MCs). The citrate carrier (CiC) and the carnitine/acylcarnitine transporter (CACT) are two members of the MCs family involved in fatty acid metabolism. By conveying acetyl-coenzyme A, in the form of citrate, from the mitochondria to the cytosol, CiC contributes to fatty acid and cholesterol synthesis; CACT allows fatty acid oxidation, transporting cytosolic fatty acids, in the form of acylcarnitines, into the mitochondrial matrix. Fatty acid synthesis and oxidation are inversely regulated so that when fatty acid synthesis is activated, the catabolism of fatty acids is turned-off. Malonyl-CoA, produced by acetyl-coenzyme A carboxylase, a key enzyme of cytosolic fatty acid synthesis, represents a regulator of both metabolic pathways. CiC and CACT activity and expression are regulated by different nutritional and hormonal conditions. Defects in the corresponding genes have been directly linked to various human diseases. This review will assess the current understanding of CiC and CACT regulation; underlining their roles in physio-pathological conditions. Emphasis will be placed on the molecular basis of the regulation of CiC and CACT associated with fatty acid metabolism. PMID:27231907

  10. Identification and Pharmacological Characterization of Multiple Allosteric Binding Sites on the Free Fatty Acid 1 Receptor

    PubMed Central

    Lin, Daniel C.-H.; Guo, Qi; Luo, Jian; Zhang, Jane; Nguyen, Kathy; Chen, Michael; Tran, Thanh; Dransfield, Paul J.; Brown, Sean P.; Houze, Jonathan; Vimolratana, Marc; Jiao, Xian Yun; Wang, Yingcai; Birdsall, Nigel J. M.

    2012-01-01

    Activation of FFA1 (GPR40), a member of G protein-coupling receptor family A, is mediated by medium- and long-chain fatty acids and leads to amplification of glucose-stimulated insulin secretion, suggesting a potential role for free fatty acid 1 (FFA1) as a target for type 2 diabetes. It was assumed previously that there is a single binding site for fatty acids and synthetic FFA1 agonists. However, using members of two chemical series of partial and full agonists that have been identified, radioligand binding interaction studies revealed that the full agonists do not bind to the same site as the partial agonists but exhibit positive heterotropic cooperativity. Analysis of functional data reveals positive functional cooperativity between the full agonists and partial agonists in various functional assays (in vitro and ex vivo) and also in vivo. Furthermore, the endogenous fatty acid docosahexaenoic acid (DHA) shows negative or neutral cooperativity with members of both series of agonists in binding assays but displays positive cooperativity in functional assays. Another synthetic agonist is allosteric with members of both agonist series, but apparently competitive with DHA. Therefore, there appear to be three allosterically linked binding sites on FFA1 with agonists specific for each of these sites. Activation of free fatty acid 1 receptor (FFAR1) by each of these agonists is differentially affected by mutations of two arginine residues, previously found to be important for FFAR1 binding and activation. These ligands with their high potencies and strong positive functional cooperativity with endogenous fatty acids, demonstrated in vitro and in vivo, have the potential to deliver therapeutic benefits. PMID:22859723

  11. Identification and pharmacological characterization of multiple allosteric binding sites on the free fatty acid 1 receptor.

    PubMed

    Lin, Daniel C-H; Guo, Qi; Luo, Jian; Zhang, Jane; Nguyen, Kathy; Chen, Michael; Tran, Thanh; Dransfield, Paul J; Brown, Sean P; Houze, Jonathan; Vimolratana, Marc; Jiao, Xian Yun; Wang, Yingcai; Birdsall, Nigel J M; Swaminath, Gayathri

    2012-11-01

    Activation of FFA1 (GPR40), a member of G protein-coupling receptor family A, is mediated by medium- and long-chain fatty acids and leads to amplification of glucose-stimulated insulin secretion, suggesting a potential role for free fatty acid 1 (FFA1) as a target for type 2 diabetes. It was assumed previously that there is a single binding site for fatty acids and synthetic FFA1 agonists. However, using members of two chemical series of partial and full agonists that have been identified, radioligand binding interaction studies revealed that the full agonists do not bind to the same site as the partial agonists but exhibit positive heterotropic cooperativity. Analysis of functional data reveals positive functional cooperativity between the full agonists and partial agonists in various functional assays (in vitro and ex vivo) and also in vivo. Furthermore, the endogenous fatty acid docosahexaenoic acid (DHA) shows negative or neutral cooperativity with members of both series of agonists in binding assays but displays positive cooperativity in functional assays. Another synthetic agonist is allosteric with members of both agonist series, but apparently competitive with DHA. Therefore, there appear to be three allosterically linked binding sites on FFA1 with agonists specific for each of these sites. Activation of free fatty acid 1 receptor (FFAR1) by each of these agonists is differentially affected by mutations of two arginine residues, previously found to be important for FFAR1 binding and activation. These ligands with their high potencies and strong positive functional cooperativity with endogenous fatty acids, demonstrated in vitro and in vivo, have the potential to deliver therapeutic benefits. PMID:22859723

  12. Community Involvement as an Effective Institutional Control at the Weldon Spring Site, a U.S. Department of Energy Office of Legacy Management Site

    SciTech Connect

    Deyo, Y.E.; Pauling, T.

    2006-07-01

    The U.S. Department of Energy (DOE) Weldon Spring Site Remedial Action Project (WSSRAP) was conducted for the purpose of remediating a portion of a former trinitrotoluene and dinitrotoluene production plant that was operational from 1941 to 1945 and a former uranium refinery that was operational from 1957 to 1966. Surface remediation activities concluded in 2001 with the completion of a 45-acre (.18 square kilometer) on-site engineered disposal facility. Long-term surveillance and maintenance activities at the site were officially transferred to the DOE Office of Legacy Management in 2003. The Weldon Spring Site is located within the St. Louis, Missouri, metropolitan area (population 3 million). DOE's close relationship with surrounding land owners created a need for innovative solutions to long-term surveillance and maintenance issues at the site. Through a Secretarial proclamation, a plan was established for development of a comprehensive public involvement and education program. This program would act as an institutional control to communicate the historical legacy of the site and would make information available about contamination present at the site to guide people in making decisions about appropriate site activities. In August 2002, the Weldon Spring Site Interpretive Center opened to the public with exhibits about the history of the area, the remediation work that was completed, and a site information repository that is available to visitors. In addition, the Hamburg Trail for hiking and biking was constructed as a joint DOE/MDC effort. The 8-mile trail travels through both DOE and MDC property; a series of historical markers posted along its length to communicate the history of the area and the remediation work that was done as part of WSSRAP activities. A ramp and viewing platform with informational plaques were constructed on the disposal cell to provide an additional mechanism for public education. With a basic marketing program, site visitor-ship has

  13. In Situ Oxalic Acid Injection to Accelerate Arsenic Remediation at a Superfund Site in New Jersey

    PubMed Central

    Wovkulich, Karen; Stute, Martin; Mailloux, Brian J.; Keimowitz, Alison R.; Ross, James; Bostick, Benjamin; Sun, Jing; Chillrud, Steven N.

    2015-01-01

    Arsenic is a prevalent contaminant at a large number of US Superfund sites; establishing techniques that accelerate As remediation could benefit many sites. Hundreds of tons of As were released into the environment by the Vineland Chemical Co. in southern New Jersey during its manufacturing lifetime (1949–1994), resulting in extensive contamination of surface and subsurface soils and sediments, groundwater, and the downstream watershed. Despite substantial intervention at this Superfund site, sufficient aquifer cleanup could require many decades if based on traditional pump and treat technologies only. Laboratory column experiments have suggested that oxalic acid addition to contaminated aquifer solids could promote significant As release from the solid phase. To evaluate the potential of chemical additions to increase As release in situ and boost treatment efficiency, a forced gradient pilot scale study was conducted on the Vineland site. During spring/summer 2009, oxalic acid and bromide tracer were injected into a small portion (~50 m2) of the site for 3 months. Groundwater samples indicate that introduction of oxalic acid led to increased As release. Between 2.9 and 3.6 kg of As were removed from the sampled wells as a result of the oxalic acid treatment during the 3-month injection. A comparison of As concentrations on sediment cores collected before and after treatment and analyzed using X-ray fluorescence spectroscopy suggested reduction in As concentrations of ~36% (median difference) to 48% (mean difference). While further study is necessary, the addition of oxalic acid shows potential for accelerating treatment of a highly contaminated site and decreasing the As remediation time-scale. PMID:25598701

  14. Yarrowia lipolytica AAL genes are involved in peroxisomal fatty acid activation.

    PubMed

    Dulermo, Rémi; Gamboa-Meléndez, Heber; Ledesma-Amaro, Rodrigo; Thevenieau, France; Nicaud, Jean-Marc

    2016-07-01

    In yeast, β-oxidation of fatty acids (FAs) essentially takes place in peroxisomes, and FA activation must precede FA oxidation. In Saccharomyces cerevisiae, a single fatty-acyl–CoA-synthetase, ScFaa2p, mediates peroxisomal FA activation. We have previously shown that this reaction also exists in the oleaginous yeast Yarrowia lipolytica; however, the protein involved in this process remains unknown. Here, we found that proteins, named Aal proteins (Acyl/Aryl-CoA-ligases), resembling the 4-coumarate–CoA-ligase-like enzymes found in plants are involved in peroxisomal FA activation in Y. lipolytica; Y. lipolytica has 10 AAL genes, eight of which are upregulated by oleate. All the Aal proteins contain a PTS1-type peroxisomal targeting sequence (A/SKL), suggesting a peroxisomal localization. The function of the Aal proteins was analyzed using the faa1Δant1Δ mutant strain, which demonstrates neither cytoplasmic FA activation (direct result of FAA1 deletion) nor peroxisomal FA activation (indirect result of ANT1 deletion, a gene coding an ATP transporter). This strain is thus highly sensitive to external FA levels and unable to store external FAs in lipid bodies (LBs). Whereas the overexpression of (cytoplasmic) AAL1ΔPTS1 was able to partially complement the growth defect observed in the faa1Δant1Δ mutant on short-, medium- and long-chain FA media, the presence of Aal2p to Aal10p only allowed growth on the short-chain FA medium. Additionally, partial LB formation was observed in the oleate medium for strains overexpressing Aal1ΔPTS1p, Aal4ΔPTS1p, Aal7ΔPTS1p, and Aal8ΔPTS1p. Finally, an analysis of the FA content of cells grown in the oleate medium suggested that Aal4p and Aal6p present substrate specificity for C16:1 and/or C18:0. PMID:27067366

  15. Strategic planning model for achieving stakeholder involvement in environmental at DOE weapons complex sites

    SciTech Connect

    Weber, G.

    1994-12-31

    Within today`s reality a public manager often needs to develop cooperative relationships among a number of individual, program, and organizational stakeholders to accomplish particular projects, programs, or policies. A DOE site manager charged with accomplishing environmental restoration and conversion at former weapons production sites is no exception. Important reasons for this include the technical and political complexity of the clean-up problem; limits on the funding, authority, and other resources available to DOE; authority, responsibilities, and interests of other stakeholders; and the ever present potential for conflict among stakeholders, and power of any one to hinder, if not halt, the clean-up process if conflicts aren`t managed and cooperative relationships established and maintained.

  16. Pathways Involved in the Synergistic Activation of Macrophages by Lipoteichoic Acid and Hemoglobin

    PubMed Central

    Cox, Kathleen H.; Cox, Michelle E.; Woo-Rasberry, Virginia; Hasty, David L.

    2012-01-01

    Lipoteichoic acid (LTA) is a Gram-positive cell surface molecule that is found in both a cell-bound form and cell-free form in the host during an infection. Hemoglobin (Hb) can synergize with LTA, a TLR2 ligand, to potently activate macrophage innate immune responses in a TLR2- and TLR4-dependent way. At low levels of LTA, the presence of Hb can result in a 200-fold increase in the secretion of IL-6 following macrophage activation. Six hours after activation, the macrophage genes that are most highly up-regulated by LTA plus Hb activation compared to LTA alone are cytokines, chemokines, receptors and interferon-regulated genes. Several of these genes exhibit a unique TLR4-dependent increase in mRNA levels that continued to rise more than eight hours after stimulation. This prolonged increase in mRNA levels could be the result of an extended period of NF-κB nuclear localization and the concurrent absence of the NF-κB inhibitor, IκBα, after stimulation with LTA plus Hb. Dynasore inhibition experiments indicate that an endocytosis-dependent pathway is required for the TLR4-dependent up-regulation of IL-6 secretion following activation with LTA plus Hb. In addition, interferon-β mRNA is present after activation with LTA plus Hb, suggesting that the TRIF/TRAM-dependent pathway may be involved. Hb alone can elicit the TLR4-dependent secretion of TNF-α from macrophages, so it may be the TLR4 ligand. Hb also led to secretion of high mobility group box 1 protein (HMGB1), which synergized with LTA to increase secretion of IL-6. The activation of both the TLR2 and TLR4 pathways by LTA plus Hb leads to an enhanced innate immune response. PMID:23071790

  17. Reactive oxygen species are involved in gibberellin/abscisic acid signaling in barley aleurone cells.

    PubMed

    Ishibashi, Yushi; Tawaratsumida, Tomoya; Kondo, Koji; Kasa, Shinsuke; Sakamoto, Masatsugu; Aoki, Nozomi; Zheng, Shao-Hui; Yuasa, Takashi; Iwaya-Inoue, Mari

    2012-04-01

    Reactive oxygen species (ROS) act as signal molecules for a variety of processes in plants. However, many questions about the roles of ROS in plants remain to be clarified. Here, we report the role of ROS in gibberellin (GA) and abscisic acid (ABA) signaling in barley (Hordeum vulgare) aleurone cells. The production of hydrogen peroxide (H2O2), a type of ROS, was induced by GA in aleurone cells but suppressed by ABA. Furthermore, exogenous H2O2 appeared to promote the induction of α-amylases by GA. In contrast, antioxidants suppressed the induction of α-amylases. Therefore, H2O2 seems to function in GA and ABA signaling, and in regulation of α-amylase production, in aleurone cells. To identify the target of H2O2 in GA and ABA signaling, we analyzed the interrelationships between H2O2 and DELLA proteins Slender1 (SLN1), GA-regulated Myb transcription factor (GAmyb), and ABA-responsive protein kinase (PKABA) and their roles in GA and ABA signaling in aleurone cells. In the presence of GA, exogenous H2O2 had little effect on the degradation of SLN1, the primary transcriptional repressor mediating GA signaling, but it promoted the production of the mRNA encoding GAMyb, which acts downstream of SLN1 and involves induction of α-amylase mRNA. Additionally, H2O2 suppressed the production of PKABA mRNA, which is induced by ABA:PKABA represses the production of GAMyb mRNA. From these observations, we concluded that H2O2 released the repression of GAMyb mRNA by PKABA and consequently promoted the production of α-amylase mRNA, thus suggesting that the H2O2 generated by GA in aleurone cells is a signal molecule that antagonizes ABA signaling. PMID:22291200

  18. Phosphoproteomics Identified an NS5A Phosphorylation Site Involved in Hepatitis C Virus Replication.

    PubMed

    Chong, Weng Man; Hsu, Shih-Chin; Kao, Wei-Ting; Lo, Chieh-Wen; Lee, Kuan-Ying; Shao, Jheng-Syuan; Chen, Yi-Hung; Chang, Justin; Chen, Steve S-L; Yu, Ming-Jiun

    2016-02-19

    The non-structural protein 5A (NS5A) is a hepatitis C virus (HCV) protein indispensable for the viral life cycle. Many prior papers have pinpointed several serine residues in the low complexity sequence I region of NS5A responsible for NS5A phosphorylation; however, the functions of specific phosphorylation sites remained obscure. Using phosphoproteomics, we identified three phosphorylation sites (serines 222, 235, and 238) in the NS5A low complexity sequence I region. Reporter virus and replicon assays using phosphorylation-ablated alanine mutants of these sites showed that Ser-235 dominated over Ser-222 and Ser-238 in HCV replication. Immunoblotting using an Ser-235 phosphorylation-specific antibody showed a time-dependent increase in Ser-235 phosphorylation that correlated with the viral replication activity. Ser-235 phosphorylated NS5A co-localized with double-stranded RNA, consistent with its role in HCV replication. Mechanistically, Ser-235 phosphorylation probably promotes the replication complex formation via increasing NS5A interaction with the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein. Casein kinase Iα (CKIα) directly phosphorylated Ser-235 in vitro. Inhibition of CKIα reduced Ser-235 phosphorylation and the HCV RNA levels in the infected cells. We concluded that NS5A Ser-235 phosphorylated by CKIα probably promotes HCV replication via increasing NS5A interaction with the 33-kDa vesicle-associated membrane protein-associated protein. PMID:26702051

  19. LIME TREATMENT LAGOONS TECHNOLOGY FOR TREATING ACID MINE DRAINAGE FROM TWO MINING SITES

    EPA Science Inventory

    Runoff and drainage from active and inactive mines are someof the most environmentally damaging land uses i the US. Acid Mine drainage (AMD) from mining sites across the country requires treatment because of high metal concentrations that exceed regulatory standards for safe disc...

  20. PHYSICAL SOLUTIONS FOR ACID ROCK DRAINAGE AT REMOTE SITES DEMONSTRATION PROJECT

    EPA Science Inventory

    This report summarizes the results of Mine Waste Technology Program, Activity III, Project 42, Physical Solutions for Acid Rock Drainage at Remote Sites, funded by the U.S. Environmental Protection Agency (EPA) and jointly administered by EPA and the U.S. Department of Energy. A...

  1. Proposed framework for cleanup and site restoration following a terrorist incident involving radioactive material.

    PubMed

    Conklin, W Craig

    2005-11-01

    Cleanup following a terrorism incident involving a radiological dispersal device (RDD) or improvised nuclear device (IND) is likely to be technically challenging, costly, and politically charged. Lessons learned from the Top Officials 2 exercise and the increased threat of terrorist use of an RDD or IND have driven federal officials to push for an agreed-upon process for determining appropriate cleanup levels. State and local authorities generally have the ultimate responsibility for final public health decisions in their jurisdictions. In response to terrorist attacks, local authorities are likely to request federal assistance in assessing the risk and establishing appropriate cleanup levels. It is realistic to expect local and state requests for significant federal assistance in planning and implementing recovery operations. State and local authorities may desire "shared accountability" with the federal government in setting the appropriate cleanup levels. Government officials at all levels will face pressure to say how clean is clean enough and how quickly people can re-enter affected areas. Issues arising include (1) the nature of the relationship between the federal, state, and local leadership involved in the recovery efforts and (2) where the funding for recovery comes from. Many agencies, including the U.S. Environmental Protection Agency (EPA), the U.S. Nuclear Regulatory Commission (NRC), and the U.S. Department of Energy (DOE) have long been involved in cleanup activities involving radioactive materials. These agencies have recognized the need for a participatory process and realize the need to remain flexible when faced with possible unprecedented environmental challenges following a terrorist attack. Currently, the Department of Homeland Security has a committee process underway, with participation of the EPA, NRC, DOE, and other federal agencies, to try to resolve these issues and to begin engaging state, local, and tribal governments, and others as

  2. Site and chirality selective chemical modifications of boron nitride nanotubes (BNNTs) via Lewis acid-base interactions.

    PubMed

    Sundaram, Rajashabala; Scheiner, Steve; Roy, Ajit K; Kar, Tapas

    2015-02-01

    The pristine BNNTs contain both Lewis acid (boron) and Lewis base (nitrogen) centers at their surface. Interactions of ammonia and borane molecules, representatives of Lewis base and acid as adsorbates respectively, with matching sites at the surface of BNNTs, have been explored in the present DFT study. Adsorption energies suggest stronger chemisorption (about 15-20 kcal mol(-1)) of borane than ammonia (about 5-10 kcal mol(-1)) in both armchair (4,4) and zigzag (8,0) variants of the tube. NH3 favors (8,0) over the (4,4) tube, whereas BH3 exhibits the opposite preference, indicating some chirality dependence on acid-base interactions. A new feature of bonding is found in BH3/AlH3-BNNTs (at the edge site) complexes, where one hydrogen of the guest molecule is involved in three-center two-electron bonding, in addition to dative covalent bond (N: → B). This interaction causes a reversal of electron flow from borane/alane to BNNT, making the tube an electron acceptor, suggesting tailoring of electronic properties could be possible by varying strength of incoming Lewis acids. On the contrary, BNNTs always behave as electron acceptor in ammonia complexes. IR, XPS and NMR spectra show some characteristic features of complexes and can help experimentalists to identify not only structures of such complexes but also the location of the guest molecules and design second functionalizations. Interaction with several other neutral BF3, BCl3, BH2CH3 and ionic CH3(+) acids as well as amino group (CH3NH2 and NH2COOH) were also studied. The strongest interaction (>100 kcal mol(-1)) is found in BNNT-CH3(+) complexes and H-bonds are the only source of stability of NH2COOH-BNNT complexes. PMID:25559141

  3. Proton-Binding Sites of Acid-Sensing Ion Channel 1

    PubMed Central

    Ishikita, Hiroshi

    2011-01-01

    Acid-sensing ion channels (ASICs) are proton-gated cation channels that exist throughout the mammalian central and peripheral nervous systems. ASIC1 is the most abundant of all the ASICs and is likely to modulate synaptic transmission. Identifying the proton-binding sites of ASCI1 is required to elucidate its pH-sensing mechanism. By using the crystal structure of ASIC1, the protonation states of each titratable site of ASIC1 were calculated by solving the Poisson-Boltzmann equation under conditions wherein the protonation states of all these sites are simultaneously in equilibrium. Four acidic-acidic residue pairs—Asp238-Asp350, Glu220-Asp408, Glu239-Asp346, and Glu80-Glu417—were found to be highly protonated. In particular, the Glu80-Glu417 pair in the inner pore was completely protonated and possessed 2 H+, implying its possible importance as a proton-binding site. The pKa of Glu239, which forms a pair with a possible pH-sensing site Asp346, differs among each homo-trimer subunit due to the different H-bond pattern of Thr237 in the different protein conformations of the subunits. His74 possessed a pKa of ≈6–7. Conservation of His74 in the proton-sensitive ASIC3 that lacks a residue corresponding to Asp346 may suggest its possible pH-sensing role in proton-sensitive ASICs. PMID:21340031

  4. Site-directed mutagenesis identifies a tyrosine radical involved in the photosynthesized oxygen-evolving system

    SciTech Connect

    Debus, R.J.; Barry, B.A.; Babcock, G.T.; McIntosh, L.

    1988-01-01

    Photosynthetic oxygen evolution takes place in the thylakoid protein complex known as photosystem II. The reaction center core of this photosystem, where photochemistry occurs, is a heterodimer of homologous polypeptides called D1 and D2. Besides chlorophyll and quinone, photosystem II contains other organic cofactors, including two known as Z and D. Z transfers electrons from the site of water oxidation to the oxidized reaction center primary donor, P/sub 680//sup +/, while D /center dot//sup +/ gives rise to the dark-stable EPR spectrum known as signal II. D/center dot//sup +/ has recently been shown to be a tyrosine radical. Z is probably a second tyrosine located in a similar environment. Indirect evidence indicates that Z and D are associated with the D1 and E2 polypeptides, respectively. To identify the specific tyrosine residue corresponding to D, the authors have changed Tyr-160 of the D2 polypeptide to phenylalanine by site-directed mutagenesis of a psbD gene in the cyanobacterium Synechocystis 6803. The resulting mutant grows photosynthetically, but it lacks the EPR signal of D/center dot//sup +/. The authors conclude that D is Tyr-160 of the D2 polypeptide. They suggest that the C/sub 2/ symmetry in photosystem II extends beyond P/sub 680/ to its immediate electron donor and conclude that Z is Try-161 of the D1 polypeptide.

  5. Involvement of a Stat3 binding site in inflammation-induced enteric apelin expression.

    PubMed

    Han, Song; Wang, Guiyun; Qi, Xiang; Englander, Ella W; Greeley, George H

    2008-11-01

    Apelin is the endogenous ligand for the APJ receptor; both are expressed in the gastrointestinal tract. Experimental colitis in rodents and inflammatory bowel disease in humans are associated with increased intestinal apelin production. Our aim was to use LPS and proinflammatory cytokine-treated (IL-6 and IFN-gamma) rodents or enteric cells to identify signaling mechanisms underlying inflammation-induced enteric apelin expression. LPS, IL-6, or IFN-gamma treatment of rodents increased enteric apelin expression. Pharmacological blockade of Jak/Stat signaling or IL-6 antibody administration inhibited elevations in enteric apelin expression. Transient transfection experiments showed that LPS, IL-6, or IFN-gamma increased apelin expression by stimulation of apelin promoter activity, and blockade of Jak/Stat signaling abolished elevations in apelin promoter activity. A chromatin immunoprecipitation assay showed that IL-6 induced binding of phospho-Stat3 to a putative Stat3 site in the apelin promoter; mutation of this site abrogated the LPS-induced elevation in apelin promoter activity. Together, our findings indicate that binding of phospho-Stat3 to the apelin promoter is the final step underlying proinflammatory cytokine-induced enteric apelin expression during intestinal inflammation. PMID:18818315

  6. Cox17 Protein Is an Auxiliary Factor Involved in the Control of the Mitochondrial Contact Site and Cristae Organizing System*

    PubMed Central

    Chojnacka, Magdalena; Gornicka, Agnieszka; Oeljeklaus, Silke; Warscheid, Bettina; Chacinska, Agnieszka

    2015-01-01

    The mitochondrial contact site and cristae organizing system (MICOS) is a recently discovered protein complex that is crucial for establishing and maintaining the proper inner membrane architecture and contacts with the outer membrane of mitochondria. The ways in which the MICOS complex is assembled and its integrity is regulated remain elusive. Here, we report a direct link between Cox17, a protein involved in the assembly of cytochrome c oxidase, and the MICOS complex. Cox17 interacts with Mic60, thereby modulating MICOS complex integrity. This interaction does not involve Sco1, a partner of Cox17 in transferring copper ions to cytochrome c oxidase. However, the Cox17-MICOS interaction is regulated by copper ions. We propose that Cox17 is a newly identified factor involved in maintaining the architecture of the MICOS complex. PMID:25918166

  7. Chicoric acid binds to two sites and decreases the activity of the YopH bacterial virulence factor

    PubMed Central

    Kuban-Jankowska, Alicja; Sahu, Kamlesh K.; Gorska, Magdalena; Tuszynski, Jack A.; Wozniak, Michal

    2016-01-01

    Chicoric acid (CA) is a phenolic compound present in dietary supplements with a large spectrum of biological properties reported ranging from antioxidant, to antiviral, to immunostimulatory properties. Due to the fact that chicoric acid promotes phagocytic activity and was reported as an allosteric inhibitor of the PTP1B phosphatase, we examined the effect of CA on YopH phosphatase from pathogenic bacteria, which block phagocytic processes of a host cell. We performed computational studies of chicoric acid binding to YopH as well as validation experiments with recombinant enzymes. In addition, we performed similar studies for caffeic and chlorogenic acids to compare the results. Docking experiments demonstrated that, from the tested compounds, only CA binds to both catalytic and secondary binding sites of YopH. Our experimental results showed that CA reduces activity of recombinant YopH phosphatase from Yersinia enterocolitica and human CD45 phosphatase. The inhibition caused by CA was irreversible and did not induce oxidation of catalytic cysteine. We proposed that inactivation of YopH induced by CA is involved with allosteric inhibition by interacting with essential regions responsible for ligand binding. PMID:26735581

  8. Chicoric acid binds to two sites and decreases the activity of the YopH bacterial virulence factor.

    PubMed

    Kuban-Jankowska, Alicja; Sahu, Kamlesh K; Gorska, Magdalena; Tuszynski, Jack A; Wozniak, Michal

    2016-01-19

    Chicoric acid (CA) is a phenolic compound present in dietary supplements with a large spectrum of biological properties reported ranging from antioxidant, to antiviral, to immunostimulatory properties. Due to the fact that chicoric acid promotes phagocytic activity and was reported as an allosteric inhibitor of the PTP1B phosphatase, we examined the effect of CA on YopH phosphatase from pathogenic bacteria, which block phagocytic processes of a host cell. We performed computational studies of chicoric acid binding to YopH as well as validation experiments with recombinant enzymes. In addition, we performed similar studies for caffeic and chlorogenic acids to compare the results. Docking experiments demonstrated that, from the tested compounds, only CA binds to both catalytic and secondary binding sites of YopH. Our experimental results showed that CA reduces activity of recombinant YopH phosphatase from Yersinia enterocolitica and human CD45 phosphatase. The inhibition caused by CA was irreversible and did not induce oxidation of catalytic cysteine. We proposed that inactivation of YopH induced by CA is involved with allosteric inhibition by interacting with essential regions responsible for ligand binding. PMID:26735581

  9. Function of heterologous Mycobacterium tuberculosis InhA, a type 2 fatty acid synthase enzyme involved in extending C20 fatty acids to C60-to-C90 mycolic acids, during de novo lipoic acid synthesis in Saccharomyces cerevisiae.

    PubMed

    Gurvitz, Aner; Hiltunen, J Kalervo; Kastaniotis, Alexander J

    2008-08-01

    We describe the physiological function of heterologously expressed Mycobacterium tuberculosis InhA during de novo lipoic acid synthesis in yeast (Saccharomyces cerevisiae) mitochondria. InhA, representing 2-trans-enoyl-acyl carrier protein reductase and the target for the front-line antituberculous drug isoniazid, is involved in the activity of dissociative type 2 fatty acid synthase (FASII) that extends associative type 1 fatty acid synthase (FASI)-derived C(20) fatty acids to form C(60)-to-C(90) mycolic acids. Mycolic acids are major constituents of the protective layer around the pathogen that contribute to virulence and resistance to certain antimicrobials. Unlike FASI, FASII is thought to be incapable of de novo biosynthesis of fatty acids. Here, the genes for InhA (Rv1484) and four similar proteins (Rv0927c, Rv3485c, Rv3530c, and Rv3559c) were expressed in S. cerevisiae etr1Delta cells lacking mitochondrial 2-trans-enoyl-thioester reductase activity. The phenotype of the yeast mutants includes the inability to produce sufficient levels of lipoic acid, form mitochondrial cytochromes, respire, or grow on nonfermentable carbon sources. Yeast etr1Delta cells expressing mitochondrial InhA were able to respire, grow on glycerol, and produce lipoic acid. Commensurate with a role in mitochondrial de novo fatty acid biosynthesis, InhA could accept in vivo much shorter acyl-thioesters (C(4) to C(8)) than was previously thought (>C(12)). Moreover, InhA functioned in the absence of AcpM or protein-protein interactions with its native FASII partners KasA, KasB, FabD, and FabH. None of the four proteins similar to InhA complemented the yeast mutant phenotype. We discuss the implications of our findings with reference to lipoic acid synthesis in M. tuberculosis and the potential use of yeast FASII mutants for investigating the physiological function of drug-targeted pathogen enzymes involved in fatty acid biosynthesis. PMID:18552191

  10. The rice OsLpa1 gene encodse a novel protein involved in phytic acid metabolism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The rice low phytic acid 1 (OsLpa1) gene was originally identified using a forward genetics approach. Mutation of this gene resulted in a 45% reduction in rice seed phytic acid with a molar-equivalent increase in inorganic phosphorus; however, the rice lpa1 mutant does not appear to differ significa...

  11. Determination of the amino acid residue involved in [3H]beta-funaltrexamine covalent binding in the cloned rat mu-opioid receptor.

    PubMed

    Chen, C; Yin, J; Riel, J K; DesJarlais, R L; Raveglia, L F; Zhu, J; Liu-Chen, L Y

    1996-08-30

    We previously demonstrated that [3H]beta-funaltrexamine ([3H]beta-FNA) labeled the rat mu opioid receptor expressed in Chinese hamster ovary cells with high specificity, and [3H]beta-FNA-labeled receptors migrated as one broad band with a mass of 80 kDa. In this study, we determined the region and then the amino acid residue of the mu receptor involved in the covalent binding of [3H]beta-FNA. [3H]beta-FNA-labeled receptors were solubilized and purified to approximately 10% purity by immunoaffinity chromatography with antibodies against a C-terminal domain peptide. The site of covalent bond formation was determined to be within Ala206-Met243 by CNBr cleavage of partially purified labeled mu receptors and determinations of sizes of labeled receptor fragments. The amino acid residue of beta-FNA covalent incorporation was then determined by site-directed mutagenesis studies within this region. Mutation of Lys233 to Ala, Arg, His, and Leu completely eliminated covalent binding of [3H]beta-FNA, although these mutants bound beta-FNA with high affinity. Mutations of other amino acid residues did not affect covalent binding of [3H]beta-FNA. These results indicate that [3H]beta-FNA binds covalently to Lys233. Since [3H]beta-FNA is a rigid molecule, the information will be very useful for molecular modeling of interaction between morphinans and the mu receptor. PMID:8702924

  12. Functional groups of sialic acids involved in binding to siglecs (sialoadhesins) deduced from interactions with synthetic analogues.

    PubMed

    Kelm, S; Brossmer, R; Isecke, R; Gross, H J; Strenge, K; Schauer, R

    1998-08-01

    The siglecs, formerly called sialoadhesins, are a family of I-type lectins binding to sialic acids on the cell surface. Five members of this family have been identified: sialoadhesin, myelin-associated glycoprotein (MAG), Schwann cell myelin protein (SMP), CD22 and CD33. We have investigated the relevance of substituents at position C-9 and in the N-acetyl group of N-acetylneuraminic acid, using a series of synthetic sialic-acid analogues either on resialylated human erythrocytes or as free alpha-glycosides in hapten inhibition. All five siglecs require the hydroxy group at C-9 for binding, suggesting hydrogen bonding of this substituent with the binding site. Remarkable differences were found among the proteins in their specificity for modifications of the N-acetyl group. Whereas sialoadhesin, MAG and SMP do not tolerate a hydroxy group as in N-glycolylneuraminic acid, they bind to halogenated acetyl residues. In the case of MAG, N-fluoroacetylneuraminic acid is bound about 17-fold better than N-acetylneuraminic acid. In contrast, human and murine CD22 both show good affinity for N-glycolylneuraminic acid, but only human CD22 bound the halogenated compounds. In conclusion, our data indicate that interactions of the hydroxy group at position 9 and the N-acyl substituent contribute significantly to the binding strength. PMID:9738906

  13. Calcitonin/calcitonin gene-related peptide transcription unit: tissue-specific expression involves selective use of alternative polyadenylation sites.

    PubMed Central

    Amara, S G; Evans, R M; Rosenfeld, M G

    1984-01-01

    Different 3' coding exons in the rat calcitonin gene are used to generate distinct mRNAs encoding either the hormone calcitonin in thyroidal C-cells or a new neuropeptide referred to as calcitonin gene-related peptide in neuronal tissue, indicating the RNA processing regulation is one strategy used in tissue-specific regulation of gene expression in the brain. Although the two mRNAs use the same transcriptional initiation site and have identical 5' terminal sequences, their 3' termini are distinct. The polyadenylation sites for calcitonin and calcitonin gene-related peptide mRNAs are located at the end of the exons 4 and 6, respectively. Termination of transcription after the calcitonin exon does not dictate the production of calcitonin mRNA, because transcription proceeds through both calcitonin and calcitonin gene-related peptide exons irrespective of which mRNA is ultimately produced. In isolated nuclei, both polyadenylation sites appear to be utilized; however, the proximal (calcitonin) site is preferentially used in nuclei from tissues producing calcitonin mRNA. These data suggest that the mechanism dictating production of each mRNA involves the selective use of alternative polyadenylation sites. Images PMID:6334229

  14. Site-specific factor involved in the editing of the psbL mRNA in tobacco plastids.

    PubMed Central

    Chaudhuri, S; Carrer, H; Maliga, P

    1995-01-01

    In tobacco plastids, functional psbL mRNA is created by editing an ACG codon to an AUG translation initiation codon. To determine if editing may occur in a chimeric mRNA, the N-terminal part of psbL containing the editing site was translationally fused with the aadA and kan bacterial genes. The chimeric constructs were introduced into the tobacco plastid genome by targeted gene insertion. Editing of the chimeric mRNAs indicated that the 98 nt fragment spanning the psbL editing site contains all cis information required for editing. Expression of the chimeric gene transcripts led to a significant decrease in the editing efficiency of the endogenous psbL mRNA. However, the efficiency of editing in the transplastomic lines was unchanged for four sites in the rpoB and ndhB mRNAs. Reduced efficiency of psbL editing, but not of the other four sites, in the transplastomic lines indicates depletion of psbL-specific editing factor(s). This finding implicates the involvement of site-specific factors in editing of plastid mRNAs in higher plants. Images PMID:7796820

  15. Site-Specific Radiofluorination of Biomolecules with 8-[(18)F]-Fluorooctanoic Acid Catalyzed by Lipoic Acid Ligase.

    PubMed

    Drake, Christopher R; Sevillano, Natalia; Truillet, Charles; Craik, Charles S; VanBrocklin, Henry F; Evans, Michael J

    2016-06-17

    New methodologies for site-specifically radiolabeling proteins with (18)F are required to generate high quality radiotracers for preclinical and clinical applications with positron emission tomography. Herein, we report an approach by which we use lipoic acid ligase (LplA) to conjugate [(18)F]-fluorooctanoic acid to an antibody fragment bearing the peptide substrate of LplA. The mild conditions of the reaction preserve antibody immunoreactivity, and the efficiency of LplA allows for >90% yield even with very small amounts of peptidic precursor (1-10 nmol). These features are advantageous compared to the current gold standard in the field. Moreover, the methodology introduces a new application for an important tool in chemical biology. PMID:27008570

  16. Antidepressant-like effects of ferulic acid: involvement of serotonergic and norepinergic systems.

    PubMed

    Chen, Jianliang; Lin, Dan; Zhang, Chong; Li, Gaowen; Zhang, Nianping; Ruan, Lina; Yan, Qizhi; Li, Jianxin; Yu, Xuefeng; Xie, Xupei; Pang, Cong; Cao, Liang; Pan, Jianchun; Xu, Ying

    2015-02-01

    Ferulic acid is a polyphenol that has antioxidant, anti-inflammatory and anticancer properties. The present study analyzed the antidepressant-like potential of ferulic acid using two well-validated mouse models of despair test, tail suspension and forced swim tests. The results suggested that ferulic acid treatment at doses of 10, 20, 40 and 80 mg/kg (p.o.) significantly reduced the immobility time in both of these two tests. These doses that affected the depressive-like behaviors did now show any effect on locomotion counts. The further neurochemical assays suggested that ferulic acid increased monoamine neurotransmitter levels in the brain regions that are relative to mood disorders: the hippocampus and frontal cortex. The increased tend to serotonin and norepinephrine was also found in the hypothalamus after higher dose of ferulic acid treatment. The subsequent study suggested that monoamine oxidase A (MAO-A) activity was inhibited in the frontal cortex and hippocampus when treatment with 40 and 80 mg/kg ferulic acid; while MAO-B activity did not change significantly. The current study provides the first lines of evidence that serotonin and norepinephrine, but not dopamine levels were elevated in mouse hippocampus and frontal cortex after ferulic acid treatment. These changes may be attributable to the inhibition of MAO-A activities in the same brain regions. PMID:25483788

  17. Identification of a new site in the S1 ligand binding region of the NMDA receptor NR2A subunit involved in receptor activation by glutamate.

    PubMed

    Lummis, Sarah C R; Fletcher, Elizabeth J; Green, Tim

    2002-03-01

    Activation of N-methyl-d-aspartate (NMDA) receptors requires the binding of both glutamate and glycine to independent sites on the receptor. These ligands bind to NR2 and NR1 subunits respectively. Ligand binding residues are located in two non-contiguous domains, S1 and S2, which have been implicated in glutamate binding in other ionotropic glutamate receptor subunits. To further define the amino acids through which glutamate activates the receptor, we generated single-site mutations to the NR2A subunit, and expressed them with wild type NR1 in HEK 293 cells. Using calcium imaging and whole cell patch clamp we determined glutamate and glycine potencies. Of the eight residues mutated we identified five (E413, K484, A508, G685 and G688), whose mutation leads to a large reduction (from 4- to 1000-fold) in glutamate potency, consistent with a role for these residues in receptor activation by glutamate. The potency of glycine was largely unchanged by these mutations. Thus our results extend the knowledge base of residues involved in NMDA receptor function and identifies a new site in S1, in the region of A508, that has a role in receptor activation by glutamate. PMID:11955515

  18. Metabotropic glutamate receptors are involved in the detection of IMP and L-amino acids by mouse taste sensory cells.

    PubMed

    Pal Choudhuri, S; Delay, R J; Delay, E R

    2016-03-01

    G-protein-coupled receptors are thought to be involved in the detection of umami and L-amino acid taste. These include the heterodimer taste receptor type 1 member 1 (T1r1)+taste receptor type 1 member 3 (T1r3), taste and brain variants of mGluR4 and mGluR1, and calcium sensors. While several studies suggest T1r1+T1r3 is a broadly tuned lLamino acid receptor, little is known about the function of metabotropic glutamate receptors (mGluRs) in L-amino acid taste transduction. Calcium imaging of isolated taste sensory cells (TSCs) of T1r3-GFP and T1r3 knock-out (T1r3 KO) mice was performed using the ratiometric dye Fura 2 AM to investigate the role of different mGluRs in detecting various L-amino acids and inosine 5' monophosphate (IMP). Using agonists selective for various mGluRs such as (RS)-3,5-dihydroxyphenylglycine (DHPG) (an mGluR1 agonist) and L-(+)-2-amino-4-phosphonobutyric acid (l-AP4) (an mGluR4 agonist), we evaluated TSCs to determine if they might respond to these agonists, IMP, and three L-amino acids (monopotassium L-glutamate, L-serine and L-arginine). Additionally, we used selective antagonists against different mGluRs such as (RS)-L-aminoindan-1,5-dicarboxylic acid (AIDA) (an mGluR1 antagonist), and (RS)-α-methylserine-O-phosphate (MSOP) (an mGluR4 antagonist) to determine if they can block responses elicited by these L-amino acids and IMP. We found that L-amino acid- and IMP-responsive cells also responded to each agonist. Antagonists for mGluR4 and mGluR1 significantly blocked the responses elicited by IMP and each of the L-amino acids. Collectively, these data provide evidence for the involvement of taste and brain variants of mGluR1 and mGluR4 in L-amino acid and IMP taste responses in mice, and support the concept that multiple receptors contribute to IMP and L-amino acid taste. PMID:26701297

  19. Involvement of Acetobacter orientalis in the production of lactobionic acid in Caucasian yogurt ("Caspian Sea yogurt") in Japan.

    PubMed

    Kiryu, T; Kiso, T; Nakano, H; Ooe, K; Kimura, T; Murakami, H

    2009-01-01

    Lactobionic acid was first found in a Caucasian fermented milk product popularly known as "Caspian Sea yogurt" in Japan. The presence of lactobionic acid in the fermented milk was indicated by the results of both high-performance anion-exchange chromatographic analysis with pulsed amperometric detection and mass spectrometric analysis. Thereafter, the acid was purified from the yogurt and analyzed by nuclear magnetic resonance. A substantial amount of lactobionic acid was found to be accumulated in the upper layer of the yogurt, especially within 10 mm from the surface. A total of 45 mg of lactobionic acid per 100 g of the upper yogurt layer was collected after 4 d of fermentation. The annual intake of lactobionic acid in individuals consuming 100 g of the yogurt every day would be 0.5 to 1.0 g. A lactose-oxidizing bacterium was isolated from the fermented milk and was identified as Acetobacter orientalis. Washed A. orientalis cells oxidized monosaccharides such as d-glucose at considerable rates, although their activities for substrates such as lactose, maltose, and cellobiose were much lower. When A. orientalis cells were cultivated in cow's milk, they exhibited lactose-oxidizing activity, suggesting that this bacterium was the main organism involved in the production of lactobionic acid in the yogurt. PMID:19109260

  20. Zinc-induced oligomerization of zinc α2 glycoprotein reveals multiple fatty acid-binding sites.

    PubMed

    Zahid, Henna; Miah, Layeque; Lau, Andy M; Brochard, Lea; Hati, Debolina; Bui, Tam T T; Drake, Alex F; Gor, Jayesh; Perkins, Stephen J; McDermott, Lindsay C

    2016-01-01

    Zinc α2 glycoprotein (ZAG) is an adipokine with a class I MHC protein fold and is associated with obesity and diabetes. Although its intrinsic ligand remains unknown, ZAG binds the dansylated C11 fatty acid 11-(dansylamino)undecanoic acid (DAUDA) in the groove between the α1 and α2 domains. The surface of ZAG has approximately 15 weak zinc-binding sites deemed responsible for precipitation from human plasma. In the present study the functional significance of these metal sites was investigated. Analytical ultracentrifugation (AUC) and CD showed that zinc, but not other divalent metals, causes ZAG to oligomerize in solution. Thus ZAG dimers and trimers were observed in the presence of 1 and 2 mM zinc. Molecular modelling of X-ray scattering curves and sedimentation coefficients indicated a progressive stacking of ZAG monomers, suggesting that the ZAG groove may be occluded in these. Using fluorescence-detected sedimentation velocity, these ZAG-zinc oligomers were again observed in the presence of the fluorescent boron dipyrromethene fatty acid C16-BODIPY (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid). Fluorescence spectroscopy confirmed that ZAG binds C16-BODIPY. ZAG binding to C16-BODIPY, but not to DAUDA, was reduced by increased zinc concentrations. We conclude that the lipid-binding groove in ZAG contains at least two distinct fatty acid-binding sites for DAUDA and C16-BODIPY, similar to the multiple lipid binding seen in the structurally related immune protein CD1c. In addition, because high concentrations of zinc occur in the pancreas, the perturbation of these multiple lipid-binding sites by zinc may be significant in Type 2 diabetes where dysregulation of ZAG and zinc homoeostasis occurs. PMID:26487699

  1. Characterization of Naphthaleneacetic Acid Binding to Receptor Sites on Cellular Membranes of Maize Coleoptile Tissue 1

    PubMed Central

    Ray, Peter M.; Dohrmann, Ulrike; Hertel, Rainer

    1977-01-01

    Characteristics of and optimum conditions for saturable (“specific”) binding of [14C]naphthaleneacetic acid to sites located on membranous particles from maize (Zea mays L.) coleoptiles are described. Most, if not all, of the specific binding appears to be due to a single kinetic class of binding sites having a KD of 5 to 7 × 10−7m for naphthalene-1-acetic acid (NAA). Binding of NAA is insensitive to high monovalent salt concentrations, indicating that binding is not primarily ionic. However, specific binding is inhibited by Mg2+ or Ca2+ above 5 mm. Specific binding is improved by organic acids, especially citrate. Binding is heat-labile and is sensitive to agents that act either on proteins or on lipids. Specific binding is reversibly inactivated by reducing agents such as dithioerythritol; a reducible group, possibly a disulfide group, may be located at the binding site and required for its function. The affinity of the specific binding sites for auxins is modified by an unidentified dialyzable, heat-stable, apparently amphoteric, organic factor (“supernatant factor”) found in maize tissue. PMID:16659851

  2. Site-specific integration of adeno-associated virus involves partial duplication of the target locus

    PubMed Central

    Henckaerts, Els; Dutheil, Nathalie; Zeltner, Nadja; Kattman, Steven; Kohlbrenner, Erik; Ward, Peter; Clément, Nathalie; Rebollo, Patricia; Kennedy, Marion; Keller, Gordon M.; Linden, R. Michael

    2009-01-01

    A variety of viruses establish latency by integrating their genome into the host genome. The integration event generally occurs in a nonspecific manner, precluding the prediction of functional consequences from resulting disruptions of affected host genes. The nonpathogenic adeno-associated virus (AAV) is unique in its ability to stably integrate in a site-specific manner into the human MBS85 gene. To gain a better understanding of the integration mechanism and the consequences of MBS85 disruption, we analyzed the molecular structure of AAV integrants in various latently infected human cell lines. Our study led to the observation that AAV integration causes an extensive but partial duplication of the target gene. Intriguingly, the molecular organization of the integrant leaves the possibility that a functional copy of the disrupted target gene could potentially be preserved despite the resulting rearrangements. A latently infected, Mbs85-targeted mouse ES cell line was generated to study the functional consequences of the observed duplication-based integration mechanism. AAV-modified ES cell lines continued to self-renew, maintained their multilineage differentiation potential and contributed successfully to mouse development when injected into blastocysts. Thus, our study reveals a viral strategy for targeted genome addition with the apparent absence of functional consequences. PMID:19372372

  3. 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.

  4. Mutation-selection models of coding sequence evolution with site-heterogeneous amino acid fitness profiles.

    PubMed

    Rodrigue, Nicolas; Philippe, Hervé; Lartillot, Nicolas

    2010-03-01

    Modeling the interplay between mutation and selection at the molecular level is key to evolutionary studies. To this end, codon-based evolutionary models have been proposed as pertinent means of studying long-range evolutionary patterns and are widely used. However, these approaches have not yet consolidated results from amino acid level phylogenetic studies showing that selection acting on proteins displays strong site-specific effects, which translate into heterogeneous amino acid propensities across the columns of alignments; related codon-level studies have instead focused on either modeling a single selective context for all codon columns, or a separate selective context for each codon column, with the former strategy deemed too simplistic and the latter deemed overparameterized. Here, we integrate recent developments in nonparametric statistical approaches to propose a probabilistic model that accounts for the heterogeneity of amino acid fitness profiles across the coding positions of a gene. We apply the model to a dozen real protein-coding gene alignments and find it to produce biologically plausible inferences, for instance, as pertaining to site-specific amino acid constraints, as well as distributions of scaled selection coefficients. In their account of mutational features as well as the heterogeneous regimes of selection at the amino acid level, the modeling approaches studied here can form a backdrop for several extensions, accounting for other selective features, for variable population size, or for subtleties of mutational features, all with parameterizations couched within population-genetic theory. PMID:20176949

  5. Site-Specific Characterization of d-Amino Acid Containing Peptide Epimers by Ion Mobility Spectrometry

    PubMed Central

    2013-01-01

    Traditionally, the d-amino acid containing peptide (DAACP) candidate can be discovered by observing the differences of biological activity and chromatographic retention time between the synthetic peptides and naturally occurring peptides. However, it is difficult to determine the exact position of d-amino acid in the DAACP candidates. Herein, we developed a novel site-specific strategy to rapidly and precisely localize d-amino acids in peptides by ion mobility spectrometry (IMS) analysis of mass spectrometry (MS)-generated epimeric fragment ions. Briefly, the d/l-peptide epimers were separated by online reversed-phase liquid chromatography and fragmented by collision-induced dissociation (CID), followed by IMS analysis. The epimeric fragment ions resulting from d/l-peptide epimers exhibit conformational differences, thus showing different mobilities in IMS. The arrival time shift between the epimeric fragment ions was used as criteria to localize the d-amino acid substitution. The utility of this strategy was demonstrated by analysis of peptide epimers with different molecular sizes, [d-Trp]-melanocyte-stimulating hormone, [d-Ala]-deltorphin, [d-Phe]-achatin-I, and their counterparts that contain all-l amino acids. Furthermore, the crustacean hyperglycemia hormones (CHHs, 8.5 kDa) were isolated from the American lobster Homarus americanus and identified by integration of MS-based bottom-up and top-down sequencing approaches. The IMS data acquired using our novel site-specific strategy localized the site of isomerization of l- to d-Phe at the third residue of the CHHs from the N-terminus. Collectively, this study demonstrates a new method for discovery of DAACPs using IMS technique with the ability to localize d-amino acid residues. PMID:24328107

  6. Root-Shoot Signaling crosstalk involved in the shoot growth promoting action of rhizospheric humic acids

    PubMed Central

    Olaetxea, Maite; Mora, Verónica; García, Andrés Calderin; Santos, Leandro Azevedo; Baigorri, Roberto; Fuentes, Marta; Garnica, María; Berbara, Ricardo Luis Louro; Zamarreño, Angel Maria; Garcia-Mina, Jose M.

    2016-01-01

    ABSTRACT Numerous studies have shown the ability of humic substances to improve plant development. This action is normally reflected in an enhancement of crop yields and quality. However, the mechanisms responsible for this action of humic substances remain rather unknown. Our studies have shown that the shoot promoting action of sedimentary humic acids is dependent of its ability to increase root hydraulic conductivity through signaling pathways related to ABA, which in turn is affected in roots by humic acids in an IAA-NO dependent way. Furthermore, these studies also indicate that the primary action of humic acids in roots might also be physical, resulting from a transient mild stress caused by humic acids associated with a fouling-cleaning cycle of wall cell pores. Finally the role of alternative signal molecules, such as ROS, and corresponding signaling pathways are also discussed and modeled in the context of the above-mentioned framework. PMID:26966789

  7. Peripheral mechanisms involved in the pressor and bradycardic effects of centrally administered arachidonic acid.

    PubMed

    Aydin, Cenk; Yalcin, Murat

    2008-06-01

    In the current study, we aimed to determine the cardiovascular effects of arachidonic acid and peripheral mechanisms mediated these effects in normotensive conscious rats. Studies were performed in male Sprague Dawley rats. Arachidonic acid was injected intracerebroventricularly (i.c.v.) at the doses of 75, 150 or 300 microg and it caused dose- and time-dependent increase in mean arterial pressure and decrease in heart rate in normal conditions. Maximal effects were observed 10 min after 150 and 300 microg dose of arachidonic acid and lasted within 30 min. In order to evaluate the role of main peripheral hormonal mechanisms in those cardiovascular effects, plasma adrenaline, noradrenaline, vasopressin levels and renin activity were measured after arachidonic acid (150 microg; i.c.v.) injection. Centrally injected arachidonic acid increased plasma levels of all these hormones and renin activity. Intravenous pretreatments with prazosin (0.5 mg/kg), an alpha1 adrenoceptor antagonist, [beta-mercapto-beta,beta-cyclopentamethylenepropionyl1, O-Me-Tyr2-Arg8]-vasopressin (10 microg/kg), a vasopressin V1 receptor antagonist, or saralasin (250 microg/kg), an angiotensin II receptor antagonist, partially blocked the pressor response to arachidonic acid (150 microg; i.c.v.) while combined administration of these three antagonists completely abolished the effect. Moreover, both individual and combined antagonist pretreatments fully blocked the bradycardic effect of arachidonic acid. In conclusion, our findings show that centrally administered arachidonic acid increases mean arterial pressure and decreases heart rate in normotensive conscious rats and the increases in plasma adrenaline, noradrenaline, vasopressin levels and renin activity appear to mediate the cardiovascular effects of the drug. PMID:18571395

  8. Two-step mechanism involving active-site conformational changes regulates human telomerase DNA binding.

    PubMed

    Tomlinson, Christopher G; Moye, Aaron L; Holien, Jessica K; Parker, Michael W; Cohen, Scott B; Bryan, Tracy M

    2015-01-15

    The ribonucleoprotein enzyme telomerase maintains telomeres and is essential for cellular immortality in most cancers. Insight into the telomerase mechanism can be gained from syndromes such as dyskeratosis congenita, in which mutation of telomerase components manifests in telomere dysfunction. We carried out detailed kinetic and thermodynamic analyses of wild-type telomerase and two disease-associated mutations in the reverse transcriptase domain. Differences in dissociation rates between primers with different 3' ends were independent of DNA affinities, revealing that initial binding of telomerase to telomeric DNA occurs through a previously undescribed two-step mechanism involving enzyme conformational changes. Both mutations affected DNA binding, but through different mechanisms: P704S specifically affected protein conformational changes during DNA binding, whereas R865H showed defects in binding to the 3' region of the DNA. To gain further insight at the structural level, we generated the first homology model of the human telomerase reverse transcriptase domain; the positions of P704S and R865H corroborate their observed mechanistic defects, providing validation for the structural model. Our data reveal the importance of protein interactions with the 3' end of telomeric DNA and the role of protein conformational change in telomerase DNA binding, and highlight naturally occurring disease mutations as a rich source of mechanistic insight. PMID:25365545

  9. Phg2, a Kinase Involved in Adhesion and Focal Site Modeling in Dictyostelium

    PubMed Central

    Gebbie, Leigh; Benghezal, Mohammed; Cornillon, Sophie; Froquet, Romain; Cherix, Nathalie; Malbouyres, Marilyne; Lefkir, Yaya; Grangeasse, Christophe; Fache, Sébastien; Dalous, Jérémie; Brückert, Franz; Letourneur, François; Cosson, Pierre

    2004-01-01

    The amoeba Dictyostelium is a simple genetic system for analyzing substrate adhesion, motility and phagocytosis. A new adhesion-defective mutant named phg2 was isolated in this system, and PHG2 encodes a novel serine/threonine kinase with a ras-binding domain. We compared the phenotype of phg2 null cells to other previously isolated adhesion mutants to evaluate the specific role of each gene product. Phg1, Phg2, myosin VII, and talin all play similar roles in cellular adhesion. Like myosin VII and talin, Phg2 also is involved in the organization of the actin cytoskeleton. In addition, phg2 mutant cells have defects in the organization of the actin cytoskeleton at the cell-substrate interface, and in cell motility. Because these last two defects are not seen in phg1, myoVII, or talin mutants, this suggests a specific role for Phg2 in the control of local actin polymerization/depolymerization. This study establishes a functional hierarchy in the roles of Phg1, Phg2, myosinVII, and talin in cellular adhesion, actin cytoskeleton organization, and motility. PMID:15194808

  10. Hygroscopic Behavior of Multicomponent Aerosols Involving NaCl and Dicarboxylic Acids.

    PubMed

    Peng, Chao; Jing, Bo; Guo, Yu-Cong; Zhang, Yun-Hong; Ge, Mao-Fa

    2016-02-25

    Atmospheric aerosols are usually complex mixtures of inorganic and organic compounds. The hygroscopicity of mixed particles is closely related to their chemical composition and interactions between components, which is still poorly understood. In this study, the hygroscopic properties of submicron particles composed of NaCl and dicarboxylic acids including oxalic acid (OA), malonic acid (MA), and succinic acid (SA) with various mass ratios are investigated with a hygroscopicity tandem differential mobility analyzer (HTDMA) system. Both the Zdanovskii-Stokes-Robinson (ZSR) method and extended aerosol inorganics model (E-AIM) are applied to predict the water uptake behaviors of sodium chloride/dicarboxylic acid mixtures. For NaCl/OA mixed particles, the measured growth factors were significantly lower than predictions from the model methods, indicating a change in particle composition caused by chloride depletion. The hygroscopic growth of NaCl/MA particles was well described by E-AIM, and that of NaCl/SA particles was dependent upon mixing ratio. Compared with model predictions, it was determined that water uptake of the NaCl/OA mixture could be enhanced and could be closer to the predictions by addition of levoglucosan or malonic acid, which retained water even at low relative humidity (RH), leading to inhibition of HCl evaporation during dehydration. These results demonstrate that the coexisting hygroscopic species have a strong influence on the phase state of particles, thus affecting chemical interactions between inorganic and organic compounds as well as the overall hygroscopicity of mixed particles. PMID:26820230

  11. Genomic Analysis of Genes Involved in the Biosynthesis of Very Long Chain Polyunsaturated Fatty Acids in Thraustochytrium sp. 26185.

    PubMed

    Zhao, Xianming; Dauenpen, Meesapyodsuk; Qu, Cunmin; Qiu, Xiao

    2016-09-01

    Thraustochytrium sp. 26185 is a marine protist that can produce a large amount of docosahexaenoic acid (DHA, 22:6n-3), an ω3 very long chain polyunsaturated fatty acid (VLCPUFA) of nutritional importance. However, the mechanism of how this fatty acid is synthesized and assembled into the storage lipid triacylglycerol is unclear. Here we report sequencing of the whole genome and genomic analysis of genes involved in the biosynthesis and assembly of the fatty acids in this species. Genome sequencing produced a total of 2,418,734,139 bp clean sequences with about 62 fold genome coverage. Annotation of the genome sequences revealed 10,797 coding genes. Among them, 10,216 genes could be assigned into 25 KOG classes where 451 genes were specifically assigned to the group of lipid transport and metabolism. Detailed analysis of these genes revealed co-existence of both aerobic pathway and anaerobic pathways for the biosynthesis of DHA in this species. However, in the aerobic pathway, a key gene encoding stearate Δ9 desaturase introducing the first double bond to long chain saturated fatty acid 18:0 was missing from the genome. Genomic survey of genes involved in the acyl trafficking among glycerolipids showed that, unlike plants, this protist did not possess phosphatidylcholine:diacylglycerol cholinephosphotransferase, an important enzyme in bridging two types of glycerolipids, diacylglycerols (DAG) and phosphatidylcholines (PtdCho). These results shed new insight on the biosynthesis and assembly of VLCPUFA in the Thraustochytrium. PMID:27514858

  12. FT-IR characterization of the acidic and basic sites on a nanostructured aluminum nitride surface

    SciTech Connect

    Baraton, M.I.; Chen, X.; Gonsalves, K.E.

    1997-12-31

    A nanostructured aluminum nitride powder prepared by sol-gel type chemical synthesis is analyzed by Fourier transform infrared spectrometry. The surface acidic and basic sites are probed out by adsorption of several organic molecules. Resulting from the unavoidable presence of oxygen, the aluminum nitride surface is an oxinitride layer in fact, and its surface chemistry should present some analogies with alumina. Therefore, a thorough comparison between the acido-basicity of aluminum nitride and aluminum oxide is discussed. The remaining nitrogen atoms in the first atomic layer modify the acidity-basicity relative balance and reveals the specificity of the aluminum nitride surface.

  13. Involvement of salicylic acid, glutathione and protein S-thiolation in plant cell death-mediated defence response of Mesembryanthemum crystallinum against Botrytis cinerea.

    PubMed

    Kuźniak, Elżbieta; Kaźmierczak, Andrzej; Wielanek, Marzena; Głowacki, Rafał; Kornas, Andrzej

    2013-02-01

    The response of Mesembryanthemum crystallinum plants performing C3 photosynthesis and crassulacean acid metabolism (CAM) to the non-host necrotrophic pathogen Botrytis cinerea was analyzed at the local and systemic levels. The induction of programmed cell death, lignin and callose deposition, changes in salicylic acid, glutathione and cysteinylglycine pools as well as the content of thiolated proteins were studied. The infected C3 and CAM plants exhibited hypersensitive-like defence response, however fluorescence staining with acridine orange and ethidium bromide revealed programmed cell death events in C3 plants only. The local immune response was not related to callose and lignin deposition. In the infected plants, salicylic acid, glutathione and cysteinylglycine, the first product of glutathione catabolism, as well as protein S-thiolation, predominantly S-glutathionylation, contributed to local defence at sites of inoculation. They (except protein thiolation) were also active in the establishment of systemic acclimation response monitored in the non-treated upper leaves. The extent to which they were involved in the local and systemic responses induced by B. cinerea differed in C3 and CAM plants. The accumulation of free salicylic acid, both in treated and upper leaves of the infected plants, was much more pronounced in CAM plants. The results have been discussed with respect to redox regulations in defence against necrotrophic pathogens and to stress acclimation. PMID:23228550

  14. Nicotinic receptor involvement in regulation of functions of mouse neutrophils from inflammatory site.

    PubMed

    Safronova, Valentina G; Vulfius, Catherine A; Shelukhina, Irina V; Mal'tseva, Valentina N; Berezhnov, Alexey V; Fedotova, Eugeniya I; Miftahova, Regina G; Kryukova, Elena V; Grinevich, Andrey A; Tsetlin, Victor I

    2016-07-01

    Participation of nicotinic acetylcholine receptors (nAChRs) in functioning of polymorphonuclear neutrophils (PMNs) isolated from inflammatory site of mice and expression of different nAChR subunits were studied. Nicotine and acetylcholine (ACh) modified respiratory burst induced by a chemotactic peptide N-formyl-MLF in neutrophils of male (but not female) mice. Antagonists of nAChRs α-cobratoxin (αCTX), α-conotoxins MII and [A10L]PnIA at concentrations of 0.01-5μM, 0.2μM and 1μM, respectively, eliminated nAChR agonist effects. ACh also affected adhesion of PMNs, this effect was also prevented by αCTX (100nM) and MII (1nM). Neutrophils of female mice after chronic nicotine consumption acquired sensitivity to nAChR agonists. Changes of free intracellular Ca(2+) concentration in neutrophils under the action of nAChR ligands were analyzed. In cells with no Ca(2+) oscillations and relatively low resting level of intracellular Ca(2+), nicotine triggered Ca(2+)-spikes, the lag of the response shortened with increasing nicotine concentration. A nicotinic antagonist caramiphen strongly decreased the effect of nicotine. RT-PCR analysis revealed mRNAs of α2, α3, α4, α5, α6, α7, α9, β2, β3, and β4 nAChR subunits. Specific binding of [(125)I]-α-bungarotoxin was demonstrated. Thus in view of the effects and binding characteristics the results obtained suggest a regulatory role of α7, α3β2 or α6* nAChR types in specific functions of PMNs. PMID:26965141

  15. Central estrogen action sites involved in prepubertal restraint of pulsatile luteinizing hormone release in female rats

    PubMed Central

    UENOYAMA, Yoshihisa; TANAKA, Akira; TAKASE, Kenji; YAMADA, Shunji; PHENG, Vutha; INOUE, Naoko; MAEDA, Kei-ichiro; TSUKAMURA, Hiroko

    2015-01-01

    The present study aimed to determine estrogen feedback action sites to mediate prepubertal restraint of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) release in female rats. Wistar-Imamichi strain rats were ovariectomized (OVX) and received a local estradiol-17β (estradiol) or cholesterol microimplant in several brain areas, such as the medial preoptic area (mPOA), paraventricular nucleus, ventromedial nucleus and arcuate nucleus (ARC), at 20 or 35 days of age. Six days after receiving the estradiol microimplant, animals were bled to detect LH pulses at 26 or 41 days of age, representing the pre- or postpubertal period, respectively. Estradiol microimplants in the mPOA or ARC, but not in other brain regions, suppressed LH pulses in prepubertal OVX rats. Apparent LH pulses were found in the postpubertal period in all animals bearing estradiol or cholesterol implants. It is unlikely that pubertal changes in responsiveness to estrogen are due to a change in estrogen receptor (ER) expression, because the number of ERα-immunoreactive cells and mRNA levels of Esr1, Esr2 and Gpr30 in the mPOA and ARC were comparable between the pre- and postpubertal periods. In addition, kisspeptin or GnRH injection overrode estradiol-dependent prepubertal LH suppression, suggesting that estrogen inhibits the kisspeptin-GnRH cascade during the prepubertal period. Thus, estrogen-responsive neurons located in the mPOA and ARC may play key roles in estrogen-dependent prepubertal restraint of GnRH/LH secretion in female rats. PMID:26004302

  16. The control of acid mine drainage at the Summitville Mine Superfund Site

    SciTech Connect

    Ketellapper, V.L.; Williams, L.O.

    1996-11-01

    The Summitville Mine Superfund Site is located about 25 miles south of Del Norte, Colorado, in Rio Grande County. Occurring at an average elevation of 11,500 feet in the San Juan Mountain Range, the mine site is located two miles east of the Continental Divide. Mining at Summitville has occurred since 1870. The mine was most recently operated by Summitville Consolidated Mining Company, Inc. (SCMCI) as an open pit gold mine with extraction by means of a cyanide leaching process. In December of 1992, SCMCI declared bankruptcy and vacated the mine site. At that time, the US Environmental Protection Agency (EPA) took over operations of the water treatment facilities to prevent a catastrophic release of cyanide and metal-laden water from the mine site. Due to high operational costs of water treatment (approximately $50,000 per day), EPA established a goal to minimize active water treatment by reducing or eliminating acid mine drainage (AMD). All of the sources of AMD generation on the mine site were evaluated and prioritized. Of the twelve areas identified as sources of AMD, the Cropsy Waste Pile, the Summitville Dam Impoundment, the Beaver Mud Dump, the Reynolds and Chandler adits, and the Mine Pits were consider to be the most significant contributors to the generation of metal-laden acidic (low pH) water. A two part plan was developed to control AMD from the most significant sources. The first part was initiated immediately to control AMD being released from the Site. This part focused on improving the efficiency of the water treatment facilities and controlling the AMD discharges from the mine drainage adits. The second part of the plan was aimed at reducing the AMD generated in groundwater and surface water runoff from the mine wastes. A lined and capped repository located in the mine pits for acid generating mining waste and water treatment plant sludge was found to be the most feasible alternative.

  17. Polymorphisms involving gain or loss of CpG sites are significantly enriched in trait-associated SNPs

    PubMed Central

    Zhou, Dan; Li, Zhenli; Yu, Dan; Wan, Ledong; Zhu, Yimin; Lai, Maode; Zhang, Dandan

    2015-01-01

    Some single nucleotide polymorphisms (SNPs) influence the existence of CpG sites, the basis of DNA modification such as methylation and hydroxymethylation. These polymorphisms can lead to gain or loss of CpG sites and were defined as CpG site related SNPs (cgSNPs) in this study. The cgSNPs change DNA sequence and might potentially affect DNA modification such as methylation. However, the functional consequence of cgSNPs is poorly understood. We observed that a considerable proportion (23.0%) of common variants were cgSNPs in human genome. Mutations involving loss of CpG sites were associated with reduced levels of methylation (~20.2%) using The Cancer Genome Atlas (TCGA) data. Using public databases (SCAN and seeQTL) of expression quantitative trait loci (eQTLs), we found that the cgSNPs were significantly enriched in eQTLs via logistic regression and simulation test. Furthermore, we observed that cgSNPs were more likely to be trait-associated loci especially cancers using a catalog of published genome-wide association studies (GWAS) recorded by National Human Genome Research Institute (NHGRI). Our results indicated that cgSNP might be meaningful as annotation either in SNP functional prediction or in screening for trait-associated SNPs. PMID:26503467

  18. Generation of Food-Grade Recombinant Lactic Acid Bacterium Strains by Site-Specific Recombination

    PubMed Central

    Martín, M. Cruz; Alonso, Juan C.; Suárez, Juan E.; Alvarez, Miguel A.

    2000-01-01

    The construction of a delivery and clearing system for the generation of food-grade recombinant lactic acid bacterium strains, based on the use of an integrase (Int) and a resolvo-invertase (β-recombinase) and their respective target sites (attP-attB and six, respectively) is reported. The delivery system contains a heterologous replication origin and antibiotic resistance markers surrounded by two directly oriented six sites, a multiple cloning site where passenger DNA could be inserted (e.g., the cI gene of bacteriophage A2), the int gene, and the attP site of phage A2. The clearing system provides a plasmid-borne gene encoding β-recombinase. The nonreplicative vector-borne delivery system was transformed into Lactobacillus casei ATCC 393 and, by site-specific recombination, integrated as a single copy in an orientation- and Int-dependent manner into the attB site present in the genome of the host strain. The transfer of the clearing system into this strain, with the subsequent expression of the β-recombinase, led to site-specific DNA resolution of the non-food-grade DNA. These methods were validated by the construction of a stable food-grade L. casei ATCC 393-derived strain completely immune to phage A2 infection during milk fermentation. PMID:10831443

  19. Estimates of cloud water deposition at Mountain Acid Deposition Program sites in the Appalachian Mountains.

    PubMed

    Baumgardner, Ralph E; Isil, Selma S; Lavery, Thomas F; Rogers, Christopher M; Mohnen, Volker A

    2003-03-01

    Cloud water deposition was estimated at three high-elevation sites in the Appalachian Mountains of the eastern United States (Whiteface Mountain, NY; Whitetop Mountain, VA; and Clingman's Dome, TN) from 1994 through 1999 as part of the Mountain Acid Deposition Program (MADPro). This paper provides a summary of cloud water chemistry, cloud liquid water content, cloud frequency, estimates of cloud water deposition of sulfur and nitrogen species, and estimates of total deposition of sulfur and nitrogen at these sites. Other cloud studies in the Appalachians and their comparison to MADPro are also summarized. Whiteface Mountain exhibited the lowest mean and median concentrations of sulfur and nitrogen ions in cloud water, while Clingman's Dome exhibited the highest mean and median concentrations. This geographic gradient is partly an effect of the different meteorological conditions experienced at northern versus southern sites in addition to the difference in pollution content of air masses reaching the sites. All sites measured seasonal cloud water deposition rates of SO4(2-) greater than 50 kg/ha and NO3(-) rates of greater than 25 kg/ha. These high-elevation sites experienced additional deposition loading of SO4(2-) and NO3(-) on the order of 6-20 times greater compared with lower elevation Clean Air Status and Trends Network (CASTNet) sites. Approximately 80-90% of this extra loading is from cloud deposition. PMID:12661689

  20. Distinguishing two groups of flavin reductases by analyzing the protonation state of an active site carboxylic acid.

    PubMed

    Dumit, Verónica I; Cortez, Néstor; Matthias Ullmann, G

    2011-07-01

    Flavin-containing reductases are involved in a wide variety of physiological reactions such as photosynthesis, nitric oxide synthesis, and detoxification of foreign compounds, including therapeutic drugs. Ferredoxin-NADP(H)-reductase (FNR) is the prototypical enzyme of this family. The fold of this protein is highly conserved and occurs as one domain of several multidomain enzymes such as the members of the diflavin reductase family. The enzymes of this family have emerged as fusion of a FNR and a flavodoxin. Although the active sites of these enzymes are very similar, different enzymes function in opposite directions, that is, some reduce oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)) and some oxidize reduced nicotinamide adenine dinucleotide phosphate (NADPH). In this work, we analyze the protonation behavior of titratable residues of these enzymes through electrostatic calculations. We find that a highly conserved carboxylic acid in the active site shows a different titration behavior in different flavin reductases. This residue is deprotonated in flavin reductases present in plastids, but protonated in bacterial counterparts and in diflavin reductases. The protonation state of the carboxylic acid may also influence substrate binding. The physiological substrate for plastidic enzymes is NADP(+), but it is NADPH for the other mentioned reductases. In this article, we discuss the relevance of the environment of this residue for its protonation and its importance in catalysis. Our results allow to reinterpret and explain experimental data. PMID:21538544

  1. HIV-1 Nef Induces Proinflammatory State in Macrophages through Its Acidic Cluster Domain: Involvement of TNF Alpha Receptor Associated Factor 2

    PubMed Central

    Fiorucci, Gianna; Vaccari, Gabriele; Acconcia, Filippo; Chiarabelli, Cristiano; Leone, Stefano; Noto, Alessia; Horenkamp, Florian A.; Manrique, Santiago; Romeo, Giovanna; Polticelli, Fabio; Geyer, Matthias; Affabris, Elisabetta

    2011-01-01

    Background HIV-1 Nef is a virulence factor that plays multiple roles during HIV replication. Recently, it has been described that Nef intersects the CD40 signalling in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit, activate and render T lymphocytes susceptible to HIV infection. The engagement of CD40 by CD40L induces the activation of different signalling cascades that require the recruitment of specific tumor necrosis factor receptor-associated factors (i.e. TRAFs). We hypothesized that TRAFs might be involved in the rapid activation of NF-κB, MAPKs and IRF-3 that were previously described in Nef-treated macrophages to induce the synthesis and secretion of proinflammatory cytokines, chemokines and IFNβ to activate STAT1, -2 and -3. Methodology/Principal Findings Searching for possible TRAF binding sites on Nef, we found a TRAF2 consensus binding site in the AQEEEE sequence encompassing the conserved four-glutamate acidic cluster. Here we show that all the signalling effects we observed in Nef treated macrophages depend on the integrity of the acidic cluster. In addition, Nef was able to interact in vitro with TRAF2, but not TRAF6, and this interaction involved the acidic cluster. Finally silencing experiments in THP-1 monocytic cells indicate that both TRAF2 and, surprisingly, TRAF6 are required for the Nef-induced tyrosine phosphorylation of STAT1 and STAT2. Conclusions Results reported here revealed TRAF2 as a new possible cellular interactor of Nef and highlighted that in monocytes/macrophages this viral protein is able to manipulate both the TRAF/NF-κB and TRAF/IRF-3 signalling axes, thereby inducing the synthesis of proinflammatory cytokines and chemokines as well as IFNβ. PMID:21886773

  2. Novel Hydroxycinnamoyl-Coenzyme A Quinate Transferase Genes from Artichoke Are Involved in the Synthesis of Chlorogenic Acid1[W

    PubMed Central

    Sonnante, Gabriella; D'Amore, Rosalinda; Blanco, Emanuela; Pierri, Ciro L.; De Palma, Monica; Luo, Jie; Tucci, Marina; Martin, Cathie

    2010-01-01

    Artichoke (Cynara cardunculus subsp. scolymus) extracts have high antioxidant capacity, due primarily to flavonoids and phenolic acids, particularly chlorogenic acid (5-caffeoylquinic acid [CGA]), dicaffeoylquinic acids, and caffeic acid, which are abundant in flower bracts and bioavailable to humans in the diet. The synthesis of CGA can occur following different routes in plant species, and hydroxycinnamoyl-coenzyme A transferases are important enzymes in these pathways. Here, we report on the isolation and characterization of two novel genes both encoding hydroxycinnamoyl-coenzyme A quinate transferases (HQT) from artichoke. The recombinant proteins (HQT1 and HQT2) were assayed after expression in Escherichia coli, and both showed higher affinity for quinate over shikimate. Their preferences for acyl donors, caffeoyl-coenzyme A or p-coumaroyl-coenzyme A, were examined. Modeling and docking analyses were used to propose possible pockets and residues involved in determining substrate specificities in the HQT enzyme family. Quantitative real-time polymerase chain reaction analysis of gene expression indicated that HQT1 might be more directly associated with CGA content. Transient and stable expression of HQT1 in Nicotiana resulted in a higher production of CGA and cynarin (1,3-dicaffeoylquinic acid). These findings suggest that several isoforms of HQT contribute to the synthesis of CGA in artichoke according to physiological needs and possibly following various metabolic routes. PMID:20431089

  3. Long-chain acyl-CoA synthetase in fatty acid metabolism involved in liver and other diseases: An update

    PubMed Central

    Yan, Sheng; Yang, Xue-Feng; Liu, Hao-Lei; Fu, Nian; Ouyang, Yan; Qing, Kai

    2015-01-01

    Long-chain acyl-CoA synthetase (ACSL) family members include five different ACSL isoforms, each encoded by a separate gene and have multiple spliced variants. ACSLs on endoplasmic reticulum and mitochondrial outer membrance catalyze fatty acids with chain lengths from 12 to 20 carbon atoms to form acyl-CoAs, which are lipid metabolic intermediates and involved in fatty acid metabolism, membrane modifications and various physiological processes. Gain- or loss-of-function studies have shown that the expression of individual ACSL isoforms can alter the distribution and amount of intracellular fatty acids. Changes in the types and amounts of fatty acids, in turn, can alter the expression of intracellular ACSLs. ACSL family members affect not only the proliferation of normal cells, but the proliferation of malignant tumor cells. They also regulate cell apoptosis through different signaling pathways and molecular mechanisms. ACSL members have individual functions in fatty acid metabolism in different types of cells depending on substrate preferences, subcellular location and tissue specificity, thus contributing to liver diseases and metabolic diseases, such as fatty liver disease, obesity, atherosclerosis and diabetes. They are also linked to neurological disorders and other diseases. However, the mechanisms are unclear. This review addresses new findings in the classification and properties of ACSLs and the fatty acid metabolism-associated effects of ACSLs in diseases. PMID:25834313

  4. Attenuated murine cytomegalovirus binds to N-acetylglucosamine, and shift to virulence may involve recognition of sialic acids.

    PubMed Central

    Ravindranath, R M; Graves, M C

    1990-01-01

    Treatment of cells with lectins specific for N-acetylglucosamine (GlcNAc) blocked infection by mouse cytomegalovirus (MCMV), and GlcNAc pretreatment of the lectin blocked this effect. MCMV failed to infect N-acetylglucosaminidase (GlcNAcase)-treated mouse embryo fibroblasts (MEF). GlcNAc and GlcNAc-containing synthetic oligosaccharides directly inhibited viral infectivity. Ulex lectin inhibition of infection was shown to be due to inhibition of surface adsorption of 35S-labeled virus. Also, GlcNAcase eluted 35S-labeled virus adsorbed to MEF at 4 degrees C and inhibited plaque formation if added after adsorption at this temperature. These findings indicate that GlcNAc binding is involved in attachment rather than in some later step in infection. High-performance thin-layer chromatography overlay of [35S]MCMV indicated that it binds to a GlcNAc-containing asialoglycolipid. Analogous experiments indicated that MCMV made virulent by in vivo salivary gland passage binds to sialic acids in addition to GlcNAc. Treatment of MEF with sialic acid-binding lectins blocked infectivity. Incubation of virus with sialic acids also prevented infection. N-acetylneuraminic acid was 10(3)-fold more potent than N-glycolylneuraminic acid. Sialidase-treated target cells were not efficiently infected by the virus. Thus, MCMV binds to GlcNAc on the cell surface, and the shift to virulence (by in vivo salivary gland passage) correlates with viral recognition of sialic acids. Images PMID:2170680

  5. Possible Segregated Ice at the Phoenix Landing Site: Was Liquid Water Involved?

    NASA Astrophysics Data System (ADS)

    Stoker, C.; Blaney, D.; Hecht, M.; Catling, D.; Pike, W. T.; Mellon, M.; Kounaves, S.; Lemmon, M.

    2008-12-01

    Lander cameras on the Phoenix mission revealed polygonal terrain at the landing site. Areas identified by topography within the work area of the arm included a polygon and a surrounding trough. Two trenches were dug, the first (Goldilocks) on the shoulder of a trough area exposed a bright, hard material and the second (Snow white) in the center of the polygon exposed hard material, but with multispectral properties indistinguishable from soil. Visibile-NIR spectra of the Goldilocks bright material are consistent with slightly dusty ice. When first exposed, a 2 cm chunk of material broke off and was observed to completely disappear in 3 sols, an implied sublmation rate of 100 micrometers per hour. We hypothesize that the Goldilocks bright material is segregated ice. The material is hard, localized, has distinct edges, and was initially covered with only 3 cm of soil, thus was 2cm shallower than the hard layer in the Snow white trench in spite of a more south-facing exposure. A trench dug 40 cm further south of Goldilocks, with similar orientation, reached 18 cm depth without encountering hard material. Plausible mechanisms for emplacement of segregated ice include liquid water pooled into a thermally-produced crack analogous to terrestrial ice wedge polygon formation, snowparticles depositing preferentially in the troughs, and vapor deposition preferentially into cracks (D. Fisher, Icarus 179, 387, 2005). Mission observations were performed relevant to evaluating these formation mechanisms. Wet chemistry analyses of soils suggest they contain Mg(ClO4)2, a soluble hygroscopic salt with a eutectic freezing point of /- 68C. If liquid water moved though the soil and formed the bright deposit in Goldilocks trench, a higher concentration of perchlorate would be expected in the area of the ice. Mg(ClO4)2. 6 H2O would crystallize when the salty water froze, forming white rhombohedral crystals. After scraping away the surface soil, approximately 500 cm2of bright material was

  6. Disulfide bridge regulates ligand-binding site selectivity in liver bile acid-binding proteins.

    PubMed

    Cogliati, Clelia; Tomaselli, Simona; Assfalg, Michael; Pedò, Massimo; Ferranti, Pasquale; Zetta, Lucia; Molinari, Henriette; Ragona, Laura

    2009-10-01

    Bile acid-binding proteins (BABPs) are cytosolic lipid chaperones that play central roles in driving bile flow, as well as in the adaptation to various pathological conditions, contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Understanding the mode of binding of bile acids with their cytoplasmic transporters is a key issue in providing a model for the mechanism of their transfer from the cytoplasm to the nucleus, for delivery to nuclear receptors. A number of factors have been shown to modulate bile salt selectivity, stoichiometry, and affinity of binding to BABPs, e.g. chemistry of the ligand, protein plasticity and, possibly, the formation of disulfide bridges. Here, the effects of the presence of a naturally occurring disulfide bridge on liver BABP ligand-binding properties and backbone dynamics have been investigated by NMR. Interestingly, the disulfide bridge does not modify the protein-binding stoichiometry, but has a key role in modulating recognition at both sites, inducing site selectivity for glycocholic and glycochenodeoxycholic acid. Protein conformational changes following the introduction of a disulfide bridge are small and located around the inner binding site, whereas significant changes in backbone motions are observed for several residues distributed over the entire protein, both in the apo form and in the holo form. Site selectivity appears, therefore, to be dependent on protein mobility rather than being governed by steric factors. The detected properties further establish a parallelism with the behaviour of human ileal BABP, substantiating the proposal that BABPs have parallel functions in hepatocytes and enterocytes. PMID:19754879

  7. Site-specific analysis of protein hydration based on unnatural amino acid fluorescence.

    PubMed

    Amaro, Mariana; Brezovský, Jan; Kováčová, Silvia; Sýkora, Jan; Bednář, David; Němec, Václav; Lišková, Veronika; Kurumbang, Nagendra Prasad; Beerens, Koen; Chaloupková, Radka; Paruch, Kamil; Hof, Martin; Damborský, Jiří

    2015-04-22

    Hydration of proteins profoundly affects their functions. We describe a simple and general method for site-specific analysis of protein hydration based on the in vivo incorporation of fluorescent unnatural amino acids and their analysis by steady-state fluorescence spectroscopy. Using this method, we investigate the hydration of functionally important regions of dehalogenases. The experimental results are compared to findings from molecular dynamics simulations. PMID:25815779

  8. Multifunctional oxidosqualene cyclases and cytochrome P450 involved in the biosynthesis of apple fruit triterpenic acids.

    PubMed

    Andre, Christelle M; Legay, Sylvain; Deleruelle, Amélie; Nieuwenhuizen, Niels; Punter, Matthew; Brendolise, Cyril; Cooney, Janine M; Lateur, Marc; Hausman, Jean-François; Larondelle, Yvan; Laing, William A

    2016-09-01

    Apple (Malus × domestica) accumulates bioactive ursane-, oleanane-, and lupane-type triterpenes in its fruit cuticle, but their biosynthetic pathway is still poorly understood. We used a homology-based approach to identify and functionally characterize two new oxidosqualene cyclases (MdOSC4 and MdOSC5) and one cytochrome P450 (CYP716A175). The gene expression patterns of these enzymes and of previously described oxidosqualene cyclases were further studied in 20 apple cultivars with contrasting triterpene profiles. MdOSC4 encodes a multifunctional oxidosqualene cyclase producing an oleanane-type triterpene, putatively identified as germanicol, as well as β-amyrin and lupeol, in the proportion 82 : 14 : 4. MdOSC5 cyclizes 2,3-oxidosqualene into lupeol and β-amyrin at a ratio of 95 : 5. CYP716A175 catalyses the C-28 oxidation of α-amyrin, β-amyrin, lupeol and germanicol, producing ursolic acid, oleanolic acid, betulinic acid, and putatively morolic acid. The gene expression of MdOSC1 was linked to the concentrations of ursolic and oleanolic acid, whereas the expression of MdOSC5 was correlated with the concentrations of betulinic acid and its caffeate derivatives. Two new multifuntional triterpene synthases as well as a multifunctional triterpene C-28 oxidase were identified in Malus × domestica. This study also suggests that MdOSC1 and MdOSC5 are key genes in apple fruit triterpene biosynthesis. PMID:27214242

  9. Magnesium Lewis Acid Assisted Oxidative Bromoetherification Involving Bromine Transfer from Alkyl Bromides with Aldehydes by Umpolung of Bromide.

    PubMed

    Moriyama, Katsuhiko; Nishinohara, Chihiro; Togo, Hideo

    2016-08-16

    An oxidative bromoetherification involving a bromine transfer from alkyl bromides upon reacting them with aldehydes in a Grignard reaction with a concurrent oxidation of bromide was developed to provide substituted tetrahydrofurans in high yields. This reaction, which proceeds through two types of bromine transfer, was promoted by the addition of a Brønsted acid. Mechanistic studies suggested that a magnesium Lewis acid activates hypobromate, which is generated in situ from the reaction of bromide and Oxone to improve the electrophilicity of the bromonium ion (Br(+) ) for the oxidative bromoetherification of alkenyl alcohols. Furthermore, the magnesium Lewis acid catalyzed oxidative bromoetherification of an alkenyl alcohol proceeded to provide a cyclization product in 92 % yield. PMID:27304660

  10. Involvement of a universal amino acid synthesis impediment in cytoplasmic male sterility in pepper.

    PubMed

    Fang, Xianping; Fu, Hong-Fei; Gong, Zhen-Hui; Chai, Wei-Guo

    2016-01-01

    To explore the mechanisms of pepper (Capsicum annuum L.) cytoplasmic male sterility (CMS), we studied the different maturation processes of sterile and fertile pepper anthers. A paraffin section analysis of the sterile anthers indicated an abnormality of the tapetal layer and an over-vacuolization of the cells. The quantitative proteomics results showed that the expression of histidinol dehydrogenase (HDH), dihydroxy-acid dehydratase (DAD), aspartate aminotransferase (ATAAT), cysteine synthase (CS), delta-1-pyrroline-5-carboxylate synthase (P5CS), and glutamate synthetase (GS) in the amino acid synthesis pathway decreased by more than 1.5-fold. Furthermore, the mRNA and protein expression levels of DAD, ATAAT, CS and P5CS showed a 2- to 16-fold increase in the maintainer line anthers. We also found that most of the amino acid content levels decreased to varying degrees during the anther tapetum period of the sterile line, whereas these levels increased in the maintainer line. The results of our study indicate that during pepper anther development, changes in amino acid synthesis are significant and accompany abnormal tapetum maturity, which is most likely an important cause of male sterility in pepper. PMID:26987793

  11. Fatty acid biosynthesis is involved in the production of hepatitis B virus particles.

    PubMed

    Okamura, Hitomi; Nio, Yasunori; Akahori, Yuichi; Kim, Sulyi; Watashi, Koichi; Wakita, Takaji; Hijikata, Makoto

    2016-06-17

    Hepatitis B virus (HBV) proliferates in hepatocytes after infection, but the host factors that contribute to the HBV lifecycle are poorly understood at the molecular level. We investigated whether fatty acid biosynthesis (FABS), which was recently reported to contribute to the genomic replication of hepatitis C virus, plays a role in HBV proliferation. We examined the effects of inhibitors of the enzymes in the FABS pathway on the HBV lifecycle by using recombinant HBV-producing cultured cells and found that the extracellular HBV DNA level, reflecting HBV particle production, was decreased by treatment with inhibitors suppressed the synthesis of long-chain saturated fatty acids with little cytotoxicity. The reduced HBV DNA level was reversed when palmitic acid, which is the product of fatty acid synthase (FAS) during FABS, was used simultaneously with the inhibitor. We also observed that the amount of intracellular HBV DNA in the cells was increased by FAS inhibitor treatment, suggesting that FABS is associated with HBV particle production but not its genome replication. This suggests that FABS might be a potent target for anti-HBV drug with a mode of action different from current HBV therapy. PMID:27178211

  12. Involvement of a universal amino acid synthesis impediment in cytoplasmic male sterility in pepper

    PubMed Central

    Fang, Xianping; Fu, Hong-Fei; Gong, Zhen-Hui; Chai, Wei-Guo

    2016-01-01

    To explore the mechanisms of pepper (Capsicum annuum L.) cytoplasmic male sterility (CMS), we studied the different maturation processes of sterile and fertile pepper anthers. A paraffin section analysis of the sterile anthers indicated an abnormality of the tapetal layer and an over-vacuolization of the cells. The quantitative proteomics results showed that the expression of histidinol dehydrogenase (HDH), dihydroxy-acid dehydratase (DAD), aspartate aminotransferase (ATAAT), cysteine synthase (CS), delta-1-pyrroline-5-carboxylate synthase (P5CS), and glutamate synthetase (GS) in the amino acid synthesis pathway decreased by more than 1.5-fold. Furthermore, the mRNA and protein expression levels of DAD, ATAAT, CS and P5CS showed a 2- to 16-fold increase in the maintainer line anthers. We also found that most of the amino acid content levels decreased to varying degrees during the anther tapetum period of the sterile line, whereas these levels increased in the maintainer line. The results of our study indicate that during pepper anther development, changes in amino acid synthesis are significant and accompany abnormal tapetum maturity, which is most likely an important cause of male sterility in pepper. PMID:26987793

  13. Mechanisms of perfluoroalkyl acid (PFAA) toxicity: Involvement of peroxisome proliferator activator receptor alpha (PPAR) molecular signals.

    EPA Science Inventory

    Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) are members of a family of environmentally persistent perfluorinated compounds and are found in the serum of wildlife and humans. PFOS and PFOA are developmentally toxic in rats and mice. Exposure in utero reduces...

  14. Characterization of an N-system amino acid transporter expressed in retina and its involvement in glutamine transport.

    PubMed

    Gu, S; Roderick, H L; Camacho, P; Jiang, J X

    2001-06-29

    We report here on the characterization of a mouse N-system amino acid transporter protein, which is involved in the transport of glutamine. This protein of 485 amino acids shares 52% sequence homology with an N-system amino acid transporter, mouse N-system amino acid transporter (mNAT) and its orthologs. Because this protein shares a high degree of sequence homology and functional similarity to mNAT, we named it mNAT2. mNAT2 is predominately expressed in the retina and to a slightly lesser extent in the brain. In the retina, it is located in the axons of ganglion cells in the nerve fiber layer and in the bundles of the optic nerve. Functional analysis of mNAT2 expressed in Xenopus oocytes revealed that the strongest transport activities were specific for l-glutamine. In addition, mNAT2 is a Na(+)- and pH-dependent, high affinity transporter and partially tolerates substitution of Na(+) by Li(+). Additionally, mNAT2 functions as a carrier-mediated transporter that facilitates efflux. The unique expression pattern and selective glutamine transport properties of mNAT2 suggest that it plays a specific role in the uptake of glutamine involved in the generation of the neurotransmitter glutamate in retina. PMID:11325958

  15. Two separate key enzymes and two pathway-specific transcription factors are involved in fusaric acid biosynthesis in Fusarium fujikuroi.

    PubMed

    Studt, Lena; Janevska, Slavica; Niehaus, Eva-Maria; Burkhardt, Immo; Arndt, Birgit; Sieber, Christian M K; Humpf, Hans-Ulrich; Dickschat, Jeroen S; Tudzynski, Bettina

    2016-03-01

    Fusaric acid (FSA) is a mycotoxin produced by several fusaria, including the rice pathogen Fusarium fujikuroi. Genes involved in FSA biosynthesis were previously identified as a cluster containing a polyketide synthase (PKS)-encoding (FUB1) and four additional genes (FUB2-FUB5). However, the biosynthetic steps leading to FSA as well as the origin of the nitrogen atom, which is incorporated into the polyketide backbone, remained unknown. In this study, seven additional cluster genes (FUB6-FUB12) were identified via manipulation of the global regulator FfSge1. The extended FUB gene cluster encodes two Zn(II)2 Cys6 transcription factors: Fub10 positively regulates expression of all FUB genes, whereas Fub12 is involved in the formation of the two FSA derivatives, i.e. dehydrofusaric acid and fusarinolic acid, serving as a detoxification mechanism. The major facilitator superfamily transporter Fub11 functions in the export of FSA out of the cell and is essential when FSA levels become critical. Next to Fub1, a second key enzyme was identified, the non-canonical non-ribosomal peptide synthetase Fub8. Chemical analyses of generated mutant strains allowed for the identification of a triketide as PKS product and the proposition of an FSA biosynthetic pathway, thereby unravelling the unique formation of a hybrid metabolite consisting of this triketide and an amino acid moiety. PMID:26662839

  16. Ionization-site effects on the photofragmentation of chloro- and bromoacetic acid molecules

    NASA Astrophysics Data System (ADS)

    Levola, Helena; Itälä, Eero; Schlesier, Kim; Kooser, Kuno; Laine, Sanna; Laksman, Joakim; Ha, Dang Trinh; Rachlew, Elisabeth; Tarkanovskaja, Marta; Tanzer, Katrin; Kukk, Edwin

    2015-12-01

    Fragmentation of gas-phase chloro- and bromoacetic acid samples, particularly its dependency on the atomic site of the initial core ionization, was studied in photoelectron-photoion-photoion coincidence (PEPIPICO) measurements. The fragmentation was investigated after ionizing carbon 1 s and bromine 3 d or chlorine 2 p core orbitals. It was observed that the samples had many similar fragmentation pathways and that their relative weights depended strongly on the initial ionization site. Additional Auger PEPIPICO measurements revealed a clear dependence of fragment pair intensities on the kinetic energy of the emitted Auger electrons. The modeled and measured Auger electron spectra indicated that the average internal energy of the molecule was larger following the carbon 1 s core-hole decay than after the decay of the halogen core hole. This difference in the internal energies was found to be the source of the site-dependent photofragmentation behavior.

  17. Remaining Sites Verification Package for the 120-B-1, 105-B Battery Acid Sump, Waste Site Reclassification Form 2006-057

    SciTech Connect

    L. M. Dittmer

    2006-09-25

    The 120-B-1 waste site, located in the 100-BC-1 Operable Unit of the Hanford Site, consisted of a concrete battery acid sump that was used from 1944 to 1969 to neutralize the spent sulfuric acid from lead cell batteries of emergency power packs and the emergency lighting system. The battery acid sump was associated with the 105-B Reactor Building and was located adjacent to the building's northwest corner. The results of verification sampling demonstrated that residual contaminant concentrations do not preclude any future uses and allow for unrestricted use of shallow zone soils. The results also showed that residual contaminant concentrations are protective of groundwater and the Columbia River.

  18. Involved-Site Image-Guided Intensity Modulated Versus 3D Conformal Radiation Therapy in Early Stage Supradiaphragmatic Hodgkin Lymphoma

    SciTech Connect

    Filippi, Andrea Riccardo; Ciammella, Patrizia; Piva, Cristina; Ragona, Riccardo; Botto, Barbara; Gavarotti, Paolo; Merli, Francesco; Vitolo, Umberto; Iotti, Cinzia; Ricardi, Umberto

    2014-06-01

    Purpose: Image-guided intensity modulated radiation therapy (IG-IMRT) allows for margin reduction and highly conformal dose distribution, with consistent advantages in sparing of normal tissues. The purpose of this retrospective study was to compare involved-site IG-IMRT with involved-site 3D conformal RT (3D-CRT) in the treatment of early stage Hodgkin lymphoma (HL) involving the mediastinum, with efficacy and toxicity as primary clinical endpoints. Methods and Materials: We analyzed 90 stage IIA HL patients treated with either involved-site 3D-CRT or IG-IMRT between 2005 and 2012 in 2 different institutions. Inclusion criteria were favorable or unfavorable disease (according to European Organization for Research and Treatment of Cancer criteria), complete response after 3 to 4 cycles of an adriamycin- bleomycin-vinblastine-dacarbazine (ABVD) regimen plus 30 Gy as total radiation dose. Exclusion criteria were chemotherapy other than ABVD, partial response after ABVD, total radiation dose other than 30 Gy. Clinical endpoints were relapse-free survival (RFS) and acute toxicity. Results: Forty-nine patients were treated with 3D-CRT (54.4%) and 41 with IG-IMRT (45.6%). Median follow-up time was 54.2 months for 3D-CRT and 24.1 months for IG-IMRT. No differences in RFS were observed between the 2 groups, with 1 relapse each. Three-year RFS was 98.7% for 3D-CRT and 100% for IG-IMRT. Grade 2 toxicity events, mainly mucositis, were recorded in 32.7% of 3D-CRT patients (16 of 49) and in 9.8% of IG-IMRT patients (4 of 41). IG-IMRT was significantly associated with a lower incidence of grade 2 acute toxicity (P=.043). Conclusions: RFS rates at 3 years were extremely high in both groups, albeit the median follow-up time is different. Acute tolerance profiles were better for IG-IMRT than for 3D-CRT. Our preliminary results support the clinical safety and efficacy of advanced RT planning and delivery techniques in patients affected with early stage HL, achieving complete

  19. Structural and Functional Characterization of PseC, an Aminotransferase Involved in the Biosynthesis of Pseudaminic Acid, an Essential Flagellar Modification in Helicobacter Pylori

    SciTech Connect

    Schoenhofen,I.; Lunin, V.; Julien, J.; Li, Y.; Ajamian, E.; Matte, A.; Cygler, M.; Brisson, J.; Aubry, A.; et al.

    2006-01-01

    Helicobacter pylori flagellin is heavily glycosylated with the novel sialic acid-like nonulosonate, pseudaminic acid (Pse). The glycosylation process is essential for assembly of functional flagellar filaments and consequent bacterial motility. As motility is a key virulence factor for this and other important pathogens, the Pse biosynthetic pathway offers potential for novel therapeutic targets. From recent NMR analyses, we determined that the conversion of UDP-a-D-GlcNAc to the central intermediate in the pathway, UDP-4-amino-4,6-dideoxy-{beta}-L-AltNAc, proceeds by formation of UDP-2-acetamido-2,6-dideoxy-{beta}-L-arabino-4-hexulose by the dehydratase/epimerase PseB (HP0840) followed with amino transfer by the aminotransferase, PseC (HP0366). The central role of PseC in the H. pylori Pse biosynthetic pathway prompted us to determine crystal structures of the native protein, its complexes with pyridoxal phosphate alone and in combination with the UDP-4-amino-4,6-dideoxy-{beta}-L-AltNAc product, the latter being converted to the external aldimine form in the enzyme's active site. In the binding site, the AltNAc sugar ring adopts a 4C1 chair conformation which is different from the predominant 1C4 form found in solution. The enzyme forms a homodimer where each monomer contributes to the active site, and these structures have permitted the identification of key residues involved in stabilization, and possibly catalysis, of the {beta}-L-arabino intermediate during the amino transfer reaction. The essential role of Lys183 in the catalytic event was confirmed by site-directed mutagenesis. This work presents for the first time a nucleotide-sugar aminotransferase co-crystallized with its natural ligand, and in conjunction with the recent functional characterization of this enzyme, will assist in elucidating the aminotransferase reaction mechanism within the Pse biosynthetic pathway.

  20. Microbial Communities in Biofilms of an Acid Mine Drainage Site Determined by Phospholipid Analysis

    NASA Astrophysics Data System (ADS)

    Das Gupta, S.; Fang, J.

    2008-12-01

    Phospholipids were extracted to determine the microbial biomass and community structure of biofims from an acid mine drainage (AMD) at the Green Valley coal mine site (GVS) in western Indiana. The distribution of specific biomarkers indicated the presence of a variety of microorganisms. Phototrophic microeukaryotes, which include Euglena mutabilis, algae, and cyanobacteria were the most dominant organisms, as indicated by the presence of polyunsaturated fatty acids. The presence of terminally methyl branched fatty acids suggests the presence of Gram-positive bacteria, and the mid-methyl branched fatty acids indicates the presence of sulfate-reducing bacteria. Fungi appear to also be an important part of the AMD microbial communities as suggested by the presence of 18:2 fatty acid. The acidophilic microeukaryotes Euglena dominated the biofilm microbial communities. These microorganisms appear to play a prominent role in the formation and preservation of stromatolites and in releasing oxygen to the atmosphere by oxygenic photosynthesis. Thus, the AMD environment comprises a host of microorganisms spreading out within the phylogenetic tree of life. Novel insights on the roles of microbial consortia in the formation and preservation of stromatolites and the production of oxygen through photosynthesis in AMD systems may have significance in the understanding of the interaction of Precambrian microbial communities in environments that produced microbially-mediated sedimentary structures and that caused oxygenation of Earth's atmosphere.

  1. Delineation of the roles of amino acids involved in the catalytic functions of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase.

    PubMed

    Vought, V; Ciccone, T; Davino, M H; Fairbairn, L; Lin, Y; Cosgrove, M S; Adams, M J; Levy, H R

    2000-12-12

    The roles of particular amino acids in substrate and coenzyme binding and catalysis of glucose-6-phosphate dehydrogenase of Leuconostoc mesenteroides have been investigated by site-directed mutagenesis, kinetic analysis, and determination of binding constants. The enzyme from this species has functional dual NADP(+)/NAD(+) specificity. Previous investigations in our laboratories determined the three-dimensional structure. Kinetic studies showed an ordered mechanism for the NADP-linked reaction while the NAD-linked reaction is random. His-240 was identified as the catalytic base, and Arg-46 was identified as important for NADP(+) but not NAD(+) binding. Mutations have been selected on the basis of the three-dimensional structure. Kinetic studies of 14 mutant enzymes are reported and kinetic mechanisms are reported for 5 mutant enzymes. Fourteen substrate or coenzyme dissociation constants have been measured for 11 mutant enzymes. Roles of particular residues are inferred from k(cat), K(m), k(cat)/K(m), K(d), and changes in kinetic mechanism. Results for enzymes K182R, K182Q, K343R, and K343Q establish Lys-182 and Lys-343 as important in binding substrate both to free enzyme and during catalysis. Studies of mutant enzymes Y415F and Y179F showed no significant contribution for Tyr-415 to substrate binding and only a small contribution for Tyr-179. Changes in kinetics for T14A, Q47E, and R46A enzymes implicate these residues, to differing extents, in coenzyme binding and discrimination between NADP(+) and NAD(+). By the same measure, Lys-343 is also involved in defining coenzyme specificity. Decrease in k(cat) and k(cat)/K(m) for the D374Q mutant enzyme defines the way Asp-374, unique to L. mesenteroides G6PD, modulates stabilization of the enzyme during catalysis by its interaction with Lys-182. The greatly reduced k(cat) values of enzymes P149V and P149G indicate the importance of the cis conformation of Pro-149 in accessing the correct transition state. PMID

  2. Involvement of a Non-Human Sialic Acid in Human Cancer

    PubMed Central

    Samraj, Annie N.; Läubli, Heinz; Varki, Nissi; Varki, Ajit

    2014-01-01

    Sialic acids are common monosaccharides that are widely expressed as outer terminal units on all vertebrate cell surfaces, and play fundamental roles in cell–cell and cell–microenvironment interactions. The predominant sialic acids on most mammalian cells are N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac). Neu5Gc is notable for its deficiency in humans due to a species-specific and species-universal inactivating deletion in the CMAH gene encoding the hydroxylase that converts CMP-Neu5Ac to CMP-Neu5Gc. However, Neu5Gc is metabolically incorporated into human tissues from dietary sources (particularly red meat), and detected at even higher levels in some human cancers. Early life exposure to Neu5Gc-containing foods in the presence of certain commensal bacteria that incorporate dietary Neu5Gc into lipooligosaccharides can lead to generation of antibodies that are also cross-reactive against Neu5Gc-containing glycans in human tissues (“xeno-autoantigens”). Such anti-Neu5Gc “xeno-autoantibodies” are found in all humans, although ranging widely in levels among individuals, and displaying diverse and variable specificities for the underlying glycan. Experimental evidence in a human-like Neu5Gc-deficient Cmah−/−mouse model shows that inflammation due to “xenosialitis” caused by this antigen–antibody interaction can promote tumor progression, suggesting a likely mechanism for the well-known epidemiological link between red meat consumption and carcinoma risk. In this review, we discuss the history of this field, mechanisms of Neu5Gc incorporation into tissues, the origin and specificities of human anti-Neu5Gc antibodies, their use as possible cancer biomarkers, implications of xenosialitis in cancer initiation and progression, and current and future approaches toward immunotherapy that could take advantage of this unusual human-specific phenomenon. PMID:24600589

  3. Characterizing Surface Acidic Sites in Mesoporous-Silica-Supported Tungsten Oxide Catalysts Using Solid State NMR and Quantum Chemistry Calculations

    SciTech Connect

    Hu, Jian Z.; Kwak, Ja Hun; Wang, Yong; Hu, Mary Y.; Turcu, Romulus VF; Peden, Charles HF

    2011-10-18

    The acidic sites in dispersed tungsten oxide supported on SBA-15 mesoporous silica were investigated using a combination of pyridine titration, both fast-, and slow-MAS {sup 15}N NMR, static {sup 2}H NMR, and quantum chemistry calculations. It is found that the bridged acidic -OH groups in surface adsorbed tungsten dimers (i.e., W-OH-W) are the Broensted acid sites. The unusually strong acidity of these Broensted acid sites is confirmed by quantum chemistry calculations. In contrast, terminal W-OH sites are very stable and only weakly acidic as are terminal Si-OH sites. Furthermore, molecular interactions between pyridine molecules and the dimer Broensted and terminal W-OH sites for dispersed tungsten oxide species is strong. This results in restricted molecular motion for the interacting pyridine molecules even at room temperature, i.e., a reorientation mainly about the molecular 2-fold axis. This restricted reorientation makes it possible to estimate the relative ratio of the Broensted (tungsten dimer) to the weakly acidic terminal W-OH sites in the catalyst using the slow-MAS {sup 1}H-{sup 15}N CP PASS method.

  4. Myogenic and metabolic feedback in cerebral autoregulation: Putative involvement of arachidonic acid-dependent pathways.

    PubMed

    Berg, Ronan M G

    2016-07-01

    The present paper presents a mechanistic model of cerebral autoregulation, in which the dual effects of the arachidonic acid metabolites 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs) on vascular smooth muscle mediate the cerebrovascular adjustments to a change in cerebral perfusion pressure (CPP). 20-HETE signalling in vascular smooth muscle mediates myogenic feedback to changes in vessel wall stretch, which may be modulated by metabolic feedback through EETs released from astrocytes and endothelial cells in response to changes in brain tissue oxygen tension. The metabolic feedback pathway is much faster than 20-HETE-dependent myogenic feedback, and the former thus initiates the cerebral autoregulatory response, while myogenic feedback comprises a relatively slower mechanism that functions to set the basal cerebrovascular tone. Therefore, assessments of dynamic cerebral autoregulation, which may provide information on the response time of the cerebrovasculature, may specifically be used to yield information on metabolic feedback mechanisms, while data based on assessments of static cerebral autoregulation represent the integrated functionality of myogenic and metabolic feedback. PMID:27241246

  5. Chemosensory signalling pathways involved in sensing of amino acids by the ghrelin cell.

    PubMed

    Vancleef, L; Van Den Broeck, T; Thijs, T; Steensels, S; Briand, L; Tack, J; Depoortere, I

    2015-01-01

    Taste receptors on enteroendocrine cells sense nutrients and transmit signals that control gut hormone release. This study aimed to investigate the amino acid (AA) sensing mechanisms of the ghrelin cell in a gastric ghrelinoma cell line, tissue segments and mice. Peptone and specific classes of amino acids stimulate ghrelin secretion in the ghrelinoma cell line. Sensing of L-Phe occurs via the CaSR, monosodium glutamate via the TAS1R1-TAS1R3 while L-Ala and peptone act via 2 different amino acid taste receptors: CaSR &TAS1R1-TAS1R3 and CaSR &GPRC6A, respectively. The stimulatory effect of peptone on ghrelin release was mimicked ex vivo in gastric but not in jejunal tissue segments, where peptone inhibited ghrelin release. The latter effect could not be blocked by receptor antagonists for CCK, GLP-1 or somatostatin. In vivo, plasma ghrelin levels were reduced both upon intragastric (peptone or L-Phe) or intravenous (L-Phe) administration, indicating that AA- sensing is not polarized and is due to inhibition of ghrelin release from the stomach or duodenum respectively. In conclusion, functional AA taste receptors regulate AA-induced ghrelin release in vitro. The effects differ between stomach and jejunum but these local nutrient sensing mechanisms are overruled in vivo by indirect mechanisms inhibiting ghrelin release. PMID:26510380

  6. Location and characteristics of enzymes involved in the breakdown of polygalacturonic acid by Bacteroides thetaiotaomicron.

    PubMed Central

    McCarthy, R E; Kotarski, S F; Salyers, A A

    1985-01-01

    When Bacteroides thetaiotaomicron is grown in medium which contains polygalacturonic acid (PGA) as the sole carbon source, two different polygalacturonases are produced: a PGA lyase (EC 4.2.2.2) and a PGA hydrolase (EC 3.2.1.15). Both enzymes are cell associated. The PGA hydrolase appears to be an inner membrane protein. The PGA lyase is a soluble protein that associates with membranes under certain conditions. The PGA lyase was purified to apparent homogeneity. It has a molecular weight (from sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of 74,000, a pH optimum of 8.7, a pI of 7.5, and a Km for PGA of 40 to 70 micrograms/ml. It requires calcium for maximal activity. The main product of this enzyme appears to be a disaccharide that contains a delta 4,5-unsaturated galacturonic acid residue. The PGA hydrolase can be solubilized from membranes with 2% Triton X-100 and has been partially purified. It has a pH optimum of 5.4 to 5.5, a pI of 4.7 to 4.9, and a Km for PGA of 350 to 400 micrograms/ml. The main product of this enzyme appears to be galacturonic acid. The specific activities of both PGA hydrolase and PGA lyase increase at the same rate when bacteria are exposed to PGA. The two enzymes therefore appear to be similarly regulated. Images PMID:3968032

  7. Chemosensory signalling pathways involved in sensing of amino acids by the ghrelin cell

    PubMed Central

    Vancleef, L.; Van Den Broeck, T.; Thijs, T.; Steensels, S.; Briand, L.; Tack, J.; Depoortere, I.

    2015-01-01

    Taste receptors on enteroendocrine cells sense nutrients and transmit signals that control gut hormone release. This study aimed to investigate the amino acid (AA) sensing mechanisms of the ghrelin cell in a gastric ghrelinoma cell line, tissue segments and mice. Peptone and specific classes of amino acids stimulate ghrelin secretion in the ghrelinoma cell line. Sensing of L-Phe occurs via the CaSR, monosodium glutamate via the TAS1R1-TAS1R3 while L-Ala and peptone act via 2 different amino acid taste receptors: CaSR & TAS1R1-TAS1R3 and CaSR & GPRC6A, respectively. The stimulatory effect of peptone on ghrelin release was mimicked ex vivo in gastric but not in jejunal tissue segments, where peptone inhibited ghrelin release. The latter effect could not be blocked by receptor antagonists for CCK, GLP-1 or somatostatin. In vivo, plasma ghrelin levels were reduced both upon intragastric (peptone or L-Phe) or intravenous (L-Phe) administration, indicating that AA- sensing is not polarized and is due to inhibition of ghrelin release from the stomach or duodenum respectively. In conclusion, functional AA taste receptors regulate AA-induced ghrelin release in vitro. The effects differ between stomach and jejunum but these local nutrient sensing mechanisms are overruled in vivo by indirect mechanisms inhibiting ghrelin release. PMID:26510380

  8. Spatio-Temporal Detection of the Thiomonas Population and the Thiomonas Arsenite Oxidase Involved in Natural Arsenite Attenuation Processes in the Carnoulès Acid Mine Drainage.

    PubMed

    Hovasse, Agnès; Bruneel, Odile; Casiot, Corinne; Desoeuvre, Angélique; Farasin, Julien; Hery, Marina; Van Dorsselaer, Alain; Carapito, Christine; Arsène-Ploetze, Florence

    2016-01-01

    The acid mine drainage (AMD) impacted creek of the Carnoulès mine (Southern France) is characterized by acid waters with a high heavy metal content. The microbial community inhabiting this AMD was extensively studied using isolation, metagenomic and metaproteomic methods, and the results showed that a natural arsenic (and iron) attenuation process involving the arsenite oxidase activity of several Thiomonas strains occurs at this site. A sensitive quantitative Selected Reaction Monitoring (SRM)-based proteomic approach was developed for detecting and quantifying the two subunits of the arsenite oxidase and RpoA of two different Thiomonas groups. Using this approach combined with FISH and pyrosequencing-based 16S rRNA gene sequence analysis, it was established here for the first time that these Thiomonas strains are ubiquitously present in minor proportions in this AMD and that they express the key enzymes involved in natural remediation processes at various locations and time points. In addition to these findings, this study also confirms that targeted proteomics applied at the community level can be used to detect weakly abundant proteins in situ. PMID:26870729

  9. Spatio-Temporal Detection of the Thiomonas Population and the Thiomonas Arsenite Oxidase Involved in Natural Arsenite Attenuation Processes in the Carnoulès Acid Mine Drainage

    PubMed Central

    Hovasse, Agnès; Bruneel, Odile; Casiot, Corinne; Desoeuvre, Angélique; Farasin, Julien; Hery, Marina; Van Dorsselaer, Alain; Carapito, Christine; Arsène-Ploetze, Florence

    2016-01-01

    The acid mine drainage (AMD) impacted creek of the Carnoulès mine (Southern France) is characterized by acid waters with a high heavy metal content. The microbial community inhabiting this AMD was extensively studied using isolation, metagenomic and metaproteomic methods, and the results showed that a natural arsenic (and iron) attenuation process involving the arsenite oxidase activity of several Thiomonas strains occurs at this site. A sensitive quantitative Selected Reaction Monitoring (SRM)-based proteomic approach was developed for detecting and quantifying the two subunits of the arsenite oxidase and RpoA of two different Thiomonas groups. Using this approach combined with FISH and pyrosequencing-based 16S rRNA gene sequence analysis, it was established here for the first time that these Thiomonas strains are ubiquitously present in minor proportions in this AMD and that they express the key enzymes involved in natural remediation processes at various locations and time points. In addition to these findings, this study also confirms that targeted proteomics applied at the community level can be used to detect weakly abundant proteins in situ. PMID:26870729

  10. The Heparan and Heparin Metabolism Pathway is Involved in Regulation of Fatty Acid Composition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Six genes involved in the heparan sulfate and heparin metabolism pathway, DSEL (dermatan sulfate epimerase-like), EXTL1 (exostoses (multiple)-like 1), HS6ST1 (heparan sulfate 6-O-sulfotransferase 1), HS6ST3 (heparan sulfate 6-O-sulfotransferase 3), NDST3 (N-deacetylase/N-sulfotransferase (heparan gl...

  11. Computational study on the roles of amino acid residues in the active site formation mechanism of blue-light photoreceptors

    NASA Astrophysics Data System (ADS)

    Sato, Ryuma; Kitoh-Nishioka, Hirotaka; Ando, Koji; Yamato, Takahisa

    2015-07-01

    To examine the functional roles of the active site methionine (M-site) and glutamic acid (E-site) residues of blue-light photoreceptors, we performed in silico mutation at the M-site in a systematic manner and focused on the hydrogen bonding between the E-site and the substrate: the cyclobutane-pyrimidine dimer (CPD). Fragment molecular orbital calculations with electron correlations demonstrated that substitution of the M-site methionine with either alanine or glutamine always destabilizes the interaction energy between the E-site and the CPD by more than 12.0 kcal/mol, indicating that the methionine and glutamic acid residues cooperatively facilitate the enzymatic reaction in the active site.

  12. Solid-state NMR investigation of acid sites in dealuminated HZSM-5 zeolite.

    PubMed

    Deng, F; Du, Y; Ye, C H

    1996-01-01

    The acid sites and the hydration behaviors of dealuminated HZSM-5 zeolites (calcined at 550, 600, 650, and 700 degrees C) were characterized by high-resolution 1H MAS, 1H{27A1} spin-echo double resonance in combination with 27A1 MAS NMR. Apart from the usually observed peaks for dealuminated zeolite HZSM-5, a narrow plus a broad peak simultaneously appears at ca. 6.9 ppm in the 1H spectra and they exhibit different decay behavior in the 1H [27A1] double-resonance experiments. The existence of the former signal indicates that Lewis acid sites may'be formed in the zeolites after calcination. By means of the spin echo double resonance technique, we observed for the first time a previously unexpected narrow signal at 5.2 ppm, which resonates on the downfield side of Bronsted acid signal (4.3 ppm) and cannot be resolved in the 1H MAS spectra. This new signal is probably due to another kind of Bronsted acid site, locating in the small cages bounded by four- and five-membered rings. Three narrow peaks at 50 ppm, 30 ppm, and 0 ppm are superimposed on a very broad signal in the 27A1 MAS NMR spectra of dried HZSM-5. The intensity of the line at 50 ppm is significantly reduced compared with that of the rehydrated sample. 27A1 MAS NMR suggests that most part of the four-coordinated framework A1 turns into a intermediate case between four- and three-coordinated A1 after the dehydration and this process is reversible upon dehydration/rehydration. While some framework A1 atoms are transformed into three-coordinated A1 species and Lewis acid sites are, thus, generated in the dealuminated zeolites. For the signal at 30 ppm, the hydration leads to a dispersion in the chemical shift or the quadrupole interaction, which broadens its linewidth in hydrated samples. PMID:8970117

  13. Docosahexaenoic Acid Ameliorates Fructose-Induced Hepatic Steatosis Involving ER Stress Response in Primary Mouse Hepatocytes

    PubMed Central

    Zheng, Jinying; Peng, Chuan; Ai, Yanbiao; Wang, Heng; Xiao, Xiaoqiu; Li, Jibin

    2016-01-01

    The increase in fructose consumption is considered to be a risk factor for developing nonalcoholic fatty liver disease (NAFLD). We investigated the effects of docosahexaenoic acid (DHA) on hepatic lipid metabolism in fructose-treated primary mouse hepatocytes, and the changes of Endoplasmic reticulum (ER) stress pathways in response to DHA treatment. The hepatocytes were treated with fructose, DHA, fructose plus DHA, tunicamycin (TM) or fructose plus 4-phenylbutyric acid (PBA) for 24 h. Intracellular triglyceride (TG) accumulation was assessed by Oil Red O staining. The mRNA expression levels and protein levels related to lipid metabolism and ER stress response were determined by real-time PCR and Western blot. Fructose treatment led to obvious TG accumulation in primary hepatocytes through increasing expression of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), two key enzymes in hepatic de novo lipogenesis. DHA ameliorates fructose-induced TG accumulation by upregulating the expression of carnitine palmitoyltransferase 1A (CPT-1α) and acyl-CoA oxidase 1 (ACOX1). DHA treatment or pretreatment with the ER stress inhibitor PBA significantly decreased TG accumulation and reduced the expression of glucose-regulated protein 78 (GRP78), total inositol-requiring kinase 1 (IRE1α) and p-IRE1α. The present results suggest that DHA protects against high fructose-induced hepatocellular lipid accumulation. The current findings also suggest that alleviating the ER stress response seems to play a role in the prevention of fructose-induced hepatic steatosis by DHA. PMID:26805874

  14. Site characterization and containment/remediation of acid mine drainage at an abandoned mine waste dump

    SciTech Connect

    Djahanguiri, F.; Snodgrass, J.; Koerth, J.

    1996-12-31

    This paper focuses on the preliminary results of laboratory tests to evaluate a new suspension grout consisting of a mixture of a naturally occurring lignite coal based wax {open_quotes}montan wax{close_quotes}, sodium bentonite {open_quotes}pure gold grout{close_quotes}, and water. The test program assesses the suitability of the grout for creating subsurface containment barriers in coal waste dump sites for acid mine seepage control to surface and ground waters. The laboratory activities evaluated the reduction in permeability that could be achieved in a coal waste dump site under optimum conditions and the compatibility of the grout with representative waste from the test site. Information on geological, geochemical and geophysical about the test site is presented. Laboratory formulation of the grout is complete and simulation of field condition is in progress. Pregrout geophysical surveys for determination of hydrogeologic conditions at the site are also completed. Based on geophysical surveys, a grout curtain is proposed which will consist of two rows of grout placement holes in an array across the seepage area toward Belt Creek in Montana, Post-grout geophysical survey will be carried out immediately after grouting work. Performance of the grout curtain will be monitored by collection of water samples from monitoring wells in the Belt Creek and seepage area.

  15. A Large-Scale Assessment of Nucleic Acids Binding Site Prediction Programs.

    PubMed

    Miao, Zhichao; Westhof, Eric

    2015-12-01

    Computational prediction of nucleic acid binding sites in proteins are necessary to disentangle functional mechanisms in most biological processes and to explore the binding mechanisms. Several strategies have been proposed, but the state-of-the-art approaches display a great diversity in i) the definition of nucleic acid binding sites; ii) the training and test datasets; iii) the algorithmic methods for the prediction strategies; iv) the performance measures and v) the distribution and availability of the prediction programs. Here we report a large-scale assessment of 19 web servers and 3 stand-alone programs on 41 datasets including more than 5000 proteins derived from 3D structures of protein-nucleic acid complexes. Well-defined binary assessment criteria (specificity, sensitivity, precision, accuracy…) are applied. We found that i) the tools have been greatly improved over the years; ii) some of the approaches suffer from theoretical defects and there is still room for sorting out the essential mechanisms of binding; iii) RNA binding and DNA binding appear to follow similar driving forces and iv) dataset bias may exist in some methods. PMID:26681179

  16. A Large-Scale Assessment of Nucleic Acids Binding Site Prediction Programs

    PubMed Central

    Miao, Zhichao; Westhof, Eric

    2015-01-01

    Computational prediction of nucleic acid binding sites in proteins are necessary to disentangle functional mechanisms in most biological processes and to explore the binding mechanisms. Several strategies have been proposed, but the state-of-the-art approaches display a great diversity in i) the definition of nucleic acid binding sites; ii) the training and test datasets; iii) the algorithmic methods for the prediction strategies; iv) the performance measures and v) the distribution and availability of the prediction programs. Here we report a large-scale assessment of 19 web servers and 3 stand-alone programs on 41 datasets including more than 5000 proteins derived from 3D structures of protein-nucleic acid complexes. Well-defined binary assessment criteria (specificity, sensitivity, precision, accuracy…) are applied. We found that i) the tools have been greatly improved over the years; ii) some of the approaches suffer from theoretical defects and there is still room for sorting out the essential mechanisms of binding; iii) RNA binding and DNA binding appear to follow similar driving forces and iv) dataset bias may exist in some methods. PMID:26681179

  17. Involvement of Sialic Acid on Endothelial Cells in Organ-Specific Lymphocyte Recirculation

    NASA Astrophysics Data System (ADS)

    Rosen, Steven D.; Singer, Mark S.; Yednock, Ted A.; Stoolman, Lloyd M.

    1985-05-01

    Mouse lymphocytes incubated on cryostat-cut sections of lymphoid organs (lymph nodes and Peyer's patches) specifically adhere to the endothelium of high endothelial venules (HEV), the specialized blood vessels to which recirculating lymphocytes attach as they migrate from the blood into the parenchyma of the lymphoid organs. Treatment of sections with sialidase eliminated the binding of lymphocytes to peripheral lymph node HEV, had no effect on binding to Peyer's patch HEV, and had an intermediate effect on mesenteric lymph node HEV. These results suggest that sialic acid on endothelial cells may be an organ-specific recognition determinant for lymphocyte attachment.

  18. Phase diagram involving the mesomorphic behavior of binary mixture of sodium oleate and orthophosphoric acid

    NASA Astrophysics Data System (ADS)

    Govindaiah, T. N.; Sreepad, H. R.

    2015-04-01

    The present investigation deals with the binary mixture of two non-mesogenic compounds, viz. sodium oleate (Naol) and orthophosphoric acid (H3PO4) which exhibits very interesting liquid crystalline smectic phases at large range of concentrations and temperature. The mixtures with concentrations ranging from 10% to 90% Naol in H3PO4 exhibit SmA, SmC, SmE and SmB phases, sequentially when the specimen is cooled from its isotropic phase. Physical properties, such as ultrasonic velocity, adiabatic compressibility and molar compressibility, show anomalous behavior at the isotropic to mesosphase transition.

  19. Genetic Incorporation of the Unnatural Amino Acid p-Acetyl Phenylalanine into Proteins for Site-Directed Spin Labeling

    PubMed Central

    Evans, Eric G.B.; Millhauser, Glenn L.

    2016-01-01

    Site-directed spin labeling (SDSL) is a powerful tool for the characterization of protein structure and dynamics; however, its application in many systems is hampered by the reliance on unique and benign cysteine substitutions for the site-specific attachment of the spin label. An elegant solution to this problem involves the use of genetically encoded unnatural amino acids (UAAs) containing reactive functional groups that are chemically orthogonal to those of the 20 amino acids found naturally in proteins. These unique functional groups can then be selectively reacted with an appropriately functionalized spin probe. In this chapter, we detail the genetic incorporation of the ketone-bearing amino acid p-acetyl phenylalanine (pAcPhe) into recombinant proteins expressed in E. coli. Incorporation of pAcPhe is followed by chemoselective reaction of the ketone side chain with a hydroxylamine-functionalized nitroxide to afford the spin-labeled side chain “K1,” and we present two protocols for successful K1 labeling of proteins bearing site-specific pAcPhe. We outline the basic requirements for pAcPhe incorporation and labeling, with an emphasis on practical aspects that must be considered by the researcher if high yields of UAA incorporation and efficient labeling reactions are to be achieved. To this end, we highlight recent advances that have led to increased yields of pAcPhe incorporation, and discuss the use of aniline-based catalysts allowing for facile conjugation of the hydroxylamine spin label under mild reaction conditions. To illustrate the utility of K1 labeling in proteins where traditional cysteine-based SDSL methods are problematic, we site-specifically K1 label the cellular prion protein at two positions in the C-terminal domain and determine the interspin distance using double electron–electron resonance EPR. Recent advances in UAA incorporation and ketone-based bioconjugation, in combination with the commercial availability of all requisite

  20. Schiff base structured acid-base cooperative dual sites in an ionic solid catalyst lead to efficient heterogeneous knoevenagel condensations.

    PubMed

    Zhang, Mingjue; Zhao, Pingping; Leng, Yan; Chen, Guojian; Wang, Jun; Huang, Jun

    2012-10-01

    An acid-base bifunctional ionic solid catalyst [PySaIm](3)PW was synthesized by the anion exchange of the ionic-liquid (IL) precursor 1-(2-salicylaldimine)pyridinium bromide ([PySaIm]Br) with the Keggin-structured sodium phosphotungstate (Na(3) PW). The catalyst was characterized by FTIR, UV/Vis, XRD, SEM, Brunauer-Emmett-Teller (BET) theory, thermogravimetric analysis, (1)H NMR spectroscopy, ESI-MS, elemental analysis, and melting points. Together with various counterparts, [PySaIm](3)PW was evaluated in Knoevenagel condensation under solvent and solvent-free conditions. The Schiff base structure attached to the IL cation of [PySaIm](3)PW involves acidic salicyl hydroxyl and basic imine, and provides a controlled nearby position for the acid-base dual sites. The high melting and insoluble properties of [PySaIm](3)PW are relative to the large volume and high valence of PW anions, as well as the intermolecular hydrogen-bonding networks among inorganic anions and IL cations. The ionic solid catalyst [PySaIm](3)PW leads to heterogeneous Knoevenagel condensations. In solvent-free condensation of benzaldehyde with ethyl cyanoacetate, it exhibits a conversion of 95.8 % and a selectivity of 100 %; the conversion is even much higher than that (78.2 %) with ethanol as a solvent. The solid catalyst has a convenient recoverability with only a slight decrease in conversion following subsequent recyclings. Furthermore, the new catalyst is highly applicable to many substrates of aromatic aldehydes with activated methylene compounds. On the basis of the characterization and reaction results, a unique acid-base cooperative mechanism within a Schiff base structure is proposed and discussed, which thoroughly explains not only the highly efficient catalytic performance of [PySaIm](3)PW, but also the lower activities of various control catalysts. PMID:22907828

  1. Modelling metal centres, acid sites and reaction mechanisms in microporous catalysts.

    PubMed

    O'Malley, Alexander J; Logsdail, A J; Sokol, A A; Catlow, C R A

    2016-07-01

    We discuss the role of QM/MM (embedded cluster) computational techniques in catalytic science, in particular their application to microporous catalysis. We describe the methodologies employed and illustrate their utility by briefly summarising work on metal centres in zeolites. We then report a detailed investigation into the behaviour of methanol at acidic sites in zeolites H-ZSM-5 and H-Y in the context of the methanol-to-hydrocarbons/olefins process. Studying key initial steps of the reaction (the adsorption and subsequent methoxylation), we probe the effect of framework topology and Brønsted acid site location on the energetics of these initial processes. We find that although methoxylation is endothermic with respect to the adsorbed system (by 17-56 kJ mol(-1) depending on the location), there are intriguing correlations between the adsorption/reaction energies and the geometries of the adsorbed species, of particular significance being the coordination of methyl hydrogens. These observations emphasise the importance of adsorbate coordination with the framework in zeolite catalysed conversions, and how this may vary with framework topology and site location, particularly suited to investigation by QM/MM techniques. PMID:27136967

  2. Involvement of the arachidonic acid cytochrome P450 epoxygenase pathway in the proliferation and invasion of human multiple myeloma cells

    PubMed Central

    Shao, Jing; Wang, Hongxiang; Yuan, Guolin; Chen, Zhichao

    2016-01-01

    Cytochrome P450 (CYP) epoxygenases and the metabolites epoxyeicosatrienoic acids (EETs) exert multiple biological effects in various malignancies. We have previously found EETs to be secreted by multiple myeloma (MM) cells and to be involved in MM angiogenesis, but the role of the arachidonic acid cytochrome P450 epoxygenase pathway in the proliferation and mobility of MM cells remains unknown. In the present study, we found that MM cell lines generated detectable levels of 11,12-EET/14,15-EET and that increased levels of EETs were found in the serum of MM patients compared to healthy donors. The addition of exogenous EETs induced significantly enhanced proliferation of MM cells, whereas 17-octadecynoic acid (17-ODYA), an inhibitor of the CYP epoxygenase pathway, inhibited the viability and proliferation of MM cells. Moreover, this inhibitory effect could be successfully reversed by exogenous EETs. 17-ODYA also inhibited the motility of MM cells in a time-dependent manner, with a reduction of the gelatinolytic activity and protein expression of the matrix metalloproteinases (MMP)-2 and MMP-9. These results suggest the CYP epoxygenase pathway to be involved in the proliferation and invasion of MM cells, for which 17-ODYA could be a promising therapeutic drug. PMID:27077015

  3. Arachidonic acid is involved in the regulation of hCG induced steroidogenesis in rat Leydig cells

    SciTech Connect

    Didolkar, A.K.; Sundaram, K.

    1987-07-27

    Phospholipase C (PLC), an enzyme involved in the hydrolysis of membrane phospholipid- phosphatidylinositol-bisphosphate to insositol triphosphate and diacylglycerol, and Phorbol 12, myristate 13, acetate (PMA) could significantly stimulate testosterone (T) secretion from Leydig cells. Arachidonic acid (AA) stimulated T secretion by about 2 fold. The steroidogenic effect of PLC and AA was biphasic. At low concentrations both PLC and AA augmented hCG induced T secretion, while at higher concentrations they inhibited steroid production. AA also had a biphasic effect on hCG induced cyclic AMP secretion. 5,8,11,14 Eicosatetrayenoic acid, a general inhibitor of AA metabolism, and Nordihydroguaiaretic acid, an inhibitor of the lipoxygenase pathway of AA metabolism, inhibited hCG induced T secretion while indomethacin, an inhibitor of cyclo-oxygenase pathway, had no effect on hCG induced T secretion. The authors conclude from these data that AA plays a role in the regulation of hCG induced steroidogenic responses in rat Leydig cells and that the metabolite(s) of AA that are involved are not cyclo-oxygenase products. 28 references, 4 figures, 2 tables.

  4. Aerobic metabolism of 4-hydroxybenzoic acid in Archaea via an unusual pathway involving an intramolecular migration (NIH shift).

    PubMed

    Fairley, D J; Boyd, D R; Sharma, N D; Allen, C C R; Morgan, P; Larkin, M J

    2002-12-01

    A novel haloarchaeal strain, Haloarcula sp. strain D1, grew aerobically on 4-hydroxybenzoic acid (4HBA) as a sole carbon and energy source and is the first member of the domain Archaea reported to do so. Unusually, D1 metabolized 4HBA via gentisic acid rather than via protocatechuic acid, hydroquinone, or catechol. Gentisate was detected in 4HBA-grown cultures, and gentisate 1,2-dioxygenase activity was induced in 4HBA-grown cells. Stoichiometric accumulation of gentisate from 4HBA was demonstrated in 4HBA-grown cell suspensions containing 2,2'-dipyridyl (which strongly inhibits gentisate 1,2-dioxygenase). To establish whether initial 1-hydroxylation of 4HBA with concomitant 1,2-carboxyl group migration to yield gentisate occurred, 2,6-dideutero-4HBA was synthesized and used as a substrate. Deuterated gentisate was recovered from cell suspensions and identified as 3-deutero-gentisate, using gas chromatography-mass spectrometry and proton nuclear magnetic resonance spectroscopy. This structural isomer would be expected only if a 1,2-carboxyl group migration had taken place, and it provides compelling evidence that the 4HBA pathway in Haloarcula sp. strain D1 involves a hydroxylation-induced intramolecular migration. To our knowledge, this is the first report of a pathway which involves such a transformation (called an NIH shift) in the domain Archaea. PMID:12450849

  5. Involvement of multiple basic amino acids in yeast ribosomal protein L1 in 5S rRNA recognition.

    PubMed

    Yeh, L C; Lee, J C

    1995-01-01

    The role of basic amino acid residues located at the C-terminal region of the yeast ribosomal protein L1 in 5S rRNA binding was characterized in vitro and in vivo. Mutant proteins containing single or multiple amino acid substitutions were generated by site-directed mutagenesis of the L1 gene carried on a plasmid. In vitro RNP formation was examined by production of the mutant protein in the presence of the RNA molecule. The thermostability of the resultant RNP was also studied. Effects of these mutations on cell viability and ribosome assembly were characterized by transformation of a conditional null L1 yeast mutant with the mutated L1 gene expressed from the plasmid. Substitution of any one of the lysine or arginine residue did not affect significantly RNA binding in vitro or cell growth in vivo. However, several mutant proteins with substitutions of two of these basic amino acids bound RNA weakly and the RNPs were less stable. Cells expressing these mutant proteins were lethal. Theoretical structural prediction of these amino acids further provided information regarding their collective contributions to RNA recognition and to interaction between the RNP and other components of the 60S ribosomal subunit. PMID:8643400

  6. Plant immunity induced by COS-OGA elicitor is a cumulative process that involves salicylic acid.

    PubMed

    van Aubel, Géraldine; Cambier, Pierre; Dieu, Marc; Van Cutsem, Pierre

    2016-06-01

    Plant innate immunity offers considerable opportunities for plant protection but beside flagellin and chitin, not many molecules and their receptors have been extensively characterized and very few have successfully reached the field. COS-OGA, an elicitor that combines cationic chitosan oligomers (COS) with anionic pectin oligomers (OGA), efficiently protected tomato (Solanum lycopersicum) grown in greenhouse against powdery mildew (Leveillula taurica). Leaf proteomic analysis of plants sprayed with COS-OGA showed accumulation of Pathogenesis-Related proteins (PR), especially subtilisin-like proteases. qRT-PCR confirmed upregulation of PR-proteins and salicylic acid (SA)-related genes while expression of jasmonic acid/ethylene-associated genes was not modified. SA concentration and class III peroxidase activity were increased in leaves and appeared to be a cumulative process dependent on the number of sprayings with the elicitor. These results suggest a systemic acquired resistance (SAR) mechanism of action of the COS-OGA elicitor and highlight the importance of repeated applications to ensure efficient protection against disease. PMID:27095400

  7. Structure of a Bacterial ABC Transporter Involved in the Import of an Acidic Polysaccharide Alginate.

    PubMed

    Maruyama, Yukie; Itoh, Takafumi; Kaneko, Ai; Nishitani, Yu; Mikami, Bunzo; Hashimoto, Wataru; Murata, Kousaku

    2015-09-01

    The acidic polysaccharide alginate represents a promising marine biomass for the microbial production of biofuels, although the molecular and structural characteristics of alginate transporters remain to be clarified. In Sphingomonas sp. A1, the ATP-binding cassette transporter AlgM1M2SS is responsible for the import of alginate across the cytoplasmic membrane. Here, we present the substrate-transport characteristics and quaternary structure of AlgM1M2SS. The addition of poly- or oligoalginate enhanced the ATPase activity of reconstituted AlgM1M2SS coupled with one of the periplasmic solute-binding proteins, AlgQ1 or AlgQ2. External fluorescence-labeled oligoalginates were specifically imported into AlgM1M2SS-containing proteoliposomes in the presence of AlgQ2, ATP, and Mg(2+). The crystal structure of AlgQ2-bound AlgM1M2SS adopts an inward-facing conformation. The interaction between AlgQ2 and AlgM1M2SS induces the formation of an alginate-binding tunnel-like structure accessible to the solvent. The translocation route inside the transmembrane domains contains charged residues suitable for the import of acidic saccharides. PMID:26235029

  8. The fatty acid transport protein Fat1p is involved in the export of fatty acids from lipid bodies in Yarrowia lipolytica.

    PubMed

    Dulermo, Rémi; Gamboa-Meléndez, Heber; Dulermo, Thierry; Thevenieau, France; Nicaud, Jean-Marc

    2014-09-01

    In order to live, cells need to import different molecules, such as sugars, amino acids or lipids, using transporters. In Saccharomyces cerevisiae, the ScFAT1 gene encodes the long-chain fatty acid transporter; however, the transport of fatty acids (FAs) in the oleaginous yeast Yarrowia lipolytica has not yet been studied. In contrast to what has previously been found for ΔScfat1 strains, ΔYlfat1 yeast was still able to grow on substrates containing short-, medium- or long-chain FAs. We observed a notable difference in cell lipid content between wild-type (WT) and deletion mutant strains after 24 h of culture in minimal oleate medium: in the WT strain, lipids represented 24% of cell dry weight (CDW), while they accounted for 37% of CDW in the ΔYlfat1 strain. This result indicates that YlFat1p is not involved in cell lipid uptake. Moreover, we also observed that fatty acid remobilisation was decreased in the ΔYlfat1 strain and that fluorescence-tagged YlFat1p proteins localised to the interfaces between lipid bodies, which suggests that YlFat1p may play a role in the export of FAs from lipid bodies. PMID:24945074

  9. Mutational Studies on Resurrected Ancestral Proteins Reveal Conservation of Site-Specific Amino Acid Preferences throughout Evolutionary History

    PubMed Central

    Risso, Valeria A.; Manssour-Triedo, Fadia; Delgado-Delgado, Asunción; Arco, Rocio; Barroso-delJesus, Alicia; Ingles-Prieto, Alvaro; Godoy-Ruiz, Raquel; Gavira, Jose A.; Gaucher, Eric A.; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M.

    2015-01-01

    Local protein interactions (“molecular context” effects) dictate amino acid replacements and can be described in terms of site-specific, energetic preferences for any different amino acid. It has been recently debated whether these preferences remain approximately constant during evolution or whether, due to coevolution of sites, they change strongly. Such research highlights an unresolved and fundamental issue with far-reaching implications for phylogenetic analysis and molecular evolution modeling. Here, we take advantage of the recent availability of phenotypically supported laboratory resurrections of Precambrian thioredoxins and β-lactamases to experimentally address the change of site-specific amino acid preferences over long geological timescales. Extensive mutational analyses support the notion that evolutionary adjustment to a new amino acid may occur, but to a large extent this is insufficient to erase the primitive preference for amino acid replacements. Generally, site-specific amino acid preferences appear to remain conserved throughout evolutionary history despite local sequence divergence. We show such preference conservation to be readily understandable in molecular terms and we provide crystallographic evidence for an intriguing structural-switch mechanism: Energetic preference for an ancestral amino acid in a modern protein can be linked to reorganization upon mutation to the ancestral local structure around the mutated site. Finally, we point out that site-specific preference conservation naturally leads to one plausible evolutionary explanation for the existence of intragenic global suppressor mutations. PMID:25392342

  10. Mutational studies on resurrected ancestral proteins reveal conservation of site-specific amino acid preferences throughout evolutionary history.

    PubMed

    Risso, Valeria A; Manssour-Triedo, Fadia; Delgado-Delgado, Asunción; Arco, Rocio; Barroso-delJesus, Alicia; Ingles-Prieto, Alvaro; Godoy-Ruiz, Raquel; Gavira, Jose A; Gaucher, Eric A; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M

    2015-02-01

    Local protein interactions ("molecular context" effects) dictate amino acid replacements and can be described in terms of site-specific, energetic preferences for any different amino acid. It has been recently debated whether these preferences remain approximately constant during evolution or whether, due to coevolution of sites, they change strongly. Such research highlights an unresolved and fundamental issue with far-reaching implications for phylogenetic analysis and molecular evolution modeling. Here, we take advantage of the recent availability of phenotypically supported laboratory resurrections of Precambrian thioredoxins and β-lactamases to experimentally address the change of site-specific amino acid preferences over long geological timescales. Extensive mutational analyses support the notion that evolutionary adjustment to a new amino acid may occur, but to a large extent this is insufficient to erase the primitive preference for amino acid replacements. Generally, site-specific amino acid preferences appear to remain conserved throughout evolutionary history despite local sequence divergence. We show such preference conservation to be readily understandable in molecular terms and we provide crystallographic evidence for an intriguing structural-switch mechanism: Energetic preference for an ancestral amino acid in a modern protein can be linked to reorganization upon mutation to the ancestral local structure around the mutated site. Finally, we point out that site-specific preference conservation naturally leads to one plausible evolutionary explanation for the existence of intragenic global suppressor mutations. PMID:25392342

  11. DNA Methylation Perturbations in Genes Involved in Polyunsaturated Fatty Acid Biosynthesis Associated with Depression and Suicide Risk

    PubMed Central

    Haghighi, Fatemeh; Galfalvy, Hanga; Chen, Sean; Huang, Yung-yu; Cooper, Thomas B.; Burke, Ainsley K.; Oquendo, Maria A.; Mann, J. John; Sublette, M. Elizabeth

    2015-01-01

    Polyunsaturated fatty acid (PUFA) status has been associated with neuropsychiatric disorders, including depression and risk of suicide. Long-chain PUFAs (LC-PUFAs) are obtained in the diet or produced by sequential desaturation and elongation of shorter-chain precursor fatty acids linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3). We compared DNA methylation patterns in genes involved in LC-PUFA biosynthesis in major depressive disorder (MDD) with (n = 22) and without (n = 39) history of suicide attempt, and age- and sex-matched healthy volunteers (n = 59). Plasma levels of selected PUFAs along the LC-PUFA biosynthesis pathway were determined by transesterification and gas chromatography. CpG methylation levels for the main human LC-PUFA biosynthetic genes, fatty acid desaturases 1 (Fads1) and 2 (Fads2), and elongation of very long-chain fatty acids protein 5 (Elovl5), were assayed by bisulfite pyrosequencing. Associations between PUFA levels and diagnosis or suicide attempt status did not survive correction for multiple testing. However, MDD diagnosis and suicide attempts were significantly associated with DNA methylation in Elovl5 gene regulatory regions. Also the relative roles of PUFA levels and DNA methylation with respect to diagnostic and suicide attempt status were determined by least absolute shrinkage and selection operator logistic regression analyses. We found that PUFA associations with suicide attempt status were explained by effects of Elovl5 DNA methylation within the regulatory regions. The observed link between plasma PUFA levels, DNA methylation, and suicide risk may have implications for modulation of disease-associated epigenetic marks by nutritional intervention. PMID:25972837

  12. DNA methylation perturbations in genes involved in polyunsaturated Fatty Acid biosynthesis associated with depression and suicide risk.

    PubMed

    Haghighi, Fatemeh; Galfalvy, Hanga; Chen, Sean; Huang, Yung-Yu; Cooper, Thomas B; Burke, Ainsley K; Oquendo, Maria A; Mann, J John; Sublette, M Elizabeth

    2015-01-01

    Polyunsaturated fatty acid (PUFA) status has been associated with neuropsychiatric disorders, including depression and risk of suicide. Long-chain PUFAs (LC-PUFAs) are obtained in the diet or produced by sequential desaturation and elongation of shorter-chain precursor fatty acids linoleic acid (LA, 18:2n-6) and α-linolenic acid (ALA, 18:3n-3). We compared DNA methylation patterns in genes involved in LC-PUFA biosynthesis in major depressive disorder (MDD) with (n = 22) and without (n = 39) history of suicide attempt, and age- and sex-matched healthy volunteers (n = 59). Plasma levels of selected PUFAs along the LC-PUFA biosynthesis pathway were determined by transesterification and gas chromatography. CpG methylation levels for the main human LC-PUFA biosynthetic genes, fatty acid desaturases 1 (Fads1) and 2 (Fads2), and elongation of very long-chain fatty acids protein 5 (Elovl5), were assayed by bisulfite pyrosequencing. Associations between PUFA levels and diagnosis or suicide attempt status did not survive correction for multiple testing. However, MDD diagnosis and suicide attempts were significantly associated with DNA methylation in Elovl5 gene regulatory regions. Also the relative roles of PUFA levels and DNA methylation with respect to diagnostic and suicide attempt status were determined by least absolute shrinkage and selection operator logistic regression analyses. We found that PUFA associations with suicide attempt status were explained by effects of Elovl5 DNA methylation within the regulatory regions. The observed link between plasma PUFA levels, DNA methylation, and suicide risk may have implications for modulation of disease-associated epigenetic marks by nutritional intervention. PMID:25972837

  13. Involvement of tryptophan residues at the coenzyme A binding site of carbon monoxide dehydrogenase from Clostridium thermoaceticum.

    PubMed

    Shanmugasundaram, T; Kumar, G K; Wood, H G

    1988-08-23

    Carbon monoxide dehydrogenase (CODH) from Clostridium thermoaceticum plays a central role in the newly discovered acetyl-CoA pathway [Wood, H.G., Ragsdale, S.W., & Pezacka, E. (1986) FEMS Microbiol. Rev. 39, 345-362]. The enzyme catalyzes the formation of acetyl-CoA from methyl, carbonyl, and CoA groups, and it has specific binding sites for these moieties. In this study, we have determined the role of tryptophans at these subsites. N-Bromosuccinimide (NBS) oxidation of the exposed and reactive tryptophans (5 out of a total of approximately 20) of CODH at pH 5.5 results in the partial inactivation of the exchange reaction (approximately 50%) involving carbon monoxide and the carbonyl group of the acetyl-CoA. Also, about 70% of the acetyl-CoA synthesis was abolished as a result of NBS modification. The presence of CoA (10 microM) produced complete protection against the partial inhibition of the exchange activity and the overall synthesis of acetyl-CoA caused by NBS. Additionally, none of the exposed tryptophans of CODH was modified in the presence of CoA. Ligands such as the methyl or the carbonyl groups did not afford protection against these inactivations or the modification of the exposed tryptophans. A significant fraction of the accessible fluorescence of CODH was shielded in the presence of CoA against acrylamide quenching. On the basis of these observations, it appears that certain tryptophans are involved at or near the CoA binding site of CODH. PMID:3219350

  14. Crystal Structure and Identification of Two Key Amino Acids Involved in AI-2 Production and Biofilm Formation in Streptococcus suis LuxS

    PubMed Central

    Wang, Yang; Yi, Li; Wang, Shaohui; Fan, Hongjie; Ding, Chan; Mao, Xiang; Lu, Chengping

    2015-01-01

    Streptococcus suis has emerged as an important zoonotic pathogen that causes meningitis, arthritis, septicemia and even sudden death in pigs and humans. Quorum sensing is the signaling network for cell-to-cell communication that bacterial cells can use to monitor their own population density through production and exchange of signal molecules. S-Ribosylhomocysteinase (LuxS) is the key enzyme involved in the activated methyl cycle. Autoinducer 2 (AI-2) is the adduct of borate and a ribose derivative and is produced from S-adenosylhomocysteine (SAH). AI-2 can mediate interspecies communication and in some species facilitate the bacterial behavior regulation such as biofilm formation and virulence in both Gram-positive and Gram-negative bacteria. Here, we reported the overexpression, purification and crystallographic structure of LuxS from S. suis. Our results showed the catalytically active LuxS exists as a homodimer in solution. Inductively coupled plasma-mass spectrometry (ICP-MS) revealed the presence of Zn2+ in LuxS. Although the core structure shares the similar topology with LuxS proteins from other bacterial species, structural analyses and comparative amino acid sequence alignments identified two key amino acid differences in S. suis LuxS, Phe80 and His87, which are located near the substrate binding site. The results of site-directed mutagenesis and enzymology studies confirmed that these two residues affect the catalytic activity of the enzyme. These in vitro results were corroborated in vivo by expression of the LuxS variants in a S. suis ΔluxS strain. The single and two amino acid of LuxS variant decreased AI-2 production and biofilm formation significantly compared to that of the parent strain. Our findings highlight the importance of key LuxS residues that influence the AI-2 production and biofilm formation in S.suis. PMID:26484864

  15. Crystal Structure and Identification of Two Key Amino Acids Involved in AI-2 Production and Biofilm Formation in Streptococcus suis LuxS.

    PubMed

    Wang, Yang; Yi, Li; Wang, Shaohui; Fan, Hongjie; Ding, Chan; Mao, Xiang; Lu, Chengping

    2015-01-01

    Streptococcus suis has emerged as an important zoonotic pathogen that causes meningitis, arthritis, septicemia and even sudden death in pigs and humans. Quorum sensing is the signaling network for cell-to-cell communication that bacterial cells can use to monitor their own population density through production and exchange of signal molecules. S-Ribosylhomocysteinase (LuxS) is the key enzyme involved in the activated methyl cycle. Autoinducer 2 (AI-2) is the adduct of borate and a ribose derivative and is produced from S-adenosylhomocysteine (SAH). AI-2 can mediate interspecies communication and in some species facilitate the bacterial behavior regulation such as biofilm formation and virulence in both Gram-positive and Gram-negative bacteria. Here, we reported the overexpression, purification and crystallographic structure of LuxS from S. suis. Our results showed the catalytically active LuxS exists as a homodimer in solution. Inductively coupled plasma-mass spectrometry (ICP-MS) revealed the presence of Zn2+ in LuxS. Although the core structure shares the similar topology with LuxS proteins from other bacterial species, structural analyses and comparative amino acid sequence alignments identified two key amino acid differences in S. suis LuxS, Phe80 and His87, which are located near the substrate binding site. The results of site-directed mutagenesis and enzymology studies confirmed that these two residues affect the catalytic activity of the enzyme. These in vitro results were corroborated in vivo by expression of the LuxS variants in a S. suis ΔluxS strain. The single and two amino acid of LuxS variant decreased AI-2 production and biofilm formation significantly compared to that of the parent strain. Our findings highlight the importance of key LuxS residues that influence the AI-2 production and biofilm formation in S.suis. PMID:26484864

  16. REMOVING SLUDGE HEELS FROM SAVANNAH RIVER SITE WASTE TANKS BY OXALIC ACID DISSOLUTION

    SciTech Connect

    Poirier, M; David Herman, D; Fernando Fondeur, F; John Pareizs, J; Michael Hay, M; Bruce Wiersma, B; Kim Crapse, K; Thomas Peters, T; Samuel Fink, S; Donald Thaxton, D

    2009-03-01

    The Savannah River Site (SRS) will remove sludge as part of waste tank closure operations. Typically the bulk sludge is removed by mixing it with supernate to produce a slurry, and transporting the slurry to a downstream tank for processing. Experience shows that a residual heel may remain in the tank that cannot be removed by this conventional technique. In the past, SRS used oxalic acid solutions to disperse or dissolve the sludge heel to complete the waste removal. To better understand the actual conditions of oxalic acid cleaning of waste from carbon steel tanks, the authors developed and conducted an experimental program to determine its effectiveness in dissolving sludge, the hydrogen generation rate, the generation rate of other gases, the carbon steel corrosion rate, the impact of mixing on chemical cleaning, the impact of temperature, and the types of precipitates formed during the neutralization process. The test samples included actual SRS sludge and simulated SRS sludge. The authors performed the simulated waste tests at 25, 50, and 75 C by adding 8 wt % oxalic acid to the sludge over seven days. They conducted the actual waste tests at 50 and 75 C by adding 8 wt % oxalic acid to the sludge as a single batch. Following the testing, SRS conducted chemical cleaning with oxalic acid in two waste tanks. In Tank 5F, the oxalic acid (8 wt %) addition occurred over seven days, followed by inhibited water to ensure the tank contained enough liquid to operate the mixer pumps. The tank temperature during oxalic acid addition and dissolution was approximately 45 C. The authors analyzed samples from the chemical cleaning process and compared it with test data. The conclusions from the work are: (1) Oxalic acid addition proved effective in dissolving sludge heels in the simulant demonstration, the actual waste demonstration, and in SRS Tank 5F. (2) The oxalic acid dissolved {approx} 100% of the uranium, {approx} 100% of the iron, and {approx} 40% of the manganese

  17. Effect of α-linolenic acid and DHA intake on lipogenesis and gene expression involved in fatty acid metabolism in growing-finishing pigs.

    PubMed

    De Tonnac, A; Labussière, E; Vincent, A; Mourot, J

    2016-07-01

    The regulation of lipogenesis mechanisms related to consumption of n-3 PUFA is poorly understood. The aim of the present study was to find out whether α-linolenic acid (ALA) or DHA uptake can have an effect on activities and gene expressions of enzymes involved in lipid metabolism in the liver, subcutaneous adipose tissue and longissimus dorsi (LD) muscle of growing-finishing pigs. Six groups of ten pigs received one of six experimental diets supplemented with rapeseed oil in the control diet, extruded linseed, microalgae or a mixture of both to implement different levels of ALA and DHA with the same content in total n-3. Results were analysed for linear and quadratic effects of DHA intake. The results showed that activities of malic enzyme (ME) and fatty acid synthase (FAS) decreased linearly in the liver with dietary DHA. Although the expression of the genes of these enzymes and their activities were poorly correlated, ME and FAS expressions also decreased linearly with DHA intake. The intake of DHA down-regulates the expressions of other genes involved in fatty acid (FA) metabolism in some tissues of pigs, such as fatty acid desaturase 2 and sterol-regulatory element binding transcription factor 1 in the liver and 2,4-dienoyl CoA reductase 2 in the LD muscle. FA oxidation in the LD muscle and FA synthesis decreased in the liver with increasing amount of dietary DHA, whereas a retroconversion of DHA into EPA seems to be set up in this last tissue. PMID:27181335

  18. Cloning and characterization of a locus encoding an indolepyruvate decarboxylase involved in indole-3-acetic acid synthesis in Erwinia herbicola.

    PubMed Central

    Brandl, M T; Lindow, S E

    1996-01-01

    Erwinia herbicola 299R synthesizes indole-3-acetic acid (IAA) primarily by the indole-3-pyruvic acid pathway. A gene involved in the biosynthesis of IAA was cloned from strain 299R. This gene (ipdC) conferred the synthesis of indole-3-acetaldehyde and tryptophol upon Escherichia coli DH5 alpha in cultures supplemented with L-tryptophan. The deduced amino acid sequence of the gene product has high similarity to that of the indolepyruvate decarboxylase of Enterobacter cloacae. Regions within pyruvate decarboxylases of various fungal and plant species also exhibited considerable homology to portions of this gene. This gene therefore presumably encodes an indolepyruvate decarboxylase (IpdC) which catalyzes the conversion of indole-3-pyruvic acid to indole-3-acetaldehyde. Insertions of Tn3-spice within ipdC abolished the ability of strain 299R to synthesize indole-3-acetaldehyde and tryptophol and reduced its IAA production in tryptophan-supplemented minimal medium by approximately 10-fold, thus providing genetic evidence for the role of the indolepyruvate pathway in IAA synthesis in this strain. An ipdC probe hybridized strongly with the genomic DNA of all E. herbicola strains tested in Southern hybridization studies, suggesting that the indolepyruvate pathway is common in this species. Maximum parsimony analysis revealed that the ipdC gene is highly conserved within this group and that strains of diverse geographic origin were very similar with respect to ipdC. PMID:8900003

  19. Effect of Polyunsaturated Fatty Acids on Homocysteine Metabolism through Regulating the Gene Expressions Involved in Methionine Metabolism

    PubMed Central

    Huang, Tao; Hu, Xiaojie; Khan, Nicholas; Yang, Jing; Li, Duo

    2013-01-01

    The objective was to investigate the regulatory effect of polyunsaturated fatty acids (PUFAs) on mRNA expression of key genes involved in homocysteine (Hcy) metabolism. Eighty male Sprague Dawley rats were randomly divided into eight groups. The oils were orally administered daily for 8 weeks. Plasma Hcy, phospholipids fatty acids, and mRNA expression were determined. Compared with the control group, plasma Hcy was significantly decreased in the 22:6n-3 and conjugated linoleic acid (CLA) groups; mRNA expression of Mthfr was significantly upregulated in the 22:6n-3, 20:5n-3, and 18:3n-3 groups and downregulated in the 18:2n-6 and stearolic acid (SO) groups. Mat1a was upregulated in the 22:6n-3, 20:5n-3, 18:3n-3, and CLA groups. In addition, Cbs was upregulated in the 22:6n-3, 20:5n-3, 18:3n-3 and CLA groups while downregulated in 18:2n-6 and SO groups. Dietary 22:6n-3 and CLA decrease the plasma concentration of Hcy. mRNA expression of Mthfr, Mat1a, Cbs and Pemt, Gnmt, Mtrr, and Bad is upregulated by n-3 PUFA and downregulated by n-6 PUFA. CLA upregulates mRNA expression of Mat1a and Cbs. PMID:23766724

  20. In situ detection of salicylic acid binding sites in plant tissues.

    PubMed

    Liu, Jing-Wen; Deng, Da-Yi; Yu, Ying; Liu, Fang-Fei; Lin, Bi-Xia; Cao, Yu-Juan; Hu, Xiao-Gang; Wu, Jian-Zhong

    2015-02-01

    The determination of hormone-binding sites in plants is essential in understanding the mechanisms behind hormone function. Salicylic acid (SA) is an important plant hormone that regulates responses to biotic and abiotic stresses. In order to label SA-binding sites in plant tissues, a quantum dots (QDs) probe functionalized with a SA moiety was successfully synthesized by coupling CdSe QDs capped with 3-mercaptopropionic acid (MPA) to 4-amino-2-hydroxybenzoic acid (PAS), using 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide (EDC) as the coupling agent. The probe was then characterized by dynamic light scattering and transmission electron microscopy, as well as UV/vis and fluorescence spectrophotometry. The results confirmed the successful conjugation of PAS to CdSe QDs and revealed that the conjugates maintained the properties of the original QDs, with small core diameters and adequate dispersal in solution. The PAS-CdSe QDs were used to detect SA-binding sites in mung bean and Arabidopsis thaliana seedlings in vitro and in vivo. The PAS-CdSe QDs were effectively transported into plant tissues and specifically bound to SA receptors in vivo. In addition, the effects of the PAS-CdSe QDs on cytosolic Ca(2+) levels in the tips of A. thaliana seedlings were investigated. Both SA and PAS-CdSe QDs had similar effects on the trend in cytosolic-free Ca(2+) concentrations, suggesting that the PAS-CdSe QDs maintained the bioactivity of SA. To summarize, PAS-CdSe QDs have high potential as a fluorescent probe for the in vitro/in vivo labeling and imaging of SA receptors in plants. PMID:24833131

  1. Studies on the acidity of mordenite and ZSM-5. 1. Determination of Broensted acid site concentrations in mordenite and ZSM-5 by conductometric titration

    SciTech Connect

    Crocker, M.; Herold, R.H.M.; Sonnemans, M.H.W.; Emeis, C.A.; Wilson, A.E.; Moolen, J.N. van der )

    1993-01-14

    The Broensted acidity of H-mordenite and H-ZSM-5 samples of varying proton concentration has been studied using aqueous conductometric titration, IR spectroscopy, and aqueous potentiometric titration. Good agreement is observed between Broensted acid site concentrations determined by conductometric titration and IR measurements, while indirect potentiometric titration affords acid site concentrations consistently lower than those measured using the conductometric technique. This finding is rationalized on the basis that, in a conductometric titration, all the accessible Broensted acid sites are direct;y titrated, whereas in the potentiometric procedure utilized, only those protons which can be ion-exchanged out of the zeolite are titrated. After allowing for the presence of extraframework aluminum in the zeolites (determined by [sup 27]Al NMR), the measured acidity for H-mordenite is found to increase linearly with increasing Al content within the range 0-1.5 mmol Al/g but appears to reach a limiting value at higher Al concentrations. For H-ZSM-5, the experimentally determined number of Broensted acid sites is also found to be linearly dependent on the Al molar fraction within the range measured (0-1.20 mmol Al/g). For both series of zeolite samples, the measured acidity is generally found to be less than the theoretical maximum calculated on the basis of an H[sup +]/Al ratio of 1. 43 refs., 10 figs., 4 tabs.

  2. Alcohol fermentation of sweet potato. 1. Acid hydrolysis and factors involved

    SciTech Connect

    Azhar, A.; Hamdy, M.K.

    1981-04-01

    Factors affecting acid hydrolysis of sweet potato powder (SPP) to fermentable sugars were examined. These include HCl concentration, temperature, time, and levels of SPP. Maximum reducing sugar, reported as dextrose equivalent (DE), was detected after 24 min hydrolysis (1% SPP) in 0.034N HCl heated at 154 degrees celcius. These samples also had 3.43% hydroxymethylfurfural (HMF) based on dry weight. A high level of HMF (9.2%) was detected in 1% SPP heated at 154 degrees C in 0.10N HCl for 18 min. The lowest concentration of HMF formed (1.8%), at maximal DE of 61%, was established in samples containing 5% SPP and heated at 154 degrees C in 0.034N HCl for 48 min. Aqueous extracts of uncured SPP, examined by HPLC, contained glucose, fructose and sucrose, butdegreaded SPP had only glucose and fructose. Products of degraded SPP, under appropriate conditions, could be used for alcohol fermentation. (Refs. 18).

  3. Involvements of chloride ion in decolorization of Acid Orange 7 by activated peroxydisulfate or peroxymonosulfate oxidation.

    PubMed

    Wang, Ping; Yang, Shiying; Shan, Liang; Niu, Rui; Shao, Xueting

    2011-01-01

    The effects of chloride anion (Cl-) (up to 1.0 mol/L) on the decolorization of a model compound, azo dye Acid Orange 7 (AO7), by sulfate radical (SO4-*) based-peroxydisulfate (PS) or peroxymonosulfate (PMS) oxidation under various activated conditions (UV254 nm/PS, Thermal (70 degrees C/PS, UV254 nm/PMS, Co2+/PMS) were investigated. Methanol and NH4+ were used as quenching reagents to determine the contributions of active chlorine species (dichloride radical (Cl2-*) and hypochlorous acid (HClO)). The results indicated that the effects of Cl- on the reaction mechanism were different under various activated conditions. For UV/PS and Thermal/PS, the inhibition tendency became more clear as the Cl- concentration increased, probably due to the reaction between Cl- and SO4-* and the generation of Cl2-* or HCIO. For UV/PMS, Cl- did not exhibit inhibition when the concentration was below 0.1 mol/L. As Cl- concentration reached to 1.0 mol/L, the decolorization rate of AO7 was, however, accelerated, possibly because PMS directly reacts with Cl- to form HClO. For Co2+/PMS, Cl- exhibited a significant inhibiting effect even at low concentration (< or = 0.01 mol/L). When Cl- concentration exceeded 0.1 mol/L, the activation of PMS by Co2+ was almost completely inhibited. Under this condition, HClO maybe played a major role in decolorization of AO7. The results implicated that chloride ion is an important factor in SO4(-*) -based degradation of organic contamination in chloride-containing water. PMID:22432303

  4. Synergistic antidepressant-like effect of ferulic acid in combination with piperine: involvement of monoaminergic system.

    PubMed

    Li, Gaowen; Ruan, Lina; Chen, Ruijie; Wang, Renye; Xie, Xupei; Zhang, Meixi; Chen, Lichao; Yan, Qizhi; Reed, Miranda; Chen, Jiechun; Xu, Ying; Pan, Jianchun; Huang, Wu

    2015-12-01

    The lifetime prevalence rate for major depressive disorder (MDD) is approximately 17 % for most developed countries around the world. Dietary polyphenols are currently used as an adjuvant therapy to accelerate the therapeutic efficacy on depression. Ferulic acid (FA) or 4-hydroxy-3-methoxy-cinnamic acid (Fig. 1a) is a main polyphenolic component of Chinese herb Radix Angelicae Sinensis, which is found to have antidepressant-like effects through regulating serotonergic and noradrenergic function. The present study examined the synergistic effect of low doses of FA combined with subthreshold dose of piperine, a bioavailability enhancer, on depression-like behaviors in mice, and investigated the possible mechanism. The administration of FA, even in the highest dose tested, reduced immobility time by 60 % in the tail suspension and forced swimming tests (TST and FST) in mice when compared to control. The maximal antidepressant-like effect of FA was obtained with 200 mg/kg. In addition, piperine only produced a weak antidepressant-like effect in the TST and FST. However, the evidence from the interaction analysis suggested a synergistic effect when low doses of FA were combined with a subthreshold dose of piperine. Further neurochemical evidence such as monoamine levels in the frontal cortex, hippocampus, and hypothalamus and measurements of monoamine oxidase activity also supported a synergistic effect of FA and piperine in the enhancement of monoaminergic function. This finding supports the concept that the combination strategy might be an alternative therapy in the treatment of psychiatric disorders with high efficacy and low side effects. PMID:26220010

  5. Characterization of purple acid phosphatases involved in extracellular dNTP utilization in Stylosanthes.

    PubMed

    Liu, Pan-Dao; Xue, Ying-Bin; Chen, Zhi-Jian; Liu, Guo-Dao; Tian, Jiang

    2016-07-01

    Stylo (Stylosanthes spp.) is a pasture legume predominant in tropical and subtropical areas, where low phosphorus (P) availability is a major constraint for plant growth. Therefore, stylo might exhibit superior utilization of the P pool on acid soils, particularly organic P. However, little is known about mechanisms of inorganic phosphate (Pi) acquisition employed by stylo. In this study, the utilization of extracellular deoxy-ribonucleotide triphosphate (dNTP) and the underlying physiological and molecular mechanisms were examined for two stylo genotypes with contrasting P efficiency. Results showed that the P-efficient genotype, TPRC2001-1, was superior to the P-inefficient genotype, Fine-stem, when using dNTP as the sole P source. This was reflected by a higher dry weight and total P content for TPRC2001-1 than for Fine-stem, which was correlated with higher root-associated acid phosphatase (APase) activities in TPRC2001-1 under low P conditions. Subsequently, three PAP members were cloned from TPRC2001-1: SgPAP7, SgPAP10, and SgPAP26 Expression levels of these three SgPAPs were up-regulated by Pi starvation in stylo roots. Furthermore, there was a higher abundance of transcripts of SgPAP7 and SgPAP10 in TPRC2001-1 than in Fine-stem. Subcellular localization analysis demonstrated that these three SgPAPs were localized on the plasma membrane. Overexpression of these three SgPAPs could result in significantly increased root-associated APase activities, and thus extracellular dNTP utilization in bean hairy roots. Taken together, the results herein suggest that SgPAP7, SgPAP10, and SgPAP26 may differentially contribute to root-associated APase activities, and thus control extracellular dNTP utilization in stylo. PMID:27194738

  6. Involvement of Intermediate Sulfur Species in Biological Reduction of Elemental Sulfur under Acidic, Hydrothermal Conditions

    PubMed Central

    Druschel, Gregory K.

    2013-01-01

    The thermoacidophile and obligate elemental sulfur (S80)-reducing anaerobe Acidilobus sulfurireducens 18D70 does not associate with bulk solid-phase sulfur during S80-dependent batch culture growth. Cyclic voltammetry indicated the production of hydrogen sulfide (H2S) as well as polysulfides after 1 day of batch growth of the organism at pH 3.0 and 81°C. The production of polysulfide is likely due to the abiotic reaction between S80 and the biologically produced H2S, as evinced by a rapid cessation of polysulfide formation when the growth temperature was decreased, inhibiting the biological production of sulfide. After an additional 5 days of growth, nanoparticulate S80 was detected in the cultivation medium, a result of the hydrolysis of polysulfides in acidic medium. To examine whether soluble polysulfides and/or nanoparticulate S80 can serve as terminal electron acceptors (TEA) supporting the growth of A. sulfurireducens, total sulfide concentration and cell density were monitored in batch cultures with S80 provided as a solid phase in the medium or with S80 sequestered in dialysis tubing. The rates of sulfide production in 7-day-old cultures with S80 sequestered in dialysis tubing with pore sizes of 12 to 14 kDa and 6 to 8 kDa were 55% and 22%, respectively, of that of cultures with S80 provided as a solid phase in the medium. These results indicate that the TEA existed in a range of particle sizes that affected its ability to diffuse through dialysis tubing of different pore sizes. Dynamic light scattering revealed that S80 particles generated through polysulfide rapidly grew in size, a rate which was influenced by the pH of the medium and the presence of organic carbon. Thus, S80 particles formed through abiological hydrolysis of polysulfide under acidic conditions appeared to serve as a growth-promoting TEA for A. sulfurireducens. PMID:23335768

  7. CsSAD: a fatty acid desaturase gene involved in abiotic resistance in Camellia sinensis (L.).

    PubMed

    Ding, Z T; Shen, J Z; Pan, L L; Wang, Y U; Li, Y S; Wang, Y; Sun, H W

    2016-01-01

    Tea (Camellia sinensis L.) is a thermophilic evergreen woody plant that has poor cold tolerance. The SAD gene plays a key role in regulating fatty acid synthesis and membrane lipid fluidity in response to temperature change. In this study, full-length SAD cDNA was cloned from tea leaves using rapid amplification of cDNA ends and polymerase chain reaction (PCR)-based methods. Sequence analysis demonstrated that CsSAD had a high similarity to other corresponding cDNAs. At 25°C, the CsSAD transcriptional level was highest in the leaf and lowest in the stem, but there was no obvious difference between the root and stem organs. CsSAD expression was investigated by reverse transcription-PCR, which showed that CsSAD was upregulated at 4° and -5°C. At 25°C, CsSAD was induced by polyethylene glycol, abscisic acid, and wounding, and a similar trend was observed at 4°C, but the mean expression level at 4°C was lower than that at 25°C. Under natural cold acclimation, the 'CsCr05' variety's CsSAD expression level increased before decreasing. The CsSAD expression level in variety 'CsCr06' showed no obvious change at first, but rapidly increased to a maximum when the temperature was very low. Our study demonstrates that CsSAD is upregulated in response to different abiotic conditions, and that it is important to study the stress resistance of the tea plant, particularly in response to low temperature, drought, and wounding. PMID:26985937

  8. The structure of a purple acid phosphatase involved in plant growth and pathogen defence exhibits a novel immunoglobulin-like fold

    PubMed Central

    Antonyuk, Svetlana Vladimirovna; Olczak, Mariusz; Olczak, Teresa; Ciuraszkiewicz, Justyna; Strange, Richard William

    2014-01-01

    Phosphatases function in the production, transport and recycling of inorganic phosphorus, which is crucial for cellular metabolism and bioenergetics, as well as in bacterial killing, since they are able to generate reactive oxygen species via Fenton chemistry. Diphosphonucleotide phosphatase/phosphodiesterase (PPD1), a glycoprotein plant purple acid phosphatase (PAP) from yellow lupin seeds, contains a bimetallic Fe–Mn catalytic site which is most active at acidic pH. Unlike other plant PAPs, PPD1 cleaves the pyrophosphate bond in diphosphonucleotides and the phosphodiester bond in various phosphodiesters. The homohexameric organization of PPD1, as revealed by a 1.65 Å resolution crystal structure and confirmed by solution X-ray scattering, is unique among plant PAPs, for which only homodimers have previously been reported. A phosphate anion is bound in a bidentate fashion at the active site, bridging the Fe and Mn atoms in a binding mode similar to that previously reported for sweet potato PAP, which suggests that common features occur in their catalytic mechanisms. The N-terminal domain of PPD1 has an unexpected and unique fibronectin type III-like fold that is absent in other plant PAPs. Here, the in vitro DNA-cleavage activity of PPD1 is demonstrated and it is proposed that the fibronectin III-like domain, which ‘overhangs’ the active site, is involved in DNA selectivity, binding and activation. The degradation of DNA by PPD1 implies a role for PPD1 in plant growth and repair and in pathogen defence. PMID:25075326

  9. Enhanced concentrations of citric acid in spring aerosols collected at the Gosan background site in East Asia

    NASA Astrophysics Data System (ADS)

    Jung, Jinsang; Kawamura, Kimitaka

    2011-09-01

    In order to investigate water-soluble dicarboxylic acids and related compounds in the aerosol samples under the Asian continent outflow, total suspended particle (TSP) samples ( n = 32) were collected at the Gosan site in Jeju Island over 2-5 days integration during 23 March-1 June 2007 and 16-24 April 2008. The samples were analyzed for water-soluble dicarboxylic acids, ketocarboxylic acids, and α-dicarbonyls using a capillary gas chromatography technique. We found elevated concentrations of atmospheric citric acid (range: 20-320 ng m -3) in the TSP samples during mid- to late April of 2007 and 2008. To specify the sources of citric acid, dicarboxylic acids and related compounds were measured in the pollen sample collected at the Gosan site (Pollen_Gosan), authentic pollen samples from Japanese cedar ( Cryptomeria) (Pollen_cedar) and Japanese cypress ( Chamaecyparis obtusa) (Pollen_cypress), and tangerine fruit produced from Jeju Island. Citric acid (2790 ng in unit mg of pollen mass) was found as most abundant species in the Pollen_Gosan, followed by oxalic acid (2390 ng mg -1). Although citric acid was not detected in the Pollen_cedar and Pollen_cypress as major species, it was found as a dominant species in the tangerine juice while malic acid was detected as major species in the tangerine peel, followed by oxalic and citric acids. Since Japanese cedar trees are planted around tangerine farms to prevent strong winds from the Pacific Ocean, citric acid that may be directly emitted from tangerine is likely adsorbed on pollens emitted from Japanese cedar and then transported to the Gosan site. Much lower malic/citric acid ratios obtained under cloudy condition than clear condition suggest that malic acid may rapidly decompose to lower molecular weight compounds such as oxalic and malonic acids (

  10. The species- and site-specific acid-base properties of penicillamine and its homodisulfide

    NASA Astrophysics Data System (ADS)

    Mirzahosseini, Arash; Szilvay, András; Noszál, Béla

    2014-08-01

    Penicillamine, penicillamine disulfide and 4 related compounds were studied by 1H NMR-pH titrations and case-tailored evaluation methods. The resulting acid-base properties are quantified in terms of 14 macroscopic and 28 microscopic protonation constants and the concomitant 7 interactivity parameters. The species- and site-specific basicities are interpreted by means of inductive and shielding effects through various intra- and intermolecular comparisons. The thiolate basicities determined this way are key parameters and exclusive means for the prediction of thiolate oxidizabilities and chelate forming properties in order to understand and influence chelation therapy and oxidative stress at the molecular level.

  11. Expert Radiation Oncologist Interpretations of Involved-Site Radiation Therapy Guidelines in the Management of Hodgkin Lymphoma

    SciTech Connect

    Hoppe, Bradford S.; Hoppe, Richard T.

    2015-05-01

    Purpose: Recently, involved-site radiation therapy (ISRT) guidelines have been developed and published to replace the previous concept of involved-field radiation therapy for patients with lymphoma. However, these ISRT guidelines may be interpreted in different ways, posing difficulties for prospective clinical trials. This study reports survey results regarding interpretation of the ISRT guidelines. Methods and Materials: Forty-four expert lymphoma radiation oncologists were asked to participate in a survey that included 7 different cases associated with 9 questions. The questions pertained to ISRT contouring and asked respondents to choose between 2 different answers (no “correct” answer) and a third write-in option allowed. Results: Fifty-two percent of those surveyed responded to the questionnaire. Among those who responded, 72% have practiced for >10 years, 46% have treated >20 Hodgkin lymphoma cases annually, and 100% were familiar with the ISRT concept. Among the 9 questions associated with the 7 cases, 3 had concordance among the expert radiation oncologists of greater than 70%. Six of the questions had less than 70% concordance (range, 56%-67%). Conclusions: Even among expert radiation oncologists, interpretation of ISRT guidelines is variable. Further guidance for ISRT field design will be needed to reduce variability among practicing physicians.

  12. His-311 and Arg-559 are key residues involved in fatty acid oxygenation in pathogen-inducible oxygenase.

    PubMed

    Koszelak-Rosenblum, Mary; Krol, Adam C; Simmons, Danielle M; Goulah, Christopher C; Wroblewski, Liliana; Malkowski, Michael G

    2008-09-01

    Pathogen-inducible oxygenase (PIOX) oxygenates fatty acids into 2R-hydroperoxides. PIOX belongs to the fatty acid alpha-dioxygenase family, which exhibits homology to cyclooxygenase enzymes (COX-1 and COX-2). Although these enzymes share common catalytic features, including the use of a tyrosine radical during catalysis, little is known about other residues involved in the dioxygenase reaction of PIOX. We generated a model of linoleic acid (LA) bound to PIOX based on computational sequence alignment and secondary structure predictions with COX-1 and experimental observations that governed the placement of carbon-2 of LA below the catalytic Tyr-379. Examination of the model identified His-311, Arg-558, and Arg-559 as potential molecular determinants of the dioxygenase reaction. Substitutions at His-311 and Arg-559 resulted in mutant constructs that retained virtually no oxygenase activity, whereas substitutions of Arg-558 caused only moderate decreases in activity. Arg-559 mutant constructs exhibited increases of greater than 140-fold in Km, whereas no substantial change in Km was observed for His-311 or Arg-558 mutant constructs. Thermal shift assays used to measure ligand binding affinity show that the binding of LA is significantly reduced in a Y379F/R559A mutant construct compared with that observed for Y379F/R558A construct. Although Oryza sativa PIOX exhibited oxygenase activity against a variety of 14-20-carbon fatty acids, the enzyme did not oxygenate substrates containing modifications at the carboxylate, carbon-1, or carbon-2. Taken together, these data suggest that Arg-559 is required for high affinity binding of substrates to PIOX, whereas His-311 is involved in optimally aligning carbon-2 below Tyr-379 for catalysis. PMID:18596034

  13. Chronic caffeine or theophylline exposure reduces gamma-aminobutyric acid/benzodiazepine receptor site interactions.

    PubMed

    Roca, D J; Schiller, G D; Farb, D H

    1988-05-01

    Methylxanthines, such as caffeine and theophylline, are adenosine receptor antagonists that exert dramatic effects upon the behavior of vertebrate animals by increasing attentiveness, anxiety, and convulsive activity. Benzodiazepines, such as flunitrazepam, generally exert behavioral effects that are opposite to those of methylxanthines. We report the finding that chronic exposure of embryonic brain neurons to caffeine or theophylline reduces the ability of gamma-aminobutyric acid (GABA) to potentiate the binding of [3H]flunitrazepam to the GABA/benzodiazepine receptor. This theophylline-induced "uncoupling" of GABA- and benzodiazepine-binding site allosteric interactions is blocked by chloroadenosine, an adenosine receptor agonist, indicating that the chronic effects of theophylline are mediated by a site that resembles an adenosine receptor. We speculate that adverse central nervous system effects of long-term exposure to methylxanthines such as in caffeine-containing beverages or theophylline-containing medications may be exerted by a cell-mediated modification of the GABAA receptor. PMID:2835648

  14. Locations of the three primary binding sites for long-chain fatty acids on bovine serum albumin

    SciTech Connect

    Hamilton, J.A.; Era, S.; Bhamidipati, S.P. ); Reed, R.G. )

    1991-03-15

    Binding of {sup 13}C-enriched oleic acid to bovine serum albumin and to three large proteolytic fragments of albumin - two complementary fragments corresponding to the two halved of albumin and one fragment corresponding to the carboxyl-terminal domain - yielded unique patterns of NMR resonances (chemical shifts and relative intensities) that were used to identify the locations of binding of the first 5 mol of oleic acid to the multidomain albumin molecule. The first 3 mol of oleic acid added to intact albumin generated three distinct NMR resonances as a result of simultaneous binding of oleic acid to three heterogeneous sites (primary sites). This distribution suggests albumin to be a less symmetrical binding molecule than theoretical models predict. This work also demonstrates the power of NMR for the study of microenvironments of individual fatty acid binding sites in specific domain.

  15. SAVANNAH RIVER SITE TANK CLEANING: CORROSION RATE FOR ONE VERSUS EIGHT PERCENT OXALIC ACID SOLUTION

    SciTech Connect

    Ketusky, E.; Subramanian, K.

    2011-01-20

    Until recently, the use of oxalic acid for chemically cleaning the Savannah River Site (SRS) radioactive waste tanks focused on using concentrated 4 and 8-wt% solutions. Recent testing and research on applicable dissolution mechanisms have concluded that under appropriate conditions, dilute solutions of oxalic acid (i.e., 1-wt%) may be more effective. Based on the need to maximize cleaning effectiveness, coupled with the need to minimize downstream impacts, SRS is now developing plans for using a 1-wt% oxalic acid solution. A technology gap associated with using a 1-wt% oxalic acid solution was a dearth of suitable corrosion data. Assuming oxalic acid's passivation of carbon steel was proportional to the free oxalate concentration, the general corrosion rate (CR) from a 1-wt% solution may not be bound by those from 8-wt%. Therefore, after developing the test strategy and plan, the corrosion testing was performed. Starting with the envisioned process specific baseline solvent, a 1-wt% oxalic acid solution, with sludge (limited to Purex type sludge-simulant for this initial effort) at 75 C and agitated, the corrosion rate (CR) was determined from the measured weight loss of the exposed coupon. Environmental variations tested were: (a) Inclusion of sludge in the test vessel or assuming a pure oxalic acid solution; (b) acid solution temperature maintained at 75 or 45 C; and (c) agitation of the acid solution or stagnant. Application of select electrochemical testing (EC) explored the impact of each variation on the passivation mechanisms and confirmed the CR. The 1-wt% results were then compared to those from the 8-wt%. The immersion coupons showed that the maximum time averaged CR for a 1-wt% solution with sludge was less than 25-mils/yr for all conditions. For an agitated 8-wt% solution with sludge, the maximum time averaged CR was about 30-mils/yr at 50 C, and 86-mils/yr at 75 C. Both the 1-wt% and the 8-wt% testing demonstrated that if the sludge was removed from

  16. Heavy metal binding to heparin disaccharides. I. Iduronic acid is the main binding site.

    PubMed

    Whitfield, D M; Choay, J; Sarkar, B

    1992-06-01

    As model compounds for Ni(II)-binding heparin-like compounds isolated from human kidneys (Templeton, D.M. & Sarkar, B. (1985) Biochem. J. 230 35-42.), we investigated two disaccharides--4-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-2,5-anhydro- D-mannitol, disodium salt (1a), and 4-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-6-O- sulfo-2,5-anhydro-D-mannitol, trisodium salt (1b)--that were isolated from heparin after nitrous acid hydrolysis and reduction. The monosulfate (1a) was active whereas the disulfate (1b) was inactive in a high-performance liquid chromatography (HPLC) binding assay with the tracer ions 63Ni(II) 54Mn(II), 65Zn(II), and 109Cd(II). This result is in accord with the isolation of two 67Cu(II) and 63Ni(II) binding fractions from a complete pool of nitrous-acid-derived heparin disaccharides using sulfate gradients and a MonoQ anion exchange column on an FPLC system. One was identified as compound (1a) and the other as a tetrasulfated trisaccharide by high resolution FAB-MS, NMR and HPLC-PAD. Similarly, two synthetic disaccharides-methyl, 2-O-sulfo-4-O-(alpha-L-idopyranosyluronic acid)-2-deoxy-2-sulfamide-alpha-D-glucosamine, trisodium salt [IdopA2S(alpha 1,4)GlcNS alpha Me, 2a], and 2-O-sulfo-4-O-(alpha-L-idopyranosyluronic acid)-2-deoxy-2-sulfamide-6-O-sulfo- alpha-D-glucosamine, tetrasodium salt [IdopA2S (alpha 1,4)GlcNS6S alpha Me, 2b]--were shown to bind tracer amounts of 63Ni and 67Cu using chromatographic assays. Subsequently, 1H NMR titrations of 1a, 1b, 2a, and 2b with Zn (OAc)2 were analyzed to yield 1:1 Zn(II)-binding constants of 472 +/- 59, 698 +/- 120, 8,758 +/- 2,237 and 20,100 +/- 5,598 M-1, respectively. The values for 2a and 2b suggest chelation. It is suggested that the idopyranosiduronic acid residue is the major metal binding site. NMR evidence for this hypothesis comes from marked 1H and 13C chemical shift changes to the iduronic acid resonances after addition of diamagnetic Zn(II) ions. PMID:1643264

  17. SuccinSite: a computational tool for the prediction of protein succinylation sites by exploiting the amino acid patterns and properties.

    PubMed

    Hasan, Md Mehedi; Yang, Shiping; Zhou, Yuan; Mollah, Md Nurul Haque

    2016-03-01

    Lysine succinylation is an emerging protein post-translational modification, which plays an important role in regulating the cellular processes in both eukaryotic and prokaryotic cells. However, the succinylation modification site is particularly difficult to detect because the experimental technologies used are often time-consuming and costly. Thus, an accurate computational method for predicting succinylation sites may help researchers towards designing their experiments and to understand the molecular mechanism of succinylation. In this study, a novel computational tool termed SuccinSite has been developed to predict protein succinylation sites by incorporating three sequence encodings, i.e., k-spaced amino acid pairs, binary and amino acid index properties. Then, the random forest classifier was trained with these encodings to build the predictor. The SuccinSite predictor achieves an AUC score of 0.802 in the 5-fold cross-validation set and performs significantly better than existing predictors on a comprehensive independent test set. Furthermore, informative features and predominant rules (i.e. feature combinations) were extracted from the trained random forest model for an improved interpretation of the predictor. Finally, we also compiled a database covering 4411 experimentally verified succinylation proteins with 12 456 lysine succinylation sites. Taken together, these results suggest that SuccinSite would be a helpful computational resource for succinylation sites prediction. The web-server, datasets, source code and database are freely available at http://systbio.cau.edu.cn/SuccinSite/. PMID:26739209

  18. The effect of the distance between acidic site and basic site immobilized on mesoporous solid on the activity in catalyzing aldol condensation

    NASA Astrophysics Data System (ADS)

    Yu, Xiaofang; Yu, Xiaobo; Wu, Shujie; Liu, Bo; Liu, Heng; Guan, Jingqi; Kan, Qiubin

    2011-02-01

    Acid-base bifunctional heterogeneous catalysts containing carboxylic and amine groups, which were immobilized at defined distance from one another on the mesoporous solid were synthesized by immobilizing lysine onto carboxyl-SBA-15. The obtained materials were characterized by X-ray diffraction (XRD), N 2 adsorption, Fourier-transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), scanning electron micrographs (SEM), transmission electron micrographs (TEM), elemental analysis, and back titration. Proximal-C-A-SBA-15 with a proximal acid-base distance was more active than maximum-C-A-SBA-15 with a maximum acid-base distance in aldol condensation reaction between acetone and various aldehydes. It appears that the distance between acidic site and basic site immobilized on mesoporous solid should be an essential factor for catalysis optimization.

  19. Glutamine Involvement in Nitrogen Control of Gibberellic Acid Production in Gibberella fujikuroi

    PubMed Central

    Muñoz, Gastón A.; Agosin, Eduardo

    1993-01-01

    When the fungus Gibberella fujikuroi ATCC 12616 was grown in fermentor cultures, both intracellular kaurene biosynthetic activities and extracellular GA3 accumulation reached high levels when exogenous nitrogen was depleted in the culture. Similar patterns were exhibited by several nonrelated enzymatic activities, such as formamidase and urease, suggesting that all are subject to nitrogen regulation. The behavior of the enzymes involved in nitrogen assimilation (glutamine synthetase, glutamate dehydrogenase, and glutamate synthase) during fungal growth in different nitrogen sources suggests that glutamine is the final product of nitrogen assimilation in G. fujikuroi. When ammonium or glutamine was added to hormone-producing cultures, extracellular GA3 did not accumulate. However, when the conversion of ammonium into glutamine was inhibited by L-methionine-DL-sulfoximine, only glutamine maintained this effect. These results suggest that glutamine may well be the metabolite effector in nitrogen repression of GA3 synthesis, as well as in other nonrelated enzymatic activities in G. fujikuroi. PMID:16349128

  20. Arachidonic acid release from rat Leydig cells: the involvement of G protein, phospholipase A2 and regulation of cAMP production.

    PubMed

    Ronco, A M; Moraga, P F; Llanos, M N

    2002-01-01

    We have previously demonstrated that the release of arachidonic acid (AA) from human chorionic gonadotropin (hCG)-stimulated Leydig cells occurs in a dose- and time-dependent manner. In addition, the amount of AA released was dependent on the hormone-receptor interaction and the concentration of LH-hCG binding sites on the cell surface. The present study was conducted to evaluate the involvement of phospholipase A(2) (PLA(2)) and G proteins in AA release from hormonally stimulated rat Leydig cells, and the possible role of this fatty acid in cAMP production. Cells were first prelabelled with [(14)C]AA to incorporate the fatty acid into cell phospholipids, and then treated in different ways to evaluate AA release. hCG (25 mIU) increased the release of AA to 180+/-12% when compared with AA released from control cells, arbitrarily set as 100%. Mepacrine and parabromophenacyl bromide (pBpB), two PLA(2) inhibitors, decreased the hormone-stimulated AA release to 85+/-9 and 70+/-24% respectively. Conversely, melittin, a PLA(2) stimulator, increased the release of AA up to 200% over control. The inhibitory effect of mepacrine on the release of AA was evident in hCG-treated Leydig cells, but not in the melittin-treated cells. To determine if the release of AA was also mediated through a G protein, cells were first permeabilized and subsequently treated with pertussis toxin or GTPgammaS, a non-hydrolyzable analog of GTP. Results demonstrate that GTPgammaS was able to induce a similar level of the release of AA as hCG. In addition, pertussis toxin completely abolished the stimulatory effect of hCG on the release of AA, indicating that a member of the G(i) family was involved in the hCG-dependent release of AA. Cells treated with PLA(2) inhibitors did not modify cAMP production, but exogenously added AA significantly reduced cAMP production from hCG-treated Leydig cells, in a manner dependent on the concentration of AA and hCG. Results presented here suggest an involvement of

  1. Lactic acid bacteria and yeasts involved in the fermentation ofamabere amaruranu, a Kenyan fermented milk.

    PubMed

    Nyambane, Bitutu; Thari, William M; Wangoh, John; Njage, Patrick M K

    2014-11-01

    Indigenous fermented milk products contain microbiota composed of technologically important species and strains which are gradually getting lost with new technologies. We investigated the microbial diversity inamabere amaruranu, a traditionally fermented milk product from Kenya. Sixteen samples of the product from different containers were obtained. One hundred and twenty isolates of lactic acid bacteria (LAB) and 67 strains of yeasts were identified using API 50 CH and API 20 C AUX identification kits, respectively. The average pH of all the traditional fermented samples was 4.00 ± 0.93. Lactobacilli, yeasts, and molds as well asEnterobacteriaceae counts from the plastic containers were significantly higher (P < 0.05) than those from gourd.Enterobacteriaceae were below 1.00 ± 1.11 log10 cfu/mL in products from the gourds and 2.17 ± 1.92 log10 cfu/mL from the plastic containers. The LAB species were identified asStreptococcus thermophilus (25%),Lactobacillus plantarum (20%), andLeuconostoc mesenteroides (20%). The predominant yeasts wereSaccharomyces cerevisiae (25%),Trichosporum mucoides (15%),Candida famata (10%), andCandida albicans (10%). The type of vessel used for fermentation had no significant influence on the type of isolated and identified species. The diverse mixture of LAB and yeasts microflora forms a potential consortium for further product innovation inamabere amaruranu and other fermented milk products. PMID:25493187

  2. The Molecular Basis of Leukocyte Adhesion Involving Phosphatidic Acid and Phospholipase D*

    PubMed Central

    Speranza, Francis; Mahankali, Madhu; Henkels, Karen M.; Gomez-Cambronero, Julian

    2014-01-01

    Defining how leukocytes adhere to solid surfaces, such as capillary beds, and the subsequent migration through the extracellular matrix, is a central biological issue. We show here that phospholipase D (PLD) and its enzymatic reaction product, phosphatidic acid (PA), regulate cell adhesion of immune cells (macrophages and neutrophils) to collagen and have defined the underlying molecular mechanism in a spatio-temporal manner that coincides with PLD activity timing. A rapid (t½ = 4 min) and transient activation of the PLD1 isoform occurs upon adhesion, and a slower (t½ = 7.5 min) but prolonged (>30 min) activation occurs for PLD2. Importantly, PA directly binds to actin-related protein 3 (Arp3) at EC50 = 22 nm, whereas control phosphatidylcholine did not bind. PA-activated Arp3 hastens actin nucleation with a kinetics of t½ = 3 min at 300 nm (compared with controls of no PA, t½ = 5 min). Thus, PLD and PA are intrinsic components of cell adhesion, which reinforce each other in a positive feedback loop and react from cues from their respective solid substrates. In nascent adhesion, PLD1 is key, whereas a sustained adhesion in mature or established focal points is dependent upon PLD2, PA, and Arp3. A prolonged adhesion could effectively counteract the reversible intrinsic nature of this cellular process and constitute a key player in chronic inflammation. PMID:25187519

  3. Alcohol fermentation of sweet potato - 1. Acid hydrolysis and factors involved

    SciTech Connect

    Azhar, A.; Hamdy, M.K.

    1981-04-01

    Factors affecting acid hydrolysis of sweet potato powder (SPP) to fermentable sugars were examined. These include HCl concentration, temperature, time, and levels of SPP. Maximum reducing sugar, reported as dextrose equivalent (DE), was detected after 24 min hydrolysis (1% SPP) in 0.034N HCl heated at 154/degree/C. These samples also had 3.43% hydroxymethylfurfural (HMF) based on dry weight. A high level of HMF (9.2%) was detected in 1% SPP heated at 154/degree/C in 0.10N HCl for 18 min. The lowest concentration of HMF formed (1.8%), at maximal DE of 61%, was established in samples containing 5% SPP and heated at 154/degree/C in 0.034N HCl for 48 min. Aqueous extracts of uncured SPP, examined by High Performance Liquid Chromatography, contained glucose, fructose and sucrose, but degraded SPP had only glucose and fructose. Products of degraded SPP, Under appropriate conditions, could be used for alcohol fermentation. 18 refs.

  4. The involvement of L-type amino acid transporters in theanine transport.

    PubMed

    Yamamoto, Sachiko; Kimura, Toru; Tachiki, Takashi; Anzai, Naohiko; Sakurai, Takuya; Ushimaru, Makoto

    2012-01-01

    L-Theanine has favorable physiological effects in terms of human health, but the mechanisms that transport it to its target organs or cells are not completely defined. To identify the major transport mechanisms of L-theanine, we screened for candidate transporters of L-3H-theanine in several mammal cell lines that intrinsically express multiple transporters with various specificities. All of the cells tested, T24, HepG2, COS1, 293A, Neuro2a, and HuH7, absorbed L-3H-theanine. Uptake was significantly inhibited by the addition of L-leucine and by a specific inhibitor of the system L transport system, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). L-3H-Theanine uptake occurred mostly independently of Na+. These results indicate that L-theanine was taken up via a system L like transport system in all of the cells tested. Additionally, in experiments using cells stably expressing two system L isoforms, LAT1 and LAT2, we found that the two isoforms mediated L-theanine transport to similar extents. Taken together, our results indicate that L-theanine is transported mostly via the system L transport pathway and its isoforms. PMID:23221699

  5. On the early toxic effect of quinolinic acid: involvement of RAGE.

    PubMed

    Cuevas, Elvis; Lantz, Susan; Newport, Glenn; Divine, Becky; Wu, Qiangen; Paule, Merle G; Tobón-Velasco, J César; Ali, Syed F; Santamaría, Abel

    2010-04-26

    Quinolinic acid (QUIN)-induced toxicity is characterized by N-methyl-d-aspartate receptors over-activation, excitotoxicity and oxidative damage. The characterization of toxic cascades produced by QUIN during the first hours after its striatal infusion is relevant for understanding toxic mechanisms. The role of the receptor-for-advanced-glycation-end-products (RAGE) in the early toxic pattern induced by QUIN was evaluated. RAGE expression - assessed by Western blot analysis and immunofluorescence - was enhanced in the striata of QUIN-lesioned rats at 2h post-lesion. QUIN-induced RAGE up-regulation was accompanied by expression of a RAGE target molecule, nuclear factor kappa B (NF-kappaB), and genes encoding for different enzymes. Other toxic markers linked to RAGE activation were increased by QUIN, including NO formation, premature glial response, lactate dehydrogenase leakage, mitochondrial dysfunction and nuclear condensation. Our results suggest that RAGE up-regulation may play a role in the early stages of QUIN toxicity. PMID:20223279

  6. Involvement of abscisic acid in correlative control of flower abscission in soybean

    SciTech Connect

    Yarrow, G.L.

    1985-01-01

    Studies were carried out in three parts: (1) analysis of endogenous abscisic acid (ABA) in abscising and non-abscising flowers, (2) partitioning of radio-labelled ABA and photoassimilates within the soybean raceme, and (3) shading experiments, wherein endogenous levels, metabolism and partitioning of ABA were determined. Endogenous concentrations of ABA failed to show any consistent relationship to abscission of soybean flowers. Partitioning of radiolabelled ABA and photoassimilates displayed consistently higher sink strengths (% DPM) for both /sup 3/H-ABA and /sup 14/C-photoassimilates for non-abscising flowers than for abscising flowers within control racemes. Shading flowers with aluminum foil, 48 hrs prior to sampling, resulted in lowered endogenous ABA concentrations at 12, 17 and 22 days after anthesis (DAA), but not at 0 or 4 DAA. No differences were found in the catabolism of /sup 3/H-ABA between shaded (abscising) and non-shaded (non-abscising) flowers. Reduced partitioning of ABA and photoassimilates to shaded flowers resulted when shades were applied at 0, 4, 12, and 17 DAA, but not at 22 DAA.

  7. Peripheral mechanisms of neuropathic pain – involvement of lysophosphatidic acid receptor-mediated demyelination

    PubMed Central

    Ueda, Hiroshi

    2008-01-01

    Recent advances in pain research provide a clear picture for the molecular mechanisms of acute pain; substantial information concerning plasticity that occurs during neuropathic pain has also become available. The peripheral mechanisms responsible for neuropathic pain are found in the altered gene/protein expression of primary sensory neurons. With damage to peripheral sensory fibers, a variety of changes in pain-related gene expression take place in dorsal root ganglion neurons. These changes, or plasticity, might underlie unique neuropathic pain-specific phenotype modifications – decreased unmyelinated-fiber functions, but increased myelinated A-fiber functions. Another characteristic change is observed in allodynia, the functional change of tactile to nociceptive perception. Throughout a series of studies, using novel nociceptive tests to characterize sensory-fiber or pain modality-specific nociceptive behaviors, it was demonstrated that communication between innocuous and noxious sensory fibers might play a role in allodynia mechanisms. Because neuropathic pain in peripheral and central demyelinating diseases develops as a result of aberrant myelination in experimental animals, demyelination seems to be a key mechanism of plasticity in neuropathic pain. More recently, we discovered that lysophosphatidic acid receptor activation initiates neuropathic pain, as well as possible peripheral mechanims of demyelination after nerve injury. These results lead to further hypotheses of physical communication between innocuous Aβ- and noxious C- or Aδ-fibers to influence the molecular mechanisms of allodynia. PMID:18377664

  8. Influence of Turning and Environmental Contamination on the Dynamics of Populations of Lactic Acid and Acetic Acid Bacteria Involved in Spontaneous Cocoa Bean Heap Fermentation in Ghana▿

    PubMed Central

    Camu, Nicholas; González, Ángel; De Winter, Tom; Van Schoor, Ann; De Bruyne, Katrien; Vandamme, Peter; Takrama, Jemmy S.; Addo, Solomon K.; De Vuyst, Luc

    2008-01-01

    The influence of turning and environmental contamination on six spontaneous cocoa bean heap fermentations performed in Ghana was studied through a multiphasic approach, encompassing both microbiological (culture-dependent and culture-independent techniques) and metabolite target analyses. A sensory analysis of chocolate made from the fermented, dried beans was performed as well. Only four clusters were found among the isolates of acetic acid bacteria (AAB) identified: Acetobacter pasteurianus, Acetobacter ghanensis, Acetobacter senegalensis, and a potential new Acetobacter lovaniensis-like species. Two main clusters were identified among the lactic acid bacteria (LAB) isolated, namely, Lactobacillus plantarum and Lactobacillus fermentum. No differences in biodiversity of LAB and AAB were seen for fermentations carried out at the farm and factory sites, indicating the cocoa pod surfaces and not the general environment as the main inoculum for spontaneous cocoa bean heap fermentation. Turning of the heaps enhanced aeration and increased the relative population size of AAB and the production of acetic acid. This in turn gave a more sour taste to chocolate made from these beans. Bitterness was reduced through losses of polyphenols and alkaloids upon fermentation and cocoa bean processing. PMID:17993565

  9. Hatching success in salamanders and chorus frogs at two sites in Colorado, USA: Effects of acidic deposition and climate

    USGS Publications Warehouse

    Muths, E.; Campbell, D.H.; Corn, P.S.

    2003-01-01

    The snowpack in the vicinity of the Mount Zirkel Wilderness Area is among the most acidic in the western United States. We analyzed water chemistry and examined hatching success in tiger salamanders and chorus frogs at ponds there and at nearby Rabbit Ears Pass (Dumont) to determine whether acid deposition affects amphibians or their breeding habitats at these potentially sensitive locations. We found a wide range of acid neutralizing capacity among ponds within sites; the minimum pH recorded during the experiment was 5.4 at one of 12 ponds with all others at pH ??? 5.7. At Dumont, hatching success for chorus frogs was greater in ponds with low acid neutralizing capacity; however, lowest pHs were >5.8. At current levels of acid deposition, weather and pond characteristics are likely more important than acidity in influencing hatching success in amphibian larvae at these sites.

  10. Evolution of nuclear retinoic acid receptor alpha (RARα) phosphorylation sites. Serine gain provides fine-tuned regulation.

    PubMed

    Samarut, Eric; Amal, Ismail; Markov, Gabriel V; Stote, Roland; Dejaegere, Annick; Laudet, Vincent; Rochette-Egly, Cécile

    2011-07-01

    The human nuclear retinoic acid (RA) receptor alpha (hRARα) is a ligand-dependent transcriptional regulator, which is controlled by a phosphorylation cascade. The cascade starts with the RA-induced phosphorylation of a serine residue located in the ligand-binding domain, S(LBD), allowing the recruitment of the cdk7/cyclin H/MAT1 subcomplex of TFIIH through the docking of cyclin H. It ends by the subsequent phosphorylation by cdk7 of an other serine located in the N-terminal domain, S(NTD). Here, we show that this cascade relies on an increase in the flexibility of the domain involved in cyclin H binding, subsequently to the phosphorylation of S(LBD). Owing to the functional importance of RARα in several vertebrate species, we investigated whether the phosphorylation cascade was conserved in zebrafish (Danio rerio), which expresses two RARα genes: RARα-A and RARα-B. We found that in zebrafish RARαs, S(LBD) is absent, whereas S(NTD) is conserved and phosphorylated. Therefore, we analyzed the pattern of conservation of the phosphorylation sites and traced back their evolution. We found that S(LBD) is most often absent outside mammalian RARα and appears late during vertebrate evolution. In contrast, S(NTD) is conserved, indicating that the phosphorylation of this functional site has been under ancient high selection constraint. This suggests that, during evolution, different regulatory circuits control RARα activity. PMID:21297158

  11. Characterization of enzymes involved in formation of ethyl esters of long-chain fatty acids in humans.

    PubMed

    Diczfalusy, M A; Björkhem, I; Einarsson, C; Hillebrant, C G; Alexson, S E

    2001-07-01

    Elevated fatty acid ethyl ester (FAEE) concentrations have been detected in postmortem organs from alcoholics and patients acutely intoxicated by alcohol, and FAEE have been implicated as mediators of ethanol-induced organ damage. The formation of FAEE is catalyzed by acyl-coenzyme A:ethanol O-acyltransferase (AEAT) and by FAEE synthase, which utilize acyl-CoA and free fatty acids, respectively, as substrates. Because little is known about the capacity of various human tissues to synthesize and hydrolyze FAEE, we investigated formation of FAEE by AEAT and FAEE synthase in tissue homogenates from human gastric ventricular and duodenal mucosa, pancreas, liver, heart, lung, and adipose tissue, gallbladder mucosa, and in serum. Liver, duodenal mucosa, and pancreas were found to have the highest capacities to synthesize FAEE, mainly due to AEAT. FAEE hydrolyzing activity was highest in liver and pancreas, but hardly detectable in adipose tissue or heart. Because fatty acids and alcohol are absorbed by the intestinal mucosa, intestine may be a major site of FAEE synthesis, and FAEE may be delivered via the circulation to other organs and taken up by lipoprotein receptor-mediated uptake. A very low rate of FAEE hydrolysis was detected in heart and adipose tissue, which probably accounts for the previously observed accumulation of FAEE in these organs. PMID:11441128

  12. Mechanisms involved in the effects of phenidone, diclofenac and ethacrynic acid in rat uterus in vitro.

    PubMed

    Pérez Vallina, J R; Cantabrana, B; Hidalgo, A

    1991-01-01

    1. The effects of phenidone (P, 10(-4)-10(-3) M), sodium diclofenac (D, 10(-5)-10(-4) M) and ethacrynic acid (E, 10(-5)-10(-4) M), proposed as inhibitors of eicosanoid synthesis, on the contractions of rat uterus induced by several agonists have been studied. 2. P, D and E inhibit the motility induced by oxytocin (4 mU/ml) (IC50: 4.62 x 10(-4), 2.55 x 10(-4) and 2.98 x 10(-5) M, respectively). 3. P (10(-3) M), D (10(-4) M) and E (10(-4) M) also inhibit the contraction induced by methacholine (10(-5) M), prostaglandin F2a (10(-6) M) and CaCl2 (6 mM), and relaxed, in a dose-dependent way, the tonic component of contraction to KCl (60 mM) (IC50: 5.81 x 10(-4), 6.67 x 10(-5) and 7.55 x 10(-5) M, respectively). 4. The CaCl2 (0.1-10 mM) reverted the relaxation of KCl contraction produced by P, but not by D or E. None of the inhibitions on CaCl2 (6 mM) are reverted by Bay K 8644. 5. D and E also relaxed the tonic contraction to vanadate (10(-4) M) in uterus incubated in calcium free solution P, enhances the vanadate-induced contractions. PMID:1714410

  13. Autoinducer-2 properties of kimchi are associated with lactic acid bacteria involved in its fermentation.

    PubMed

    Park, Hyunjoon; Shin, Heuynkil; Lee, Kyuyeon; Holzapfel, Wilhelm

    2016-05-16

    Bacteria use the cell density-dependent quorum signalling system to regulate particular gene expressions. In food microbiology, signalling is well known for its relation to (foodborne) pathogenicity, food spoilage, and biofilm formation. Quorum quenching and inhibition are thus being considered as a feasible approach in food preservation and safety. In the case of the luxS-mediated universal quorum sensing using autoinducer-2 (AI-2), however, it could be a different issue. Several studies have reported a luxS AI-2 synthase homologue in numerous bacteria, comprising both pathogens and beneficial strains. A recent study has shown the AI-2 signal to restore the balance of the major phyla of the gut microbiota in antibiotic-induced dysbiosis. We measured the AI-2 activity of the lactic fermented food, kimchi, and found different AI-2 signalling intensities. In order to trace the origin of the signal production, we obtained 229 lactic acid bacterial isolates from the kimchi samples, and detected the AI-2 properties of each isolate using a modified AI-2 bioluminescence assay. Our results showed isolates of dominant species of the genera Lactobacillus, Weissella and Leuconostoc which either produced or inhibited the AI-2 signal. No isolate of the dominant species Lactobacillus sakei (75 isolates) and Lactobacillus curvatus (28 isolates) showed AI-2 producing activity, while AI-2 inhibition could not be detected for any of the 31 Lactobacillus plantarum isolates. These results suggest the AI-2 activity of kimchi to result from the interaction of the associated microbial food cultures (MFCs) during fermentation. Thus far, only sparse information is available on AI-2 signalling interaction in fermented food, however, we suggest that fermented food may be a supplier of AI-2 signalling molecules via typical MFCs. PMID:26977818

  14. Gintonin enhances performance of mice in rotarod test: Involvement of lysophosphatidic acid receptors and catecholamine release.

    PubMed

    Lee, Byung-Hwan; Kim, Jisu; Lee, Ra Mi; Choi, Sun-Hye; Kim, Hyeon-Joong; Hwang, Sung-Hee; Lee, Myung Koo; Bae, Chun-Sik; Kim, Hyoung-Chun; Rhim, Hyewon; Lim, Kiwon; Nah, Seung-Yeol

    2016-01-26

    Ginseng has a long history of use as a tonic for restoration of vigor. One example of ginseng-derived tonic effect is that it can improve physical stamina under conditions of stress. However, the active ingredient and the underlying molecular mechanism responsible for the ergogenic effect are unknown. Recent studies show that ginseng contains a novel ingredient, gintonin, which consists of a unique class of herbal-medicine lysophosphatidic acids (LPAs). Gintonin activates G protein-coupled LPA receptors to produce a transient [Ca(2+)]i signal, which is coupled to diverse intra- and inter-cellular signal transduction pathways that stimulate hormone or neurotransmitter release. However, relatively little is known about how gintonin-mediated cellular modulation is linked to physical endurance. In the present study, systemic administration of gintonin, but not ginsenosides, in fasted mice increased blood glucose concentrations in a dose-dependent manner. Gintonin treatment elevated blood glucose to a maximum level after 30min. This elevation in blood glucose level could be abrogated by the LPA1/3 receptor antagonist, Ki16425, or the β-adrenergic receptor antagonist, propranolol. Furthermore, gintonin-dependent enhanced performance of fasted mice in rotarod test was likewise abrogated by Ki16425. Gintonin also elevated plasma epinephrine and norepinephrine concentrations. The present study shows that gintonin mediates catecholamine release through activation of the LPA receptor and that activation of the β-adrenergic receptor is coupled to liver glycogenolysis, thereby increasing the supply of glucose and enhancing performance in the rotarod test. Thus, gintonin acts via the LPA-catecholamine-glycogenolysis axis, representing a candidate mechanism that can explain how ginseng treatment enhances physical stamina. PMID:26706688

  15. Enzymes involved in a novel anaerobic cyclohexane carboxylic acid degradation pathway.

    PubMed

    Kung, Johannes W; Meier, Anne-Katrin; Mergelsberg, Mario; Boll, Matthias

    2014-10-01

    The anaerobic degradation of cyclohexane carboxylic acid (CHC) has so far been studied only in Rhodopseudomonas palustris, in which CHC is activated to cyclohexanoyl coenzyme A (cyclohexanoyl-CoA [CHCoA]) and then dehydrogenated to cyclohex-1-ene-1-carboxyl-CoA (CHeneCoA). This intermediate is further degraded by reactions of the R. palustris-specific benzoyl-CoA degradation pathway of aromatic compounds. However, CHeneCoA is not an intermediate in the degradation of aromatic compounds in all other known anaerobic bacteria; consequently, degradation of CHC was mostly unknown in anaerobic bacteria. We identified a previously unknown CHC degradation pathway in the Fe(III)-reducing Geobacter metallireducens by determining the following CHC-induced in vitro activities: (i) the activation of CHC to CHCoA by a succinyl-CoA:CHC CoA transferase, (ii) the 1,2-dehydrogenation of CHCoA to CHeneCoA by CHCoA dehydrogenase, and (iii) the unusual 1,4-dehydrogenation of CHeneCoA to cyclohex-1,5-diene-1-carboxyl-CoA. This last represents a previously unknown joint intermediate of the CHC and aromatic compound degradation pathway in bacteria other than R. palustris. The enzymes catalyzing the three reactions were purified and characterized as specific enzymes after heterologous expression of the encoding genes. Quantitative reverse transcription-PCR revealed that expression of these genes was highly induced during growth with CHC but not with benzoate. The newly identified CHC degradation pathway is suggested to be present in nearly all CHC-degrading anaerobic bacteria, including denitrifying, Fe(III)-reducing, sulfate-reducing, and fermenting bacteria. Remarkably, all three CHC degradation pathways always link CHC catabolism to the catabolic pathways of aromatic compounds. We propose that the capacity to use CHC as a carbon source evolved from already-existing aromatic compound degradation pathways. PMID:25112478

  16. SIRT1/Atg5/autophagy are involved in the antiatherosclerosis effects of ursolic acid.

    PubMed

    Jiang, Qixiao; Hao, Ranran; Wang, Wencheng; Gao, Hui; Wang, Chunbo

    2016-09-01

    The purpose of this study was to investigate the antiatherosclerosis effects of ursolic acid (UA) in high-fat diet-fed quails (Coturnix coturnix) and potential mechanism. Quails were treated with high-fat diet (14 % pork oil, 1 % cholesterol w/w) with or without UA (50, 150, or 300 mg/kg/day) for 10 weeks. Serum lipid profile was assessed at 0, 4.5, and 10 weeks. After 10 weeks, serum antioxidant status and morphology of aorta were assessed. Additionally, human umbilical vein endothelial cells (HUVECs) were exposed to 100 μg/ml oxidized low-density lipoprotein (ox-LDL) for 24 h, with or without pretreatment with UA (5, 10 or 20 μM) for 16 h, autophagy inhibitor 3-MA 5 mM for 2 h, or SIRT1 inhibitor EX-527 10 μM for 2 h. Cell viability and oxidative stress status were assessed and autophagy status was determined. Acetylation of lysine residue on Atg5 was assessed with immunoprecipitation. In results, high-fat diet negatively affected serum lipid profile and antioxidant status in quails and induced significant histological changes. Cotreatment with UA remarkably alleviated such changes. In HUVECs, ox-LDL treatment induced significant cytotoxicity along with oxidative stress, while UA cotreatment alleviated such changes significantly. UA treatment induced autophagy, enhanced SIRT1 expression, and decreased acetylation of lysine residue on Atg5. Cotreatment with 3-MA or EX-527 effectively abolished UA's protective effects. In summary, UA exerted antiatherosclerosis effects in quails and protected HUVECs from ox-LDL induced cytotoxicity, and the mechanism is associated with increased SIRT1 expression, decreased Atg5 acetylation on lysine residue, and increased autophagy. PMID:27514536

  17. Structural and Functional Characterization of CRM1-Nup214 Interactions Reveals Multiple FG-Binding Sites Involved in Nuclear Export.

    PubMed

    Port, Sarah A; Monecke, Thomas; Dickmanns, Achim; Spillner, Christiane; Hofele, Romina; Urlaub, Henning; Ficner, Ralf; Kehlenbach, Ralph H

    2015-10-27

    CRM1 is the major nuclear export receptor. During translocation through the nuclear pore, transport complexes transiently interact with phenylalanine-glycine (FG) repeats of multiple nucleoporins. On the cytoplasmic side of the nuclear pore, CRM1 tightly interacts with the nucleoporin Nup214. Here, we present the crystal structure of a 117-amino-acid FG-repeat-containing fragment of Nup214, in complex with CRM1, Snurportin 1, and RanGTP at 2.85 Å resolution. The structure reveals eight binding sites for Nup214 FG motifs on CRM1, with intervening stretches that are loosely attached to the transport receptor. Nup214 binds to N- and C-terminal regions of CRM1, thereby clamping CRM1 in a closed conformation and stabilizing the export complex. The role of conserved hydrophobic pockets for the recognition of FG motifs was analyzed in biochemical and cell-based assays. Comparative studies with RanBP3 and Nup62 shed light on specificities of CRM1-nucleoporin binding, which serves as a paradigm for transport receptor-nucleoporin interactions. PMID:26489467

  18. Diagnosis of myocardial involvement in patients with systemic myopathies with 15-(p-(I-123)iodophenyl) pentadecanoic acid (IPPA) SPECT

    SciTech Connect

    Kropp, J.; Briele, B.; Smekal, A.V.; Hotze, A.L.; Biersack, H.J.; Koehler, U.; Zierz, St. ); Knapp, F.F. )

    1992-01-01

    Involvement of the myocardium in non-infectious myopathies presents in most cases as systolic dysfunction or a disturbed cardiac rhythm. We are interested in exploring how often cardiac involvement can be evaluated with various diagnostic techniques in patients with proven myopathy. We investigated 41 patients with myopathies of various etiology, including mitochondrial and congenital myopathies, Curshmann-Steinert disease, muscular dystrophy, and others. Myopathy was proven by muscular biopsy usually from the bicep. Fatty acid imaging was performed with 15-(p-(I-123)iodophenyl)pentadecanoic acid (IP-PA) and sequential SPECT-scintigraphy with a 180 deg. rotation starting at the 45 deg. RAO position. 190 MBq were injected at the maximal stage of a submaximal exercise. Filtered backprojection and reorientation of the slices were achieved by standard techniques. The quantitative comparison of the oblique slices (bulls-eye technique) of the SPECT-studies revealed turnover-rates as a qualitative measure of {beta}-oxidation. Serum levels of lactate (L), pyruvate (P), glucose (G) and triglycerides (TG) were measured at rest and stress. Ventricular function was investigated by radionuclide ventriculography (MUGA) at rest and under stress with Tc-99m labeled red blood cells. In addition, ECG, 24 hour-ECG, and echocardiography were also performed with standard techniques.

  19. Diagnosis of myocardial involvement in patients with systemic myopathies with 15-(p-[I-123]iodophenyl) pentadecanoic acid (IPPA) SPECT

    SciTech Connect

    Kropp, J.; Briele, B.; Smekal, A.V.; Hotze, A.L.; Biersack, H.J.; Koehler, U.; Zierz, St.; Knapp, F.F.

    1992-03-01

    Involvement of the myocardium in non-infectious myopathies presents in most cases as systolic dysfunction or a disturbed cardiac rhythm. We are interested in exploring how often cardiac involvement can be evaluated with various diagnostic techniques in patients with proven myopathy. We investigated 41 patients with myopathies of various etiology, including mitochondrial and congenital myopathies, Curshmann-Steinert disease, muscular dystrophy, and others. Myopathy was proven by muscular biopsy usually from the bicep. Fatty acid imaging was performed with 15-(p-[I-123]iodophenyl)pentadecanoic acid (IP-PA) and sequential SPECT-scintigraphy with a 180 deg. rotation starting at the 45 deg. RAO position. 190 MBq were injected at the maximal stage of a submaximal exercise. Filtered backprojection and reorientation of the slices were achieved by standard techniques. The quantitative comparison of the oblique slices (bulls-eye technique) of the SPECT-studies revealed turnover-rates as a qualitative measure of {beta}-oxidation. Serum levels of lactate (L), pyruvate (P), glucose (G) and triglycerides (TG) were measured at rest and stress. Ventricular function was investigated by radionuclide ventriculography (MUGA) at rest and under stress with Tc-99m labeled red blood cells. In addition, ECG, 24 hour-ECG, and echocardiography were also performed with standard techniques.

  20. Farnesoid X receptor signal is involved in deoxycholic acid-induced intestinal metaplasia of normal human gastric epithelial cells.

    PubMed

    Li, Shu; Chen, Xin; Zhou, Lu; Wang, Bang-Mao

    2015-11-01

    The farnesoid X receptor (FXR) signaling pathway is known to be involved in the metabolism of bile acid, glucose and lipid. In the present study, we demonstrated that 400 µmol/l deoxycholic acid (DCA) stimulation promotes the proliferation of normal human gastric epithelial cells (GES-1). In addition, DCA activated FXR and increased the expression of intestinal metaplasia genes, including caudal-related homeobox transcription factor 2 (Cdx2) and mucin 2 (MUC2). The treatment of FXR agonist GW4064/antagonist guggulsterone (Gug.) significantly increased/decreased the expression levels of FXR, Cdx2 and MUC2 protein in DCA-induced GES-1 cells. GW4064/Gug. also enhanced/reduced the nuclear factor-κB (NF-κB) activity and binding of the Cdx2 promoter region and NF-κB, the most common subunit p50 protein. Taken together, the results indicated that DCA is capable of modulating the expression of Cdx2 and the downstream MUC2 via the nuclear receptor FXR-NF-κB activity in normal gastric epithelial cells. FXR signaling pathway may therefore be involved in the intestinal metaplasia of human gastric mucosa. PMID:26324224

  1. Exploring Regulation Genes Involved in the Expression of L-Amino Acid Oxidase in Pseudoalteromonas sp. Rf-1

    PubMed Central

    Wang, Ju; Lin, Jianxun; Zhao, Minyan

    2015-01-01

    Bacterial L-amino acid oxidase (LAAO) is believed to play important biological and ecological roles in marine niches, thus attracting increasing attention to understand the regulation mechanisms underlying its production. In this study, we investigated genes involved in LAAO production in marine bacterium Pseudoalteromonas sp. Rf-1 using transposon mutagenesis. Of more than 4,000 mutants screened, 15 mutants showed significant changes in LAAO activity. Desired transposon insertion was confirmed in 12 mutants, in which disrupted genes and corresponding functionswere identified. Analysis of LAAO activity and lao gene expression revealed that GntR family transcriptional regulator, methylase, non-ribosomal peptide synthetase, TonB-dependent heme-receptor family, Na+/H+ antiporter and related arsenite permease, N-acetyltransferase GCN5, Ketol-acid reductoisomerase and SAM-dependent methytransferase, and their coding genes may be involved in either upregulation or downregulation pathway at transcriptional, posttranscriptional, translational and/or posttranslational level. The nhaD and sdmT genes were separately complemented into the corresponding mutants with abolished LAAO-activity. The complementation of either gene can restore LAAO activity and lao gene expression, demonstrating their regulatory role in LAAO biosynthesis. This study provides, for the first time, insights into the molecular mechanisms regulating LAAO production in Pseudoalteromonas sp. Rf-1, which is important to better understand biological and ecological roles of LAAO. PMID:25815733

  2. Evaluation Of Sludge Heel Dissolution Efficiency With Oxalic Acid Cleaning At Savannah River Site

    SciTech Connect

    Sudduth, Christie; Vitali, Jason; Keefer, Mark

    2014-01-08

    The chemical cleaning process baseline strategy at the Savannah River Site was revised to improve efficiency during future execution of the process based on lessons learned during previous bulk oxalic acid cleaning activities and to account for operational constraints imposed by safety basis requirements. These improvements were also intended to transcend the difficulties that arise from waste removal in higher rheological yield stress sludge tanks. Tank 12 implemented this improved strategy and the bulk oxalic acid cleaning efforts concluded in July 2013. The Tank 12 radiological removal results were similar to previous bulk oxalic acid cleaning campaigns despite the fact that Tank 12 contained higher rheological yield stress sludge that would make removal more difficult than the sludge treated in previous cleaning campaigns. No appreciable oxalate precipitation occurred during the cleaning process in Tank 12 compared to previous campaigns, which aided in the net volume reduction of 75-80%. Overall, the controls established for Tank 12 provide a template for an improved cleaning process.

  3. Solubilization and purification of the glucosyltransferase involved in the biosynthesis of teichuronic acid by fragments of Micrococcus luteus cell membranes

    SciTech Connect

    Hildebrandt, K.M.; Anderson, J.S.

    1987-05-01

    Enzymes involved in the biosynthesis of teichuronic acid have been demonstrated in cytoplasmic membrane fragments recovered from lysozyme treated Micrococcus luteus cells. Solubilization of the glucosyltransferase activity was effected with aqueous solutions of Triton X-100, Nonidet P-40, Tween 20, or Thesit. Thesit proved most amenable for recovery of glucosyltransferase activity as well as spectrophotometric protein determinations. Recovery of the glucosyltranferase activity was aided during purification by inclusion of 15% glycerol, 0.75% Thesit, 20 mM magnesium ion and 2 mM 2-mercaptoethanol in all buffers. Glucosyltransferase activity was monitored by the transfer of (/sup 14/C)glucose from UDP-(/sup 14/C)glucose to an artificial acceptor. Although the natural acceptor is presumed to be an undecaprenyl diphosphate-activated oligosaccharide, alternate acceptors such as isolated cell wall fractions containing teichuronic acid served equally well. Highly purified teichuronic acid devoid of peptidoglycan was the most effective alternate acceptor. The glucosyltransferase was purified by ammonium sulfate precipitation followed by ion exchange chromatography on DEAE-cellulose yielding an overall 200-fold increase in specific activity.

  4. Identification and physiological characterization of phosphatidic acid phosphatase enzymes involved in triacylglycerol biosynthesis in Streptomyces coelicolor

    PubMed Central

    2013-01-01

    Background Phosphatidic acid phosphatase (PAP, EC 3.1.3.4) catalyzes the dephosphorylation of phosphatidate yielding diacylglycerol (DAG), the lipid precursor for triacylglycerol (TAG) biosynthesis. Despite the importance of PAP activity in TAG producing bacteria, studies to establish its role in lipid metabolism have been so far restricted only to eukaryotes. Considering the increasing interest of bacterial TAG as a potential source of raw material for biofuel production, we have focused our studies on the identification and physiological characterization of the putative PAP present in the TAG producing bacterium Streptomyces coelicolor. Results We have identified two S. coelicolor genes, named lppα (SCO1102) and lppβ (SCO1753), encoding for functional PAP proteins. Both enzymes mediate, at least in part, the formation of DAG for neutral lipid biosynthesis. Heterologous expression of lppα and lppβ genes in E. coli resulted in enhanced PAP activity in the membrane fractions of the recombinant strains and concomitantly in higher levels of DAG. In addition, the expression of these genes in yeast complemented the temperature-sensitive growth phenotype of the PAP deficient strain GHY58 (dpp1lpp1pah1). In S. coelicolor, disruption of either lppα or lppβ had no effect on TAG accumulation; however, the simultaneous mutation of both genes provoked a drastic reduction in de novo TAG biosynthesis as well as in total TAG content. Consistently, overexpression of Lppα and Lppβ in the wild type strain of S. coelicolor led to a significant increase in TAG production. Conclusions The present study describes the identification of PAP enzymes in bacteria and provides further insights on the genetic basis for prokaryotic oiliness. Furthermore, this finding completes the whole set of enzymes required for de novo TAG biosynthesis pathway in S. coelicolor. Remarkably, the overexpression of these PAPs in Streptomyces bacteria contributes to a higher productivity of this single

  5. The amino acid substitution N136Y in Candida albicans sterol 14alpha-demethylase is involved in fluconazole resistance.

    PubMed

    Alvarez-Rueda, Nidia; Fleury, Audrey; Logé, Cédric; Pagniez, Fabrice; Robert, Estelle; Morio, Florent; Le Pape, Patrice

    2016-10-01

    Resistance to fluconazole antifungal is an ongoing impediment to a successful treatment of Candida albicans infections. One of the most prevalent mechanisms leading to azole resistance is genetic alterations of the 14α-demethylase, the target of azole antifungals, through point mutations. Site-directed mutagenesis and molecular modeling of 14α-demethylase rationalize biological data about the role of protein substitutions in the azole treatment failure. In this work, we investigated the role of N136Y substitution by site-directed mutagenesis into Pichia pastoris guided by structural analysis. Single amino acid substitutions were created by site-directed mutagenesis into P. pastoris with C. albicans ERG11 gene as template. In vitro susceptibility of P. pastoris transformants expressing wild-type and mutants to azole compounds was determined by CLSI M27-A2 and spot agar methods. The fluconazole effect on ergosterol biosynthesis was analyzed by gas chromatography-mass spectrometry. By microdilution and spot tests, N136Y transformants showed a reduced in vitro susceptibility to fluconazole compared to wild-type controls. As expected, ergosterol/lanosterol ratios were higher in N136Y transformants compared to the wild-type controls after treatment with fluconazole. Molecular modeling suggests that residue Asn136 located within the first mutation hot spot, could play a role during heme and azole binding. These results provide new insights into the structural basis for 14α-demethylase-azole interaction and could guide the design of novel azole antifungals. PMID:27143634

  6. Involvement of Arabidopsis RACK1 in Protein Translation and Its Regulation by Abscisic Acid

    SciTech Connect

    Guo, Jianjun; Wang, Shucai; Valerius, Oliver; Hall, Hardy; Zeng, Qingning; Li, Jian-Feng; Weston, David; Ellis, Brian; Chen, Jay

    2011-01-01

    Earlier studies have shown that RACK1 functions as a negative regulator of ABA responses in Arabidopsis, but the molecular mechanism of the action of RACK1 in these processes remains elusive. Global gene expression profiling revealed that approximately 40% of the genes affected by ABA treatment were affected in a similar manner by the rack1 mutation, supporting the view that RACK1 is an important regulator of ABA responses. On the other hand, co-expression analysis revealed that >80% of the genes co-expressed with RACK1 encode ribosome proteins, implying a close relationship between RACK1 s function and the ribosome complex. These results implied that the regulatory role for RACK1 in ABA responses may be partially due to its putative function in protein translation, which is one of the major cellular processes that mammalian and yeast RACK1 is involved in. Consistently, all three Arabidopsis RACK1 homologous genes, namely RACK1A, RACK1B and RACK1C, complemented the growth defects of the S. cerevisiae cpc2/rack1 mutant. In addition, RACK1 physically interacts with Arabidopsis Eukaryotic Initiation Factor 6 (eIF6), whose mammalian homologue is a key regulator of 80S ribosome assembly. Moreover, rack1 mutants displayed hypersensitivity to anisomycin, an inhibitor of protein translation, and displayed characteristics of impaired 80S functional ribosome assembly and 60S ribosomal subunit biogenesis in a ribosome profiling assay. Gene expression analysis revealed that ABA inhibits the expression of both RACK1 and eIF6. Taken together, these results suggest that RACK1 may be required for normal production of 60S and 80S ribosomes and that its action in these processes may be regulated by ABA.

  7. Mycobacterium tuberculosis Proteins Involved in Mycolic Acid Synthesis and Transport Localize Dynamically to the Old Growing Pole and Septum

    PubMed Central

    Cantaloube, Sylvain; Bonne, Mélanie; Diagne, Cheikh T.; Laval, Françoise; Daffé, Mamadou; Zerbib, Didier

    2014-01-01

    Understanding the mechanism that controls space-time coordination of elongation and division of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is critical for fighting the tubercle bacillus. Most of the numerous enzymes involved in the synthesis of Mycolic acid - Arabinogalactan-Peptidoglycan complex (MAPc) in the cell wall are essential in vivo. Using a dynamic approach, we localized Mtb enzymes belonging to the fatty acid synthase-II (FAS-II) complexes and involved in mycolic acid (MA) biosynthesis in a mycobacterial model of Mtb: M. smegmatis. Results also showed that the MA transporter MmpL3 was present in the mycobacterial envelope and was specifically and dynamically accumulated at the poles and septa during bacterial growth. This localization was due to its C-terminal domain. Moreover, the FAS-II enzymes were co-localized at the poles and septum with Wag31, the protein responsible for the polar localization of mycobacterial peptidoglycan biosynthesis. The dynamic localization of FAS-II and of the MA transporter with Wag31, at the old-growing poles and at the septum suggests that the main components of the mycomembrane may potentially be synthesized at these precise foci. This finding highlights a major difference between mycobacteria and other rod-shaped bacteria studied to date. Based on the already known polar activities of envelope biosynthesis in mycobacteria, we propose the existence of complex polar machinery devoted to the biogenesis of the entire envelope. As a result, the mycobacterial pole would represent the Achilles' heel of the bacillus at all its growing stages. PMID:24817274

  8. SITE EVALUATION OF INNOVATIVE SEMI-PASSIVE ACID MINE DRAINAGE TREATMENT TECHNOLOGIES AT THE SUMMITVILLE SUPERFUND SITE, COLORADO

    EPA Science Inventory

    The EPA SITE Program is conducting a detailed sampling and evaluation of two innovative passive mine drainage treatment technologies at the Summitville Superfund Mining site in Southern Colorado. The technologies evaluated include the Aquafix automatic hydraulic lime dispensing s...

  9. Heteroconium chaetospira induces resistance to clubroot via upregulation of host genes involved in jasmonic acid, ethylene, and auxin biosynthesis.

    PubMed

    Lahlali, Rachid; McGregor, Linda; Song, Tao; Gossen, Bruce D; Narisawa, Kazuhiko; Peng, Gary

    2014-01-01

    An endophytic fungus, Heteroconium chaetospira isolate BC2HB1 (Hc), suppressed clubroot (Plasmodiophora brassicae -Pb) on canola in growth-cabinet trials. Confocal microscopy demonstrated that Hc penetrated canola roots and colonized cortical tissues. Based on qPCR analysis, the amount of Hc DNA found in canola roots at 14 days after treatment was negatively correlated (r = 0.92, P<0.001) with the severity of clubroot at 5 weeks after treatment at a low (2×10(5) spores pot(-1)) but not high (2×10(5) spores pot(-1)) dose of pathogen inoculum. Transcript levels of nine B. napus (Bn) genes in roots treated with Hc plus Pb, Pb alone and a nontreated control were analyzed using qPCR supplemented with biochemical analysis for the activity of phenylalanine ammonia lyases (PAL). These genes encode enzymes involved in several biosynthetic pathways related potentially to plant defence. Hc plus Pb increased the activity of PAL but not that of the other two genes (BnCCR and BnOPCL) involved also in phenylpropanoid biosynthesis, relative to Pb inoculation alone. In contrast, expression of several genes involved in the jasmonic acid (BnOPR2), ethylene (BnACO), auxin (BnAAO1), and PR-2 protein (BnPR-2) biosynthesis were upregulated by 63, 48, 3, and 3 fold, respectively, by Hc plus Pb over Pb alone. This indicates that these genes may be involved in inducing resistance in canola by Hc against clubroot. The upregulation of BnAAO1 appears to be related to both pathogenesis of clubroot and induced defence mechanisms in canola roots. This is the first report on regulation of specific host genes involved in induced plant resistance by a non-mycorrhizal endophyte. PMID:24714177

  10. Amino Acids Involved in Polyphosphate Synthesis and Its Mobilization Are Distinct in Polyphosphate Kinase-1 from Mycobacterium tuberculosis

    PubMed Central

    Mittal, Payal; Karthikeyan, Subramanian; Chakraborti, Pradip K.

    2011-01-01

    Background In bacteria polyphosphates (poly-P) are involved in cellular metabolism and development especially during stress. The enzyme, principally involved in polyphosphate biosynthesis and its mobilization leading to generation of NTPs, is known as polyphosphate kinase (PPK). Principal Findings Among two genes of polyphosphate kinases present in Mycobacterium tuberculosis, we cloned and expressed PPK1 in Escherichia coli as histidine-tagged protein. This ∼86 kDa protein is capable of autophosphorylation and synthesis of poly-P as well as NTP. Among 22 conserved histidine residues, we found only His-491 is autophosphorylated and crucial for any enzymatic activity. Substitution of His-510 caused mPPK1 protein deficient but not defective in autophosphorylation, thereby contrary to earlier reports negating any role of this residue in the process. However, mutation of His-510 with either Ala or Gln affected ATP or poly-P synthesis depending on the substitution; while such effects were severe with H510A but mild with H510Q. Furthermore, mPPK1 also renders auxiliary nucleotide diphosphate kinase function by synthesizing virtually all NTPs/dNTPs from their cognate NDPs/dNDPs by utilizing poly-P as the phosphate donor albeit with varied efficiency. To assess the influence of other catalytic domain residues of mPPK1 towards its functionality, we designed mutations based on E. coli PPK1 crystal structure since it owes 68% amino acid sequence similarity with mPPK1. Interestingly, our results revealed that mutations in mPPK1 affecting poly-P synthesis always affected its ATP synthesizing ability; however, the reverse may not be true. Conclusions/Significance We conclude that amino acid residues involved in poly-P and ATP synthesizing activities of mPPK1 are distinct. Considering conserved nature of PPK1, it seems our observations have broader implications and not solely restricted to M. tuberculosis only. PMID:22110640

  11. Identification of Key Factors Involved in the Biosorption of Patulin by Inactivated Lactic Acid Bacteria (LAB) Cells

    PubMed Central

    Wang, Ling; Wang, Zhouli; Yuan, Yahong; Cai, Rui; Niu, Chen; Yue, Tianli

    2015-01-01

    The purpose of this study was to identify the key factors involved in patulin adsorption by heat-inactivated lactic acid bacteria (LAB) cells. For preventing bacterial contamination, a sterilization process was involved in the adsorption process. The effects of various physical, chemical, and enzymatic pre-treatments, simultaneous treatments, and post-treatments on the patulin adsorption performances of six LAB strains were evaluated. The pre-treated cells were characterized by scanning electron microscopy (SEM). Results showed that the removal of patulin by viable cells was mainly based on adsorption or degradation, depending on the specific strain. The adsorption abilities were widely increased by NaOH and esterification pre-treatments, and reduced by trypsin, lipase, iodate, and periodate pre-treatments. Additionally, the adsorption abilities were almost maintained at pH 2.2–4.0, and enhanced significantly at pH 4.0–6.0. The effects of sodium and magnesium ions on the adsorption abilities at pH 4 were slight and strain-specific. A lower proportion of patulin was released from the strain with higher adsorption ability. Analyses revealed that the physical structure of peptidoglycan was not a principal factor. Vicinal OH and carboxyl groups were not involved in patulin adsorption, while alkaline amino acids, thiol and ester compounds were important for patulin adsorption. Additionally, besides hydrophobic interaction, electrostatic interaction also participated in patulin adsorption, which was enhanced with the increase in pH (4.0–6.0). PMID:26581099

  12. Characteristics of fluoride in pore-water at accidental hydrofluoric acid spillage site, Gumi, Korea

    NASA Astrophysics Data System (ADS)

    Kwon, E. H.; Lee, H. A.; Lee, J.; Kim, D.; Lee, S.; Yoon, H. O.

    2015-12-01

    A leakage accident of hydrofluoric acid (HF) occurred in Gumi, South Korea at Sep. 2012. The study site is located in the borderline between a large-scale industrial complex and a rural area. The HF plume was made immediately, and moved toward the rural area through air. After the accident, 212 ha of farm land were influenced and most of crops were withered. To recover the soil, CaO was applied after six months. Although several studies have done to estimate the extension and movement of HF plume in the air and to assess the impact on human health or plant after the incident, the long-term fate of fluoride (F) in the affected soils is not identified clearly. Thus, this study aimed to understand the behavior of F in the soil after HF releasing from accident site through chemical analysis and geochemical modeling. Within the radius of 1 km of accident site, 16 pore-water and soil samples were collected. The semi-quantitative soil composition (i.e., Al, Ca, Fe, K, Mg, Mn, Na, P, Si, Ti), total F, total P, OM contents in soil, and soil pH have already been measured, and pore-water compositions are also identified. From these experimental and modeling data, we could be evaluate if impact of accident exists until now, and also could be select and identify existing form of fluoride in soil and pore-water.

  13. Involvement of ethylene in gibberellic acid-induced sulfur assimilation, photosynthetic responses, and alleviation of cadmium stress in mustard.

    PubMed

    Masood, Asim; Khan, M Iqbal R; Fatma, Mehar; Asgher, Mohd; Per, Tasir S; Khan, Nafees A

    2016-07-01

    The role of gibberellic acid (GA) or sulfur (S) in stimulation of photosynthesis is known. However, information on the involvement of ethylene in GA-induced photosynthetic responses and cadmium (Cd) tolerance is lacking. This work shows that ethylene is involved in S-assimilation, photosynthetic responses and alleviation of Cd stress by GA in mustard (Brassica juncea L.). Plants grown with 200 mg Cd kg(-1) soil were less responsive to ethylene despite high ethylene evolution and showed photosynthetic inhibition. Plants receiving 10 μM GA spraying plus 100 mg S kg(-1) soil supplementation exhibited increased S-assimilation and photosynthetic responses under Cd stress. Application of GA plus S decreased oxidative stress of plants grown with Cd and limited stress ethylene formation to the range suitable for promoting sulfur use efficiency (SUE), glutathione (GSH) production and photosynthesis. The role of ethylene in GA-induced S-assimilation and reversal of photosynthetic inhibition by Cd was substantiated by inhibiting ethylene biosynthesis with the use of aminoethoxyvinylglycine (AVG). The suppression of S-assimilation and photosynthetic responses by inhibiting ethylene in GA plus S treated plants under Cd stress indicated the involvement of ethylene in GA-induced S-assimilation and Cd stress alleviation. The outcome of the study is important to unravel the interaction between GA and ethylene and their role in Cd tolerance in plants. PMID:26998941

  14. Generation of Broensted and Lewis acid sites on the surface of silica by addition of dopant cations

    SciTech Connect

    Connell, G.; Dumesic, J.A.

    1987-06-01

    Pyridine adsorption was used to study the acidic properties of silica doped with the following cations: Sc/sup 3 +/, Mg/sup 2 +/, Fe/sup 2 +/, Fe/sup 3 +/, Zn/sup 2 +/, Al/sup 3 +/, and Ga/sup 3 +/. All samples were exposed to pyridine at 423 K and subsequently evacuated at successively higher temperatures to 723 K. Infrared spectra of the adsorbed pyridine indicated that all of these cations generated Lewis acid sites. This can be explained by the presence of coordinatively unsaturated dopant cations on the surface of silica, in accord with a model based on Pauling's electrostatic bond strength rules. The infrared frequency of the 19b band of pyridine adsorbed on these Lewis acid sites was found to increase with increasing electronegativity of the dopant cation. It is suggested that both of these quantities are related to the strength of the Lewis acid sites. Broensted acid sites were also observed by infrared spectroscopy for Sc/sup 3 +/, Al/sup 3 +/, and Ga/sup 3 +/ on silica. These dopant cations are believed to be bonded tetrahedrally on the surface of silica, generating bridging hydroxyl groups between the dopant cation and Si/sup 4 +/. As for zeolite catalysts, the proton associated with these groups and required for charge neutrality is the Broensted acid site. Finally, Broensted acid sites can also be generated on silica by highly electronegative anions, such as HPO/sub 4//sup 2 -/, which generate Broensted acidity in a different manner. 55 references.

  15. Fabrication of hydrophobic polymer foams with double acid sites on surface of macropore for conversion of carbohydrate.

    PubMed

    Pan, Jianming; Mao, Yanli; Gao, Heping; Xiong, Qingang; Qiu, Fengxian; Zhang, Tao; Niu, Xiangheng

    2016-06-01

    Herein we reported a simple and novel synthetic strategy for the fabrication of two kinds of hydrophobic polymer foam catalysts (i.e. Cr(3+)-HPFs-1-H(+) and HPFs-1-H(+)) with hierarchical porous structure, inhomogeneous acidic composition and Lewis-Brønsted double acid sites distributed on the surface, which was used to one-pot conversion of carbohydrate (such as cellulose, glucose and fructose) to a key chemical platform (i.e. 5-hydroxymethylfurfural, HMF). The water-in-oil (W/O) high internal phase emulsions (HIPEs), stabilized by both Span 80 and acidic prepolymers as analogous particles offered the acidic actives, were used as the template for simultaneous polymerization of oil phase in the presence of divinylbenzene (DVB) and styrene (St). After subsequent ion-exchange process, Lewis and Brønsted acid sites derived from exchanged Cr(3+) and H(+) ion were both fixed on the surface of cell of the catalysts. The HPFs-1-H(+) and Cr(3+)-HPFs-1-H(+) had similar hierarchical porous, hydrophobic surface and acid sites (HPFs-1-H(+) with macropores ranging from 0.1 μm to 20 μm, uniform mesopores in 14.4 nm, water contact angle of 122° and 0.614 mmolg(-1) of Brønsted acid sites, as well as Cr(3+)-HPFs-1-H(+) with macropores ranging from 0.1 μm to 20 μm, uniform mesopores in 13.3 nm, water contact angle of 136° and 0.638 mmolg(-1) of Lewis-Brønsted acid sites). It was confirmed that Lewis acid sites of catalyst had a slight influence on the HMF yield of fructose came from the function of Brønsted acid sites, and Lewis acid sites were in favor of improving the HMF yield from cellulose and glucose. This work opens up a simple and novel route to synthesize multifunctional polymeric catalysts for efficient one-pot conversion of carbohydrate to HMF. PMID:27083362

  16. Dansyl labeling to modulate the relative affinity of bile acids for the binding sites of human serum albumin.

    PubMed

    Rohacova, Jana; Sastre, German; Marin, M Luisa; Miranda, Miguel A

    2011-09-01

    Binding of natural bile acids to human serum albumin (HSA) is an important step in enterohepatic circulation and provides a measure of liver function. In this article, we report on the use of four dansyl (Dns) derivatives of cholic acid (ChA) to demonstrate a regiodifferentiation in their relative affinity for the two binding sites of HSA. Using both steady-state and time-resolved fluorescence, formation of Dns-ChA@HSA complexes was confirmed; the corresponding binding constants were determined, and their distribution between bulk solution and HSA microenvironment was estimated. By means of energy transfer from Trp to the Dns moiety, donor-acceptor distances were estimated (21-25 Å) and found to be compatible with both site 1 and site 2 occupancies. Nevertheless, titration using warfarin and ibuprofen as specific displacement probes clearly indicated that 3α- and 3β-Dns-ChA bind to HSA at site 2, whereas their C-7 regioisomers bind to HSA at site 1. Furthermore, the C-3-labeled compounds are displaced by lithocholic acid, whereas they are insensitive to ChA, confirming the assumption that the former binds to HSA at site 2. Thus, Dns labeling provides a useful tool to modulate the relative affinity of ChA to the major binding sites of HSA and, in combination with other fluorescent ChA analogs, to mimic the binding behavior of natural bile acids. PMID:21797258

  17. Role of Criegee Intermediates in Formation of Sulfuric Acid at BVOCs-rich Cape Corsica Site

    NASA Astrophysics Data System (ADS)

    Kukui, A.; Dusanter, S.; Sauvage, S.; Gros, V.; Bourrianne, T.; Sellegri, K.; Wang, J.; Colomb, A.; Pichon, J. M.; Chen, H.; Kalogridis, C.; Zannoni, N.; Bonsang, B.; Michoud, V.; Locoge, N.; Leonardis, T.

    2015-12-01

    Oxidation of SO2 in reactions with stabilised Criegee Intermediates (sCI) was suggested as an additional source of gaseous sulfuric acid (H2SO4) in the atmosphere, complementary to the conventional H2SO4 formation in reaction of SO2 with OH radicals. Evaluation of the importance of this additional source is complicated due to large uncertainty in the mechanism and rate constants for the reactions of different sCI with SO2, water vapor and other atmospheric species. Here we present an evaluation of the role of sCI in H2SO4 production at remote site on Cape Corsica near the North tip of Corsica Island (Ersa station, Western Mediterranean). In July 2013 comprehensive field observations including gas phase (OH and RO2 radicals, H2SO4, VOCs, NOx, SO2, others) and aerosol measurements were conducted at this site in the frame of ChArMEx project. During the field campaign the site was strongly influenced by local emissions of biogenic volatile compounds (BVOCs), including isoprene and terpenes, forming different sCI in reactions with ozone and, hence, presenting additional source of H2SO4 via sCI+SO2. However, this additional source of H2SO4 at the Ersa site was found to be insignificant. The observed concentrations of H2SO4 were found to be in good agreement with those estimated from the H2SO4 condensation sink and the production of H2SO4 only in the reaction of OH with SO2, without accounting for any additional H2SO4 source. Using the BVOCs observations we present estimation of the upper limit for the rate constants of H2SO4 production via reactions of different sCI with SO2.

  18. Methane to acetic acid over Cu-exchanged zeolites: mechanistic insights from a site-specific carbonylation reaction.

    PubMed

    Narsimhan, Karthik; Michaelis, Vladimir K; Mathies, Guinevere; Gunther, William R; Griffin, Robert G; Román-Leshkov, Yuriy

    2015-02-11

    The selective low temperature oxidation of methane is an attractive yet challenging pathway to convert abundant natural gas into value added chemicals. Copper-exchanged ZSM-5 and mordenite (MOR) zeolites have received attention due to their ability to oxidize methane into methanol using molecular oxygen. In this work, the conversion of methane into acetic acid is demonstrated using Cu-MOR by coupling oxidation with carbonylation reactions. The carbonylation reaction, known to occur predominantly in the 8-membered ring (8MR) pockets of MOR, is used as a site-specific probe to gain insight into important mechanistic differences existing between Cu-MOR and Cu-ZSM-5 during methane oxidation. For the tandem reaction sequence, Cu-MOR generated drastically higher amounts of acetic acid when compared to Cu-ZSM-5 (22 vs 4 μmol/g). Preferential titration with sodium showed a direct correlation between the number of acid sites in the 8MR pockets in MOR and acetic acid yield, indicating that methoxy species present in the MOR side pockets undergo carbonylation. Coupled spectroscopic and reactivity measurements were used to identify the genesis of the oxidation sites and to validate the migration of methoxy species from the oxidation site to the carbonylation site. Our results indicate that the Cu(II)-O-Cu(II) sites previously associated with methane oxidation in both Cu-MOR and Cu-ZSM-5 are oxidation active but carbonylation inactive. In turn, combined UV-vis and EPR spectroscopic studies showed that a novel Cu(2+) site is formed at Cu/Al <0.2 in MOR. These sites oxidize methane and promote the migration of the product to a Brønsted acid site in the 8MR to undergo carbonylation. PMID:25562431

  19. 36C1 measurements and the hydrology of an acid injection site

    USGS Publications Warehouse

    Vourvopoulos, G.; Brahana, J.V.; Nolte, E.; Korschinek, G.; Priller, A.; Dockhorn, B.

    1990-01-01

    In an area in western Tennessee (United States), an industrial firm is injecting acidic (pH = 0.1) iron chloride into permeable zones of carbonate rocks at depths ranging from 1000 to 2200 m below land surface. Overlying the injection zone at a depth of approximately 500 m below land surface is a regional fresh-water aquifer, the Knox aquifer. A study is currently underway to investigate whether the injection wells are hydraulically isolated from the fresh-water aquifer. Drilling of a test well that will reach a total depth of 2700 m has been initiated. The 36Cl content of 15 samples from the Knox aquifer, from monitor wells in the vicinity of the injection site, and from the test well have been analyzed. ?? 1990.

  20. Identification of three critical acidic residues of poly(ADP-ribose) glycohydrolase involved in catalysis: determining the PARG catalytic domain

    PubMed Central

    Patel, Chandra N.; Koh, David W.; Jacobson, Myron K.; Oliveira, Marcos A.

    2005-01-01

    PARG [poly(ADP-ribose) glycohydrolase] catalyses the hydrolysis of α(1″→2′) or α(1‴→2″) O-glycosidic linkages of ADP-ribose polymers to produce free ADP-ribose. We investigated possible mechanistic similarities between PARG and glycosidases, which also cleave O-glycosidic linkages. Glycosidases typically utilize two acidic residues for catalysis, thus we targeted acidic residues within a conserved region of bovine PARG that has been shown to contain an inhibitor-binding site. The targeted glutamate and aspartate residues were changed to asparagine in order to minimize structural alterations. Mutants were purified and assayed for catalytic activity, as well as binding, to an immobilized PARG inhibitor to determine ability to recognize substrate. Our investigation revealed residues essential for PARG catalytic activity. Two adjacent glutamic acid residues are found in the conserved sequence Gln755-Glu-Glu757, and a third residue found in the conserved sequence Val737-Asp-Phe-Ala-Asn741. Our functional characterization of PARG residues, along with recent identification of an inhibitor-binding residue Tyr796 and a glycine-rich region Gly745-Gly-Gly747 important for PARG function, allowed us to define a PARG ‘signature sequence’ [vDFA-X3-GGg-X6–8-vQEEIRF-X3-PE-X14-E-X12-YTGYa], which we used to identify putative PARG sequences across a range of organisms. Sequence alignments, along with our mapping of PARG functional residues, suggest the presence of a conserved catalytic domain of approx. 185 residues which spans residues 610–795 in bovine PARG. PMID:15658938

  1. The reaction mechanism for the SCR process on monomer V(5+) sites and the effect of modified Brønsted acidity.

    PubMed

    Arnarson, Logi; Falsig, Hanne; Rasmussen, Søren B; Lauritsen, Jeppe V; Moses, Poul Georg

    2016-06-22

    The energetics, structures and activity of a monomeric VO3H/TiO2(001) catalyst are investigated for the selective catalytic reduction (SCR) reaction by the use of density functional theory (DFT). Furthermore we study the influences of a dopant substitute in the TiO2 support and its effects on the known properties of the SCR system such as Brønsted acidity and reducibility of vanadium. We find for the reduction part of the SCR mechanism that it involves two Ti-O-V oxygen sites. One is a hydroxyl possessing Brønsted acidity which contributes to the formation of NH4(+), while the other accepts a proton which charge stabilizes the reduced active site. In the reduction the proton is donated to the latter due to a reaction between NH3 and NO that forms a H2NNO molecule which decomposes into N2(g) and H2O(g). A dopant substitution of 10 different dopants: Si, Ge, Se, Zr, Sn, Te, Hf, V, Mo and W at each of the sites, which participate in the reaction, modifies the energetics and therefore the SCR activity. We find that Brønsted acidity is a descriptor for the SCR activity at low temperatures. Based on this descriptor we find that Zr, Hf and Sn have a positive effect as they decrease the activation energy for the SCR reaction. PMID:27297567

  2. Comparison of the autoradiographic binding distribution of [3H]-gabapentin with excitatory amino acid receptor and amino acid uptake site distributions in rat brain.

    PubMed Central

    Thurlow, R. J.; Hill, D. R.; Woodruff, G. N.

    1996-01-01

    1. Gabapentin is a novel anticonvulsant with an unknown mechanism of action. Recent homogenate binding studies with [3H]-gabapentin have suggested a structure-activity relationship similar to that shown for the amino acid transport system responsible for the uptake of large neutral amino acids (LNAA). 2. The autoradiographic binding distribution of [3H]-gabapentin in rat brain was compared with the distributions for excitatory amino acid receptor subtypes and the uptake sites for excitatory and large neutral amino acids in consecutive rat brain sections. 3. Densitometric measurement of the autoradiographic images followed by normalisation with respect to the hippocampus CA1 stratum radiatum, was carried out before comparison of each binding distribution with that of [3H]-gabapentin by linear regression analysis. The correlation coefficients observed showed no absolute correlation was observed between the binding distributions of [3H]-gabapentin and those of the excitatory amino acid receptor subtypes. The acidic and large neutral amino acid uptake site distributions demonstrated a much closer correlation to the [3H]-gabapentin binding site distribution. The correlation coefficients for D-[3H]-aspartate, L-[3H]-leucine and L-[3H]-isoleucine binding site distributions were 0.76, 0.90 and 0.88 respectively. 4. Concentration-dependent inhibition by unlabelled gabapentin of autoradiographic binding of L-[3H]-leucine and L-[3H]-isoleucine was observed, with non-specific binding levels being reached at concentrations between 10 and 100 microM. 5. Excitotoxic quinolinic acid lesion studies in rat brain caudate putamen and autoradiography were carried out for the amino acid uptake sites mentioned above. The resulting glial infiltration of the lesioned areas was visualized by autoradiography using the peripheral benzodiazepine receptor specific ligand [3H]-PK11195. A significant decrease in binding density in the lesioned area compared with sham-operated animals was observed

  3. Involvement of individual subsites and secondary substrate binding sites in multiple attack on amylose by barley alpha-amylase.

    PubMed

    Kramhøft, Birte; Bak-Jensen, Kristian Sass; Mori, Haruhide; Juge, Nathalie; Nøhr, Jane; Svensson, Birte

    2005-02-15

    Barley alpha-amylase 1 (AMY1) hydrolyzed amylose with a degree of multiple attack (DMA) of 1.9; that is, on average, 2.9 glycoside bonds are cleaved per productive enzyme-substrate encounter. Six AMY1 mutants, spanning the substrate binding cleft from subsites -6 to +4, and a fusion protein, AMY1-SBD, of AMY1 and the starch binding domain (SBD) of Aspergillus niger glucoamylase were also analyzed. DMA of the subsite -6 mutant Y105A and AMY1-SBD increased to 3.3 and 3.0, respectively. M53E, M298S, and T212W at subsites -2, +1/+2, and +4, respectively, and the double mutant Y105A/T212W had decreased DMA of 1.0-1.4. C95A (subsite -5) had a DMA similar to that of wild type. Maltoheptaose (G7) was always the major initial oligosaccharide product. Wild-type and the subsite mutants released G6 at 27-40%, G8 at 60-70%, G9 at 39-48%, and G10 at 33-44% of the G7 rate, whereas AMY1-SBD more efficiently produced G8, G9, and G10 at rates similar to, 66%, and 60% of G7, respectively. In contrast, the shorter products appeared with large individual differences: G1, 0-15%; G2, 8-43%; G3, 0-22%; and G4, 0-11% of the G7 rate. G5 was always a minor product. Multiple attack thus involves both longer translocation of substrate in the binding cleft upon the initial cleavage to produce G6-G10, essentially independent of subsite mutations, and short-distance moves resulting in individually very different rates of release of G1-G4. Accordingly, the degree of multiple attack as well as the profile of products can be manipulated by structural changes in the active site or by introduction of extra substrate binding sites. PMID:15697208

  4. Volatile aromatic hydrocarbons and dicarboxylic acid concentrations in air at an urban site in the Southwestern US

    NASA Astrophysics Data System (ADS)

    Tran, Ngoc K.; Steinberg, Spencer M.; Johnson, Brian J.

    Concentrations of benzene, toluene, ethylbenzene, o-xylene, and m- and p-xylene were measured at an urban sampling site in Las Vegas, NV by sorbent sampling followed by thermal desorption and determination by GC-PID. Simultaneously, measurements of oxalic, malonic, succinic, and adipic acids were made at the same site by collection on quartz filters, extraction, esterification, and determination by GC-FID. For the period from April 7, 1997 to June 11, 1997, 201 sets of hydrocarbon measurements and 99 sets of acid measurements were made. Additional measurements of dicarboxylic acids were made on samples that represented potential direct sources, e.g. green plants and road dust. Correlations between the hydrocarbon and CO concentrations (measured by the Clark County Health District at a nearby site) were highly significant and a strong negative correlation of hydrocarbon concentration with ozone concentration (also from the county site) was observed under quiescent atmospheric conditions. In general, dicarboxylic acid concentrations were well correlated with one another (with the exception of adipic acid) but not well correlated with hydrocarbon, CO, and ozone concentrations. Multiple sources and complex formation processes are indicated for the dicarboxylic acids.

  5. Silencing of ABCC13 transporter in wheat reveals its involvement in grain development, phytic acid accumulation and lateral root formation.

    PubMed

    Bhati, Kaushal Kumar; Alok, Anshu; Kumar, Anil; Kaur, Jagdeep; Tiwari, Siddharth; Pandey, Ajay Kumar

    2016-07-01

    Low phytic acid is a trait desired in cereal crops and can be achieved by manipulating the genes involved either in its biosynthesis or its transport in the vacuoles. Previously, we have demonstrated that the wheat TaABCC13 protein is a functional transporter, primarily involved in heavy metal tolerance, and a probable candidate gene to achieve low phytate wheat. In the current study, RNA silencing was used to knockdown the expression of TaABCC13 in order to evaluate its functional importance in wheat. Transgenic plants with significantly reduced TaABCC13 transcripts in either seeds or roots were selected for further studies. Homozygous RNAi lines K1B4 and K4G7 exhibited 34-22% reduction of the phytic acid content in the mature grains (T4 seeds). These transgenic lines were defective for spike development, as characterized by reduced grain filling and numbers of spikelets. The seeds of transgenic wheat had delayed germination, but the viability of the seedlings was unaffected. Interestingly, early emergence of lateral roots was observed in TaABCC13-silenced lines as compared to non-transgenic lines. In addition, these lines also had defects in metal uptake and development of lateral roots in the presence of cadmium stress. Our results suggest roles of TaABCC13 in lateral root initiation and enhanced sensitivity towards heavy metals. Taken together, these data demonstrate that wheat ABCC13 is functionally important for grain development and plays an important role during detoxification of heavy metals. PMID:27342224

  6. Chylomicron remnants and nonesterified fatty acids differ in their ability to inhibit genes involved in lipogenesis in rats.

    PubMed

    Kohan, Alison B; Qing, Yang; Cyphert, Holly A; Tso, Patrick; Salati, Lisa M

    2011-02-01

    Primary hepatocytes treated with nonesterified PUFA have been used as a model for analyzing the inhibitory effects of dietary polyunsaturated fats on lipogenic gene expression. Although nonesterified fatty acids play an important signaling role in starvation, they do not completely recapitulate the mechanism of dietary fat presentation to the liver, which is delivered via chylomicron remnants. To test the effect of remnant TG on lipogenic enzyme expression, chylomicron remnants were generated from the lymph of rats intubated with either safflower oil or lard. The remnants were added to the medium of primary rat hepatocytes in culture and the accumulation of mRNA for genes involved in carbohydrate and lipid metabolism was measured. Both PUFA-enriched remnants and nonesterified PUFA inhibited the expression and maturation of sterol response element binding protein-1c (SREBP-1c) and the expression of lipogenic genes regulated by this transcription factor. These remnants also inhibited the expression of glucose-6-phosphate dehydrogenase (G6PD), a gene regulated at post-transcriptional steps. In contrast, PUFA-enriched remnants did not inhibit the accumulation of mRNA for malic enzyme, glucokinase, and L-pyruvate kinase, whereas nonesterified fatty acids caused a decrease in these mRNA. These genes are regulated independently of SREBP-1c. SFA-enriched remnants did not inhibit lipogenic gene expression, which is consistent with a lack of inhibition of lipogenesis by dietary saturated fats. Thus, the inhibitory action of dietary polyunsaturated fats on lipogenesis involves a direct action of chylomicron remnants on the liver. PMID:21169224

  7. An ortholog of farA of Aspergillus nidulans is implicated in the transcriptional activation of genes involved in fatty acid utilization in the yeast Yarrowia lipolytica

    SciTech Connect

    Poopanitpan, Napapol; Kobayashi, Satoshi; Fukuda, Ryouichi; Horiuchi, Hiroyuki; Ohta, Akinori

    2010-11-26

    Research highlights: {yields} POR1 is a Yarrowia lipolytica ortholog of farA involved in fatty acid response in A. nidulans. {yields} Deletion of POR1 caused growth defects on fatty acids. {yields} {Delta}por1 strain exhibited defects in the induction of genes involved in fatty acid utilization. -- Abstract: The yeast Yarrowia lipolytica effectively utilizes hydrophobic substrates such as fatty acids and n-alkanes. To identify a gene(s) regulating fatty acid utilization in Y. lipolytica, we first studied homologous genes to OAF1 and PIP2 of Saccharomyces cerevisiae, but their disruption did not change growth on oleic acid at all. We next characterized a Y. lipolytica gene, POR1 (primary oleate regulator 1), an ortholog of farA encoding a transcriptional activator that regulates fatty acid utilization in Aspergillus nidulans. The deletion mutant of POR1 was defective in the growth on various fatty acids, but not on glucose, glycerol, or n-hexadecane. It exhibited slight defect on n-decane. The transcriptional induction of genes involved in {beta}-oxidation and peroxisome proliferation by oleate was distinctly diminished in the {Delta}por1 strains. These data suggest that POR1 encodes a transcriptional activator widely regulating fatty acid metabolism in Y. lipolytica.

  8. Site-specific acid-base properties of pholcodine and related compounds.

    PubMed

    Kovács, Z; Hosztafi, S; Noszál, B

    2006-11-01

    The acid-base properties of pholcodine, a cough-depressant agent, and related compounds including metabolites were studied by 1H NMR-pH titrations, and are characterised in terms of macroscopic and microscopic protonation constants. New N-methylated derivatives were also synthesized in order to quantitate site- and nucleus-specific protonation shifts and to unravel microscopic acid-base equilibria. The piperidine nitrogen was found to be 38 and 400 times more basic than its morpholine counterpart in pholcodine and norpholcodine, respectively. The protonation data show that the molecule of pholcodine bears an average of positive charge of 1.07 at physiological pH, preventing it from entering the central nervous system, a plausible reason for its lack of analgesic or addictive properties. The protonation constants of pholcodine and its derivatives are interpreted by comparing with related molecules of pharmaceutical interest. The pH-dependent relative concentrations of the variously protonated forms of pholcodine and morphine are depicted in distribution diagrams. PMID:17004059

  9. Hybridoma antibodies to the lipid-binding site(s) in the amino-terminal region of fibronectin inhibits binding of streptococcal lipoteichoic acid.

    PubMed

    Stanislawski, L; Courtney, H S; Simpson, W A; Hasty, D L; Beachey, E H; Robert, L; Ofek, I

    1987-08-01

    In this report, we present evidence to suggest that streptococci and lipoteichoic acid (LTA) interact with a fatty acid binding site located near the NH2-terminus of fibronectin. The evidence is based on the following observations. Antibodies directed against a synthetic peptide (residues 1-30 of the amino-terminus of fibronectin) reacted with the two thermolysin-generated peptides (24 and 28 kilodaltons [kDa]) that were adsorbed by and eluted from streptococci. The adsorption of the 24- and 28-kDa peptides to streptococci was inhibited by LTA. The two monoclonal antibodies that inhibited the binding of LTA to fibronectin reacted only with the 24- and 28-kDa fragments of fibronectin. Conversely, LTA, as well as lauric acid and oleic acid, blocked the binding of the same monoclonal antibodies to fibronectin. LTA had no effect on the binding of hybridoma antibodies directed against the collagen or cell-binding domain. PMID:3298457

  10. Spectroscopic Investigation of the Species Involved in the Rhodium-Catalyzed Oxidative Carbonylation of Toluene to Toluic Acid

    SciTech Connect

    Zakzeski, Joseph; Burton, Sarah D.; Behn, Andrew; Head-Gordon, Martin P.; Bell, Alexis T.

    2009-11-14

    A spectroscopic investigation of complexes used to catalyze the oxidative carbonylation of toluene to para-toluic acid was conducted. Rhodium complexes were analyzed by 103Rh and 13C NMR, UV-visible spectroscopy, and infrared spectroscopy. In the presence of vanadium and oxygen, the resting state of the Rh catalyst was found to exist as a Rh(III) complex with carbonyl and trifluoroacetate ligands, consistent with the structure Rh(CO)2(TFA)3. The complex exhibited a carbonyl peak with an unusual degree of shielding, which resulted in the appearance of the carbonyl peak at an unprecedented upfield position in the 13C NMR spectrum. This shielding was caused by interaction of the carbonyl group with the trifluoroacetate ligand. In the absence of oxygen, the Rh(III) complex reduced to Rh(I), and the reduced form exhibited properties resembling the catalyst precursor. Structures and spectroscopic properties calculated using Density Functional Theory were in good agreement with experimental results. The vanadium co-catalyst was similarly characterized by 51V NMR and UV-visible spectroscopy. The oxidized species corresponded to [(VO2)(TFA)]2, whereas the reduced species corresponded (VO)(TFA)2. The spectroscopic results obtained in this study confirm the identity of the species that have been proposed to be involved in the Rh-catalyzed oxidative carbonylation of toluene to toluic acid.

  11. Arabidopsis seed-specific vacuolar aquaporins are involved in maintaining seed longevity under the control of ABSCISIC ACID INSENSITIVE 3.

    PubMed

    Mao, Zhilei; Sun, Weining

    2015-08-01

    The tonoplast intrinsic proteins TIP3;1 and TIP3;2 are specifically expressed during seed maturation and localized to the seed protein storage vacuole membrane. However, the function and physiological roles of TIP3s are still largely unknown. The seed performance of TIP3 knockdown mutants was analysed using the controlled deterioration test. The tip3;1/tip3;2 double mutant was affected in seed longevity and accumulated high levels of hydrogen peroxide compared with the wild type, suggesting that TIP3s function in seed longevity. The transcription factor ABSCISIC ACID INSENSITIVE 3 (ABI3) is known to be involved in seed desiccation tolerance and seed longevity. TIP3 transcript and protein levels were significantly reduced in abi3-6 mutant seeds. TIP3;1 and TIP3;2 promoters could be activated by ABI3 in the presence of abscisic acid (ABA) in Arabidopsis protoplasts. TIP3 proteins were detected in the protoplasts transiently expressing ABI3 and in ABI3-overexpressing seedlings when treated with ABA. Furthermore, ABI3 directly binds to the RY motif of the TIP3 promoters. Therefore, seed-specific TIP3s may help maintain seed longevity under the expressional control of ABI3 during seed maturation and are members of the ABI3-mediated seed longevity pathway together with small heat shock proteins and late embryo abundant proteins. PMID:26019256

  12. Identification of IgE-binding peptide and critical amino acids of Jatropha curcas allergen involved in allergenic response.

    PubMed

    Crespo, Livia Maia; de Oliveira, Natalia Deus; Damatta, Renato Augusto; do Nascimento, Viviane Veiga; Soares, Thais Pacheco; Machado, Olga Lima Tavares

    2016-01-01

    Increasing energy demand has spurred interest in the use of biofuels. Jatropha curcas (physic nut), an inedible oilseed, is a potential source of bioenergy. The seeds, however, contain allergens such as Jat c 1, a 2S albumin that can induce hypersensitivity reactions in humans and result in allergic diseases. Recent advances in identifying and characterizing plant allergens and, in particular, their immunoglobulin E (IgE)-binding epitopes have produced a wealth of information. We identified IgE-binding regions and the critical amino acids involved in the degranulation of mast cells and the release of histamine, preliminary steps for the prevention and treatment of this allergy. Four IgE-binding regions were identified in the sequence of Jat c 1. We identified and demonstrated the fundamental role of two glutamic acid residues in IgE binding. The sequence LEKQLEEGEVGS produces a random loop on the most exposed part of Jat c 1. This region is important to the stimulation of the allergic response. The possibility of using this information to produce vaccines and other pharmacological agents for allergy treatment is discussed. PMID:27119058

  13. Key amino acid residues involved in multi-point binding interactions between brazzein, a sweet protein, and the T1R2-T1R3 human sweet receptor

    PubMed Central

    Assadi-Porter, Fariba M.; Maillet, Emeline L.; Radek, James T.; Quijada, Jeniffer; Markley, John L.; Max, Marianna

    2010-01-01

    The sweet protein brazzein activates the human sweet receptor, a heterodimeric G-protein coupled receptor (GPCR) composed of subunits T1R2 and T1R3. In order to elucidate the key amino acid(s) responsible for this interaction, we mutated residues in brazzein and each of the two subunits of the receptor. The effects of brazzein mutations were assayed by a human taste panel and by an in vitro assay involving receptor subunits expressed recombinantly in human embryonic kidney cells; the effects of the receptor mutations were assayed by the in vitro assay. We mutated surface residues of brazzein at three putative interaction sites: Site 1 (Loop43), Site 2 (N- and C-terminus and adjacent Glu36, Loop33), and Site 3 (Loop9–19). Basic residues in Site 1 and acidic residues in Site 2 were essential for positive responses from each assay. Mutation of Y39A (Site 1) greatly reduced positive responses. A bulky side chain at position 54 (Site 2), rather than a side chain with hydrogen bonding potential, was required for positive responses as was the presence of the native disulfide bond in Loop 9–19 (Site 3). Results from mutagenesis and chimeras of the receptor indicated that brazzein interacts with both T1R2 and T1R3 and that the Venus fly trap module of T1R2 is important for brazzein agonism. With one exception, all mutations of receptor residues at putative interaction sites predicted by wedge models failed to yield the expected decrease in the brazzein response. The exception, hT1R2:R217A-hT1R3, which contained a substitution in lobe 2 at the interface between the two subunits, exhibited a small selective decrease in brazzein activity. However, because the mutation was found to increase the positive cooperativity of binding by multiple ligands proposed to bind both T1R subunits (brazzein, monellin, and sucralose) but not those that bind to a single subunit (neotame and cyclamate), we suggest that this site in involved in subunit-subunit interaction rather than direct

  14. Neurologic syndrome associated with homozygous mutation at MAG sialic acid binding site.

    PubMed

    Roda, Ricardo H; FitzGibbon, Edmond J; Boucekkine, Houda; Schindler, Alice B; Blackstone, Craig

    2016-08-01

    The MAG gene encodes myelin-associated glycoprotein (MAG), an abundant protein involved in axon-glial interactions and myelination during nerve regeneration. Several members of a consanguineous family with a clinical syndrome reminiscent of Pelizaeus-Merzbacher disease and demyelinating leukodystrophy on brain MRI were recently found to harbor a homozygous missense p.Ser133Arg MAG mutation. Here, we report two brothers from a nonconsanguineous family afflicted with progressive cognitive impairment, neuropathy, ataxia, nystagmus, and gait disorder. Exome sequencing revealed the homozygous missense mutation p.Arg118His in MAG. This Arg118 residue in immunoglobulin domain 1 is critical for sialic acid binding, providing a compelling mechanistic basis for disease pathogenesis. PMID:27606346

  15. Prediction of mucin-type O-glycosylation sites in mammalian proteins using the composition of k-spaced amino acid pairs

    PubMed Central

    Chen, Yong-Zi; Tang, Yu-Rong; Sheng, Zhi-Ya; Zhang, Ziding

    2008-01-01

    Background As one of the most common protein post-translational modifications, glycosylation is involved in a variety of important biological processes. Computational identification of glycosylation sites in protein sequences becomes increasingly important in the post-genomic era. A new encoding scheme was employed to improve the prediction of mucin-type O-glycosylation sites in mammalian proteins. Results A new protein bioinformatics tool, CKSAAP_OGlySite, was developed to predict mucin-type O-glycosylation serine/threonine (S/T) sites in mammalian proteins. Using the composition of k-spaced amino acid pairs (CKSAAP) based encoding scheme, the proposed method was trained and tested in a new and stringent O-glycosylation dataset with the assistance of Support Vector Machine (SVM). When the ratio of O-glycosylation to non-glycosylation sites in training datasets was set as 1:1, 10-fold cross-validation tests showed that the proposed method yielded a high accuracy of 83.1% and 81.4% in predicting O-glycosylated S and T sites, respectively. Based on the same datasets, CKSAAP_OGlySite resulted in a higher accuracy than the conventional binary encoding based method (about +5.0%). When trained and tested in 1:5 datasets, the CKSAAP encoding showed a more significant improvement than the binary encoding. We also merged the training datasets of S and T sites and integrated the prediction of S and T sites into one single predictor (i.e. S+T predictor). Either in 1:1 or 1:5 datasets, the performance of this S+T predictor was always slightly better than those predictors where S and T sites were independently predicted, suggesting that the molecular recognition of O-glycosylated S/T sites seems to be similar and the increase of the S+T predictor's accuracy may be a result of expanded training datasets. Moreover, CKSAAP_OGlySite was also shown to have better performance when benchmarked against two existing predictors. Conclusion Because of CKSAAP encoding's ability of

  16. Diversity of acidophilic prokaryotes at two acid mine drainage sites in Turkey.

    PubMed

    Aytar, Pınar; Kay, Catherine Melanie; Mutlu, Mehmet Burçin; Çabuk, Ahmet; Johnson, David Barrie

    2015-04-01

    The biodiversity of acidophilic prokaryotes in two acidic (pH 2.8-3.05) mine drainage (AMD) sites (Balya and Çan) in Turkey was examined using a combined cultivation-based and cultivation-independent approach. The latter included analyzing microbial diversity using fluorescent in situ hybridization (FISH), terminal restriction enzyme fragment length polymorphism (`T-RFLP), and quantitative PCR (qPCR). Numbers of cultivatable heterotrophic acidophilic bacteria were over an order of magnitude greater than those of chemolithotrophic acidophiles in both AMD ponds examined. Isolates identified as strains of Acidithiobacillus ferrivorans, Acidiphilium organovorum, and Ferrimicrobium acidiphilum were isolated from the Balya AMD pond, and others identified as strains of Leptospirillum ferriphilum, Acidicapsa ligni, and Acidiphilium rubrum from Çan AMD. Other isolates were too distantly related (from analysis of their 16S rRNA genes) to be identified at the species level. Archaeal diversity in the two ponds appeared to be far more limited. T-RFLP and qPCR confirmed the presence of Ferroplasma-like prokaryotes, but no archaea were isolated from the two sites. qPCR generated semiquantitative data for genera of some of the iron-oxidizing acidophiles isolated and/or detected, suggesting the order of abundance was Leptospirillum > Ferroplasma > Acidithiobacillus (Balya AMD) and Ferroplasma > Leptospirillum > Acidithiobacillus (Çan AMD). PMID:25380633

  17. The Protonation Site of para-Dimethylaminobenzoic Acid Using Atmospheric Pressure Ionization Methods

    NASA Astrophysics Data System (ADS)

    Chai, Yunfeng; Weng, Guofeng; Shen, Shanshan; Sun, Cuirong; Pan, Yuanjiang

    2015-04-01

    The protonation site of para-dimethylaminobenzoic acid ( p-DMABA) was investigated using atmospheric pressure ionization methods (ESI and APCI) coupled with collision-induced dissociation (CID), nuclear magnetic resonance (NMR), and computational chemistry. Theoretical calculations and NMR experiments indicate that the dimethyl amino group is the preferred site of protonation both in the gas phase and aqueous solution. Protonation of p-DMABA occurs at the nitrogen atom by ESI independent of the solvents and other operation conditions under typical thermodynamic control. However, APCI produces a mixture of the nitrogen- and carbonyl oxygen-protonated p-DMABA when aprotic organic solvents (acetonitrile, acetone, and tetrahydrofuran) are used, exhibiting evident kinetic characteristics of protonation. But using protic organic solvents (methanol, ethanol, and isopropanol) in APCI still leads to the formation of thermodynamically stable N-protonated p-DMABA. These structural assignments were based on the different CID behavior of the N- and O-protonated p-DMABA. The losses of methyl radical and water are the diagnostic fragmentations of the N- and O-protonated p-DMABA, respectively. In addition, the N-protonated p-DMABA is more stable than the O-protonated p-DMABA in CID revealed by energy resolved experiments and theoretical calculations.

  18. S6K is a morphogenic protein with a mechanism involving Filamin-A phosphorylation and phosphatidic acid binding

    PubMed Central

    Henkels, Karen M.; Mallets, Elizabeth R.; Dennis, Patrick B.; Gomez-Cambronero, Julian

    2015-01-01

    Change of cell shape in vivo plays many roles that are central to life itself, such as embryonic development, inflammation, wound healing, and pathologic processes such as cancer metastasis. Nonetheless, the spatiotemporal mechanisms that control the concerted regulation of cell shape remain understudied. Here, we show that ribosomal S6K, which is normally considered a protein involved in protein translation, is a morphogenic protein. Its presence in cells alters the overall organization of the cell surface and cell circularity [(4π × area)/(perimeter)2] from 0.47 ± 0.06 units in mock-treated cells to 0.09 ± 0.03 units in S6K-overexpressing macrophages causing stellation and arborization of cell shape. This effect was partially reversed in cells expressing a kinase-inactive S6K mutant and was fully reversed in cells silenced with small interference RNA. Equally important is that S6K is itself regulated by phospholipids, specifically phosphatidic acid, whereby 300 nM 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA), but not the control 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), binds directly to S6K and causes an ∼2.9-fold increase in S6K catalytic activity. This was followed by an increase in Filamin A (FLNA) functionality as measured by phospho-FLNA (S2152) expression and by a subsequent elevation of actin nucleation. This reliance of S6K on phosphatidic acid (PA), a curvature-inducing phospholipid, explained the extra-large perimeter of cells that overexpressed S6K. Furthermore, the diversity of the response to S6K in several unrelated cell types (fibroblasts, leukocytes, and invasive cancer cells) that we report here indicates the existence of an underlying common mechanism in mammalian cells. This new signaling set, PA-S6K-FLNA-actin, sheds light for the first time into the morphogenic pathway of cytoskeletal structures that are crucial for adhesion and cell locomotion during inflammation and metastasis.—Henkels, K. M., Mallets, E. R., Dennis, P. B

  19. Chemical characteristics and sources of organic acids in precipitation at a semi-urban site in Southwest China

    NASA Astrophysics Data System (ADS)

    Zhang, Y. L.; Lee, X. Q.; Cao, F.

    2011-01-01

    In order to investigate the chemical characteristics and sources of organic acids in precipitation in Southwest China, 105 rainwater samples were collected at a semi-urban site in Anshun from June 2007 to June 2008. Organic acids and major anions were analyzed along with pH and electrical conductivity. The pH values varied from 3.57 to 7.09 for all the rainfall events sampled, with an average of 4.67 which was typical acidic value. Formic, acetic and oxalic acids were found to be the predominant carboxylic acids and their volume weighted average (VWA) concentrations were 8.77, 6.93 and 2.84 μmol l -1, respectively. These organic acids were estimated to account for 8.1% to the total free acidity (TFA) in precipitation. The concentrations of the majority organic acids at studied site had a clear seasonal pattern, reaching higher levels during the non-growing season than those in growing season, which was attributed to dilution effect of heavy rainfall during the growing season. The seasonal variation of wet deposition flux of these organic acids confirmed higher source strength of biogenic emissions from vegetation during the growing season. Formic-to-acetic acids ratio (F/A), an indicator of primary versus secondary sources of these organic acids, suggested that primary sources from vehicular emission, biomass burning, soil and vegetation emissions were dominant sources. In addition, the lowest concentrations of organic acids were found under type S, when air masses originated from the marine (South China Sea) during Southern Asian Monsoon period. And the highest concentrations were observed in precipitation events from Northeast China (type NE), prevailing mostly during winter with the lowest rainfall.

  20. Polyunsaturated fatty acid biosynthesis is involved in phenylephrine-mediated calcium release in vascular smooth muscle cells.

    PubMed

    Irvine, Nicola A; Lillycrop, Karen A; Fielding, Barbara; Torrens, Christopher; Hanson, Mark A; Burdge, Graham C

    2015-10-01

    Stimulation of vascular smooth muscle (VSM) α1-adrenoceptors induces myosin phosphorylation and vasoconstriction via mobilisation of intracellular calcium and production of specific eicosanoids. Polyunsaturated fatty acid (PUFA) biosynthesis in VSM cells is involved, although the precise mechanism is not known. To address this, we characterised PUFA biosynthesis in VSM cells and determined its role in intracellular calcium release and eicosanoid production. Murine VSM cells converted 18:2n-6 to longer chain PUFA including 22:5n-6. Δ6 (D6d) and Δ5 (D5d) desaturase, and elongase (Elovl) 5 were expressed. Elovl2 was not detected in human, mouse or rat VSM cells, or in rat or mouse aortae, but tit was not associated with hypermethylation of its promoter. D6d or D5d inhibition reduced 18:3n-6 and 20:4n-6 synthesis, respectively, and induced concentration-related decrease in phenylephrine-mediated calcium release, and in PGE2 and PGF2α secretion. Together these findings suggest that PUFA biosynthesis in VSM cells is involved in calcium release associated with vasoconstriction. PMID:26324193

  1. Involvement of the neuronal phosphotyrosine signal adaptor N-Shc in kainic acid-induced epileptiform activity

    PubMed Central

    Baba, Shiro; Onga, Kazuko; Kakizawa, Sho; Ohyama, Kyoji; Yasuda, Kunihiko; Otsubo, Hiroshi; Scott, Brian W.; Burnham, W. McIntyre; Matsuo, Takayuki; Nagata, Izumi; Mori, Nozomu

    2016-01-01

    BDNF-TrkB signaling is implicated in experimental seizures and epilepsy. However, the downstream signaling involved in the epileptiform activity caused by TrkB receptor activation is still unknown. The aim of the present study was to determine whether TrkB-mediated N-Shc signal transduction was involved in kainic acid (KA)-induced epileptiform activity. We investigated KA-induced behavioral seizures, epileptiform activities and neuronal cell loss in hippocampus between N-Shc deficient and control mice. There was a significant reduction in seizure severity and the frequency of epileptiform discharges in N-Shc deficient mice, as compared with wild-type and C57BL/6 mice. KA-induced neuronal cell loss in the CA3 of hippocampus was also inhibited in N-Shc deficient mice. This study demonstrates that the activation of N-Shc signaling pathway contributes to an acute KA-induced epileptiform activity and neuronal cell loss in the hippocampus. We propose that the N-Shc-mediated signaling pathway could provide a potential target for the novel therapeutic approaches of epilepsy. PMID:27273072

  2. Involvement of aberrant DNA methylation on reduced expression of lysophosphatidic acid receptor-1 gene in rat tumor cell lines

    SciTech Connect

    Tsujiuchi, Toshifumi . E-mail: ttujiuch@life.kindai.ac.jp; Shimizu, Kyoko; Onishi, Mariko; Sugata, Eriko; Fujii, Hiromasa; Mori, Toshio; Honoki, Kanya; Fukushima, Nobuyuki

    2006-10-27

    Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. Recently, it has been reported that alterations of LPA receptor expression might be important in the malignant transformation of tumor cells. Therefore, to assess an involvement of DNA methylation in reduced expression of the LPA receptor-1 (lpa1) gene, we investigated the expression of the lpa1 gene and its DNA methylation patterns in rat tumor cell lines. Both rat brain-derived neuroblastoma B103 and liver-derived hepatoma RH7777 cells used in this study indicated no expression of lpa1. For the analysis of methylation status, bisulfite sequencing was performed with B103 and RH7777 cells, comparing with other lpa1 expressed cells and normal tissues of brain and liver. The lpa1 expressed cells and tissues were all unmethylated in this region of lpa1. In contrast, both B103 and RH7777 cells were highly methylated, correlating with reduced expression of the lpa1. Treatment with 5-aza 2'-deoxycytidine induced expression of lpa1 gene in B103 and RH7777 cells after 24 h. In RH7777 cells treated with 5-aza 2'-deoxycytidine, stress fiber formation was also observed in response to LPA in RH7777 cells, but not in untreated RH7777 cells. These results suggest that aberrant DNA methylation of the lpa1 gene may be involved in its reduced expression in rat tumor cells.

  3. Histone Modification Is Involved in Okadaic Acid (OA) Induced DNA Damage Response and G2-M Transition Arrest in Maize.

    PubMed

    Zhang, Hao; Wang, Pu; Hou, Haoli; Wen, Huan; Zhou, Hong; Gao, Fei; Wu, Jinping; Qiu, Zhengming; Li, Lijia

    2016-01-01

    Histone modifications are involved in regulation of chromatin structure. To investigate the relationship between chromatin modification and cell cycle regulation during plant cell proliferation, Okadaic acid (OA), a specific inhibitor of serine/threonine protein phosphatase, was applied in this study. The results showed that OA caused the cell cycle arrest at preprophase, leading to seedling growth inhibition. Western blotting assay revealed that the spatial distribution of phosphorylation of Ser10 histone H3 tails (H3S10ph) signals was altered under OA treatment. Reactive oxygen species (ROS) was found to be at higher levels and TdT-mediated dUTP nick end labeling (TUNEL) assay displayed DNA breaks happened at the chromatin after treatment with OA, companied with an increase in the acetylation of histone H4 at lysine 5 (H4K5ac) level. From these observations, we speculated that the alteration of the spatial distribution of H3S10ph and the level of H4K5ac was involved in the procedure that OA induced DNA breaks and G2-M arrested by the accumulation of ROS, and that the histone H3S10ph and H4K5ac might facilitate DNA repair by their association with the chromatin decondensation. PMID:27196101

  4. Histone Modification Is Involved in Okadaic Acid (OA) Induced DNA Damage Response and G2-M Transition Arrest in Maize

    PubMed Central

    Zhang, Hao; Wang, Pu; Hou, Haoli; Wen, Huan; Zhou, Hong; Gao, Fei; Wu, Jinping; Qiu, Zhengming; Li, Lijia

    2016-01-01

    Histone modifications are involved in regulation of chromatin structure. To investigate the relationship between chromatin modification and cell cycle regulation during plant cell proliferation, Okadaic acid (OA), a specific inhibitor of serine/threonine protein phosphatase, was applied in this study. The results showed that OA caused the cell cycle arrest at preprophase, leading to seedling growth inhibition. Western blotting assay revealed that the spatial distribution of phosphorylation of Ser10 histone H3 tails (H3S10ph) signals was altered under OA treatment. Reactive oxygen species (ROS) was found to be at higher levels and TdT-mediated dUTP nick end labeling (TUNEL) assay displayed DNA breaks happened at the chromatin after treatment with OA, companied with an increase in the acetylation of histone H4 at lysine 5 (H4K5ac) level. From these observations, we speculated that the alteration of the spatial distribution of H3S10ph and the level of H4K5ac was involved in the procedure that OA induced DNA breaks and G2-M arrested by the accumulation of ROS, and that the histone H3S10ph and H4K5ac might facilitate DNA repair by their association with the chromatin decondensation. PMID:27196101

  5. Involvement of the neuronal phosphotyrosine signal adaptor N-Shc in kainic acid-induced epileptiform activity.

    PubMed

    Baba, Shiro; Onga, Kazuko; Kakizawa, Sho; Ohyama, Kyoji; Yasuda, Kunihiko; Otsubo, Hiroshi; Scott, Brian W; Burnham, W McIntyre; Matsuo, Takayuki; Nagata, Izumi; Mori, Nozomu

    2016-01-01

    BDNF-TrkB signaling is implicated in experimental seizures and epilepsy. However, the downstream signaling involved in the epileptiform activity caused by TrkB receptor activation is still unknown. The aim of the present study was to determine whether TrkB-mediated N-Shc signal transduction was involved in kainic acid (KA)-induced epileptiform activity. We investigated KA-induced behavioral seizures, epileptiform activities and neuronal cell loss in hippocampus between N-Shc deficient and control mice. There was a significant reduction in seizure severity and the frequency of epileptiform discharges in N-Shc deficient mice, as compared with wild-type and C57BL/6 mice. KA-induced neuronal cell loss in the CA3 of hippocampus was also inhibited in N-Shc deficient mice. This study demonstrates that the activation of N-Shc signaling pathway contributes to an acute KA-induced epileptiform activity and neuronal cell loss in the hippocampus. We propose that the N-Shc-mediated signaling pathway could provide a potential target for the novel therapeutic approaches of epilepsy. PMID:27273072

  6. Site-specific incorporation of probes into RNA polymerase by unnatural-amino-acid mutagenesis and Staudinger-Bertozzi ligation

    PubMed Central

    Chakraborty, Anirban; Mazumder, Abhishek; Lin, Miaoxin; Hasemeyer, Adam; Xu, Qumiao; Wang, Dongye; Ebright, Yon W.; Ebright, Richard H.

    2015-01-01

    Summary A three-step procedure comprising (i) unnatural-amino-acid mutagenesis with 4-azido-phenylalanine, (ii) Staudinger-Bertozzi ligation with a probe-phosphine derivative, and (iii) in vitro reconstitution of RNA polymerase (RNAP) enables the efficient site-specific incorporation of a fluorescent probe, a spin label, a crosslinking agent, a cleaving agent, an affinity tag, or any other biochemical or biophysical probe, at any site of interest in RNAP. Straightforward extensions of the procedure enable the efficient site-specific incorporation of two or more different probes in two or more different subunits of RNAP. We present protocols for synthesis of probe-phosphine derivatives, preparation of RNAP subunits and the transcription initiation factor σ, unnatural amino acid mutagenesis of RNAP subunits and σ, Staudinger ligation with unnatural-amino-acid-containing RNAP subunits and σ, quantitation of labelling efficiency and labelling specificity, and reconstitution of RNAP. PMID:25665560

  7. Retinoic Acid, GABA-ergic, and TGF-β Signaling Systems Are Involved in Human Cleft Palate Fibroblast Phenotype

    PubMed Central

    Baroni, Tiziano; Bellucci, Catia; Lilli, Cinzia; Pezzetti, Furio; Carinci, Francesco; Becchetti, Ennio; Carinci, Paolo; Stabellini, Giordano; Calvitti, Mario; Lumare, Eleonora; Bodo, Maria

    2006-01-01

    During embryogenesis, a complex interplay between extracellular matrix (ECM) molecules, regulatory molecules, and growth factors mediates morphogenetic processes involved in palatogenesis. Transforming growth factor-β (TGF-β), retinoic acid (RA), and γ-aminobutyric acid (GABA)ergic signaling systems are also potentially involved. Using [3H]glucosamine and [35S]methionine incorporation, anion exchange chromatography, semiquantitative radioactive RT-PCR, and a TGF-β binding assay, we aimed to verify the presence of phenotypic differences between primary cultures of secondary palate (SP) fibroblasts from 2-year-old subjects with familial nonsyndromic cleft lip and/or palate (CLP-SP fibroblasts) and age-matched normal SP (N-SP) fibroblasts. The effects of RA—which, at pharmacologic doses, induces cleft palate in newborns of many species—were also studied. We found an altered ECM production in CLP-SP fibroblasts that synthesized and secreted more glycosaminoglycans (GAGs) and fibronectin (FN) compared with N-SP cells. In CLP-SP cells, TGF-β3 mRNA expression and TGF-β receptor number were higher and RA receptor-α (RARA) gene expression was increased. Moreover, we demonstrated for the first time that GABA receptor (GABRB3) mRNA expression was upregulated in human CLP-SP fibroblasts. In N-SP and CLP-SP fibroblasts, RA decreased GAG and FN secretion and increased TGF-β3 mRNA expression but reduced the number of TGF-β receptors. TGF-β receptor type I mRNA expression was decreased, TGF-β receptor type II was increased, and TGF-β receptor type III was not affected. RA treatment increased RARA gene expression in both cell populations but upregulated GABRB3 mRNA expression only in N-SP cells. These results show that CLP-SP fibroblasts compared with N-SP fibroblasts exhibit an abnormal phenotype in vitro and respond differently to RA treatment, and suggest that altered crosstalk between RA, GABAergic, and TGF-β signaling systems could be involved in human cleft

  8. The three transfer RNAs occupying the A, P and E sites on the ribosome are involved in viral programmed -1 ribosomal frameshift

    PubMed Central

    Léger, Mélissa; Dulude, Dominic; Steinberg, Sergey V.; Brakier-Gingras, Léa

    2007-01-01

    The -1 programmed ribosomal frameshifts (PRF), which are used by many viruses, occur at a heptanucleotide slippery sequence and are currently thought to involve the tRNAs interacting with the ribosomal P- and A-site codons. We investigated here whether the tRNA occupying the ribosomal E site that precedes a slippery site influences -1 PRF. Using the human immunodeficiency virus type 1 (HIV-1) frameshift region, we found that mutating the E-site codon altered the -1 PRF efficiency. When the HIV-1 slippery sequence was replaced with other viral slippery sequences, mutating the E-site codon also altered the -1 PRF efficiency. Because HIV-1 -1 PRF can be recapitulated in bacteria, we used a bacterial ribosome system to select, by random mutagenesis, 16S ribosomal RNA (rRNA) mutations that modify the expression of a reporter requiring HIV-1 -1 PRF. Three mutants were isolated, which are located in helices 21 and 22 of 16S rRNA, a region involved in translocation and E-site tRNA binding. We propose a novel model where -1 PRF is triggered by an incomplete translocation and depends not only on the tRNAs interacting with the P- and A-site codons, but also on the tRNA occupying the E site. PMID:17704133

  9. Low molecular weight (C1-C10) monocarboxylic acids, dissolved organic carbon and major inorganic ions in alpine snow pit sequence from a high mountain site, central Japan

    NASA Astrophysics Data System (ADS)

    Kawamura, Kimitaka; Matsumoto, Kohei; Tachibana, Eri; Aoki, Kazuma

    2012-12-01

    Snowpack samples were collected from a snow pit sequence (6 m in depth) at the Murodo-Daira site near the summit of Mt. Tateyama, central Japan, an outflow region of Asian dusts. The snow samples were analyzed for a homologous series of low molecular weight normal (C1-C10) and branched (iC4-iC6) monocarboxylic acids as well as aromatic (benzoic) and hydroxy (glycolic and lactic) acids, together with major inorganic ions and dissolved organic carbon (DOC). The molecular distributions of organic acids were characterized by a predominance of acetic (range 7.8-76.4 ng g-1-snow, av. 34.8 ng g-1) or formic acid (2.6-48.1 ng g-1, 27.7 ng g-1), followed by propionic acid (0.6-5.2 ng g-1, 2.8 ng g-1). Concentrations of normal organic acids generally decreased with an increase in carbon chain length, although nonanoic acid (C9) showed a maximum in the range of C5-C10. Higher concentrations were found in the snowpack samples containing dust layer. Benzoic acid (0.18-4.1 ng g-1, 1.4 ng g-1) showed positive correlation with nitrate (r = 0.70), sulfate (0.67), Na+ (0.78), Ca2+ (0.86) and Mg+ (0.75), suggesting that this aromatic acid is involved with anthropogenic sources and Asian dusts. Higher concentrations of Ca2+ and SO42- were found in the dusty snow samples. We found a weak positive correlation (r = 0.43) between formic acid and Ca2+, suggesting that gaseous formic acid may react with Asian dusts in the atmosphere during long-range transport. However, acetic acid did not show any positive correlations with major inorganic ions. Hydroxyacids (0.03-5.7 ng g-1, 1.5 ng g-1) were more abundant in the granular and dusty snow. Total monocarboxylic acids (16-130 ng g-1, 74 ng g-1) were found to account for 1-6% of DOC (270-1500 ng g-1, 630 ng g-1) in the snow samples.

  10. Expression of Genes Involved in Iron and Sulfur Respiration in a Novel Thermophilic Crenarchaeon Isolated from Acid-Sulfate-Chloride Geothermal Systems

    NASA Astrophysics Data System (ADS)

    Kozubal, M.; Macur, R.; Inskeep, W. P.

    2007-12-01

    Acidic geothermal springs within Yellowstone National Park (YNP) provide an excellent opportunity to study microbial populations and their relationship with geochemical processes such as redox cycling and biomineralization of iron. Fourteen acid-sulfate-chloride (ASC) and acid-sulfate (AS) geothermal springs located in (YNP) have been extensively characterized for aqueous chemistry, solid phase mineral deposition and microbial diversity and distribution. The oxidation of Fe(II) with oxygen as an electron acceptor is exergonic under these conditions, consequently, Fe(II) may be an important electron donor driving primary production in ASC and AS habitats, and products of biomineralization (e.g. Fe[III]-oxides of varying crystallinity and structure, as well as jarosite in some cases) are common in the outflow channels of these environments. Recently, we isolated a novel Metallosphaera-like microorganism (Metallosphaera strain MK1) from an ASC spring in Norris Geyser Basin, YNP. Clone libraries (16S rRNA gene) from multiple sites suggest that microorganisms closely related to strain MK1 (between 98-100 percent similarity) dominate many spring locations between 55-80 C. The in situ abiotic oxidation rate of Fe(II) has been shown to be very slow in these systems and Metallosphaera strain MK1 has been directly implicated in biotic Fe(II) oxidation. Metallosphaera strain MK1 has been submitted for full genome sequencing and is yielding gene sequences related to the terminal oxidases SOXABC and SOXM super-complex. In addition, sequences from a recently characterized terminal oxidase FOX complex involved in Fe(II) and pyrite oxidation from Sulfolobus metallicus have been found in Metallosphaera strain MK1. A protein complex analogous to Metallosphaera sedula has been identified in strain MK1 and this complex has also been expressed in cells grown on pyrite and Fe(II). Other sequences identified in Metallosphaera strain MK1 that are involved in respiration are the TQO

  11. Site-specific protein adducts of 4-hydroxy-2(E)-nonenal in human THP-1 monocytic cells: Protein carbonylation is diminished by ascorbic acid

    PubMed Central

    Chavez, Juan; Chung, Woon-Gye; Miranda, Cristobal L.; Singhal, Mudita; Stevens, Jan F.; Maier, Claudia S.

    2010-01-01

    The protein targets and sites of modification by 4-hydroxy-2(E)-nonenal (HNE) in human monocytic THP-1 cells after exogenous exposure to HNE were examined using a multi-pronged proteomic approach involving electrophoretic, immunoblotting and mass spectrometric methods. Immunoblot analysis using monoclonal anti-HNE antibodies showed several proteins as targets of HNE adduction. Pretreatment of THP-1 cells with ascorbic acid resulted in reduced levels of HNE-protein adducts. Biotinylation of Michael-type HNE adducts using an aldehyde-reactive hydroxylamine-functionalized probe (aldehyde-reactive probe, ARP) and subsequent enrichment facilitated the identification and site-specific assignment of the modifications by LC-MS/MS analysis. Sixteen proteins were unequivocally identified as targets of HNE adduction and eighteen sites of HNE modification at Cys and His residues were assigned. HNE exposure of THP-1 cells resulted in the modification of proteins involved in cytoskeleton organization and regulation, proteins associated with stress responses and enzymes of the glycolytic and other metabolic pathways. This study yielded the first evidence of site-specific adduction of HNE to Cys-295 in tubulin α-1B chain, Cys-351 and Cys-499 in α-actinin-4, Cys-328 in vimentin, Cys-369 in D-3-phosphoglycerate dehydrogenase and His-246 in aldolase A. PMID:20043646

  12. Investigation of prototypal MOFs consisting of polyhedral cages with accessible Lewis-acid sites for quinoline synthesis.

    PubMed

    Gao, Wen-Yang; Leng, Kunyue; Cash, Lindsay; Chrzanowski, Matthew; Stackhouse, Chavis A; Sun, Yinyong; Ma, Shengqian

    2015-03-21

    A series of prototypal metal-organic frameworks (MOFs) consisting of polyhedral cages with accessible Lewis-acid sites, have been systematically investigated for Friedländer annulation reaction, a straightforward approach to synthesizing quinoline and its derivatives. Amongst them MMCF-2 demonstrates significantly enhanced catalytic activity compared with the benchmark MOFs, HKUST-1 and MOF-505, as a result of a high-density of accessible Cu(II) Lewis acid sites and large window size in the cuboctahedral cage-based nanoreactor of MMCF-2. PMID:25693429

  13. Site-specific deletion in cauliflower mosaic virus DNA: possible involvement of RNA splicing and reverse transcription

    PubMed Central

    Hirochika, Hirohiko; Takatsuji, Hiroshi; Ubasawa, Aiko; Ikeda, Joh-E

    1985-01-01

    A frequent site-specific deletion was observed in the life cycle of cauliflower mosaic virus (S strain). Analysis of the sequence around the deletion site and the parental sequence implied that the deletion was promoted at sequences similar to the donor and acceptor consensus sequences of RNA splicing, designated as the deletion donor and acceptor sequences, respectively. To elucidate the mechanism of this site-specific deletion, point mutations were introduced into the deletion donor sequence (GT to GG or GA transversion). Deletion at the original deletion donor site did not occur in these mutants, instead, new (cryptic) donor sites were activated. All of these activated cryptic sites had sequences similar to the splicing consensus sequence. In all cases except one, the original deletion acceptor site was used. These results can be most readily explained by postulating that the site-specific deletion occurs by reverse transcription of spliced viral RNA. This frequent site-specific deletion was not observed in other strains. For a virus which replicates by reverse transcription, a mechanism to regulate the rate of splicing is required to ensure the intactness of the viral genome. We discuss the possibility that the S strain has a mutation in this regulatory mechanism. ImagesFig. 1.Fig. 3.Fig. 5.Fig. 7. PMID:16453624

  14. Oleanolic Acid Induces Differentiation of Neural Stem Cells to Neurons: An Involvement of Transcription Factor Nkx-2.5

    PubMed Central

    Ning, You; Huang, Jianhua; Kalionis, Bill; Bian, Qin; Dong, Jingcheng; Wu, Junzhen; Tai, Xiantao; Xia, Shijin; Shen, Ziyin

    2015-01-01

    Neural stem cells (NSCs) harbor the potential to differentiate into neurons, astrocytes, and oligodendrocytes under normal conditions and/or in response to tissue damage. NSCs open a new way of treatment of the injured central nervous system and neurodegenerative disorders. Thus far, few drugs have been developed for controlling NSC functions. Here, the effect as well as mechanism of oleanolic acid (OA), a pentacyclic triterpenoid, on NSC function was investigated. We found OA significantly inhibited neurosphere formation in a dose-dependent manner and achieved a maximum effect at 10 nM. OA also reduced 5-ethynyl-2′-deoxyuridine (EdU) incorporation into NSCs, which was indicative of inhibited NSC proliferation. Western blotting analysis revealed the protein levels of neuron-specific marker tubulin-βIII (TuJ1) and Mash1 were increased whilst the astrocyte-specific marker glial fibrillary acidic protein (GFAP) decreased. Immunofluorescence analysis showed OA significantly elevated the percentage of TuJ1-positive cells and reduced GFAP-positive cells. Using DNA microarray analysis, 183 genes were differentially regulated by OA. Through transcription factor binding site analyses of the upstream regulatory sequences of these genes, 87 genes were predicted to share a common motif for Nkx-2.5 binding. Finally, small interfering RNA (siRNA) methodology was used to silence Nkx-2.5 expression and found silence of Nkx-2.5 alone did not change the expression of TuJ-1 and the percentage of TuJ-1-positive cells. But in combination of OA treatment and silence of Nkx-2.5, most effects of OA on NSCs were abolished. These results indicated that OA is an effective inducer for NSCs differentiation into neurons at least partially by Nkx-2.5-dependent mechanism. PMID:26240574

  15. Oleanolic Acid Induces Differentiation of Neural Stem Cells to Neurons: An Involvement of Transcription Factor Nkx-2.5.

    PubMed

    Ning, You; Huang, Jianhua; Kalionis, Bill; Bian, Qin; Dong, Jingcheng; Wu, Junzhen; Tai, Xiantao; Xia, Shijin; Shen, Ziyin

    2015-01-01

    Neural stem cells (NSCs) harbor the potential to differentiate into neurons, astrocytes, and oligodendrocytes under normal conditions and/or in response to tissue damage. NSCs open a new way of treatment of the injured central nervous system and neurodegenerative disorders. Thus far, few drugs have been developed for controlling NSC functions. Here, the effect as well as mechanism of oleanolic acid (OA), a pentacyclic triterpenoid, on NSC function was investigated. We found OA significantly inhibited neurosphere formation in a dose-dependent manner and achieved a maximum effect at 10 nM. OA also reduced 5-ethynyl-2'-deoxyuridine (EdU) incorporation into NSCs, which was indicative of inhibited NSC proliferation. Western blotting analysis revealed the protein levels of neuron-specific marker tubulin-βIII (TuJ1) and Mash1 were increased whilst the astrocyte-specific marker glial fibrillary acidic protein (GFAP) decreased. Immunofluorescence analysis showed OA significantly elevated the percentage of TuJ1-positive cells and reduced GFAP-positive cells. Using DNA microarray analysis, 183 genes were differentially regulated by OA. Through transcription factor binding site analyses of the upstream regulatory sequences of these genes, 87 genes were predicted to share a common motif for Nkx-2.5 binding. Finally, small interfering RNA (siRNA) methodology was used to silence Nkx-2.5 expression and found silence of Nkx-2.5 alone did not change the expression of TuJ-1 and the percentage of TuJ-1-positive cells. But in combination of OA treatment and silence of Nkx-2.5, most effects of OA on NSCs were abolished. These results indicated that OA is an effective inducer for NSCs differentiation into neurons at least partially by Nkx-2.5-dependent mechanism. PMID:26240574

  16. IDENTIFICATION AND FUNCTIONAL CHARACTERIZATION OF THE MOSS PHYSCOMITRELLA PATENS DELTA5-DESATURASE GENE INVOLVED IN ARACHIDONIC AND EICOSAPENTAENOIC ACID BIOSYNTHESIS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The moss Physcomitrella patens contains high levels of arachidonic acid and lesser amounts of eicosapentaenoic acid. In general, these C20 polyunsaturated fatty acids are synthesized from linoleic and alpha-linolenic acids, respectively, by a series of reactions catalyzed by a delta6-desaturase, an ...

  17. An Arabidopsis mitochondria-localized RRL protein mediates abscisic acid signal transduction through mitochondrial retrograde regulation involving ABI4

    PubMed Central

    Yao, Xuan; Li, Juanjuan; Liu, Jianping; Liu, Kede

    2015-01-01

    The molecular mechanisms of abscisic acid (ABA) signalling have been studied for many years; however, how mitochondria-localized proteins play roles in ABA signalling remains unclear. Here an Arabidopsis mitochondria-localized protein RRL (RETARDED ROOT GROWTH-LIKE) was shown to function in ABA signalling. A previous study had revealed that the Arabidopsis mitochondria-localized protein RRG (RETARDED ROOT GROWTH) is required for cell division in the root meristem. RRL shares 54% and 57% identity at the nucleotide and amino acid sequences, respectively, with RRG; nevertheless, RRL shows a different function in Arabidopsis. In this study, disruption of RRL decreased ABA sensitivity whereas overexpression of RRL increased ABA sensitivity during seed germination and seedling growth. High expression levels of RRL were found in germinating seeds and developing seedlings, as revealed by β-glucuronidase (GUS) staining of ProRRL–GUS transgenic lines. The analyses of the structure and function of mitochondria in the knockout rrl mutant showed that the disruption of RRL causes extensively internally vacuolated mitochondria and reduced ABA-stimulated reactive oxygen species (ROS) production. Previous studies have revealed that the expression of alternative oxidase (AOX) in the alternative respiratory pathway is increased by mitochondrial retrograde regulation to regain ROS levels when the mitochondrial electron transport chain is impaired. The APETALA2 (AP2)-type transcription factor ABI4 is a regulator of ALTERNATIVE OXIDASE1a (AOX1a) in mitochondrial retrograde signalling. This study showed that ABA-induced AOX1a and ABI4 expression was inhibited in the rrl mutant, suggesting that RRL is probably involved in ABI4-mediated mitochondrial retrograde signalling. Furthermore, the results revealed that ABI4 is a downstream regulatory factor in RRL-mediated ABA signalling in seed germination and seedling growth. PMID:26163700

  18. In Candida parapsilosis the ATC1 Gene Encodes for an Acid Trehalase Involved in Trehalose Hydrolysis, Stress Resistance and Virulence

    PubMed Central

    Sánchez-Fresneda, Ruth; Martínez-Esparza, María; Maicas, Sergi; Argüelles, Juan-Carlos; Valentín, Eulogio

    2014-01-01

    An ORF named CPAR2-208980 on contig 005809 was identified by screening a Candida parapsilosis genome data base. Its 67% identity with the acid trehalase sequence from C. albicans (ATC1) led us to designate it CpATC1. Homozygous mutants that lack acid trehalase activity were constructed by gene disruption at the two CpATC1 chromosomal alleles. Phenotypic characterization showed that atc1Δ null cells were unable to grow on exogenous trehalose as carbon source, and also displayed higher resistance to environmental challenges, such as saline exposure (1.2 M NaCl), heat shock (42°C) and both mild and severe oxidative stress (5 and 50 mM H2O2). Significant amounts of intracellular trehalose were specifically stored in response to the thermal upshift in both wild type and mutant strains. Analysis of their antioxidant activities revealed that catalase was only triggered in response to heat shock in atc1Δ cells, whereas glutathione reductase was activated upon mild oxidative stress in wild type and reintegrant strains, and in response to the whole set of stress treatments in the homozygous mutant. Furthermore, yeast cells with double CpATC1 deletion were significantly attenuated in non-mammalian infection models, suggesting that CpATC1 is required for the pathobiology of the fungus. Our results demonstrate the involvement of CpAtc1 protein in the physiological hydrolysis of external trehalose in C. parapsilosis, where it also plays a major role in stress resistance and virulence. PMID:24922533

  19. [In vivo study of bacterial invasion in root planed and citric acid treated radicular surfaces of periodontally involved human teeth].

    PubMed

    Ito, K; Arai, N; Otogoto, J; Murai, S

    1989-06-01

    The purpose of this study was to investigate whether plaque bacteria invade the exposed radicular dentin after root planing or chemical root treatment in vivo. Eighteen caries-free human periodontally involved teeth with hopeless prognoses were studied. Fourteen teeth were scaled and root planed with hand curette type scalers. The proximal surface of each treated tooth was designated as the RP surface. The remaining half of the proximal surface was treated with citric acid (pH 1.0) for 3 minutes and was designated as the CA surface. Four untreated teeth served as controls. After 4 weeks, the teeth were extracted, and were processed for light microscopy and for scanning electron microscopy concerning bacterial invasion into the supragingival radicular dentin. The following results were obtained. 1. Radicular cementum was present on most untreated tooth surfaces. However, bacteria were never seen in the dentinal tubules. 2. Bacterial invasion into the dentinal tubules was observed in five of the 10 proximal surfaces (50% of the RP surfaces) and in nine of the 10 proximal surfaces (90% of the CA surfaces). 3. The depth (9.5 +/- 24.1 microns vs 84.6 +/- 136.3 microns) and percentage (0.8 +/- 2.1% vs 20.3 +/- 17.3%) of bacterial invasion in the dentinal tubules of the RP surfaces was lower than that of the CA surfaces. 4. Cocci and short rods were present in the supragingival dentinal tubules. 5. Since CA surfaces may accelerate bacterial invasion the citric acid treatment might be harmful in patients with inadequate plaque control. PMID:2700200

  20. A Model for the Active-Site Formation Process in DMSO Reductase Family Molybdenum Enzymes Involving Oxido-Alcoholato and Oxido-Thiolato Molybdenum(VI) Core Structures.

    PubMed

    Sugimoto, Hideki; Sato, Masanori; Asano, Kaori; Suzuki, Takeyuki; Mieda, Kaoru; Ogura, Takashi; Matsumoto, Takashi; Giles, Logan J; Pokhrel, Amrit; Kirk, Martin L; Itoh, Shinobu

    2016-02-15

    New bis(ene-1,2-dithiolato)-oxido-alcoholato molybdenum(VI) and -oxido-thiolato molybdenum(VI) anionic complexes, denoted as [Mo(VI)O(ER)L2](-) (E = O, S; L = dimethoxycarboxylate-1,2-ethylenedithiolate), were obtained from the reaction of the corresponding dioxido-molybdenum(VI) precursor complex with either an alcohol or a thiol in the presence of an organic acid (e.g., 10-camphorsulfonic acid) at low temperature. The [Mo(VI)O(ER)L2](-) complexes were isolated and characterized, and the structure of [Mo(VI)O(OEt)L2](-) was determined by X-ray crystallography. The Mo(VI) center in [Mo(VI)O(OEt)L2](-) exhibits a distorted octahedral geometry with the two ene-1,2-dithiolate ligands being symmetry inequivalent. The computed structure of [Mo(VI)O(SR)L2](-) is essentially identical to that of [Mo(VI)O(OR)L2](-). The electronic structures of the resulting molybdenum(VI) complexes were evaluated using electronic absorption spectroscopy and bonding calculations. The nature of the distorted O(h) geometry in these [Mo(VI)O(EEt)L2](-) complexes results in a lowest unoccupied molecular orbital wave function that possesses strong π* interactions between the Mo(d(xy)) orbital and the cis S(p(z)) orbital localized on one sulfur donor from a single ene-1,2-dithiolate ligand. The presence of a covalent Mo-S(dithiolene) bonding interaction in these monooxido Mo(VI) compounds contributes to their low-energy ligand-to-metal charge transfer transitions. A second important d-p π bonding interaction derives from the ∼180° O(oxo)-Mo-E-C dihedral angle involving the alcoholate and thiolate donors, and this contributes to ancillary ligand contributions to the electronic structure of these species. The formation of [Mo(VI)O(OEt)L2](-) and [Mo(VI)O(SEt)L2](-) from the dioxidomolybdenum(VI) precursor may be regarded as a model for the active-site formation process that occurs in the dimethyl sulfoxide reductase family of pyranopterin molybdenum enzymes. PMID:26816115

  1. Regulation of the Tyrosine Biosynthetic Enzymes in Salmonella typhimurium: Analysis of the Involvement of Tyrosyl-Transfer Ribonucleic Acid and Tyrosyl-Transfer Ribonucleic Acid Synthetase1

    PubMed Central

    Heinonen, J.; Artz, S. W.; Zalkin, H.

    1972-01-01

    Mutants of Salmonella typhimurium were isolated that require tyrosine for growth because of an altered tyrosyl-transfer ribonucleic acid (tRNA) synthetase. Extracts of one strain (JK10) contain a labile enzyme with decreased ability to transfer tyrosine to tRNATyr and a higher Km for tyrosine than the wild-type enzyme. Strain JK10 maintains repressed levels of the tyrosine biosynthetic enzymes when the growth rate is restricted due to limitation of charged tRNATyr. Several second-site revertants of strain JK10 exhibit temperature-sensitive growth due to partially repaired, heat-labile tyrosyl-tRNA synthetase. The tyrosine biosynthetic enzymes are not derepressed in thermosensitive strains grown at the restrictive temperature. A class of tyrosine regulatory mutants, designated tyrR, contains normal levels of tyrosyl-tRNA synthetase and tRNATyr. These results suggest that charging of tRNATyr is not necessary for repression. This conclusion is substantiated by the finding that 4-aminophenylalanine, a tyrosine analogue which causes repression of the tyrosine biosynthetic enzymes, is not attached to tRNATyr in vivo, nor does it inhibit the attachment reaction in vitro. A combined regulatory effect due to the simultaneous presence of tyrS and tyrR mutations in the same strain was detected. The possibility of direct participation of tyrosyl-tRNA synthetase in tyrosine regulation is discussed. PMID:4404819

  2. Early smelter sites: A neglected chapter in the history and geography of acid rain in the United States

    NASA Astrophysics Data System (ADS)

    Quinn, M.-L.

    Dominant spatial and temporal theories of acid rain in the U.S. are identified, followed by brief comments on how historical data have generally been used in modern acid rain research. A frequently-cited 1982 article by E.B. Cowling is examined, one that has influenced much thinking on the history of acid rain. The article overlooks early American smelters, however, and the role they played in the true history and geography of acid rain in the United States. Continuing with this theme, a connection is established between acid rain and turn-of-the-century smelter smoke problems. Literature on the latter subject is discussed, and American and German examples are given. A beginning is then made on writing acid rain's neglected chapter, focusing on Tennessee's Copper Basin (Ducktown District) where copper smelting dates back to the 1850s. A short historical overview of this area's smelting operations is given, with particular attention to the air pollution and other environmental problems resulting from large emissions of sulfur dioxide. Five additional early smelter sites for potential study are mentioned as well. The paper concludes with some observations regarding the way in which expanded research of early smelter sites could affect the general perception of acid rain in the U.S. It is also suggested that such research might contribute to a better atmosphere for making decisions and policies pertaining to the phenomenon as it exists today.

  3. Isotope geochemistry of waters affected by acid mine drainage in old labour sites (SE, Spain).

    NASA Astrophysics Data System (ADS)

    Pérez-Sirvent, Carmen; Martinez-Sanchez, Maria Jose; Garcia-Lorenzo, Maria Luz; Agudo, Ines; Hernandez-Cordoba, Manuel; Recio, Clemente

    2015-04-01

    The ore deposits of this zone have iron, lead and zinc as the main metal components. Iron is present in oxides, hydroxides, sulfides, sulfates, carbonates, and silicates; lead and zinc occur in sulfides (galena and sphalerite, respectively), carbonates, sulfates, and lead or zinc-bearing (manganese, iron) oxides. Mining started with the Romans and activity peaked in the second half of the 19th century and throughout the 20th century until the 1980's. From 1940 to 1957, mineral concentration was made by froth flotation and, prior to this, by gravimetric techniques. The mining wastes, or tailings, with a very fine particle size were deposited inland (tailings dams) and, since 1957, huge releases were made in directly the sea coast. The objective of this work was to evaluate processes affecting waters from abandoned mine sites by way of stable isotopic analysis, particularly H and O stable isotopes from water and S and O from dissolved sulfates. Several common chemical and physical processes, such as evaporation, water-rock interaction and mixing could alter water isotopic composition. Evaporation, which causes an enrichment in δD and δ18O in the residual water, is an important process in semiarid areas. The results obtained indicate that, for sites near the coast, waters are meteoric, and marine infiltration only takes place in the deepest layers near the shore or if water remains stagnated in sediments with low permeability. The main source of sulfate was the oxidation of sulfides, resulting in the liberation of acid, sulfate and metals. In order to assess the mechanism responsible for sulfide oxidation, the stoichiometric isotope balance model and the general isotope balance model were tested, suggesting that the oxidation via Fe3+ was predominant in the surface, and controlled by A. ferrooxidans, while at depth, sulfate reduction occurred.

  4. Cross-talk between glucocorticoid and retinoic acid signals involving glucocorticoid receptor interaction with the homoeodomain protein Pbx1.

    PubMed Central

    Subramaniam, Nanthakumar; Campión, Javier; Rafter, Ingalill; Okret, Sam

    2003-01-01

    Glucocorticoid (GC) signalling influences the response of the cell to a number of other signals via a mechanism referred to as 'cross-talk'. This cross-talk may act at several levels, including an interaction between the transcription factors involved in the signalling pathways. In the present paper, we demonstrate a novel functional interaction between GC and all- trans -retinoic acid (RA) signalling. We show that, in P19 embryonal carcinoma cells, GCs potentiate RA-induced expression of the murine Hoxb -1 gene through an autoregulatory element, b1-ARE, recognized by the Pbx1 and HOXB1 homoeodomain proteins. The synergistic effect of GC did not involve GC receptor (GR) binding to the b1-ARE, and the GC-GR complex alone was unable to activate transcription via the element. Furthermore, the ability of the GR to transactivate was not required, excluding expression of a GC-induced protein as the mechanism for the GC/RA synergy. Additional transfection experiments showed that the Pbx1/HOXB1 heterodimer was the target for the GC effect. Furthermore, functional dissection of the GR demonstrated that the DNA-binding domain (DBD) of the GR was required for the synergy. A physical interaction between the GR and Pbx1 proteins was demonstrated in vivo by co-immunoprecipitation experiments. These results are compatible with a model in which the GC/RA synergy is mediated by a direct interaction between the GR and Pbx1. On the basis of the ubiquitous expression of both GR and Pbx1, a number of genes regulated by Pbx are likely to be important targets for GC-mediated 'cross-talk'. PMID:12487626

  5. Notch stimulates growth by direct regulation of genes involved in the control of glycolysis and the tricarboxylic acid cycle.

    PubMed

    Slaninova, Vera; Krafcikova, Michaela; Perez-Gomez, Raquel; Steffal, Pavel; Trantirek, Lukas; Bray, Sarah J; Krejci, Alena

    2016-02-01

    Glycolytic shift is a characteristic feature of rapidly proliferating cells, such as cells during development and during immune response or cancer cells, as well as of stem cells. It results in increased glycolysis uncoupled from mitochondrial respiration, also known as the Warburg effect. Notch signalling is active in contexts where cells undergo glycolytic shift. We decided to test whether metabolic genes are direct transcriptional targets of Notch signalling and whether upregulation of metabolic genes can help Notch to induce tissue growth under physiological conditions and in conditions of Notch-induced hyperplasia. We show that genes mediating cellular metabolic changes towards the Warburg effect are direct transcriptional targets of Notch signalling. They include genes encoding proteins involved in glucose uptake, glycolysis, lactate to pyruvate conversion and repression of the tricarboxylic acid cycle. The direct transcriptional upregulation of metabolic genes is PI3K/Akt independent and occurs not only in cells with overactivated Notch but also in cells with endogenous levels of Notch signalling and in vivo. Even a short pulse of Notch activity is able to elicit long-lasting metabolic changes resembling the Warburg effect. Loss of Notch signalling in Drosophila wing discs as well as in human microvascular cells leads to downregulation of glycolytic genes. Notch-driven tissue overgrowth can be rescued by downregulation of genes for glucose metabolism. Notch activity is able to support growth of wing during nutrient-deprivation conditions, independent of the growth of the rest of the body. Notch is active in situations that involve metabolic reprogramming, and the direct regulation of metabolic genes may be a common mechanism that helps Notch to exert its effects in target tissues. PMID:26887408

  6. ZmABA2, an interacting protein of ZmMPK5, is involved in abscisic acid biosynthesis and functions.

    PubMed

    Ma, Fangfang; Ni, Lan; Liu, Libo; Li, Xi; Zhang, Huan; Zhang, Aying; Tan, Mingpu; Jiang, Mingyi

    2016-02-01

    In maize (Zea mays), the mitogen-activated protein kinase ZmMPK5 has been shown to be involved in abscisic acid (ABA)-induced antioxidant defence and to enhance the tolerance of plants to drought, salt stress and oxidative stress. However, the underlying molecular mechanisms are poorly understood. Here, using ZmMPK5 as bait in yeast two-hybrid screening, a protein interacting with ZmMPK5 named ZmABA2, which belongs to a member of the short-chain dehydrogenase/reductase family, was identified. Pull-down assay and bimolecular fluorescence complementation analysis and co-immunoprecipitation test confirmed that ZmMPK5 interacts with ZmABA2 in vitro and in vivo. Phosphorylation of Ser173 in ZmABA2 by ZmMPK5 was shown to increase the activity of ZmABA2 and the protein stability. Various abiotic stimuli induced the expression of ZmABA2 in leaves of maize plants. Pharmacological, biochemical and molecular biology and genetic analyses showed that both ZmMPK5 and ZmABA2 coordinately regulate the content of ABA. Overexpression of ZmABA2 in tobacco plants was found to elevate the content of ABA, regulate seed germination and root growth under drought and salt stress and enhance the tolerance of tobacco plants to drought and salt stress. These results suggest that ZmABA2 is a direct target of ZmMPK5 and is involved in ABA biosynthesis and functions. PMID:26096642

  7. Notch stimulates growth by direct regulation of genes involved in the control of glycolysis and the tricarboxylic acid cycle

    PubMed Central

    Slaninova, Vera; Krafcikova, Michaela; Perez-Gomez, Raquel; Steffal, Pavel; Trantirek, Lukas; Bray, Sarah J.

    2016-01-01

    Glycolytic shift is a characteristic feature of rapidly proliferating cells, such as cells during development and during immune response or cancer cells, as well as of stem cells. It results in increased glycolysis uncoupled from mitochondrial respiration, also known as the Warburg effect. Notch signalling is active in contexts where cells undergo glycolytic shift. We decided to test whether metabolic genes are direct transcriptional targets of Notch signalling and whether upregulation of metabolic genes can help Notch to induce tissue growth under physiological conditions and in conditions of Notch-induced hyperplasia. We show that genes mediating cellular metabolic changes towards the Warburg effect are direct transcriptional targets of Notch signalling. They include genes encoding proteins involved in glucose uptake, glycolysis, lactate to pyruvate conversion and repression of the tricarboxylic acid cycle. The direct transcriptional upregulation of metabolic genes is PI3K/Akt independent and occurs not only in cells with overactivated Notch but also in cells with endogenous levels of Notch signalling and in vivo. Even a short pulse of Notch activity is able to elicit long-lasting metabolic changes resembling the Warburg effect. Loss of Notch signalling in Drosophila wing discs as well as in human microvascular cells leads to downregulation of glycolytic genes. Notch-driven tissue overgrowth can be rescued by downregulation of genes for glucose metabolism. Notch activity is able to support growth of wing during nutrient-deprivation conditions, independent of the growth of the rest of the body. Notch is active in situations that involve metabolic reprogramming, and the direct regulation of metabolic genes may be a common mechanism that helps Notch to exert its effects in target tissues. PMID:26887408

  8. TGD4 involved in endoplasmic reticulum-to-chloroplast lipid trafficking is a phosphatidic acid binding protein

    SciTech Connect

    Wang Z.; Xu C.; Benning, C.

    2012-05-01

    The synthesis of galactoglycerolipids, which are prevalent in photosynthetic membranes, involves enzymes at the endoplasmic reticulum (ER) and the chloroplast envelope membranes. Genetic analysis of trigalactosyldiacylglycerol (TGD) proteins in Arabidopsis has demonstrated their role in polar lipid transfer from the ER to the chloroplast. The TGD1, 2, and 3 proteins resemble components of a bacterial-type ATP-binding cassette (ABC) transporter, with TGD1 representing the permease, TGD2 the substrate binding protein, and TGD3 the ATPase. However, the function of the TGD4 protein in this process is less clear and its location in plant cells remains to be firmly determined. The predicted C-terminal {beta}-barrel structure of TGD4 is weakly similar to proteins of the outer cell membrane of Gram-negative bacteria. Here, we show that, like TGD2, the TGD4 protein when fused to DsRED specifically binds phosphatidic acid (PtdOH). As previously shown for tgd1 mutants, tgd4 mutants have elevated PtdOH content, probably in extraplastidic membranes. Using highly purified and specific antibodies to probe different cell fractions, we demonstrated that the TGD4 protein was present in the outer envelope membrane of chloroplasts, where it appeared to be deeply buried within the membrane except for the N-terminus, which was found to be exposed to the cytosol. It is proposed that TGD4 is either directly involved in the transfer of polar lipids, possibly PtdOH, from the ER to the outer chloroplast envelope membrane or in the transfer of PtdOH through the outer envelope membrane.

  9. Cross-talk between glucocorticoid and retinoic acid signals involving glucocorticoid receptor interaction with the homoeodomain protein Pbx1.

    PubMed

    Subramaniam, Nanthakumar; Campión, Javier; Rafter, Ingalill; Okret, Sam

    2003-03-15

    Glucocorticoid (GC) signalling influences the response of the cell to a number of other signals via a mechanism referred to as 'cross-talk'. This cross-talk may act at several levels, including an interaction between the transcription factors involved in the signalling pathways. In the present paper, we demonstrate a novel functional interaction between GC and all- trans -retinoic acid (RA) signalling. We show that, in P19 embryonal carcinoma cells, GCs potentiate RA-induced expression of the murine Hoxb -1 gene through an autoregulatory element, b1-ARE, recognized by the Pbx1 and HOXB1 homoeodomain proteins. The synergistic effect of GC did not involve GC receptor (GR) binding to the b1-ARE, and the GC-GR complex alone was unable to activate transcription via the element. Furthermore, the ability of the GR to transactivate was not required, excluding expression of a GC-induced protein as the mechanism for the GC/RA synergy. Additional transfection experiments showed that the Pbx1/HOXB1 heterodimer was the target for the GC effect. Furthermore, functional dissection of the GR demonstrated that the DNA-binding domain (DBD) of the GR was required for the synergy. A physical interaction between the GR and Pbx1 proteins was demonstrated in vivo by co-immunoprecipitation experiments. These results are compatible with a model in which the GC/RA synergy is mediated by a direct interaction between the GR and Pbx1. On the basis of the ubiquitous expression of both GR and Pbx1, a number of genes regulated by Pbx are likely to be important targets for GC-mediated 'cross-talk'. PMID:12487626

  10. The apparent absence of involvement of biotin in the vitamin K-dependent carboxylation of glutamic acid residues of proteins.

    PubMed Central

    Friedman, P A; Shia, M A

    1977-01-01

    The mechanism of the vitamin K-dependent post-translational carboxylation of the gamma-carbon atom of glutamic acid residues in proteins remains obscure. Experiments were performed in vivo and in vitro in an attempt to establish a role for biotin in the transfer of the carboxyl group. Weanling male rats were fed on a biotin-deficient diet until severe biotin deficiency was induced. Their degree of biotin deficiency was documented by assaying for liver acetyl-CoA carboxylase activity, which was about 15% of normal. However, one-stage and two-stage prothrombin times measured on the plasmas were normal. In addition, the liver microsomal fraction did not contain any more prothrombin precursor than did that of normal rat liver. Experiments were done in vitro in which vitamin K-dependent fixing of 14CO2 was measured in the liver microsomal fraction from vitamin K-deficient male rats in the presence or absence of avidin. No evidence for an avidin-sensitive critical biotin-containing site was obtained. Thus neither series of experiments suggests a role for biotin; the data are compatible with carboxyl transfer occurring either through a carboxylated vitamin K intermediate; or via a yet to be identified intermediate, or perhaps via CO2 itself. PMID:17395

  11. Iodine 125-lysergic acid diethylamide binds to a novel serotonergic site on rat choroid plexus epithelial cells

    SciTech Connect

    Yagaloff, K.A.; Hartig, P.R.

    1985-12-01

    /sup 125/I-Lysergic acid diethylamide (/sup 125/I-LSD) binds with high affinity to serotonergic sites on rat choroid plexus. These sites were localized to choroid plexus epithelial cells by use of a novel high resolution stripping film technique for light microscopic autoradiography. In membrane preparations from rat choroid plexus, the serotonergic site density was 3100 fmol/mg of protein, which is 10-fold higher than the density of any other serotonergic site in brain homogenates. The choroid plexus site exhibits a novel pharmacology that does not match the properties of 5-hydroxytryptamine-1a (5-HT1a), 5-HT1b, or 5-HT2 serotonergic sites. /sup 125/I-LSD binding to the choroid plexus site is potently inhibited by mianserin, serotonin, and (+)-LSD. Other serotonergic, dopaminergic, and adrenergic agonists and antagonists exhibit moderate to weak affinities for this site. The rat choroid plexus /sup 125/I-LSD binding site appears to represent a new type of serotonergic site which is located on non-neuronal cells in this tissue.

  12. Identification of gamma-aminobutyric acid and its binding sites in Caenorhabditis elegans

    SciTech Connect

    Schaeffer, J.M.; Bergstrom, A.R.

    1988-01-01

    Gamma-aminobutyric acid (GABA), glutamate decarboxylase and GABA-transaminase were identified in the nematode Caenorhabditis elegans. The concentration of GABA in C. elegans is approximately 10-fold lower than the concentration of GABA in rat brain. Glutamate decarboxylase and GABA-transaminase, the GABA anabolic and catabolic enzymes, are also present in C. elegans. Crude membrane fractions were prepared from C. elegans and used to study specific (/sup 3/H) GABA binding sites. GABA binds to C. elegans membranes with high affinity and low capacity. Muscimol is a competitive inhibitor of specific GABA binding with a K/sub I/ value of 120 nM. None of the other GABA agonists or antagonists inhibited greater than 40% of the specific GABA binding at concentrations up to 10/sup -4/M. Thirteen spider venoms were examined as possible GABA agonists or antagonists, the venom from Calilena agelenidae inhibits specific GABA binding with a K/sub I/ value of 6 nl/ml. These results suggest that GABA has a physiological role as a neurotransmitter in C. elegans.

  13. Hydrolysis of phosphodiesters by diiron complexes: design of nonequivalent iron sites in purple acid phosphatase models.

    PubMed

    Verge, François; Lebrun, Colette; Fontecave, Marc; Ménage, Stéphane

    2003-01-27

    New mu-oxo-diferric complexes have been designed for hydrolysis of phosphodiesters. To mimic the diiron active site of purple acid phosphatase, a combinatorial method has been used to select complexes containing two distinct iron coordination spheres. The introduction of a bidentate ligand, a substituted phenanthroline (L) into complex 1, [Fe2O(bipy)4(OH2)2](NO3)4, generates in solution the complex [Fe2O(bipy)3(L)(OH2)2](NO3)4 as shown by ESI/MS and 1H NMR studies. The latter complex was found to be 20-fold more active than complex 1. On the basis of kinetic studies, we demonstrated that the complex [Fe2O(bipy)3(L)(OH)(OH2)](NO3)3 was the active species and the reaction proceeded through the formation of a ternary complex in which one iron binds a hydroxide and the second, the substrate. At nonsaturating concentrations of the substrate, the increased activity with increased methyl substituents in L was due to an increased affinity of the complex for the substrate. The activity of [Fe2O(bipy)3(33'44'Me2-Phen)(OH2)2](NO3)4 [33'44'Me2Phen = 3,3',4,4'-dimethyl-1,10-phenanthroline] was found to be comparable to that reported for Co(III) or Ce(IV) complexes. PMID:12693232

  14. High-Temperature Decomposition of Brønsted Acid Sites in Gallium-Substituted Zeolites

    SciTech Connect

    K Al-majnouni; N Hould; W Lonergan; D Vlachos; R Lobo

    2011-12-31

    The dehydroxylation of Broensted acid sites (BAS) in Ga-substituted zeolites was investigated at temperatures up to 850 C using X-ray absorption spectroscopy (XAS), Fourier transform infrared spectroscopy (FTIR), and mass spectrometry-temperature programmed desorption (MS-TPD). X-ray absorption near-edge spectroscopy (XANES) revealed that the majority of gallium has tetrahedral coordination even after complete dehydroxylation. The interatomic gallium-oxygen distance and gallium coordination number determined by extended X-ray absorption fine structure (EXAFS) are consistent with gallium in tetrahedral coordination at low T (< 550 C). Upon heating Ga-Beta and Ga-ZSM5 to 850 C, analysis of the EXAFS showed that 70 and 80% of the gallium was still in tetrahedral coordination. The remainder of the gallium was found to be in octahedral coordination. No trigonal Ga atoms were observed. FTIR measurements carried out at similar temperatures show that the intensity of the OH vibration due to BAS has been eliminated. MS-TPD revealed that hydrogen in addition to water evolved from the samples during dehydroxylation. This shows that dehydrogenation in addition to dehydration is a mechanism that contributes to BAS decomposition. Dehydrogenation was further confirmed by exposing the sample to hydrogen to regenerate some of the BAS as monitored by FTIR and MS-TPD.

  15. Binding site discovery from nucleic acid sequences by discriminative learning of hidden Markov models

    PubMed Central

    Maaskola, Jonas; Rajewsky, Nikolaus

    2014-01-01

    We present a discriminative learning method for pattern discovery of binding sites in nucleic acid sequences based on hidden Markov models. Sets of positive and negative example sequences are mined for sequence motifs whose occurrence frequency varies between the sets. The method offers several objective functions, but we concentrate on mutual information of condition and motif occurrence. We perform a systematic comparison of our method and numerous published motif-finding tools. Our method achieves the highest motif discovery performance, while being faster than most published methods. We present case studies of data from various technologies, including ChIP-Seq, RIP-Chip and PAR-CLIP, of embryonic stem cell transcription factors and of RNA-binding proteins, demonstrating practicality and utility of the method. For the alternative splicing factor RBM10, our analysis finds motifs known to be splicing-relevant. The motif discovery method is implemented in the free software package Discrover. It is applicable to genome- and transcriptome-scale data, makes use of available repeat experiments and aside from binary contrasts also more complex data configurations can be utilized. PMID:25389269

  16. ESTIMATES OF CLOUD WATER DEPOSITION AT MOUNTAIN DEPOSITION AT MOUNTAIN ACID DEPOSITION PROGRAM SITES IN THE APPALACHIAN MOUNTAINS

    EPA Science Inventory

    Cloud water deposition was estimated at three high elevation sites in the Appalachian Mountains of the eastern United States (Whiteface Mountain, NY, Whitetop Mountain, VA, and Clingrnan's Dome, TN) from 1994 through 1999 as part of the Mountain Acid Deposition Program (MADPro). ...

  17. Non-thermal plasma-assisted NOx reduction over Na-Y zeolites: The promotional effect of acid sites

    SciTech Connect

    Kwak, Ja Hun; Peden, Charles HF; Szanyi, Janos

    2006-06-01

    The effect of acid sites on the catalytic activities of a series of H+-modified Na-Y zeolites was investigated in the non-thermal plasma assisted NOx reduction reaction using a simulated diesel engine exhaust gas mixture. The acid sites were formed by NH4+ ion exchange and subsequent heat treatment of a NaY zeolite. The catalytic activities of these H+-modified NaY zeolites significantly increased with the number of acid sites. This NOx conversion increase was correlated with the decrease in the amount of unreacted NO2. The increase in the number of acid sites did not change the NO level, it stayed constant. Temperature programmed desorption following NO2 adsorption showed the appearance of a high temperature desorption peak at 453 K in addition to the main desorption feature of 343 K observed for the base Na-Y. The results of both the IR and TPD experiments revealed the formation of crotonaldehyde, resulting from condensation reaction of adsorbed acetaldehyde. Strong adsorptions of both NOx and hydrocarbon species are proposed to be responsible for the higher catalytic activity of H+-modified Na-Y zeolites in comparison to the base NaY material

  18. Mechanistic studies of a novel C-S lyase in ergothioneine biosynthesis: the involvement of a sulfenic acid intermediate.

    PubMed

    Song, Heng; Hu, Wen; Naowarojna, Nathchar; Her, Ampon Sae; Wang, Shu; Desai, Rushil; Qin, Li; Chen, Xiaoping; Liu, Pinghua

    2015-01-01

    Ergothioneine is a histidine thio-derivative isolated in 1909. In ergothioneine biosynthesis, the combination of a mononuclear non-heme iron enzyme catalyzed oxidative C-S bond formation reaction and a PLP-mediated C-S lyase (EgtE) reaction results in a net sulfur transfer from cysteine to histidine side-chain. This demonstrates a new sulfur transfer strategy in the biosynthesis of sulfur-containing natural products. Due to difficulties associated with the overexpression of Mycobacterium smegmatis EgtE protein, the proposed EgtE functionality remained to be verified biochemically. In this study, we have successfully overexpressed and purified M. smegmatis EgtE enzyme and evaluated its activities under different in vitro conditions: C-S lyase reaction using either thioether or sulfoxide as a substrate in the presence or absence of reductants. Results from our biochemical characterizations support the assignment of sulfoxide 4 as the native EgtE substrate and the involvement of a sulfenic acid intermediate in the ergothioneine C-S lyase reaction. PMID:26149121

  19. Putrescine is involved in Arabidopsis freezing tolerance and cold acclimation by regulating abscisic acid levels in response to low temperature.

    PubMed

    Cuevas, Juan C; López-Cobollo, Rosa; Alcázar, Rubén; Zarza, Xavier; Koncz, Csaba; Altabella, Teresa; Salinas, Julio; Tiburcio, Antonio F; Ferrando, Alejandro

    2008-10-01

    The levels of endogenous polyamines have been shown to increase in plant cells challenged with low temperature; however, the functions of polyamines in the regulation of cold stress responses are unknown. Here, we show that the accumulation of putrescine under cold stress is essential for proper cold acclimation and survival at freezing temperatures because Arabidopsis (Arabidopsis thaliana) mutants defective in putrescine biosynthesis (adc1, adc2) display reduced freezing tolerance compared to wild-type plants. Genes ADC1 and ADC2 show different transcriptional profiles upon cold treatment; however, they show similar and redundant contributions to cold responses in terms of putrescine accumulation kinetics and freezing sensitivity. Our data also demonstrate that detrimental consequences of putrescine depletion during cold stress are due, at least in part, to alterations in the levels of abscisic acid (ABA). Reduced expression of NCED3, a key gene involved in ABA biosynthesis, and down-regulation of ABA-regulated genes are detected in both adc1 and adc2 mutant plants under cold stress. Complementation analysis of adc mutants with ABA and reciprocal complementation tests of the aba2-3 mutant with putrescine support the conclusion that putrescine controls the levels of ABA in response to low temperature by modulating ABA biosynthesis and gene expression. PMID:18701673

  20. Mechanistic studies of a novel C-S lyase in ergothioneine biosynthesis: the involvement of a sulfenic acid intermediate

    PubMed Central

    Song, Heng; Hu, Wen; Naowarojna, Nathchar; Her, Ampon Sae; Wang, Shu; Desai, Rushil; Qin, Li; Chen, Xiaoping; Liu, Pinghua

    2015-01-01

    Ergothioneine is a histidine thio-derivative isolated in 1909. In ergothioneine biosynthesis, the combination of a mononuclear non-heme iron enzyme catalyzed oxidative C-S bond formation reaction and a PLP-mediated C-S lyase (EgtE) reaction results in a net sulfur transfer from cysteine to histidine side-chain. This demonstrates a new sulfur transfer strategy in the biosynthesis of sulfur-containing natural products. Due to difficulties associated with the overexpression of Mycobacterium smegmatis EgtE protein, the proposed EgtE functionality remained to be verified biochemically. In this study, we have successfully overexpressed and purified M. smegmatis EgtE enzyme and evaluated its activities under different in vitro conditions: C-S lyase reaction using either thioether or sulfoxide as a substrate in the presence or absence of reductants. Results from our biochemical characterizations support the assignment of sulfoxide 4 as the native EgtE substrate and the involvement of a sulfenic acid intermediate in the ergothioneine C-S lyase reaction. PMID:26149121

  1. The Cytotoxicity of Benzaldehyde Nitrogen Mustard-2-Pyridine Carboxylic Acid Hydrazone Being Involved in Topoisomerase IIα Inhibition

    PubMed Central

    Fu, Yun; Zhou, Sufeng; Liu, Youxun; Yang, Yingli; Sun, Xingzhi; Li, Changzheng

    2014-01-01

    The antitumor property of iron chelators and aromatic nitrogen mustard derivatives has been well documented. Combination of the two pharmacophores in one molecule in drug designation is worth to be explored. We reported previously the syntheses and preliminary cytotoxicity evaluation of benzaldehyde nitrogen mustard pyridine carboxyl acid hydrazones (BNMPH) as extended study, more tumor cell lines (IC50 for HepG2: 26.1 ± 3.5 μM , HCT-116: 57.5 ± 5.3 μM, K562: 48.2 ± 4.0 μM, and PC-12: 19.4 ± 2.2 μM) were used to investigate its cytotoxicity and potential mechanism. In vitro experimental data showed that the BNMPH chelating Fe2+ caused a large number of ROS formations which led to DNA cleavage, and this was further supported by comet assay, implying that ROS might be involved in the cytotoxicity of BNMPH. The ROS induced changes of apoptosis related genes, but the TFR1 and NDRG1 metastatic genes were not obviously regulated, prompting that BNMPH might not be able to deprive Fe2+ of ribonucleotide reductase. The BNMPH induced S phase arrest was different from that of iron chelators (G1) and alkylating agents (G2). BNMPH also exhibited its inhibition of human topoisomerase IIα. Those revealed that the cytotoxic mechanism of the BNMPH could stem from both the topoisomerase II inhibition, ROS generation and DNA alkylation. PMID:24995306

  2. The BRPF2/BRD1-MOZ complex is involved in retinoic acid-induced differentiation of embryonic stem cells.

    PubMed

    Cho, Hye In; Kim, Min Seong; Jang, Yeun Kyu

    2016-08-01

    The scaffold protein BRPF2 (also called BRD1), a key component of histone acetyltransferase complexes, plays an important role in embryonic development, but its function in the differentiation of embryonic stem cells (ESCs) remains unknown. In the present study, we investigated whether BRPF2 is involved in mouse ESC differentiation. BRPF2 depletion resulted in abnormal formation of embryoid bodies, downregulation of differentiation-associated genes, and persistent maintenance of alkaline phosphatase activity even after retinoic acid-induced differentiation, indicating impaired differentiation of BRPF2-depleted ESCs. We also found reduced global acetylation of histone H3 lysine 14 (H3K14) in BRPF2-depleted ESCs, irrespective of differentiation status. Further, co-immunoprecipitation analysis revealed a physical association between BRPF2 and the histone acetyltransferase MOZ in differentiated ESCs, suggesting the role of BRPF2-MOZ complexes in ESC differentiation. Together, these results suggest that BRPF2-MOZ complexes play an important role in the differentiation of ESCs via H3K14 acetylation. PMID:27256846

  3. Involvement of apoptotic cell death and cell cycle perturbation in retinoic acid-induced cleft palate in mice

    SciTech Connect

    Okano, Junko . E-mail: okajun@anat1.med.kyoto-u.ac.jp; Suzuki, Shigehiko; Shiota, Kohei

    2007-05-15

    Retinoic acid (RA), a metabolite of vitamin A, plays a key role in a variety of biological processes and is essential for normal embryonic development. On the other hand, exogenous RA could cause cleft palate in offspring when it is given to pregnant animals at either the early or late phases of palatogenesis, but the pathogenetic mechanism of cleft palate caused by excess RA remains not fully elucidated. The aim of the present study was to investigate the effects of excess of RA on early palatogenesis in mouse fetuses and analyze the teratogenic mechanism, especially at the stage prior to palatal shelf elevation. We gave all-trans RA (100 mg/kg) orally to E11.5 ICR pregnant mice and observed the changes occurring in the palatal shelves of their fetuses. It was found that apoptotic cell death increased not only in the epithelium of the palatal shelves but also in the tongue primordium, which might affect tongue withdrawal movement during palatogenesis and impair the horizontal elevation of palatal shelves. In addition, RA was found to prevent the G{sub 1}/S progression of palatal mesenchymal cells through upregulation of p21 {sup Cip1}, leading to Rb hypophospholylation. Thus, RA appears to cause G{sub 1} arrest in palatal mesenchymal cells in a similar manner as in various cancer and embryonic cells. It is likely that apoptotic cell death and cell cycle disruption are involved in cleft palate formation induced by RA.

  4. NPR1-dependent salicylic acid signaling is not involved in elevated CO2-induced heat stress tolerance in Arabidopsis thaliana

    PubMed Central

    Ahammed, Golam Jalal; Li, Xin; Yu, Jingquan; Shi, Kai

    2015-01-01

    Elevated CO2 can protect plants from heat stress (HS); however, the underlying mechanisms are largely unknown. Here, we used a set of Arabidopsis mutants such as salicylic acid (SA) signaling mutants nonexpressor of pathogenesis-related gene 1 (npr1-1 and npr1-5) and heat-shock proteins (HSPs) mutants (hsp21 and hsp70-1) to understand the requirement of SA signaling and HSPs in elevated CO2-induced HS tolerance. Under ambient CO2 (380 µmol mol−1) conditions, HS (42°C, 24 h) drastically decreased maximum photochemical efficiency of PSII (Fv/Fm) in all studied plant groups. Enrichment of CO2 (800 µmol mol−1) with HS remarkably increased the Fv/Fm value in all plant groups except hsp70-1, indicating that NPR1-dependent SA signaling is not involved in the elevated CO2-induced HS tolerance. These results also suggest an essentiality of HSP70-1, but not HSP21 in elevated CO2-induced HS mitigation. PMID:25874349

  5. Bile Acid Signaling Is Involved in the Neurological Decline in a Murine Model of Acute Liver Failure.

    PubMed

    McMillin, Matthew; Frampton, Gabriel; Quinn, Matthew; Ashfaq, Samir; de los Santos, Mario; Grant, Stephanie; DeMorrow, Sharon

    2016-02-01

    Hepatic encephalopathy is a serious neurological complication of liver failure. Serum bile acids are elevated after liver damage and may disrupt the blood-brain barrier and enter the brain. Our aim was to assess the role of serum bile acids in the neurological complications after acute liver failure. C57Bl/6 or cytochrome p450 7A1 knockout (Cyp7A1(-/-)) mice were fed a control, cholestyramine-containing, or bile acid-containing diet before azoxymethane (AOM)-induced acute liver failure. In parallel, mice were given an intracerebroventricular infusion of farnesoid X receptor (FXR) Vivo-morpholino before AOM injection. Liver damage, neurological decline, and molecular analyses of bile acid signaling were performed. Total bile acid levels were increased in the cortex of AOM-treated mice. Reducing serum bile acids via cholestyramine feeding or using Cyp7A1(-/-) mice reduced bile acid levels and delayed AOM-induced neurological decline, whereas cholic acid or deoxycholic acid feeding worsened AOM-induced neurological decline. The expression of bile acid signaling machinery apical sodium-dependent bile acid transporter, FXR, and small heterodimer partner increased in the frontal cortex, and blocking FXR signaling delayed AOM-induced neurological decline. In conclusion, circulating bile acids may play a pathological role during hepatic encephalopathy, although precisely how they dysregulate normal brain function is unknown. Strategies to minimize serum bile acid concentrations may reduce the severity of neurological complications associated with liver failure. PMID:26683664

  6. Temperature-induced intersite charge transfer involving Cr ions in A-site-ordered perovskites ACu(3)Cr(4)O(12) (A=La and Y).

    PubMed

    Zhang, Shoubao; Saito, Takashi; Mizumaki, Masaichiro; Shimakawa, Yuichi

    2014-07-28

    Changes in the valence state of transition-metal ions in oxides drastically modify the chemical and physical properties of the compounds. Intersite charge transfer (ISCT), which involves simultaneous changes in the valence states of two valence-variable transition-metal cations at different crystallographic sites, further expands opportunities to show multifunctional properties. To explore new ISCT materials, we focus on A-site-ordered perovskite-structure oxides with the chemical formula AA'3 B4 O12 , which contain different transition-metal cations at the square-planar A' and octahedral B sites. We have obtained new A-site-ordered perovskites LaCu3 Cr4 O12 and YCu3 Cr4 O12 by synthesis under high-pressure and high-temperature conditions and found that they showed temperature-induced ISCT between A'-site Cu and B-site Cr ions. The compounds are the first examples of those, in which Cr ions are involved in temperature-induced ISCT. In contrast to the previously reported ISCT compounds, LaCu3 Cr4 O12 and YCu3 Cr4 O12 showed positive-thermal-expansion-like volume changes at the ISCT transition. PMID:24975031

  7. The two active sites in human branched-chain alpha-keto acid dehydrogenase operate independently without an obligatory alternating-site mechanism.

    PubMed

    Li, Jun; Machius, Mischa; Chuang, Jacinta L; Wynn, R Max; Chuang, David T

    2007-04-20

    A long standing controversy is whether an alternating activesite mechanism occurs during catalysis in thiamine diphosphate (ThDP)-dependent enzymes. We address this question by investigating the ThDP-dependent decarboxylase/dehydrogenase (E1b) component of the mitochondrial branched-chain alpha-keto acid dehydrogenase complex (BCKDC). Our crystal structure reveals that conformations of the two active sites in the human E1b heterotetramer harboring the reaction intermediate are identical. Acidic residues in the core of the E1b heterotetramer, which align with the proton-wire residues proposed to participate in active-site communication in the related pyruvate dehydrogenase from Bacillus stearothermophilus, are mutated. Enzyme kinetic data show that, except in a few cases because of protein misfolding, these alterations are largely without effect on overall activity of BCKDC, ruling out the requirement of a proton-relay mechanism in E1b. BCKDC overall activity is nullified at 50% phosphorylation of E1b, but it is restored to nearly half of the pre-phosphorylation level after dissociation and reconstitution of BCKDC with the same phosphorylated E1b. The results suggest that the abolition of overall activity likely results from the specific geometry of the half-phosphorylated E1b in the BCKDC assembly and not due to a disruption of the alternating active-site mechanism. Finally, we show that a mutant E1b containing only one functional active site exhibits half of the wild-type BCKDC activity, which directly argues against the obligatory communication between active sites. The above results provide evidence that the two active sites in the E1b heterotetramer operate independently during the ThDP-dependent decarboxylation reaction. PMID:17329260

  8. Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid

    PubMed Central

    Kwon, Inchan; Choi, Eun Sil

    2016-01-01

    Multiple-site-specific incorporation of a noncanonical amino acid into a recombinant protein would be a very useful technique to generate multiple chemical handles for bioconjugation and multivalent binding sites for the enhanced interaction. Previously combination of a mutant yeast phenylalanyl-tRNA synthetase variant and the yeast phenylalanyl-tRNA containing the AAA anticodon was used to incorporate a noncanonical amino acid into multiple UUU phenylalanine (Phe) codons in a site-specific manner. However, due to the less selective codon recognition of the AAA anticodon, there was significant misincorporation of a noncanonical amino acid into unwanted UUC Phe codons. To enhance codon selectivity, we explored degenerate leucine (Leu) codons instead of Phe degenerate codons. Combined use of the mutant yeast phenylalanyl-tRNA containing the CAA anticodon and the yPheRS_naph variant allowed incorporation of a phenylalanine analog, 2-naphthylalanine, into murine dihydrofolate reductase in response to multiple UUG Leu codons, but not to other Leu codon sites. Despite the moderate UUG codon occupancy by 2-naphthylalaine, these results successfully demonstrated that the concept of forced ambiguity of the genetic code can be achieved for the Leu codons, available for multiple-site-specific incorporation. PMID:27028506

  9. Evidence for the Involvement of Xenobiotic-responsive Nuclear Receptors in Transcriptional Effects Upon Perfluoroalkyl Acid Exposure in Diverse Species.

    EPA Science Inventory

    Humans and other species have detectable body burdens of a number of perfluorinated alkyl acids (PFAA) including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). In mouse and rat liver these compounds elicit transcriptional and phenotypic effects similar to pe...

  10. Evidence for the Involvement of Xenobiotic-response Nuclear Receptors in Transcriptional Effects Upon Perfluroroalkyl Acid Exposure in Diverse Species

    EPA Science Inventory

    Humans and ecological species have been found to have detectable body burdens of a number of perfluorinated alkyl acids (PFAA) including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). In mouse and rat liver these compounds elicit transcriptional and phenotyp...

  11. Schistosoma mansoni: possible involvement of protein kinase C in linoleic acid-induced proteolytic enzyme release from cercariae.

    PubMed

    Matsumura, K; Mitsui, Y; Sato, K; Sakamoto, M; Aoki, Y

    1991-04-01

    The possible involvement of protein kinase C and Ca2+ metabolism in the proteolytic enzyme release from schistosome cercariae was studied. Cercariae were placed in dechlorinated tap water containing 0.37 mM calcium in the small glass petri dish and exposed to the stimuli (linoleic acid, phorbol esters, and Ca2+ ionophore) with or without inhibitors of protein kinase C or Ca2+ metabolism. The proteolytic activity of incubation medium of cercariae thus treated was measured by the azocoll assay. The penetration response of cercariae induced by linoleic acid, a physiological stimulus, was mimicked by phorbol esters. When exposed to phorbol esters, 0.02 to 2 microM of 12-O-tetradecanoylphorbol-13-acetate (TPA) and 0.2 to 2 microM of phorbol-12,13-dibutyrate (PDBu), cercariae ceased the swimming movement, began a rhythmic thrusting of the anterior tip of the parasite, and released the proteolytic enzyme, but they did not shed the tails. Lowering Ca2+ in water by addition of 5 mM ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), phorbol ester-induced release of enzyme was completely inhibited. Phorbol ester-induced release of enzyme was partially inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C, at a concentration of 100 microM. H-7 alone, at a concentration of 100 microM, did not affect the swimming movement of cercariae. The cercariae were stimulated to release the enzyme by high concentrations (10 and 100 microM) of the Ca2+ ionophore, A23187, but enzyme was not released by low concentrations (0.5 and 1 microM) of this drug. Cercariae exposed to A23187 behaved differently from those exposed to phorbol esters. They ceased swimming, showed strong muscle contraction, and shed their tail. A23187 stimulated cercariae to release the enzyme in the water containing 5 mM EGTA. A23187-induced enzyme release was not inhibited by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin

  12. Effects of sex and site on amino acid metabolism enzyme gene expression and activity in rat white adipose tissue

    PubMed Central

    Arriarán, Sofía; Agnelli, Silvia; Remesar, Xavier; Fernández-López, José Antonio

    2015-01-01

    Background and Objectives. White adipose tissue (WAT) shows marked sex- and diet-dependent differences. However, our metabolic knowledge of WAT, especially on amino acid metabolism, is considerably limited. In the present study, we compared the influence of sex on the amino acid metabolism profile of the four main WAT sites, focused on the paths related to ammonium handling and the urea cycle, as a way to estimate the extent of WAT implication on body amino-nitrogen metabolism. Experimental Design. Adult female and male rats were maintained, undisturbed, under standard conditions for one month. After killing them under isoflurane anesthesia. WAT sites were dissected and weighed. Subcutaneous, perigonadal, retroperitoneal and mesenteric WAT were analyzed for amino acid metabolism gene expression and enzyme activities. Results. There was a considerable stability of the urea cycle activities and expressions, irrespective of sex, and with only limited influence of site. Urea cycle was more resilient to change than other site-specialized metabolic pathways. The control of WAT urea cycle was probably related to the provision of arginine/citrulline, as deduced from the enzyme activity profiles. These data support a generalized role of WAT in overall amino-N handling. In contrast, sex markedly affected WAT ammonium-centered amino acid metabolism in a site-related way, with relatively higher emphasis in males’ subcutaneous WAT. Conclusions. We found that WAT has an active amino acid metabolism. Its gene expressions were lower than those of glucose-lipid interactions, but the differences were quantitatively less important than usually reported. The effects of sex on urea cycle enzymes expression and activity were limited, in contrast with the wider variations observed in other metabolic pathways. The results agree with a centralized control of urea cycle operation affecting the adipose organ as a whole. PMID:26587356

  13. Effects of sex and site on amino acid metabolism enzyme gene expression and activity in rat white adipose tissue.

    PubMed

    Arriarán, Sofía; Agnelli, Silvia; Remesar, Xavier; Fernández-López, José Antonio; Alemany, Marià

    2015-01-01

    Background and Objectives. White adipose tissue (WAT) shows marked sex- and diet-dependent differences. However, our metabolic knowledge of WAT, especially on amino acid metabolism, is considerably limited. In the present study, we compared the influence of sex on the amino acid metabolism profile of the four main WAT sites, focused on the paths related to ammonium handling and the urea cycle, as a way to estimate the extent of WAT implication on body amino-nitrogen metabolism. Experimental Design. Adult female and male rats were maintained, undisturbed, under standard conditions for one month. After killing them under isoflurane anesthesia. WAT sites were dissected and weighed. Subcutaneous, perigonadal, retroperitoneal and mesenteric WAT were analyzed for amino acid metabolism gene expression and enzyme activities. Results. There was a considerable stability of the urea cycle activities and expressions, irrespective of sex, and with only limited influence of site. Urea cycle was more resilient to change than other site-specialized metabolic pathways. The control of WAT urea cycle was probably related to the provision of arginine/citrulline, as deduced from the enzyme activity profiles. These data support a generalized role of WAT in overall amino-N handling. In contrast, sex markedly affected WAT ammonium-centered amino acid metabolism in a site-related way, with relatively higher emphasis in males' subcutaneous WAT. Conclusions. We found that WAT has an active amino acid metabolism. Its gene expressions were lower than those of glucose-lipid interactions, but the differences were quantitatively less important than usually reported. The effects of sex on urea cycle enzymes expression and activity were limited, in contrast with the wider variations observed in other metabolic pathways. The results agree with a centralized control of urea cycle operation affecting the adipose organ as a whole. PMID:26587356

  14. Role of GlnR in Acid-Mediated Repression of Genes Encoding Proteins Involved in Glutamine and Glutamate Metabolism in Streptococcus mutans▿ †

    PubMed Central

    Chen , Pei-Min; Chen, Yi-Ywan M.; Yu, Sung-Liang; Sher, Singh; Lai, Chern-Hsiung; Chia, Jean-San

    2010-01-01

    The acid tolerance response (ATR) is one of the major virulence traits of Streptococcus mutans. In this study, the role of GlnR in acid-mediated gene repression that affects the adaptive ATR in S. mutans was investigated. Using a whole-genome microarray and in silico analyses, we demonstrated that GlnR and the GlnR box (ATGTNAN7TNACAT) were involved in the transcriptional repression of clusters of genes encoding proteins involved in glutamine and glutamate metabolism under acidic challenge. Reverse transcription-PCR (RT-PCR) analysis revealed that the coordinated regulation of the GlnR regulon occurred 5 min after acid treatment and that prolonged acid exposure (30 min) resulted in further reduction in expression. A lower level but consistent reduction in response to acidic pH was also observed in chemostat-grown cells, confirming the negative regulation of GlnR. The repression by GlnR through the GlnR box in response to acidic pH was further confirmed in the citBZC operon, containing genes encoding the first three enzymes in the glutamine/glutamate biosynthesis pathway. The survival rate of the GlnR-deficient mutant at pH 2.8 was more than 10-fold lower than that in the wild-type strain 45 min after acid treatment, suggesting that the GlnR regulon participates in S. mutans ATR. It is hypothesized that downregulation of the synthesis of the amino acid precursors in response to acid challenge would promote citrate metabolism to pyruvate, with the consumption of H+ and potential ATP synthesis. Such regulation will ensure an optimal acid adaption in S. mutans. PMID:20173059

  15. Mutational analysis of nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of critical amino acid residues involved in exopolysaccharide alginate synthesis.

    PubMed Central

    Sundin, G W; Shankar, S; Chakrabarty, A M

    1996-01-01

    We report the utilization of site-directed and random mutagenesis procedures in the gene encoding nucleoside diphosphate kinase (ndk) from Pseudomonas aeruginosa in order to examine the role of Ndk in the production of alginate by this organism. Cellular levels of the 16-kDa form of the Ndk enzyme are greatly reduced in P. aeruginosa 8830 with a knockout mutation in the algR2 gene (8830R2::Cm); this strain is also defective in the production of the exopolysaccharide alginate. In this study, we isolated four mutations in ndk (Ala-14-->Pro [Ala14Pro], Gly21Val, His117Gln, and Ala125Arg) which resulted in the loss of Ndk biochemical activity; hyperexpression of any of these four mutant genes did not restore alginate production to 8830R2::Cm. We identified six additional amino acid residues (Ser-43, Ala-56, Ser-69, Glu-80, Gly-91, and Asp-135) whose alteration resulted in the inability of Ndk to complement alginate production. After hyperproduction in 8830R2::Cm, it was determined that each of these six mutant Ndks was biochemically active. However, in four cases, the in vivo levels of Ndk were reduced, which consequently affected the growth of 8830R2::Cm in the presence of Tween 20. Two mutant Ndk proteins which could not complement the alginate synthesis defect in 8830R2::Cm were not affected in any characteristic examined in the present study. All of the mutant Ndks characterized which were still biochemically active formed membrane complexes with Pk, resulting in GTP synthesis. Two of the four Ndk activity mutants (His117Gln and Ala125Arg) identified were capable of being truncated to 12 kDa and formed a membrane complex with Pk; however, the complexes formed were inactive for GTP synthesis. The other two Ndk activity mutants could be truncated to 12 kDa but were not detected in membrane fractions. These results further our understanding of the role of Ndk in alginate synthesis and identify amino acid residues in Ndk which have not previously been studied as

  16. A nuclear magnetic resonance-based structural rationale for contrasting stoichiometry and ligand binding site(s) in fatty acid-binding proteins.

    PubMed

    He, Yan; Estephan, Rima; Yang, Xiaomin; Vela, Adriana; Wang, Hsin; Bernard, Cédric; Stark, Ruth E

    2011-03-01

    Liver fatty acid-binding protein (LFABP) is a 14 kDa cytosolic polypeptide, differing from other family members in the number of ligand binding sites, the diversity of bound ligands, and the transfer of fatty acid(s) to membranes primarily via aqueous diffusion rather than direct collisional interactions. Distinct two-dimensional (1)H-(15)N nuclear magnetic resonance (NMR) signals indicative of slowly exchanging LFABP assemblies formed during stepwise ligand titration were exploited, without determining the protein-ligand complex structures, to yield the stoichiometries for the bound ligands, their locations within the protein binding cavity, the sequence of ligand occupation, and the corresponding protein structural accommodations. Chemical shifts were monitored for wild-type LFABP and an R122L/S124A mutant in which electrostatic interactions viewed as being essential to fatty acid binding were removed. For wild-type LFABP, the results compared favorably with the data for previous tertiary structures of oleate-bound wild-type LFABP in crystals and in solution: there are two oleates, one U-shaped ligand that positions the long hydrophobic chain deep within the cavity and another extended structure with the hydrophobic chain facing the cavity and the carboxylate group lying close to the protein surface. The NMR titration validated a prior hypothesis that the first oleate to enter the cavity occupies the internal protein site. In contrast, (1)H and (15)N chemical shift changes supported only one liganded oleate for R122L/S124A LFABP, at an intermediate location within the protein cavity. A rationale based on protein sequence and electrostatics was developed to explain the stoichiometry and binding site trends for LFABPs and to put these findings into context within the larger protein family. PMID:21226535

  17. Effects of peroxisome proliferators on rat liver phospholipids: sphingomyelin degradation may be involved in hepatotoxic mechanism of perfluorodecanoic acid.

    PubMed

    Adinehzadeh, M; Reo, N V

    1998-05-01

    Perfluorooctanoic acid (PFOA), perfluorodecanoic acid (PFDA), clofibrate, di(2-ethylhexyl)phthalate (DEHP), and Wy-14,643 represent a class of compounds known as peroxisome proliferators (PPs). Such compounds induce biogenesis of liver peroxisomes and cause a varying degree of hepatotoxicity and carcinogenesis in rodents. We examined the effects of these PPs on rat hepatic lipids and phospholipid profiles using phosphorus-31 NMR spectroscopy. All PPs caused a 25-57% increase in hepatic phospholipid content, while all but clofibrate increased the total lipid content by 26-156%. Treatments also influenced the composition of liver phospholipids. Phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEth) contents were significantly increased in all treatment groups. Most notably, PFDA caused the largest increase in PtdCho and PtdEth content (ca. 70%), while PFOA and Wy-14,643 were the only test compounds that influenced the PtdCho:PtdEth ratio. PFDA also caused an ca. 30% decrease in sphingomyelin (SphM) from 24 to 120 h postdose. SphM is a key lipid in signal transduction processes involved in apoptosis. Hydrolysis of SphM can be mediated through the action of tumor necrosis factor (TNF-alpha). We measured the TNF-alpha concentrations in rat sera at 24 h post-PFDA-exposure and found an 8-fold increase relative to vehicle-treated controls. These data demonstrate that an increase in the serum TNF-alpha level correlates with the time frame for the observed reduction in hepatic SphM. PFOA, a structurally similar compound, had no effect on hepatic SphM content, nor did it affect the serum TNF-alpha concentration. These effects may be related to differences in the tumorigenicity associated with these compounds. We postulate that PFDA activates the SphM signal transduction pathway via the release of TNF-alpha. This then stimulates cytotoxic responses and processes of apoptosis and may suppress cell proliferative and mitogenic responses. PMID:9585473

  18. Site- and kinase-specific phosphorylation-mediated activation of SLAC1, a guard cell anion channel stimulated by abscisic acid.

    PubMed

    Maierhofer, Tobias; Diekmann, Marion; Offenborn, Jan Niklas; Lind, Christof; Bauer, Hubert; Hashimoto, Kenji; S Al-Rasheid, Khaled A; Luan, Sheng; Kudla, Jörg; Geiger, Dietmar; Hedrich, Rainer

    2014-09-01

    Under drought stress, abscisic acid (ABA) triggers closure of leaf cell pores called stomata, which are formed by two specialized cells called guard cells in plant epidermis. Two pathways downstream of ABA stimulate phosphorylation of the S-type anion channels SLAC1 (slow anion channel associated 1) and SLAH3 (SLAC1 homolog 3), which causes these channels to open, reducing guard cell volume and triggering stomatal closure. One branch involves OST1 (open stomata 1), a calcium-independent SnRK2-type kinase, and the other branch involves calcium-dependent protein kinases of the CPK (calcium-dependent protein kinase) family. We used coexpression analyses in Xenopus oocytes to show that the calcineurin B-like (CBL) calcium sensors CBL1 and CBL9 and their interacting protein kinase CIPK23 also triggered SLAC1 and SLAH3 opening. We analyzed whether regulation of SLAC1 opening by these different families of kinases involved the same or different sites on SLAC1 by measuring channel conductance of SLAC1 with mutations in the putative phosphorylation sites in the amino or carboxyl termini coexpressed with specific kinases in Xenopus oocytes. SLAC1 mutants lacking the OST1-phosphorylated site were still activated by CPK or by CBL/CIPK complexes. Phosphorylation and activation of SLAC1 by any of the kinases were inhibited by the phosphatase ABI1 (ABA insensitive 1), which is inactivated in response to ABA signaling. These findings identified CBL/CIPK complexes as potential regulators of stomatal aperture through S-type anion channels and indicated that phosphorylation at distinct sites enables SLAC1 activation by both calcium-dependent and calcium-independent pathways downstream of ABA. PMID:25205850

  19. Protons and Psalmotoxin-1 reveal nonproton ligand stimulatory sites in chicken acid-sensing ion channel

    PubMed Central

    Smith, Rachel N; Gonzales, Eric B

    2014-01-01

    Acid-sensing ion channels (ASICs) are proton-sensitive, sodium-selective channels expressed in the nervous system that sense changes in extracellular pH. These ion channels are sensitive to an increasing number of nonproton ligands that include natural venom peptides and guanidine compounds. In the case of chicken ASIC1, the spider toxin Psalmotoxin-1 (PcTx1) activates the channel, resulting in an inward current. Furthermore, a growing class of ligands containing a guanidine group has been identified that stimulate peripheral ASICs (ASIC3), but exert subtle influence on other ASIC subtypes. The effects of the guanidine compounds on cASIC1 have not been the focus of previous study. Here, we investigated the interaction of the guanidine compound 2-guanidine-4-methylquinazoline (GMQ) on cASIC1 proton activation and PcTx1 stimulation. Exposure of expressed cASIC1 to PcTx1 resulted in biphasic currents consisting of a transient peak followed by an irreversible cASIC1 PcTx1 persistent current. This cASIC1 PcTx1 persistent current may be the result of locking the cASIC1 protein into a desensitized transition state. The guanidine compound GMQ increased the apparent affinity of protons on cASIC1 and decreased the half-maximal constant of the cASIC1 steady-state desensitization profile. Furthermore, GMQ stimulated the cASIC1 PcTx1 persistent current in a concentration-dependent manner, which resulted in a non-desensitizing inward current. Our data suggests that GMQ may have multiple sites within cASIC1 and may act as a “molecular wedge” that forces the PcTx1-desensitized ASIC into an open state. Our findings indicate that guanidine compounds, such as GMQ, may alter acid-sensing ion channel activity in combination with other stimuli, and that additional ASIC subtypes (along with ASIC3) may serve to sense and mediate signals from multiple stimuli. PMID:24262969

  20. Transcriptome Profiling and Functional Analysis of Agrobacterium tumefaciens Reveals a General Conserved Response to Acidic Conditions (pH 5.5) and a Complex Acid-Mediated Signaling Involved in Agrobacterium-Plant Interactions▿

    PubMed Central

    Yuan, Ze-Chun; Liu, Pu; Saenkham, Panatda; Kerr, Kathleen; Nester, Eugene W.

    2008-01-01

    Agrobacterium tumefaciens transferred DNA (T-DNA) transfer requires that the virulence genes (vir regulon) on the tumor-inducing (Ti) plasmid be induced by plant phenolic signals in an acidic environment. Using transcriptome analysis, we found that these acidic conditions elicit two distinct responses: (i) a general and conserved response through which Agrobacterium modulates gene expression patterns to adapt to environmental acidification and (ii) a highly specialized acid-mediated signaling response involved in Agrobacterium-plant interactions. Overall, 78 genes were induced and 74 genes were repressed significantly under acidic conditions (pH 5.5) compared to neutral conditions (pH 7.0). Microarray analysis not only confirmed previously identified acid-inducible genes but also uncovered many new acid-induced genes which may be directly involved in Agrobacterium-plant interactions. These genes include virE0, virE1, virH1, and virH2. Further, the chvG-chvI two-component system, previously shown to be critical for virulence, was also induced under acid conditions. Interestingly, acidic conditions induced a type VI secretion system and a putative nonheme catalase. We provide evidence suggesting that acid-induced gene expression was independent of the VirA-VirG two-component system. Our results, together with previous data, support the hypothesis that there is three-step sequential activation of the vir regulon. This process involves a cascade regulation and hierarchical signaling pathway featuring initial direct activation of the VirA-VirG system by the acid-activated ChvG-ChvI system. Our data strengthen the notion that Agrobacterium has evolved a mechanism to perceive and subvert the acidic conditions of the rhizosphere to an important signal that initiates and directs the early virulence program, culminating in T-DNA transfer. PMID:17993523

  1. Identifying involvement of Lys251/Asp252 pair in electron transfer and associated proton transfer at the quinone reduction site of Rhodobacter capsulatus cytochrome bc1.

    PubMed

    Kuleta, Patryk; Sarewicz, Marcin; Postila, Pekka; Róg, Tomasz; Osyczka, Artur

    2016-10-01

    Describing dynamics of proton transfers in proteins is challenging, but crucial for understanding processes which use them for biological functions. In cytochrome bc1, one of the key enzymes of respiration or photosynthesis, proton transfers engage in oxidation of quinol (QH2) and reduction of quinone (Q) taking place at two distinct catalytic sites. Here we evaluated by site-directed mutagenesis the contribution of Lys251/Asp252 pair (bacterial numbering) in electron transfers and associated with it proton uptake to the quinone reduction site (Qi site). We showed that the absence of protonable group at position 251 or 252 significantly changes the equilibrium levels of electronic reactions including the Qi-site mediated oxidation of heme bH, reverse reduction of heme bH by quinol and heme bH/Qi semiquinone equilibrium. This implicates the role of H-bonding network in binding of quinone/semiquinone and defining thermodynamic properties of Q/SQ/QH2 triad. The Lys251/Asp252 proton path is disabled only when both protonable groups are removed. With just one protonable residue from this pair, the entrance of protons to the catalytic site is sustained, albeit at lower rates, indicating that protons can travel through parallel routes, possibly involving water molecules. This shows that proton paths display engineering tolerance for change as long as all the elements available for functional cooperation secure efficient proton delivery to the catalytic site. PMID:27421232

  2. Gamma-aminobutyric acid-modulated benzodiazepine binding sites in bacteria

    SciTech Connect

    Lummis, S.C.R.; Johnston, G.A.R. ); Nicoletti, G. ); Holan, G. )

    1991-01-01

    Benzodiazepine binding sites, which were once considered to exist only in higher vertebrates, are here demonstrated in the bacteria E. coli. The bacterial ({sup 3}H)diazepam binding sites are modulated by GABA; the modulation is dose dependent and is reduced at high concentrations. The most potent competitors of E.Coli ({sup 3}H)diazepam binding are those that are active in displacing ({sup 3}H)benzodiazepines from vertebrate peripheral benzodiazepine binding sites. These vertebrate sites are not modulated by GABA, in contrast to vertebrate neuronal benzodiazepine binding sites. The E.coli benzodiazepine binding sites therefore differ from both classes of vertebrate benzodiazepine binding sites; however the ligand spectrum and GABA-modulatory properties of the E.coli sites are similar to those found in insects. This intermediate type of receptor in lower species suggests a precursor for at least one class of vertebrate benzodiazepine binding sites may have existed.

  3. γ-aminobutyric acid transporter-1 is involved in anxiety-like behaviors and cognitive function in knockout mice

    PubMed Central

    GONG, XUE; SHAO, YIYE; LI, BING; CHEN, LONG; WANG, CUICUI; CHEN, YINGHUI

    2015-01-01

    The aim of the present study was to investigate the effect of γ-aminobutyric acid transporter-1 (GAT-1) on the anxiety-like behaviors and cognitive function in knockout mice. In total, 20 adult male mice were divided into two groups, namely the GAT-1 knockout (GAT-1−/−) and wild-type (WT) groups. The open field test, elevated 0-maze (EZM) and Morris water maze were used to evaluate changes in anxiety-like behaviors and cognitive function. Compared with the WT mice, GAT-1−/− mice made more entries and spent a longer time within the central area, traveling a greater distance, during the open field test (P<0.05). The EZM revealed that GAT-1−/− mice spent more time in the open sectors and made more total entries when compared with the WT mice (P<0.01). Observations from the two tests indicated reduced anxiety-like behaviors in the GAT-1−/− mice. During the learning session using a Morris water maze, the latency to find the platform was significantly longer in the GAT-1−/− mice when compared with the WT mice (P<0.01). In addition, during the probe test, the GAT-1−/− mice spent less time in the target quadrant and more time in the opposite quadrant when compared with the WT mice (P<0.01); thus, the cognitive function in the GAT-1−/− mice was impaired. Therefore, the results demonstrated that the anxiety-like behaviors were reduced and cognitive function was impaired in GAT-1 knockout mice, indicating that GAT-1 is involved in anxiety and cognitive functions. PMID:26622370

  4. Differential involvement of indole-3-acetic acid biosynthetic pathways in pathogenicity and epiphytic fitness of Erwinia herbicola pv. gypsophilae.

    PubMed

    Manulis, S; Haviv-Chesner, A; Brandl, M T; Lindow, S E; Barash, I

    1998-07-01

    Erwinia herbicola pv. gypsophilae (Ehg), which induces galls on Gypsophila paniculata, harbors two major pathways for indole-3-acetic acid (IAA) synthesis, the indole-3-acetamide (IAM) and indole-3-pyruvate (IPyA) routes, as well as cytokinin biosynthetic genes. Mutants were generated in which the various biosynthetic routes were disrupted separately or jointly in order to assess the contribution of IAA of various origins and cytokinins to pathogenicity and epiphytic fitness. Inactivation of the IAM pathway or cytokinin biosynthesis caused the largest reduction in gall size. Inactivation of the IPyA pathway caused a minor, nonsignificant decrease in pathogenicity. No further reduction in gall size was observed by the simultaneous inactivation of both IAA pathways only or in combination with that of cytokinin production. However, inactivation of the IPyA pathway caused a 14-fold reduction in the population of Ehg on bean plants. Inactivation of the IAM pathway or cytokinin production did not affect epiphytic fitness. While the apparent transcriptional activity of iaaM-inaZ fusion increased slightly in cells of Ehg on bean and gypsophila leaves, compared with that in culture, very high levels of induction were observed in cells injected into gypsophila stems. In contrast, moderate levels of induction of ipdC-inaZ in Ehg were observed on leaves of these plants and in gypsophila stems, when compared with that in culture. These results suggest that the IAM pathway is involved primarily in gall formation and support the main contribution of the IpyA pathway to the epiphytic fitness of this bacterial species. PMID:9650296

  5. The antinociceptive effects of ferulic acid on neuropathic pain: involvement of descending monoaminergic system and opioid receptors.

    PubMed

    Xu, Ying; Lin, Dan; Yu, Xuefeng; Xie, Xupei; Wang, Liqun; Lian, Lejing; Fei, Ning; Chen, Jie; Zhu, Naping; Wang, Gang; Huang, Xianfeng; Pan, Jianchun

    2016-04-12

    Neuropathic pain can be considered as a form of chronic stress that may share common neuropathological mechanism between pain and stress-related depression and respond to similar treatment. Ferulic acid (FA) is a major active component of angelica sinensis and has been reported to exert antidepressant-like effects; however, it remains unknown whether FA ameliorate chronic constriction injury (CCI)-induced neuropathic pain and the involvement of descending monoaminergic system and opioid receptors. Chronic treatment with FA (20, 40 and 80 mg/kg) ameliorated mechanical allodynia and thermal hyperalgesia in von Frey hair and hot plate tasks, accompanied by increasing spinal noradrenaline (NA) and serotonin (5-HT) levels. Subsequent study suggested that treatment of CCI animals with 40 and 80 mg/kg FA also inhibited spinal MAO-A levels. FA's effects on mechanical allodynia or thermal hyperalgesiawas blocked by 6-hydroxydopamine (6-OHDA) or p-chlorophenylalanine (PCPA) via pharmacological depletion of spinal noradrenaline or serotonin. Moreover, the anti-allodynic action of FA on mechanical stimuli was prevented by pre-treatment with beta2-adrenoceptor antagonist ICI 118,551, or by the delta-opioid receptor antagonist naltrindole. While the anti-hyperalgesia on thermal stimuli induced by FA was blocked by pre-treatment with 5-HT1A receptor antagonist WAY-100635, or with the irreversible mu-opioid receptor antagonist beta-funaltrexamine. These results suggest that the effect of FA on neuropathic pain is potentially mediated via amelioration of the descending monoaminergic system that coupled with spinal beta2- and 5-HT1A receptors and the downstream delta- and mu-opioid receptors differentially. PMID:26967251

  6. Evidence against direct involvement of phosphorylation in the activation of carnitine palmitoyltransferase by okadaic acid in rat hepatocytes.

    PubMed Central

    Guzman, M; Kolodziej, M P; Caldwell, A; Corstorphine, C G; Zammit, V A

    1994-01-01

    The mechanism of activation of mitochondrial overt carnitine palmitoyltransferase (CPT I) by treatment of hepatocytes with okadaic acid (OA) was investigated. Activation was observed when cells were permeabilized with digitonin, but not when a total membrane fraction was obtained by sonication. Both cell disruption methods preserved the activation of phosphorylase observed in OA-treated hepatocytes. Activation of CPT I was also observed in crude homogenates of OA-treated hepatocytes, but it was lost upon subsequent isolation of mitochondria from such homogenates. In all experiments, any activation observed did not depend on the presence or absence of fluoride ions in the permeabilization/homogenization media. When hepatocytes were permeabilized in the absence of fluoride and further incubated with exogenous phosphatases 1 and 2A, the OA-induced activation of CPT was not reversed, whereas the activation of glycogen phosphorylase in the same cells was rapidly reversed. Treatment of hepatocytes with OA, followed by permeabilization and incubation before assay of CPT I, demonstrated that OA had no short-term effect on the sensitivity of CPT I to malonyl-CoA, although the difference in sensitivity between cells isolated from fed and starved rats was fully preserved. Incubation of isolated mitochondria or purified mitochondrial outer membranes with cyclic AMP-dependent or AMP-activated protein kinases, under phosphorylating conditions, did not affect the activity of CPT I or its sensitivity to malonyl-CoA inhibition. Under the same conditions, the use of [32P]ATP resulted in the labelling of several outer-membrane proteins but, unlike [3H]etomoxir-labelled CPT I, none of them was specifically removed from membrane extracts by a specific polyclonal antibody to the enzyme. We conclude that the increase in overt CPT activity observed in permeabilized hepatocytes is not due to direct phosphorylation of CPT I, but may involve interactions between the mitochondrial outer

  7. Involvement of indole-3-acetic acid produced by Azospirillum brasilense in accumulating intracellular ammonium in Chlorella vulgaris.

    PubMed

    Meza, Beatriz; de-Bashan, Luz E; Bashan, Yoav

    2015-01-01

    Accumulation of intracellular ammonium and activities of the enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) were measured when the microalgae Chlorella vulgaris was immobilized in alginate with either of two wild type strains of Azospirillum brasilense or their corresponding indole-3-acetic acid (IAA)-attenuated mutants. After 48 h of immobilization, both wild types induced higher levels of intracellular ammonium in the microalgae than their respective mutants; the more IAA produced, the higher the intracellular ammonium accumulated. Accumulation of intracellular ammonium in the cells of C. vulgaris followed application of four levels of exogenous IAA reported for A. brasilense and its IAA-attenuated mutants, which had a similar pattern for the first 24 h. This effect was transient and disappeared after 48 h of incubation. Immobilization of C. vulgaris with any bacteria strain induced higher GS activity. The bacterial strains also had GS activity, comparable to the activity detected in C. vulgaris, but weaker than when immobilized with the bacteria. When net activity was calculated, the wild type always induced higher GS activity than IAA-attenuated mutants. GDH activity in most microalgae/bacteria interactions resembled GS activity. When complementing IAA-attenuated mutants with exogenous IAA, GS activity in co-immobilized cultures matched those of the wild type A. brasilense immobilized with the microalga. Similarity occurred when the net GS activity was measured, and was higher with greater quantities of exogenous IAA. It is proposed that IAA produced by A. brasilense is involved in ammonium uptake and later assimilation by C. vulgaris. PMID:25554489

  8. Effect of cannabidiol on sepsis-induced motility disturbances in mice: involvement of CB receptors and fatty acid amide hydrolase.

    PubMed

    de Filippis, D; Iuvone, T; d'amico, A; Esposito, G; Steardo, L; Herman, A G; Pelckmans, P A; de Winter, B Y; de Man, J G

    2008-08-01

    Sepsis is an inflammatory condition that is associated with reduced propulsive gastrointestinal motility (ileus). A therapeutic option to treat sepsis is to promote intestinal propulsion preventing bacterial stasis, overgrowth and translocation. Recent evidence suggests that anti-oxidants improve sepsis-induced ileus. Cannabidiol, a non-psychotropic component of Cannabis sativa, exerts strong anti-oxidant and anti-inflammatory effects without binding to cannabinoid CB(1) or CB(2) receptors. Cannabidiol also regulates the activity of fatty acid amide hydrolase (FAAH) which is the main enzyme involved in endocannabinoid breakdown and which modulates gastrointestinal motility. Because of the therapeutic potential of cannabidiol in several pathologies, we investigated its effect on sepsis-induced ileus and on cannabinoid receptor and FAAH expression in the mouse intestine. Sepsis was induced by treating mice with lipopolysaccharides for 18 h. Sepsis led to a decrease in gastric emptying and intestinal transit. Cannabidiol further reduced gastrointestinal motility in septic mice but did not affect gastrointestinal motility in control mice. A low concentration of the CB(1) antagonist AM251 did not affect gastrointestinal motility in control mice but reversed the effect of cannabidiol in septic mice. Sepsis was associated with a selective upregulation of intestinal CB(1) receptors without affecting CB(2) receptor expression and with increased FAAH expression. The increase in FAAH expression was completely reversed by cannabidiol but not affected by AM251. Our results show that sepsis leads to an imbalance of the endocannabinoid system in the mouse intestine. Despite its proven anti-oxidant and anti-inflammatory properties, cannabidiol may be of limited use for the treatment of sepsis-induced ileus. PMID:18373655

  9. The antinociceptive effects of ferulic acid on neuropathic pain: involvement of descending monoaminergic system and opioid receptors

    PubMed Central

    Xu, Ying; Lin, Dan; Yu, Xuefeng; Xie, Xupei; Wang, Liqun; Lian, Lejing; Fei, Ning; Chen, Jie; Zhu, Naping; Wang, Gang; Huang, Xianfeng; Pan, Jianchun

    2016-01-01

    Neuropathic pain can be considered as a form of chronic stress that may share common neuropathological mechanism between pain and stress-related depression and respond to similar treatment. Ferulic acid (FA) is a major active component of angelica sinensis and has been reported to exert antidepressant-like effects; however, it remains unknown whether FA ameliorate chronic constriction injury (CCI)-induced neuropathic pain and the involvement of descending monoaminergic system and opioid receptors. Chronic treatment with FA (20, 40 and 80 mg/kg) ameliorated mechanical allodynia and thermal hyperalgesia in von Frey hair and hot plate tasks, accompanied by increasing spinal noradrenaline (NA) and serotonin (5-HT) levels. Subsequent study suggested that treatment of CCI animals with 40 and 80 mg/kg FA also inhibited spinal MAO-A levels. FA's effects on mechanical allodynia or thermal hyperalgesiawas blocked by 6-hydroxydopamine (6-OHDA) or p-chlorophenylalanine (PCPA) via pharmacological depletion of spinal noradrenaline or serotonin. Moreover, the anti-allodynic action of FA on mechanical stimuli was prevented by pre-treatment with beta2-adrenoceptor antagonist ICI 118,551, or by the delta-opioid receptor antagonist naltrindole. While the anti-hyperalgesia on thermal stimuli induced by FA was blocked by pre-treatment with 5-HT1A receptor antagonist WAY-100635, or with the irreversible mu-opioid receptor antagonist beta-funaltrexamine. These results suggest that the effect of FA on neuropathic pain is potentially mediated via amelioration of the descending monoaminergic system that coupled with spinal beta2- and 5-HT1A receptors and the downstream delta- and mu-opioid receptors differentially. PMID:26967251

  10. GA3, a new gambogic acid derivative, exhibits potent antitumor activities in vitro via apoptosis-involved mechanisms

    PubMed Central

    Xie, Hua; Qin, Yu-xin; Zhou, Yun-long; Tong, Lin-jiang; Lin, Li-ping; Geng, Mei-yu; Duan, Wen-hu; Ding, Jian

    2009-01-01

    Aim: Gambogic acid (GA) is the major active ingredient of gamboge, which is secreted from a Chinese traditional medicine, Garcinia hanburyi, which possesses potent antitumor activity. GA3, a new GA derivative, has been shown to possess better water solubility than GA. The aim of the present study was to examine the antitumor activity of GA3 and the mechanism underlying it. Methods: The growth inhibition of cancer cell lines induced by GA3 was assessed using the SRB assay. DAPI staining, flow cytometry, a DNA fragment assay, and Western blot analysis were used to study the apoptotic mechanisms of GA3. Results: GA3 displayed wide cytotoxicity in diversified human cancer cell lines with a mean IC50 value of 2.15 μmol/L. GA3 was also effective against multidrug resistant cells, with an average resistance factor (RF) that was much lower than that of the reference drug, doxorubicin. Mechanistic studies revealed that GA3-induced apoptosis in HL-60 cells proceeded via both extrinsic and intrinsic pathways, with caspase-8 functioning upstream of caspase-9. In addition, GA3-driven apoptotic events were associated with up-regulation of Bax, down-regulation of Bcl-2 and cleavage of Bid. Moreover, GA3 triggered cytochrome c release from the mitochondria, in particular bypassing the involvement of the mitochondrial membrane potential. Conclusion: Better solubility and a potential anti-MDR activity, combined with a comparable antitumor efficacy, make GA3 a potential drug candidate in cancer therapy that deserves further investigation. PMID:19262558

  11. Age-related differences in messenger ribonucleic acid expression of key proteins involved in adipose cell differentiation and metabolism.

    PubMed

    Imbeault, P; Vidal, H; Tremblay, A; Vega, N; Nadeau, A; Després, J P; Mauriège, P

    2001-02-01

    This study was performed to compare the expression of key proteins [lipoprotein lipase (LPL), hormone-sensitive lipase (HSL), complement 3 (C3), and peroxisome proliferator-stimulated receptor-gamma (PPAR gamma)] involved in sc abdominal adipose tissue (AT) metabolism of young (n = 13) vs. middle-aged (n = 16) men. The sc abdominal AT-LPL activity as well as fat cell lipolysis were also measured in both groups of men. Young and middle-aged men displayed similar body weight and sc abdominal fat accumulation, measured by computed tomography. However, middle-aged men were characterized by a higher percent body fat (28 +/- 5% vs. 22 +/- 7%; P < 0.05) than young subjects. No difference between groups was observed in sc abdominal adipose tissue LPL activity. On the other hand, maximal lipolytic responses of sc abdominal adipocytes to isoproterenol (beta-adrenergic agonist) or to postadrenoceptor agents such as dibutyryl cAMP, forskolin, and theophylline were lower in middle-aged than in young men (P < 0.05). AT-LPL messenger ribonucleic acid (mRNA) levels were similar regardless of the subject's age. However, HSL, C3, and PPAR gamma mRNA levels were higher in middle-aged than in young individuals (P < 0.01-0.05). After correction for percent body fat, only HSL and C3 mRNA levels remained significantly different between groups (P < 0.05). Taken together, these results suggest that aging has an effect on the up-regulation of HSL and C3 mRNA levels, whereas PPAR gamma expression seems to be related mainly to increased adiposity. PMID:11158053

  12. Possible involvement of K+-conductance in the action of gamma-aminobutyric acid in the guinea-pig hippocampus.

    PubMed Central

    Inoue, M.; Matsuo, T.; Ogata, N.

    1985-01-01

    The mechanism underlying the action of gamma-aminobutyric acid (GABA) in the hippocampus was investigated using guinea-pig brain slices. GABA either superfused or applied directly by microiontophoresis produced a biphasic response in pyramidal cells, comprising hyperpolarizing and depolarizing components. When different concentrations of GABA were applied to the same neurone, the lower concentrations generally produced a hyperpolarization-predominant response, while higher concentrations resulted in a depolarization-predominant response. The depolarizing component of the response to GABA was augmented in a medium containing a low concentration of Cl-, relatively unaffected by a change in external K+ concentration, and blocked by picrotoxin (2 X 10(-5) M). The depolarizing response to GABA persisted in a Ca2+-free medium in which the concentration of Na+ was reduced to 13 mM. Combined application of low doses of picrotoxin and bicuculline eliminated the major part of the depolarizing component of the biphasic response to GABA and produced a relatively pure hyperpolarizing response. The reversal potential of this pharmacologically 'isolated' hyperpolarizing response to GABA was estimated, from the current-voltage relationships, to be about -90 mV and was the same as that of the hyperpolarization induced by baclofen. When the membrane was successively hyperpolarized by inward direct current (d.c.) injections, the reversal point of the 'pharmacologically isolated' hyperpolarizing response to GABA coincided with that of the post-burst hyperpolarization. Low concentrations of Cl- in the bathing medium had no noticeable effect on the hyperpolarizing component of the response to GABA, whereas it markedly increased the amplitude of the depolarizing component. These results suggest that the action of GABA in the hippocampus may involve an activation of K+ conductance. PMID:2413946

  13. Acid-Sulfate-Weathering Activity in Shergottite Sites on Mars Recorded in Grim Glasses

    NASA Technical Reports Server (NTRS)

    Rao, M. N.; Nyquist, L. E.; Ross, K.; Sutton, S. R.; Schwandt, C. S.

    2011-01-01

    Based on mass spectrometric studies of sulfur species in Shergotty and EET79001, [1] and [2] showed that sulfates and sulfides occur in different proportions in shergottites. Sulfur speciation studies in gas-rich impact-melt (GRIM) glasses in EET79001 by the XANES method [3] showed that S K-XANES spectra in GRIM glasses from Lith A indicate that S is associated with Ca and Al presumably as sulfides/sulfates whereas the XANES spectra of amorphous sulfide globules in GRIM glasses from Lith B indicate that S is associated with Fe as FeS. In these amorphous iron sulfide globules, [4] found no Ni using FE-SEM and suggested that the globules resulting from immiscible sulfide melt may not be related to the igneous iron sulfides having approximately 1-3% Ni. Furthermore, in the amorphous iron sulfides from 507 GRIM glass, [5] determined delta(sup 34)S values ranging from +3.5%o to -3.1%o using Nano-SIMS. These values plot between the delta(sup 34)S value of +5.25%o determined in the sulfate fraction in Shergotty [6] at one extreme and the value of -1.7%o obtained for igneous sulfides in EET79001 and Shergotty [7] at the other. These results suggest that the amorphous Fe-S globules likely originated by shock reduction of secondary iron sulfate phases occurring in the regolith precursor materials during impact [7]. Sulfates in the regolith materials near the basaltic shergottite sites on Mars owe their origin to surficial acid-sulfate interactions. We examine the nature of these reactions by studying the composition of the end products in altered regolith materials. For the parent material composition, we use that of the host shergottite material in which the impact glasses are situated.

  14. Effects of acidic recharge on groundwater at the St. Kevin Gulch site, Leadville, Colorado

    USGS Publications Warehouse

    Paschke, S.S.; Harrison, W.J.; Walton-Day, K.

    2001-01-01

    The acid rock drainage-affected stream of St. Kevin Gulch recharges the Quaternary sand and gravel aquifer of Tennessee Park, near Leadville, Colorado, lowering pH and contributing iron, cadmium, copper, zinc and sulphate to the ground-water system. Dissolved metal mobility is controlled by the seasonal spring runoff as well as oxidation/reduction (redox) reactions in the aquifer. Oxidizing conditions occur in the unconfined portions of the aquifer whilst sulphate-reducing conditions are found down gradient where semi-confined groundwater flow occurs beneath a natural wetland. Iron-reducing conditions occur in the transition from unconfined to semi-confined groundwater flow. Dissolved iron concentrations are low to not detectable in the alluvial fan recharge zone and increase in a down gradient direction. The effects of low-pH, metal-rich recharge are pronounced during low-flow in the fall when there is a defined area of low pH groundwater with elevated concentrations of dissolved zinc, cadmium, copper and sulphate adjacent to St. Kevin Gulch. Dissolved metal and sulphate concentrations in the recharge zone are diluted during spring runoff, although the maximum concentrations of dissolved zinc, cadmium, copper and sulphate occur at selected down gradient locations during high flow. Dissolved zinc, cadmium and copper concentrations are low to not detectable, whereas dissolved iron concentrations are greatest, in groundwater samples from the sulphate-reducing zone. Attenuation of zinc, cadmium and copper beneath the wetland suggests sulphide mineral precipitation is occurring in the semi-confined aquifer, in agreement with previous site investigations and saturation index calculations. Adsorption of dissolved zinc, cadmium and copper onto iron hydroxides is a minor attenuation process due to the low pH of the groundwater system.

  15. Proteolytic Activation of the Human Epithelial Sodium Channel by Trypsin IV and Trypsin I Involves Distinct Cleavage Sites*

    PubMed Central

    Haerteis, Silke; Krappitz, Annabel; Krappitz, Matteus; Murphy, Jane E.; Bertog, Marko; Krueger, Bettina; Nacken, Regina; Chung, Hyunjae; Hollenberg, Morley D.; Knecht, Wolfgang; Bunnett, Nigel W.; Korbmacher, Christoph

    2014-01-01

    Proteolytic activation is a unique feature of the epithelial sodium channel (ENaC). However, the underlying molecular mechanisms and the physiologically relevant proteases remain to be identified. The serine protease trypsin I can activate ENaC in vitro but is unlikely to be the physiologically relevant activating protease in ENaC-expressing tissues in vivo. Herein, we investigated whether human trypsin IV, a form of trypsin that is co-expressed in several extrapancreatic epithelial cells with ENaC, can activate human ENaC. In Xenopus laevis oocytes, we monitored proteolytic activation of ENaC currents and the appearance of γENaC cleavage products at the cell surface. We demonstrated that trypsin IV and trypsin I can stimulate ENaC heterologously expressed in oocytes. ENaC cleavage and activation by trypsin IV but not by trypsin I required a critical cleavage site (Lys-189) in the extracellular domain of the γ-subunit. In contrast, channel activation by trypsin I was prevented by mutating three putative cleavage sites (Lys-168, Lys-170, and Arg-172) in addition to mutating previously described prostasin (RKRK178), plasmin (Lys-189), and neutrophil elastase (Val-182 and Val-193) sites. Moreover, we found that trypsin IV is expressed in human renal epithelial cells and can increase ENaC-mediated sodium transport in cultured human airway epithelial cells. Thus, trypsin IV may regulate ENaC function in epithelial tissues. Our results show, for the first time, that trypsin IV can stimulate ENaC and that trypsin IV and trypsin I activate ENaC by cleavage at distinct sites. The presence of distinct cleavage sites may be important for ENaC regulation by tissue-specific proteases. PMID:24841206

  16. The ripX Locus of Bacillus subtilis Encodes a Site-Specific Recombinase Involved in Proper Chromosome Partitioning

    PubMed Central

    Sciochetti, Stephen A.; Piggot, Patrick J.; Sherratt, David J.; Blakely, Garry

    1999-01-01

    The Bacillus subtilis ripX gene encodes a protein that has 37 and 44% identity with the XerC and XerD site-specific recombinases of Escherichia coli. XerC and XerD are hypothesized to act in concert at the dif site to resolve dimeric chromosomes formed by recombination during replication. Cultures of ripX mutants contained a subpopulation of unequal-size cells held together in long chains. The chains included anucleate cells and cells with aberrantly dense or diffuse nucleoids, indicating a chromosome partitioning failure. This result is consistent with RipX having a role in the resolution of chromosome dimers in B. subtilis. Spores contain a single uninitiated chromosome, and analysis of germinated, outgrowing spores showed that the placement of FtsZ rings and septa is affected in ripX strains by the first division after the initiation of germination. The introduction of a recA mutation into ripX strains resulted in only slight modifications of the ripX phenotype, suggesting that chromosome dimers can form in a RecA-independent manner in B. subtilis. In addition to RipX, the CodV protein of B. subtilis shows extensive similarity to XerC and XerD. The RipX and CodV proteins were shown to bind in vitro to DNA containing the E. coli dif site. Together they functioned efficiently in vitro to catalyze site-specific cleavage of an artificial Holliday junction containing a dif site. Inactivation of codV alone did not cause a discernible change in phenotype, and it is speculated that RipX can substitute for CodV in vivo. PMID:10498718

  17. Screening and assessment of solidification/stabilization amendments suitable for soils of lead-acid battery contaminated site.

    PubMed

    Zhang, Zhuo; Guo, Guanlin; Teng, Yanguo; Wang, Jinsheng; Rhee, Jae Seong; Wang, Sen; Li, Fasheng

    2015-05-15

    Lead exposure via ingestion of soil and dust generally occurs at lead-acid battery manufacturing and recycling sites. Screening solidification/stabilization (S/S) amendments suitable for lead contaminated soil in an abandoned lead-acid battery factory site was conducted based on its chemical forms and environmental risks. Twelve amendments were used to immobilize the Pb in soil and assess the solidification/stabilization efficiency by toxicity leaching tests. The results indicated that three amendments, KH₂PO₄ (KP), KH₂PO₄:oyster shell power=1:1 (by mass ratio; SPP), and KH₂PO₄:sintered magnesia=1:1 (by mass ratio; KPM) had higher remediation efficiencies that led to a 92% reduction in leachable Pb with the addition of 5% amendments, while the acid soluble fraction of Pb (AS-Pb) decreased by 41-46% and the residual fraction (RS-Pb) increased by 16-25%. The S/S costs of the three selected amendments KP, SPP, and KPM could be controlled to $22.3 per ton of soil when the Pb concentration in soil ranged from 2000 to 3000 mg/kg. The results of this study demonstrated that KP, SPP, and KPM can effectively decrease bioavailability of Pb. These findings could provide basis for decision-making of S/S remediation of lead-acid battery contaminated sites. PMID:25699676

  18. Site-specific regulation of adult neurogenesis by dietary fatty acid content, vitamin E and flight exercise in European starlings.

    PubMed

    Hall, Zachary J; Bauchinger, Ulf; Gerson, Alexander R; Price, Edwin R; Langlois, Lillie A; Boyles, Michelle; Pierce, Barbara; McWilliams, Scott R; Sherry, David F; Macdougall-Shackleton, Scott A

    2014-03-01

    Exercise is known to have a strong effect on neuroproliferation in mammals ranging from rodents to humans. Recent studies have also shown that fatty acids and other dietary supplements can cause an upregulation of neurogenesis. It is not known, however, how exercise and diet interact in their effects on adult neurogenesis. We examined neuronal recruitment in multiple telencephalic sites in adult male European starlings (Sturnus vulgaris) exposed to a factorial combination of flight exercise, dietary fatty acids and antioxidants. Experimental birds were flown in a wind tunnel following a training regime that mimicked the bird's natural flight behaviour. In addition to flight exercise, we manipulated the composition of dietary fatty acids and the level of enrichment with vitamin E, an antioxidant reported to enhance neuronal recruitment. We found that all three factors - flight exercise, fatty acid composition and vitamin E enrichment - regulate neuronal recruitment in a site-specific manner. We also found a robust interaction between flight training and vitamin E enrichment at multiple sites of neuronal recruitment. Specifically, flight training was found to enhance neuronal recruitment across the telencephalon, but only in birds fed a diet with a low level of vitamin E. Conversely, dietary enrichment with vitamin E upregulated neuronal recruitment, but only in birds not flown in the wind tunnel. These findings indicate conserved modulation of adult neurogenesis by exercise and diet across vertebrate taxa and indicate possible therapeutic interventions in disorders characterized by reduced adult neurogenesis. PMID:24372878

  19. Experimentally caused proliferation of lysosomes in cultured BHK cells involving an increase of biphosphatidic acids and triglycerides.

    PubMed

    Brotherus, J; Niinioja, T; Sandelin, K; Renkonen, O

    1977-05-01

    When cultured hamster fibroblasts (BHK 21 cells) were incubated in a synthetic serum-free medium up to 4 days, they developed signs of a progressive proliferation of lysosomes. The cells became filled with vacuoles that contained polymorphic debris and showed acid phosphatase activity. The specific activities of acid protease and acid phosphatase in the cell cultures increased three- to fourfold. The process was accompanied by a marked decrease in the contents of protein, deoxyribonucleic acid, and total phospholipids of the cultures. The concentration of lysobisphosphatidic acid increased during the incubation from about 1.5% to 3-6% of the cellular phospholipids. The concentrations of two related lipids, bisphosphatidic acid and semilysobisphosphatidic acid also increased substantially. The triglyceride content of the cells increased several fold, whereas the concentration of phosphatidylcholine decreased markedly. Lysobisphosphatidic acid did not increase upon induction of vacuolization by exogenous sucrose, nor when there was an accumulation of triglyceride due to addition of oleic acid to the growth medium. These findings suggest that the formation of the bisphosphatidic acids may be specifically linked to the autolysis of the phospholipids of the cellular membranes and the formation of triglycerides associated with this process. PMID:864326

  20. Systematic identification of genes involved in metabolic acid stress resistance in yeast and their potential as cancer targets.

    PubMed

    Shin, John J; Aftab, Qurratulain; Austin, Pamela; McQueen, Jennifer A; Poon, Tak; Li, Shu Chen; Young, Barry P; Roskelley, Calvin D; Loewen, Christopher J R

    2016-09-01

    A hallmark of all primary and metastatic tumours is their high rate of glucose uptake and glycolysis. A consequence of the glycolytic phenotype is the accumulation of metabolic acid; hence, tumour cells experience considerable intracellular acid stress. To compensate, tumour cells upregulate acid pumps, which expel the metabolic acid into the surrounding tumour environment, resulting in alkalization of intracellular pH and acidification of the tumour microenvironment. Nevertheless, we have only a limited understanding of the consequences of altered intracellular pH on cell physiology, or of the genes and pathways that respond to metabolic acid stress. We have used yeast as a genetic model for metabolic acid stress with the rationale that the metabolic changes that occur in cancer that lead to intracellular acid stress are likely fundamental. Using a quantitative systems biology approach we identified 129 genes required for optimal growth under conditions of metabolic acid stress. We identified six highly conserved protein complexes with functions related to oxidative phosphorylation (mitochondrial respiratory chain complex III and IV), mitochondrial tRNA biosynthesis [glutamyl-tRNA(Gln) amidotransferase complex], histone methylation (Set1C-COMPASS), lysosome biogenesis (AP-3 adapter complex), and mRNA processing and P-body formation (PAN complex). We tested roles for two of these, AP-3 adapter complex and PAN deadenylase complex, in resistance to acid stress using a myeloid leukaemia-derived human cell line that we determined to be acid stress resistant. Loss of either complex inhibited growth of Hap1 cells at neutral pH and caused sensitivity to acid stress, indicating that AP-3 and PAN complexes are promising new targets in the treatment of cancer. Additionally, our data suggests that tumours may be genetically sensitized to acid stress and hence susceptible to acid stress-directed therapies, as many tumours accumulate mutations in mitochondrial respiratory chain

  1. Adaptive responses of Bacillus cereus ATCC14579 cells upon exposure to acid conditions involve ATPase activity to maintain their internal pH

    PubMed Central

    Senouci-Rezkallah, Khadidja; Jobin, Michel P; Schmitt, Philippe

    2015-01-01

    This study examined the involvement of ATPase activity in the acid tolerance response (ATR) of Bacillus cereus ATCC14579 strain. In the current work, B. cereus cells were grown in anaerobic chemostat culture at external pH (pHe) 7.0 or 5.5 and at a growth rate of 0.2 h−1. Population reduction and internal pH (pHi) after acid shock at pH 4.0 was examined either with or without ATPase inhibitor N,N’-dicyclohexylcarbodiimide (DCCD) and ionophores valinomycin and nigericin. Population reduction after acid shock at pH 4.0 was strongly limited in cells grown at pH 5.5 (acid-adapted cells) compared with cells grown at pH 7.0 (unadapted cells), indicating that B. cereus cells grown at low pHe were able to induce a significant ATR and Exercise-induced increase in ATPase activity. However, DCCD and ionophores had a negative effect on the ability of B. cereus cells to survive and maintain their pHi during acid shock. When acid shock was achieved after DCCD treatment, pHi was markedly dropped in unadapted and acid-adapted cells. The ATPase activity was also significantly inhibited by DCCD and ionophores in acid-adapted cells. Furthermore, transcriptional analysis revealed that atpB (ATP beta chain) transcripts was increased in acid-adapted cells compared to unadapted cells before and after acid shock. Our data demonstrate that B. cereus is able to induce an ATR during growth at low pH. These adaptations depend on the ATPase activity induction and pHi homeostasis. Our data demonstrate that the ATPase enzyme can be implicated in the cytoplasmic pH regulation and in acid tolerance of B. cereus acid-adapted cells. PMID:25740257

  2. Site-specific insertion of 3-aminotyrosine into subunit alpha2 of E. coli ribonucleotide reductase: direct evidence for involvement of Y730 and Y731 in radical propagation.

    PubMed

    Seyedsayamdost, Mohammad R; Xie, Jianming; Chan, Clement T Y; Schultz, Peter G; Stubbe, JoAnne

    2007-12-01

    E. coli ribonucleotide reductase (RNR) catalyzes the production of deoxynucleotides using complex radical chemistry. Active RNR is composed of a 1:1 complex of two subunits: alpha2 and beta2. Alpha2 binds nucleoside diphosphate substrates and deoxynucleotide/ATP allosteric effectors and is the site of nucleotide reduction. Beta2 contains the stable diiron tyrosyl radical (Y122.) cofactor that initiates deoxynucleotide formation. This process is proposed to involve reversible radical transfer over >35 A between the Y122 in beta2 and C439 in the active site of alpha2. A docking model of alpha2beta2, based on structures of the individual subunits, suggests that radical initiation involves a pathway of transient, aromatic amino acid radical intermediates, including Y730 and Y731 in alpha2. In this study the function of residues Y730 and Y731 is investigated by their site-specific replacement with 3-aminotyrosine (NH2Y). Using the in vivo suppressor tRNA/aminoacyl-tRNA synthetase method, Y730NH2Y-alpha2 and Y731NH2Y-alpha2 have been generated with high fidelity in yields of 4-6 mg/g of cell paste. These mutants have been examined by stopped flow UV-vis and EPR spectroscopies in the presence of beta2, CDP, and ATP. The results reveal formation of an NH2Y radical (NH2Y730. or NH2Y731.) in a kinetically competent fashion. Activity assays demonstrate that both NH2Y-alpha2s make deoxynucleotides. These results show that the NH2Y. can oxidize C439 suggesting a hydrogen atom transfer mechanism for the radical propagation pathway within alpha2. The observed NH2Y. may constitute the first detection of an amino acid radical intermediate in the proposed radical propagation pathway during turnover. PMID:17990884

  3. Distribution and integrated assessment of lead in an abandoned lead-acid battery site in Southwest China before redevelopment.

    PubMed

    Wang, Mei; Zhang, Chao; Zhang, Zhuo; Li, Fasheng; Guo, Guanlin

    2016-06-01

    Lead-acid battery sites have contributed enormous amounts of lead to the environment, significantly affecting its global biogeochemical cycle and leaving the potential risks to human health. An abandoned lead-acid battery site prepared for redevelopment was selected in order to study the distribution of lead in soils, plants, rhizosphere soils and soil solutions. In total, 197 samples from 77 boreholes were collected and analyzed. Single extractions by acetic acid (HOAc) were conducted to assess the bioavailability and speciation of lead in soils for comparison with the parts of the plants that are aboveground. Health risks for future residential development were evaluated by the integrated exposure uptake biokinetic (IEUBK) model. The results indicated that lead concentrations in 83% of the soil samples exceeded the Chinese Environmental Quality Standard for soil (350 mg/kg for Pb) and mainly occurred at depths between 0 and 1.5 m while accumulating at the surface of demolished construction waste and miscellaneous fill. Lead concentrations in soil solutions and HOAc extraction leachates were linked closely to the contents of aboveground Broussonetia papyrifera and Artemisia annua, two main types of local plants that were found at the site. The probability density of lead in blood (PbB) in excess of 10 µg/dL could overtake the 99% mark in the residential scenario. The findings provided a relatively integrated method to illustrate the onsite investigations and assessment for similar sites before remediation and future development from more comprehensive aspects. PMID:26921546

  4. Site-directed mutagenesis of dicarboxylic acids near the active site of Bacillus cereus 5/B/6 beta-lactamase II.

    PubMed Central

    Lim, H M; Iyer, R K; Pène, J J

    1991-01-01

    An amino acid residue functioning as a general base has been proposed to assist in the hydrolysis of beta-lactam antibiotics by the zinc-containing Bacillus cereus beta-lactamase II [Bicknell & Waley (1985) Biochemistry 24, 6876-6887]. Oligonucleotide-directed mutagenesis of cloned Bacillus cereus 5/B/6 beta-lactamase II was used in an 'in vivo' study to investigate the role of carboxy-group-containing amino acids near the active site of the enzyme. Substitution of asparagine for the wild-type aspartic acid residue at position 81 resulted in fully functional enzyme. An aspartic acid residue at position 90 is essential for beta-lactamase II to confer any detectable ampicillin and cephalosporin C resistance to Escherichia coli. Conversion of Asp90 into Asn90 or Glu90 lead to the synthesis of inactive enzyme, suggesting that the spatial position of the beta-carboxy group of Asp90 is critical for enzyme function. Images Fig. 2. Fig. 3. PMID:1904717

  5. Involvement of acyl-CoA synthetase genes in n-alkane assimilation and fatty acid utilization in yeast Yarrowia lipolytica.

    PubMed

    Tenagy; Park, Jun Seok; Iwama, Ryo; Kobayashi, Satoshi; Ohta, Akinori; Horiuchi, Hiroyuki; Fukuda, Ryouichi

    2015-06-01

    Here, we investigated the roles of YAL1 (FAA1) and FAT1 encoding acyl-CoA synthetases (ACSs) and three additional orthologs of ACS genes FAT2-FAT4 of the yeast Yarrowia lipolytica in the assimilation or utilization of n-alkanes and fatty acids. ACS deletion mutants were generated to characterize their function. The FAT1 deletion mutant exhibited decreased growth on n-alkanes of 10-18 carbons, whereas the FAA1 mutant showed growth reduction on n-alkane of 16 carbons. However, FAT2-FAT4 deletion mutants did not show any growth defects, suggesting that FAT1 and FAA1 are involved in the activation of fatty acids produced during the metabolism of n-alkanes. In contrast, deletions of FAA1 and FAT1-FAT4 conferred no defect in growth on fatty acids. The wild-type strain grew in the presence of cerulenin, an inhibitor of fatty acid synthesis, by utilizing exogenously added fatty acid or fatty acid derived from n-alkane when oleic acid or n-alkane of 18 carbons was supplemented. However, the FAA1 deletion mutant did not grow, indicating a critical role for FAA1 in the utilization of fatty acids. Fluorescent microscopic observation and biochemical analyses suggested that Fat1p is present in the peroxisome and Faa1p is localized in the cytosol and to membranes. PMID:26019148

  6. High desolvation temperature facilitates the ESI-source H/D exchange at non-labile sites of hydroxybenzoic acids and aromatic amino acids.

    PubMed

    Zherebker, Alexander; Kostyukevich, Yury; Kononikhin, Alexey; Roznyatovsky, Vitaliy A; Popov, Igor; Grishin, Yuri K; Perminova, Irina V; Nikolaev, Eugene

    2016-04-21

    Hydrogen/deuterium exchange coupled with high-resolution mass spectrometry has become a powerful analytical approach for structural investigations of complex organic matrices. Here we report the feasibility of the site-specific H/D exchange of non-labile hydrogens directly in the electrospray ionization (ESI) source, which was facilitated by an increase in the desolvation temperature from 200 °C up to 400 °C. We have found that the exchanges at non-labile sites were observed only for the model compounds capable of keto-enol tautomeric transformations (e.g., 2,3-, 2,4-dihydroxybenzoic acids, gallic acid, DOPA), and only when water was used as a solvent. We hypothesized that the detected additional exchanges were induced by the presence of hydroxyls in the sprayed water droplets generated in the negative ESI mode. It was indicative of the exchange reactions taking place in the sprayed droplets rather than in the gas phase. To support this hypothesis, the H/D exchange experiments were run in deuterated water under base-catalyzed conditions for three model compounds, which showed the most intensive exchanges in the MS experiments: DOPA, 2,4-DHB, and 5-acetylsalicylic acid. (2)H NMR spectroscopy has confirmed keto-enolic transformations of the model compounds leading to the specific labeling of the corresponding non-labile sites. We believe that the proposed technique will be useful for structural investigations of natural complex mixtures (e.g. proteins, humic substances) using site-specific H/D exchange. PMID:27002310

  7. Identity of the amino acid residues involved in C3bi binding to the I-domain supports a mosaic model to explain the broad ligand repertoire of integrin alpha M beta 2.

    PubMed

    Ustinov, Valentin A; Plow, Edward F

    2005-03-22

    Interactions between the complement degradation product C3bi and leukocyte integrin alpha(M)beta(2) are critical for host defense against foreign pathogens and in tumor cell surveillance. To gain insight into the mechanism by which the alpha(M)I-domain of the integrin interacts with C3bi, detailed mapping of the C3bi binding site was undertaken. Previous mutagenesis studies had implicated five small structural segments within the alpha(M)I-domain in recognition of this ligand. Sets of three amino acids within the five implicated segments were mutated to the corresponding alpha(L)I-domain residues. Then, within the affected mutants, single point mutations were introduced to precisely define the requisite residues. Ultimately, H148, F150, Q204, L205, R208, T211, T213, I256, P257 were identified as being critical for C3bi binding. A synthetic peptide approach confirmed the involvement of the specified residues with the complex midsegment, Q204-I215, in C3bi recognition. Furthermore, the alpha(D)I-domain, which has a low intrinsic affinity for C3bi, acquired high affinity for the ligand when the implicated residues were inserted. The residues necessary to engage C3bi reside on or adjacent to the cation binding MIDAS site of the alpha(M)I-domain. The amino acids involved in C3bi binding are distinct from those involved in interaction of previously mapped ligands with the alpha(M)I-domain. This divergence supports a mosaic model, in which different ligands engage different amino acids to bind to alpha(M)I-domain, accounting for the broad recognition capacity of integrin alpha(M)beta(2). PMID:15766265

  8. Docosahexaenoic acid modulates the enterocyte Caco-2 cell expression of MicroRNAs involved in lipid metabolism

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Consumption of the long-chain omega-3 (n-3) polyunsaturated fatty acid docosahexaenoic acid (DHA) is associated with a reduced risk of cardiovascular disease and greater chemoprevention. However, the mechanisms underlying the biologic effects of DHA remain unknown. It is well known that microRNAs (m...

  9. Modulation of fatty acid metabolism is involved in the alleviation of isoproterenol-induced rat heart failure by fenofibrate.

    PubMed

    Li, Ping; Luo, Shike; Pan, Chunji; Cheng, Xiaoshu

    2015-12-01

    Heart failure is a disease predominantly caused by an energy metabolic disorder in cardiomyocytes. The present study investigated the inhibitory effects of fenofibrate (FF) on isoproterenol (ISO)‑induced hear failure in rats, and examined the underlying mechanisms. The rats were divided into CON, ISO (HF model), FF and FF+ISO (HF animals pretreated with FF) groups. The cardiac structure and function of the rats were assessed, and contents of free fatty acids and glucose metabolic products were determined. In addition, myocardial cells were isolated from neonatal rats and used in vitro to investigate the mechanisms by which FF relieves heart failure. Western blot analysis was performed to quantify the expression levels of peroxisome proliferator‑activated receptor (PPAR)α and uncoupling protein 2 (UCP2). FF effectively alleviated the ISO‑induced cardiac structural damage, functional decline, and fatty acid and carbohydrate metabolic abnormalities. Compared with the ISO group, the serum levels of brain natriuretic peptide (BNP), free fatty acids, lactic acid and pyruvic acid were decreased in the FF animals. In the cultured myocardial cells, lactic acid and pyruvic acid contents were lower in the supernatants obtained from the FF animals, with lower levels of mitochondrial ROS production and cell necrosis, compared with the ISO group, whereas PPARα upregulation and UCP2 downregulation occurred in the FF+ISO group. The results demonstrated that FF efficiently alleviated heart failure in the ISO‑induced rat model, possibly via promoting fatty acid oxidation. PMID:26497978

  10. Two shikimate dehydrogenases, VvSDH3 and VvSDH4, are involved in gallic acid biosynthesis in grapevine

    PubMed Central

    Bontpart, Thibaut; Marlin, Thérèse; Vialet, Sandrine; Guiraud, Jean-Luc; Pinasseau, Lucie; Meudec, Emmanuelle; Sommerer, Nicolas; Cheynier, Véronique; Terrier, Nancy

    2016-01-01

    In plants, the shikimate pathway provides aromatic amino acids that are used to generate numerous secondary metabolites, including phenolic compounds. In this pathway, shikimate dehydrogenases (SDH) ‘classically’ catalyse the reversible dehydrogenation of 3-dehydroshikimate to shikimate. The capacity of SDH to produce gallic acid from shikimate pathway metabolites has not been studied in depth. In grapevine berries, gallic acid mainly accumulates as galloylated flavan-3-ols. The four grapevine SDH proteins have been produced in Escherichia coli. In vitro, VvSDH1 exhibited the highest ‘classical’ SDH activity. Two genes, VvSDH3 and VvSDH4, mainly expressed in immature berry tissues in which galloylated flavan-3-ols are accumulated, encoded enzymes with lower ‘classical’ activity but were able to produce gallic acid in vitro. The over-expression of VvSDH3 in hairy-roots increased the content of aromatic amino acids and hydroxycinnamates, but had little or no effect on molecules more distant from the shikimate pathway (stilbenoids and flavan-3-ols). In parallel, the contents of gallic acid, β-glucogallin, and galloylated flavan-3-ols were increased, attesting to the influence of this gene on gallic acid metabolism. Phylogenetic analysis from dicotyledon SDHs opens the way for the examination of genes from other plants which accumulate gallic acid-based metabolites. PMID:27241494

  11. Two shikimate dehydrogenases, VvSDH3 and VvSDH4, are involved in gallic acid biosynthesis in grapevine.

    PubMed

    Bontpart, Thibaut; Marlin, Thérèse; Vialet, Sandrine; Guiraud, Jean-Luc; Pinasseau, Lucie; Meudec, Emmanuelle; Sommerer, Nicolas; Cheynier, Véronique; Terrier, Nancy

    2016-05-01

    In plants, the shikimate pathway provides aromatic amino acids that are used to generate numerous secondary metabolites, including phenolic compounds. In this pathway, shikimate dehydrogenases (SDH) 'classically' catalyse the reversible dehydrogenation of 3-dehydroshikimate to shikimate. The capacity of SDH to produce gallic acid from shikimate pathway metabolites has not been studied in depth. In grapevine berries, gallic acid mainly accumulates as galloylated flavan-3-ols. The four grapevine SDH proteins have been produced in Escherichia coli In vitro, VvSDH1 exhibited the highest 'classical' SDH activity. Two genes, VvSDH3 and VvSDH4, mainly expressed in immature berry tissues in which galloylated flavan-3-ols are accumulated, encoded enzymes with lower 'classical' activity but were able to produce gallic acid in vitro The over-expression of VvSDH3 in hairy-roots increased the content of aromatic amino acids and hydroxycinnamates, but had little or no effect on molecules more distant from the shikimate pathway (stilbenoids and flavan-3-ols). In parallel, the contents of gallic acid, β-glucogallin, and galloylated flavan-3-ols were increased, attesting to the influence of this gene on gallic acid metabolism. Phylogenetic analysis from dicotyledon SDHs opens the way for the examination of genes from other plants which accumulate gallic acid-based metabolites. PMID:27241494

  12. Clustered Genes Involved in Cyclopiazonic Acid Production are Next to the Aflatoxin Biosynthesis Gene Cluster in Aspergillus flavus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cyclopiazonic acid (CPA), an indole-tetramic acid toxin, is produced by many species of Aspergillus and Penicillium. In addition to CPA Aspergillus flavus produces polyketide-derived carcinogenic aflatoxins (AFs). AF biosynthesis genes form a gene cluster in a subtelomeric region. Isolates of A. fla...

  13. Identification of a conserved protein involved in anaerobic unsaturated fatty acid synthesis in Neiserria gonorrhoeae: implications for facultative and obligate anaerobes that lack FabA.

    PubMed

    Isabella, Vincent M; Clark, Virginia L

    2011-10-01

    Transcriptome analysis of the facultative anaerobe, Neisseria gonorrhoeae, revealed that many genes of unknown function were induced under anaerobic conditions. Mutation of one such gene, NGO1024, encoding a protein belonging to the 2-nitropropane dioxygenase-like superfamily of proteins, was found to result in an inability of gonococci to grow anaerobically. Anaerobic growth of an NG1024 mutant was restored upon supplementation with unsaturated fatty acids (UFA), but not with the saturated fatty acid palmitate. Gonococcal fatty acid profiles confirmed that NGO1024 was involved in UFA synthesis anaerobically, but not aerobically, demonstrating that gonococci contain two distinct pathways for the production of UFAs, with a yet unidentified aerobic mechanism, and an anaerobic mechanism involving NGO1024. Expression of genes involved in classical anaerobic UFA synthesis, fabA, fabM and fabB, was toxic in gonococci and unable to complement a NGO1024 mutation, suggesting that the chemistry involved in gonococcal anaerobic UFA synthesis is distinct from that of the classical pathway. NGO1024 homologues, which we suggest naming UfaA, form a distinct lineage within the 2-nitropropane dioxygenase-like superfamily, and are found in many facultative and obligate anaerobes that produce UFAs but lack fabA, suggesting that UfaA is part of a widespread pathway involved in UFA synthesis. PMID:21895795

  14. Isoaspartic acid is present at specific sites in myelin basic protein from multiple sclerosis patients: could this represent a trigger for disease onset?

    PubMed

    Friedrich, Michael G; Hancock, Sarah E; Raftery, Mark J; Truscott, Roger J W

    2016-01-01

    Multiple sclerosis (MS) is associated with breakdown of the myelin sheath that coats neurons in the central nervous system. The cause of MS is not known, although the pathogenesis involves destruction of myelin by the immune system. It was the aim of this study to examine the abundant myelin protein, myelin basic protein (MBP), to determine if there are sites of modification that may be characteristic for MS. MBP from the cerebellum was examined from controls and MS patients across the age range using mass spectrometry and amino acid analysis. Amino acid racemization data indicated that myelin basic protein is long-lived and proteomic analysis of MBP showed it to be highly modified. A common modification of MBP was racemization of Asp and this was significantly greater in MS patients. In long-lived proteins, L-Asp and L-Asn can racemize to three other isomers, D-isoAsp, L-isoAsp and D-Asp and this is significant because isoAsp formation in peptides renders them immunogenic.Proteomic analysis revealed widespread modifications of MBP with two surface regions that are altered in MS. In particular, isoAsp was significantly elevated at these sites in MS patients. The generation of isoAsp could be responsible for eliciting an immune response to modified MBP and therefore be implicated in the etiology of MS. PMID:27519525

  15. Amino acids of the Torpedo marmorata acetylcholine receptor. cap alpha. subunit labeled by a photoaffinity ligand for the acetylcholine binding site

    SciTech Connect

    Dennis, M.; Giraudat, J.; Kotzyba-Hibert, F.; Goeldner, M.; Hirth, C.; Chang, J.Y.; Lazure, C.; Chretien, M.; Changeux, J.P.

    1988-04-05

    The acetylcholine-binding sites on the native, membrane-bound acetylcholine receptor from Torpedo marmorata were covalently labeled with the photoaffinity reagent (/sup 3/H)-p-(dimethylamino)-benzenediazonium fluoroborate (DDF) in the presence of phencyclidine by employing an energy-transfer photolysis procedure. The ..cap alpha..-chains isolated from receptor-rich membranes photolabeled in the absence or presence of carbamoylcholine were cleaved with CNBr and the radiolabeled fragments purified by high-performance liquid ch