Science.gov

Sample records for active site caused

  1. Single Active Site Mutation Causes Serious Resistance of HIV Reverse Transcriptase to Lamivudine: Insight from Multiple Molecular Dynamics Simulations.

    PubMed

    Moonsamy, Suri; Bhakat, Soumendranath; Walker, Ross C; Soliman, Mahmoud E S

    2016-03-01

    Molecular dynamics simulations, binding free energy calculations, principle component analysis (PCA), and residue interaction network analysis were employed in order to investigate the molecular mechanism of M184I single mutation which played pivotal role in making the HIV-1 reverse transcriptase (RT) totally resistant to lamivudine. Results showed that single mutations at residue 184 of RT caused (1) distortion of the orientation of lamivudine in the active site due to the steric conflict between the oxathiolane ring of lamivudine and the side chain of beta-branched amino acids Ile at position 184 which, in turn, perturbs inhibitor binding, (2) decrease in the binding affinity by (~8 kcal/mol) when compared to the wild-type, (3) variation in the overall enzyme motion as evident from the PCA for both systems, and (4) distortion of the hydrogen bonding network and atomic interactions with the inhibitor. The comprehensive analysis presented in this report can provide useful information for understanding the drug resistance mechanism against lamivudine. The results can also provide some potential clues for further design of novel inhibitors that are less susceptible to drug resistance.

  2. Complete androgen insensitivity syndrome caused by a novel splice donor site mutation and activation of a cryptic splice donor site in the androgen receptor gene.

    PubMed

    Infante, Joana B; Alvelos, Maria I; Bastos, Margarida; Carrilho, Francisco; Lemos, Manuel C

    2016-01-01

    The androgen insensitivity syndrome is an X-linked recessive genetic disorder characterized by resistance to the actions of androgens in an individual with a male karyotype. We evaluated a 34-year-old female with primary amenorrhea and a 46,XY karyotype, with normal secondary sex characteristics, absence of uterus and ovaries, intra-abdominal testis, and elevated testosterone levels. Sequence analysis of the androgen receptor (AR) gene revealed a novel splice donor site mutation in intron 4 (c.2173+2T>C). RT-PCR analysis showed that this mutation resulted in the activation of a cryptic splice donor site located in the second half of exon 4 and in the synthesis of a shorter mRNA transcript and an in-frame deletion of 41 amino acids. This novel mutation associated with a rare mechanism of abnormal splicing further expands the spectrum of mutations associated with the androgen insensitivity syndrome and may contribute to the understanding of the molecular mechanisms involved in splicing defects.

  3. Galactosemia caused by a point mutation that activates cryptic donor splice site in the galactose-1-phosphate uridyltransferase gene

    SciTech Connect

    Wadelius, C.; Lagerkvist, A. Uppsala Univ. ); Molin, A.K.; Larsson, A. ); Von Doebeln, U. )

    1993-08-01

    Galactosemia affects 1/84,000 in Sweden and is manifested in infancy when the child is exposed to galactose in the diet. If untreated there is a risk of severe early symptoms and, even with a lactose-free diet, late symptoms such as mental retardation and ovarial dysfunction may develop. In classical galactosemia, galactose-1-phosphate uridyltransferase (GALT) (EC 2.7.7.12) is defective and the normal cDNA sequence of this enzyme has been characterized. Recently eight mutations leading to galactosemia were published. Heparinized venous blood was drawn from a patient with classical galactosemia. In the cDNA from the patient examined, an insertion of 54 bp was found at position 1087. Amplification of the relevant genomic region of the patient's DNA was performed. Exon-intron boundaries and intronic sequences thus determined revealed that the 54-bp insertion was located immediately downstream of exon 10. It was further found that the patient was heterozygous for a point mutation, changing a C to a T (in 5 of 9 clones) at the second base in the intron downstream of the insertion. This alteration creates a sequence which, as well as the ordinary splice site, differs in only two positions from the consensus sequence. It was found that the mutation occurred in only one of the 20 alleles from galactosemic patients and in none of the 200 alleles from normal controls. The mutation is inherited from the mother, who also was found to express the 54-bp-long insertion at the mRNA level. Sequences from the 5[prime] end of the coding region were determined after genomic amplification, revealing a sequence identical to that reported. The mutation on the paternal allele has not been identified. 9 refs., 1 fig.

  4. List 9 - Active CERCLIS Sites:

    EPA Pesticide Factsheets

    The List 9 displays the sequence of activities undertaken at active CERCLIS sites. An active site is one at which site assessment, removal, remedial, enforcement, cost recovery, or oversight activities are being planned or conducted.

  5. Erythema ab igne: Usual site, unusual cause.

    PubMed

    Manoharan, D

    2015-04-01

    Erythema ab igne is reticular erythematous pigmented dermatoses seen in patients exposed to prolonged or repeated sub-threshold Infrared radiation inadequate to cause burns. Here, we report a case of erythema ab igne in a 40-year-old male patient seen over the abdomen due to prolonged laptop use.

  6. Salt site performance assessment activities

    SciTech Connect

    Kircher, J.F.; Gupta, S.K.

    1983-01-01

    During this year the first selection of the tools (codes) for performance assessments of potential salt sites have been tentatively selected and documented; the emphasis has shifted from code development to applications. During this period prior to detailed characterization of a salt site, the focus is on bounding calculations, sensitivity and with the data available. The development and application of improved methods for sensitivity and uncertainty analysis is a focus for the coming years activities and the subject of a following paper in these proceedings. Although the assessments to date are preliminary and based on admittedly scant data, the results indicate that suitable salt sites can be identified and repository subsystems designed which will meet the established criteria for protecting the health and safety of the public. 36 references, 5 figures, 2 tables.

  7. [Cause and prevention of surgical site infection and hypertrophic scars].

    PubMed

    Ogawa, Rei

    2012-03-01

    Surgical site infection (SSI) occurs at the site of surgery within 1 month of an operation or within 1 year of an operation if a foreign body is implanted as part of the surgery. Most SSIs (about 70%) are superficial infections involving the skin and subcutaneous tissues only. The remaining infections are more serious and can involve tissues under the skin, organs, or implanted material. Hypertrophic scars( HSs) occur frequently on particular sites, including the anterior chest wall. The anterior chest wall is frequently subjected to skin stretching caused by the natural daily movements of the body. Most cases of SSIs and HSs can be prevented by (1) suture technique modification to prevent high stretching tension and ischemia, and (2) appropriate wound care after surgery. It would be useful to avoid subjecting wounded skin to sustained mechanical force, thereby permitting the wound to rest and heal normally.

  8. Androgen receptor exon 1 mutation causes androgen insensitivity by creating phosphorylation site and inhibiting melanoma antigen-A11 activation of NH2- and carboxyl-terminal interaction-dependent transactivation.

    PubMed

    Lagarde, William H; Blackwelder, Amanda J; Minges, John T; Hnat, Andrew T; French, Frank S; Wilson, Elizabeth M

    2012-03-30

    Naturally occurring germ line mutations in the X-linked human androgen receptor (AR) gene cause incomplete masculinization of the external genitalia by disrupting AR function in males with androgen insensitivity syndrome. Almost all AR missense mutations that cause androgen insensitivity syndrome are located in the highly structured DNA and ligand binding domains. In this report we investigate the functional defect associated with an AR exon 1 missense mutation, R405S, that caused partial androgen insensitivity. The 46,XX heterozygous maternal carrier had a wild-type Arg-405 CGC allele but transmitted an AGC mutant allele coding for Ser-405. At birth, the 46,XY proband had a bifid scrotum, hypospadias, and micropenis consistent with clinical stage 3 partial androgen insensitivity. Androgen-dependent transcriptional activity of AR-R405S expressed in CV1 cells was less than wild-type AR and refractory in androgen-dependent AR NH(2)- and carboxyl interaction transcription assays that depend on the coregulator effects of melanoma antigen-A11. This mutation created a Ser-405 phosphorylation site evident by the gel migration of an AR-R405S NH(2)-terminal fragment as a double band that converted to the wild-type single band after treatment with λ-phosphatase. Detrimental effects of the R405S mutation were related to the proximity of the AR WXXLF motif (433)WHTLF(437) required for melanoma antigen-A11 and p300 to stimulate transcriptional activity associated with the AR NH(2)- and carboxyl-terminal interaction. We conclude that the coregulator effects of melanoma antigen-A11 on the AR NH(2)- and carboxyl-terminal interaction amplify the androgen-dependent transcriptional response to p300 required for normal human male sex development in utero.

  9. Normal Modes Expose Active Sites in Enzymes

    PubMed Central

    Glantz-Gashai, Yitav; Samson, Abraham O.

    2016-01-01

    Accurate prediction of active sites is an important tool in bioinformatics. Here we present an improved structure based technique to expose active sites that is based on large changes of solvent accessibility accompanying normal mode dynamics. The technique which detects EXPOsure of active SITes through normal modEs is named EXPOSITE. The technique is trained using a small 133 enzyme dataset and tested using a large 845 enzyme dataset, both with known active site residues. EXPOSITE is also tested in a benchmark protein ligand dataset (PLD) comprising 48 proteins with and without bound ligands. EXPOSITE is shown to successfully locate the active site in most instances, and is found to be more accurate than other structure-based techniques. Interestingly, in several instances, the active site does not correspond to the largest pocket. EXPOSITE is advantageous due to its high precision and paves the way for structure based prediction of active site in enzymes. PMID:28002427

  10. On the causes of geomagnetic activity

    NASA Technical Reports Server (NTRS)

    Svalgaard, L.

    1975-01-01

    The causes of geomagnetic activity are studied both theoretically in terms of the reconnection model and empirically using the am-index and interplanetary solar wind parameters. It is found that two separate mechanisms supply energy to the magnetosphere. One mechanism depends critically on the magnitude and direction of the interplanetary magnetic field. Both depend strongly on solar wind speed.

  11. Causes of evolutionary rate variation among protein sites

    PubMed Central

    Echave, Julian; Spielman, Stephanie J.; Wilke, Claus O.

    2016-01-01

    It has long been recognized that certain sites within a protein, such as sites in the protein core or catalytic residues in enzymes, are more conserved than are other sites. However, our understanding of rate variation among sites remains surprisingly limited. Recent progress to address this includes the development of a wide array of reliable methods to estimate site-specific substitution rates from sequence alignments. In addition, several molecular traits have been identified that correlate with site-specific rates, and novel mechanistic, biophysical models have been proposed to explain the observed correlations. Nonetheless, at best, current models explain approximately 60% of the observed variance, highlighting the limitations of current methods and models, and the need for new research directions. PMID:26781812

  12. Validated ligand mapping of ACE active site

    NASA Astrophysics Data System (ADS)

    Kuster, Daniel J.; Marshall, Garland R.

    2005-08-01

    Crystal structures of angiotensin-converting enzyme (ACE) complexed with three inhibitors (lisinopril, captopril, enalapril) provided experimental data for testing the validity of a prior active site model predicting the bound conformation of the inhibitors. The ACE active site model - predicted over 18 years ago using a series of potent ACE inhibitors of diverse chemical structure - was recreated using published data and commercial software. Comparison between the predicted structures of the three inhibitors bound to the active site of ACE and those determined experimentally yielded root mean square deviation (RMSD) values of 0.43-0.81 Å, among the distances defining the active site map. The bound conformations of the chemically relevant atoms were accurately deduced from the geometry of ligands, applying the assumption that the geometry of the active site groups responsible for binding and catalysis of amide hydrolysis was constrained. The mapping of bound inhibitors at the ACE active site was validated for known experimental compounds, so that the constrained conformational search methodology may be applied with confidence when no experimentally determined structure of the enzyme yet exists, but potent, diverse inhibitors are available.

  13. Magnetic Anomaly Caused By Lightning On Archaeological Sites

    NASA Astrophysics Data System (ADS)

    Verrier, V.; Mathe, P. E.; Rochette, P.; Jones, G.

    Recent developments in archeological surveying have seen the emergence of mag- netic methods, commonly involving near surface measurements of low field magnetic susceptibility. However, these technics remain inefficient to detect either deep buried features or material which susceptibility constrast with the enclosing material is too low. An alternate way to assess site location and archeological features in these cases is provided by the mapping of total magnetic field or field gradient anomalies, due to the induced or remanent magnetizations of the material. REFERENCES : Verrier, V., Rochette, P., Richard, P. and Lojou, J.Y., Paleomagnetic expertise of ground lightning impacts, in 25th Int. Conf. Lightning Protection, Rhodos, p. 166-169, 2000. When remanence dominate, the archeological signal may be complicated by the su- perimposition of an anomaly due to lightning. Locally, the electric discharge generates a strong pulse of magnetic field. The materials around an impact acquired a lightning induced remanent magnetization (LIRM) which endures as long as the materials are not removed. In order to characterize the LIRM signature, some studies were realised on struck sites where the impact point is known. In each case, the LIRM hypothesis was tested on discrete samples collected around the impact, on which the lightning magnetic field intensity was determined (Verrier, 2000). The typical LIRM anomaly was also mapped with the G-858 MagMapper. It consists in a relatively strong dipole associated with maxima that are usually not aligned SN, clearly distinguishable from the archeological signature.

  14. Corrosion Research And Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2001-01-01

    This report covers corrosion-related activities at the NASA Kennedy Space Center during the summer of 2000. The NASA Kennedy Space Center's corrosion web site, corrosion.ksc.nasa.gov, was updated with new information based on feedback over the past two years. The methodology for a two-year atmospheric exposure testing program to study the effectiveness of commercial chemicals sold for rinsing aircraft and other equipment was developed and some preliminary laboratory chemical analyses are presented.

  15. Corrosion Research and Web Site Activities

    NASA Technical Reports Server (NTRS)

    Heidersbach, Robert H.

    2002-01-01

    This report covers corrosion-related activities at the NASA Kennedy Space Center during the summer of 2000. The NASA Kennedy Space Center's corrosion web site, corrosion.ksc.nasa.gov, was updated with new information based on feedback over the past two years. The methodology for a two-year atmospheric exposure testing program to study the effectiveness of commercial chemicals sold for rinsing aircraft and other equipment was developed and some preliminary laboratory chemical analyses are presented.

  16. Promoter-proximal polyadenylation sites reduce transcription activity

    PubMed Central

    Andersen, Pia K.; Lykke-Andersen, Søren; Jensen, Torben Heick

    2012-01-01

    Gene expression relies on the functional communication between mRNA processing and transcription. We previously described the negative impact of a point-mutated splice donor (SD) site on transcription. Here we demonstrate that this mutation activates an upstream cryptic polyadenylation (CpA) site, which in turn causes reduced transcription. Functional depletion of U1 snRNP in the context of the wild-type SD triggers the same CpA event accompanied by decreased RNA levels. Thus, in accordance with recent findings, U1 snRNP can shield premature pA sites. The negative impact of unshielded pA sites on transcription requires promoter proximity, as demonstrated using artificial constructs and supported by a genome-wide data set. Importantly, transcription down-regulation can be recapitulated in a gene context devoid of splice sites by placing a functional bona fide pA site/transcription terminator within ∼500 base pairs of the promoter. In contrast, promoter-proximal positioning of a pA site-independent histone gene terminator supports high transcription levels. We propose that optimal communication between a pA site-dependent gene terminator and its promoter critically depends on gene length and that short RNA polymerase II-transcribed genes use specialized termination mechanisms to maintain high transcription levels. PMID:23028143

  17. Nest predation increases with parental activity: Separating nest site and parental activity effects

    USGS Publications Warehouse

    Martin, T.E.; Scott, J.; Menge, C.

    2000-01-01

    Alexander Skutch hypothesized that increased parental activity can increase the risk of nest predation. We tested this hypothesis using ten open-nesting bird species in Arizona, USA. Parental activity was greater during the nestling than incubation stage because parents visited the nest frequently to feed their young during the nestling stage. However, nest predation did not generally increase with parental activity between nesting stages across the ten study species. Previous investigators have found similar results. We tested whether nest site effects might yield higher predation during incubation because the most obvious sites are depredated most rapidly. We conducted experiments using nest sites from the previous year to remove parental activity. Our results showed that nest sites have highly repeatable effects on nest predation risk; poor nest sites incurred rapid predation and caused predation rates to be greater during the incubation than nestling stage. This pattern also was exhibited in a bird species with similar (i.e. controlled) parental activity between nesting stages. Once nest site effects are taken into account, nest predation shows a strong proximate increase with parental activity during the nestling stage within and across species. Parental activity and nest sites exert antagonistic influences on current estimates of nest predation between nesting stages and both must be considered in order to understand current patterns of nest predation, which is an important source of natural selection.

  18. Nest predation increases with parental activity: separating nest site and parental activity effects.

    PubMed Central

    Martin, T E; Scott, J; Menge, C

    2000-01-01

    Alexander Skutch hypothesized that increased parental activity can increase the risk of nest predation. We tested this hypothesis using ten open-nesting bird species in Arizona, USA. Parental activity was greater during the nestling than incubation stage because parents visited the nest frequently to feed their young during the nestling stage. However, nest predation did not generally increase with parental activity between nesting stages across the ten study species. Previous investigators have found similar results. We tested whether nest site effects might yield higher predation during incubation because the most obvious sites are depredated most rapidly. We conducted experiments using nest sites from the previous year to remove parental activity. Our results showed that nest sites have highly repeatable effects on nest predation risk; poor nest sites incurred rapid predation and caused predation rates to be greater during the incubation than nestling stage. This pattern also was exhibited in a bird species with similar (i.e. controlled) parental activity between nesting stages. Once nest site effects are taken into account, nest predation shows a strong proximate increase with parental activity during the nestling stage within and across species. Parental activity and nest sites exert antagonistic influences on current estimates of nest predation between nesting stages and both must be considered in order to understand current patterns of nest predation, which is an important source of natural selection. PMID:11413645

  19. Spectroscopic studies of the active site of galactose oxidase

    SciTech Connect

    Knowles, P.F.; Brown, R.D. III; Koenig, S.H.

    1995-07-19

    X-ray absorption and EPR spectroscopy have been used to probe the copper site structure in galactose oxidase at pH 4.5 and 7.0. the results suggest that there are no major differences in the structure of the tetragonal Cu(II) site at these pH values. Analysis of the extended X-ray absorption fine structure (EXAFS) indicates that four N,O scatterers are present at approximately 2 {Angstrom}; these are presumably the equatorial ligands. In addition, the EXAFS data establish that oxidative activation to produce the active-site tyrosine radical does not cause major changes in the copper coordination environment. Therefore results obtained on the one-electron reduced enzyme, containing Cu(II) but not the tyrosine radical, probably also apply to the catalytically active Cu(II)/tyrosine radical state. Solvent water exchange, inhibitor binding, and substrate binding have been probed via nuclear magnetic relaxation dispersion (NMRD) measurements. The NMRD profile of galactose oxidase is quantitatively consistent with the rapid exchange of a single, equatorial water ligand with a Cu(II)-O separation of about 2.4 {Angstrom}. Azide and cyanide displace this coordinated water. The binding of azide and the substrate dihydroxyacetone produce very similar effects on the NMRD profile of galactose oxidase, indicating that substrates also bind to the active site Cu(II) in an equatorial position.

  20. Active Sites Environmental Monitoring Program: Program plan

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1992-02-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of transuranic (TRU) waste and active low-level waste (LLW) facilities at Oak Ridge National Laboratory (ORNL) in accordance with US Department of Energy (DOE) Order 5820.2A. Active LLW facilities in Solid Waste Storage Area (SWSA) 6 include Tumulus I and Tumulus II, the Interim Waste Management Facility (IWMF), LLW silos, high-range wells, asbestos silos, and fissile wells. The tumulus pads and IWMF are aboveground, high-strength concrete pads on which concrete vaults containing metal boxes of LLW are placed; the void space between the boxes and vaults is filled with grout. Eventually, these pads and vaults will be covered by an engineered multilayered cap. All other LLW facilities in SWSA 6 are below ground. In addition, this plan includes monitoring of the Hillcut Disposal Test Facility (HDTF) in SWSA 6, even though this facility was completed prior to the data of the DOE order. In SWSA 5 North, the TRU facilities include below-grade engineered caves, high-range wells, and unlined trenches. All samples from SWSA 6 are screened for alpha and beta activity, counted for gamma-emitting isotopes, and analyzed for tritium. In addition to these analytes, samples from SWSA 5 North are analyzed for specific transuranic elements.

  1. Effects of postdosed species on preadsorbed CO on Fe(100): Adsorption site conversion caused by site competition

    SciTech Connect

    Lu, Jiongping; Albert, M.R.; Bernasek, S.L. )

    1990-07-26

    Carbon monoxide (CO) adsorption on the clean Fe(100) surface and the c(2{times}2)CO-Fe(100) surface has been studied. The effects of postdosed oxygen(O{sub 2}) and methanethiol (CH{sub 3}SH) on preadsorbed CO were examined. These studies were performed under ultrahigh-vacuum(UHV) conditions, using high-resolution electron energy loss spectroscopy (HREELS), temperature-programmed desorption spectroscopy (TPD), Auger electron spectroscopy (AES), and low-energy electron diffraction (LEED). For CO, four associative adsorption states and one dissociative adsorption state were identified. Postdosed species were found to cause 4-fold hollow site CO molecules to migrate into lower coordination sites. This type of adsorption site conversion is discussed in terms of adsorption site competition between preadsorbed species and postdosed species.

  2. [Structural regularities in activated cleavage sites of thrombin receptors].

    PubMed

    Mikhaĭlik, I V; Verevka, S V

    1999-01-01

    Comparison of thrombin receptors activation splitting sites sequences testifies to their similarity both in activation splitting sites of protein precursors and protein proteinase inhibitors reactive sites. In all these sites corresponded to effectory sites P2'-positions are placed by hydrophobic amino-acids only. The regularity defined conforms with previous thesis about the role of effectory S2'-site in regulation of the processes mediated by serine proteinases.

  3. Active Site Loop Conformation Regulates Promiscuous Activity in a Lactonase from Geobacillus kaustophilus HTA426

    PubMed Central

    Zhang, Yu; An, Jiao; Yang, Guang-Yu; Bai, Aixi; Zheng, Baisong; Lou, Zhiyong; Wu, Geng; Ye, Wei; Chen, Hai-Feng; Feng, Yan; Manco, Giuseppe

    2015-01-01

    Enzyme promiscuity is a prerequisite for fast divergent evolution of biocatalysts. A phosphotriesterase-like lactonase (PLL) from Geobacillus kaustophilus HTA426 (GkaP) exhibits main lactonase and promiscuous phosphotriesterase activities. To understand its catalytic and evolutionary mechanisms, we investigated a “hot spot” in the active site by saturation mutagenesis as well as X-ray crystallographic analyses. We found that position 99 in the active site was involved in substrate discrimination. One mutant, Y99L, exhibited 11-fold improvement over wild-type in reactivity (kcat/Km) toward the phosphotriesterase substrate ethyl-paraoxon, but showed 15-fold decrease toward the lactonase substrate δ-decanolactone, resulting in a 157-fold inversion of the substrate specificity. Structural analysis of Y99L revealed that the mutation causes a ∼6.6 Å outward shift of adjacent loop 7, which may cause increased flexibility of the active site and facilitate accommodation and/or catalysis of organophosphate substrate. This study provides for the PLL family an example of how the evolutionary route from promiscuity to specificity can derive from very few mutations, which promotes alteration in the conformational adjustment of the active site loops, in turn draws the capacity of substrate binding and activity. PMID:25706379

  4. Inferring proximity to the reconnection site via structural changes to the magnetopause caused by asymmetric reconnection.

    NASA Astrophysics Data System (ADS)

    Argall, M. R.; Chen, L. J.; Torbert, R. B.; Daughton, W. S.; Yoo, J.; Yamada, M.

    2014-12-01

    The mechanisms of field line breaking and magnetic energy dissipation that result in magnetic reconnection have yet to be determined by spacecraft observations. Many parameters have been proposed to locate the reconnection site, but they either fail to identify uniquely the reconnection site or have not been tested for asymmetric reconnection. We demonstrate that the change in magnetopause structure caused by reconnection can be used to locate and estimate proximity to the site of reconnection. Cluster observations of quiet magnetopause crossings, for which no evidence of reconnection is found, show no obvious spatial dependence of the DC electric field, while the plasma density and velocity make the transition from magnetosheath to magnetosphere values simultaneously with the tangential magnetic field (BL) reversal. Conversely, in-situ observations of several active crossings, for which signs of reconnection are evident, show that the density transition and BL reversal can occur simultaneously or be offset from one another by over 100 ion skin depths (λi) (assuming a constant magnetopause velocity), the outflow jet can occur anywhere from the BL reversal to several λi earthward of the density gradient, and the DC electric field changes sign on either side of the density gradient. Laboratory experiments and 2D and 3D particle-in-cell simulations of asymmetric reconnection reveal that the relative transition offsets are due to exhaust crossings at different proximities to the X-line. Only within the thin electron current layer surrounding the X-line do the transitions remain concurrent. We present one reconnection event during which the transitions in plasma density, DC electric field, and BL are simultaneous in two of the four Cluster spacecraft and offset in the other two spacecraft. The multiple satellite encounter allows us to examine spatial features in the region surrounding the X-line.

  5. MYST protein acetyltransferase activity requires active site lysine autoacetylation.

    PubMed

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-04

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases.

  6. MYST protein acetyltransferase activity requires active site lysine autoacetylation

    PubMed Central

    Yuan, Hua; Rossetto, Dorine; Mellert, Hestia; Dang, Weiwei; Srinivasan, Madhusudan; Johnson, Jamel; Hodawadekar, Santosh; Ding, Emily C; Speicher, Kaye; Abshiru, Nebiyu; Perry, Rocco; Wu, Jiang; Yang, Chao; Zheng, Y George; Speicher, David W; Thibault, Pierre; Verreault, Alain; Johnson, F Bradley; Berger, Shelley L; Sternglanz, Rolf; McMahon, Steven B; Côté, Jacques; Marmorstein, Ronen

    2012-01-01

    The MYST protein lysine acetyltransferases are evolutionarily conserved throughout eukaryotes and acetylate proteins to regulate diverse biological processes including gene regulation, DNA repair, cell-cycle regulation, stem cell homeostasis and development. Here, we demonstrate that MYST protein acetyltransferase activity requires active site lysine autoacetylation. The X-ray crystal structures of yeast Esa1 (yEsa1/KAT5) bound to a bisubstrate H4K16CoA inhibitor and human MOF (hMOF/KAT8/MYST1) reveal that they are autoacetylated at a strictly conserved lysine residue in MYST proteins (yEsa1-K262 and hMOF-K274) in the enzyme active site. The structure of hMOF also shows partial occupancy of K274 in the unacetylated form, revealing that the side chain reorients to a position that engages the catalytic glutamate residue and would block cognate protein substrate binding. Consistent with the structural findings, we present mass spectrometry data and biochemical experiments to demonstrate that this lysine autoacetylation on yEsa1, hMOF and its yeast orthologue, ySas2 (KAT8) occurs in solution and is required for acetylation and protein substrate binding in vitro. We also show that this autoacetylation occurs in vivo and is required for the cellular functions of these MYST proteins. These findings provide an avenue for the autoposttranslational regulation of MYST proteins that is distinct from other acetyltransferases but draws similarities to the phosphoregulation of protein kinases. PMID:22020126

  7. The active site of ribulose-bisphosphate carboxylase/oxygenase

    SciTech Connect

    Hartman, F.C.

    1991-01-01

    The active site of ribulose-bisphosphate carboxylase/oxygenase requires interacting domains of adjacent, identical subunits. Most active-site residues are located within the loop regions of an eight-stranded {beta}/{alpha}-barrel which constitutes the larger C-terminal domain; additional key residues are located within a segment of the smaller N-terminal domain which partially covers the mouth of the barrel. Site-directed mutagenesis of the gene encoding the enzyme from Rhodospirillum rubrum has been used to delineate functions of active-site residues. 6 refs., 2 figs.

  8. DOE site performance assessment activities. Radioactive Waste Technical Support Program

    SciTech Connect

    Not Available

    1990-07-01

    Information on performance assessment capabilities and activities was collected from eight DOE sites. All eight sites either currently dispose of low-level radioactive waste (LLW) or plan to dispose of LLW in the near future. A survey questionnaire was developed and sent to key individuals involved in DOE Order 5820.2A performance assessment activities at each site. The sites surveyed included: Hanford Site (Hanford), Idaho National Engineering Laboratory (INEL), Los Alamos National Laboratory (LANL), Nevada Test Site (NTS), Oak Ridge National Laboratory (ORNL), Paducah Gaseous Diffusion Plant (Paducah), Portsmouth Gaseous Diffusion Plant (Portsmouth), and Savannah River Site (SRS). The questionnaire addressed all aspects of the performance assessment process; from waste source term to dose conversion factors. This report presents the information developed from the site questionnaire and provides a comparison of site-specific performance assessment approaches, data needs, and ongoing and planned activities. All sites are engaged in completing the radioactive waste disposal facility performance assessment required by DOE Order 5820.2A. Each site has achieved various degrees of progress and have identified a set of critical needs. Within several areas, however, the sites identified common needs and questions.

  9. Savannah River Site prioritization of transition activities

    SciTech Connect

    Finley, R.H.

    1993-11-01

    Effective management of SRS conversion from primarily a production facility to other missions (or Decontamination and Decommissioning (D&D)) requires a systematic and consistent method of prioritizing the transition activities. This report discusses the design of a prioritizing method developed to achieve systematic and consistent methods of prioritizing these activities.

  10. A Disease-Causing Variant in PCNA Disrupts a Promiscuous Protein Binding Site.

    PubMed

    Duffy, Caroline M; Hilbert, Brendan J; Kelch, Brian A

    2016-03-27

    The eukaryotic DNA polymerase sliding clamp, proliferating cell nuclear antigen or PCNA, is a ring-shaped protein complex that surrounds DNA to act as a sliding platform for increasing processivity of cellular replicases and for coordinating various cellular pathways with DNA replication. A single point mutation, Ser228Ile, in the human PCNA gene was recently identified to cause a disease whose symptoms resemble those of DNA damage and repair disorders. The mutation lies near the binding site for most PCNA-interacting proteins. However, the structural consequences of the S228I mutation are unknown. Here, we describe the structure of the disease-causing variant, which reveals a large conformational change that dramatically transforms the binding pocket for PCNA client proteins. We show that the mutation markedly alters the binding energetics for some client proteins, while another, p21(CIP1), is only mildly affected. Structures of the disease variant bound to peptides derived from two PCNA partner proteins reveal that the binding pocket can adjust conformation to accommodate some ligands, indicating that the binding site is dynamic and pliable. Our work has implications for the plasticity of the binding site in PCNA and reveals how a disease mutation selectively alters interactions to a promiscuous binding site that is critical for DNA metabolism.

  11. Safety Oversight of Decommissioning Activities at DOE Nuclear Sites

    SciTech Connect

    Zull, Lawrence M.; Yeniscavich, William

    2008-01-15

    The Defense Nuclear Facilities Safety Board (Board) is an independent federal agency established by Congress in 1988 to provide nuclear safety oversight of activities at U.S. Department of Energy (DOE) defense nuclear facilities. The activities under the Board's jurisdiction include the design, construction, startup, operation, and decommissioning of defense nuclear facilities at DOE sites. This paper reviews the Board's safety oversight of decommissioning activities at DOE sites, identifies the safety problems observed, and discusses Board initiatives to improve the safety of decommissioning activities at DOE sites. The decommissioning of former defense nuclear facilities has reduced the risk of radioactive material contamination and exposure to the public and site workers. In general, efforts to perform decommissioning work at DOE defense nuclear sites have been successful, and contractors performing decommissioning work have a good safety record. Decommissioning activities have recently been completed at sites identified for closure, including the Rocky Flats Environmental Technology Site, the Fernald Closure Project, and the Miamisburg Closure Project (the Mound site). The Rocky Flats and Fernald sites, which produced plutonium parts and uranium materials for defense needs (respectively), have been turned into wildlife refuges. The Mound site, which performed R and D activities on nuclear materials, has been converted into an industrial and technology park called the Mound Advanced Technology Center. The DOE Office of Legacy Management is responsible for the long term stewardship of these former EM sites. The Board has reviewed many decommissioning activities, and noted that there are valuable lessons learned that can benefit both DOE and the contractor. As part of its ongoing safety oversight responsibilities, the Board and its staff will continue to review the safety of DOE and contractor decommissioning activities at DOE defense nuclear sites.

  12. Controlled Orientation of Active Sites in a Nanostructured Multienzyme Complex

    PubMed Central

    Lim, Sung In; Yang, Byungseop; Jung, Younghan; Cha, Jaehyun; Cho, Jinhwan; Choi, Eun-Sil; Kim, Yong Hwan; Kwon, Inchan

    2016-01-01

    Multistep cascade reactions in nature maximize reaction efficiency by co-assembling related enzymes. Such organization facilitates the processing of intermediates by downstream enzymes. Previously, the studies on multienzyme nanocomplexes assembled on DNA scaffolds demonstrated that closer interenzyme distance enhances the overall reaction efficiency. However, it remains unknown how the active site orientation controlled at nanoscale can have an effect on multienzyme reaction. Here, we show that controlled alignment of active sites promotes the multienzyme reaction efficiency. By genetic incorporation of a non-natural amino acid and two compatible bioorthogonal chemistries, we conjugated mannitol dehydrogenase to formate dehydrogenase with the defined active site arrangement with the residue-level accuracy. The study revealed that the multienzyme complex with the active sites directed towards each other exhibits four-fold higher relative efficiency enhancement in the cascade reaction and produces 60% more D-mannitol than the other complex with active sites directed away from each other. PMID:28004799

  13. Perspective: On the active site model in computational catalyst screening

    NASA Astrophysics Data System (ADS)

    Reuter, Karsten; Plaisance, Craig P.; Oberhofer, Harald; Andersen, Mie

    2017-01-01

    First-principles screening approaches exploiting energy trends in surface adsorption represent an unparalleled success story in recent computational catalysis research. Here we argue that our still limited understanding of the structure of active sites is one of the major bottlenecks towards an ever extended and reliable use of such computational screening for catalyst discovery. For low-index transition metal surfaces, the prevalently chosen high-symmetry (terrace and step) sites offered by the nominal bulk-truncated crystal lattice might be justified. For more complex surfaces and composite catalyst materials, computational screening studies will need to actively embrace a considerable uncertainty with respect to what truly are the active sites. By systematically exploring the space of possible active site motifs, such studies might eventually contribute towards a targeted design of optimized sites in future catalysts.

  14. Diffusional correlations among multiple active sites in a single enzyme.

    PubMed

    Echeverria, Carlos; Kapral, Raymond

    2014-04-07

    Simulations of the enzymatic dynamics of a model enzyme containing multiple substrate binding sites indicate the existence of diffusional correlations in the chemical reactivity of the active sites. A coarse-grain, particle-based, mesoscopic description of the system, comprising the enzyme, the substrate, the product and solvent, is constructed to study these effects. The reactive and non-reactive dynamics is followed using a hybrid scheme that combines molecular dynamics for the enzyme, substrate and product molecules with multiparticle collision dynamics for the solvent. It is found that the reactivity of an individual active site in the multiple-active-site enzyme is reduced substantially, and this effect is analyzed and attributed to diffusive competition for the substrate among the different active sites in the enzyme.

  15. Robotics at Savannah River site: activity report

    SciTech Connect

    Byrd, J.S.

    1984-09-01

    The objectives of the Robotics Technology Group at the Savannah River Laboratory are to employ modern industrial robots and to develop unique automation and robotic systems to enhance process operations at the Savannah River site (SRP and SRL). The incentives are to improve safety, reduce personnel radiation exposure, improve product quality and productivity, and to reduce operating costs. During the past year robotic systems have been installed to fill chemical dilution vials in a SRP laboratory at 772-F and remove radioactive waste materials in the SRL Californium Production Facility at 773-A. A robotic system to lubricate an extrusion press has been developed and demonstrated in the SRL robotics laboratory and is scheduled for installation at the 321-M fuel fabrication area. A mobile robot was employed by SRP for a radiation monitoring task at a waste tank top in H-Area. Several other robots are installed in the SRL robotics laboratories and application development programs are underway. The status of these applications is presented in this report.

  16. Active sites of thioredoxin reductases: why selenoproteins?

    PubMed

    Gromer, Stephan; Johansson, Linda; Bauer, Holger; Arscott, L David; Rauch, Susanne; Ballou, David P; Williams, Charles H; Schirmer, R Heiner; Arnér, Elias S J

    2003-10-28

    Selenium, an essential trace element for mammals, is incorporated into a selected class of selenoproteins as selenocysteine. All known isoenzymes of mammalian thioredoxin (Trx) reductases (TrxRs) employ selenium in the C-terminal redox center -Gly-Cys-Sec-Gly-COOH for reduction of Trx and other substrates, whereas the corresponding sequence in Drosophila melanogaster TrxR is -Ser-Cys-Cys-Ser-COOH. Surprisingly, the catalytic competence of these orthologous enzymes is similar, whereas direct Sec-to-Cys substitution of mammalian TrxR, or other selenoenzymes, yields almost inactive enzyme. TrxRs are therefore ideal for studying the biology of selenocysteine by comparative enzymology. Here we show that the serine residues flanking the C-terminal Cys residues of Drosophila TrxRs are responsible for activating the cysteines to match the catalytic efficiency of a selenocysteine-cysteine pair as in mammalian TrxR, obviating the need for selenium. This finding suggests that the occurrence of selenoenzymes, which implies that the organism is selenium-dependent, is not necessarily associated with improved enzyme efficiency. Our data suggest that the selective advantage of selenoenzymes is a broader range of substrates and a broader range of microenvironmental conditions in which enzyme activity is possible.

  17. Whole-Genome Sequencing Analysis of Methicillin-Resistant Staphylococcus simulans Causing Surgical Site Infection

    PubMed Central

    2016-01-01

    Staphylococcus simulans is a normal part of the microbiota in humans and animals and is rarely associated with human invasive infections. We present here the genome sequence of S. simulans CJ16, which caused the first case of surgical site infection. Adhesion proteins, including fibronectin-binding protein (FnbA), elastin-binding protein (EbpS), and cell wall-anchored protein (SasA, SasF, and SasH), were detected in the genome, which might promote the survival of S. simulans on human skin and pathogenesis of infections. PMID:27313298

  18. Community Update on Site Activities, July 19, 2013

    EPA Pesticide Factsheets

    In an effort to engage and inform community members interested in the New Bedford Harbor Superfund Site cleanup, EPA will be issuing periodic topic-based fact sheets that will provide background information and updates about ongoing activities.

  19. A Novel Splice-Site Mutation in the GJB2 Gene Causing Mild Postlingual Hearing Impairment

    PubMed Central

    Gandía, Marta; del Castillo, Francisco J.; Rodríguez-Álvarez, Francisco J.; Garrido, Gema; Villamar, Manuela; Calderón, Manuela; Moreno-Pelayo, Miguel A.; Moreno, Felipe; del Castillo, Ignacio

    2013-01-01

    The DFNB1 subtype of autosomal recessive, nonsyndromic hearing impairment, caused by mutations affecting the GJB2 (connection-26) gene, is highly prevalent in most populations worldwide. DFNB1 hearing impairment is mostly severe or profound and usually appears before the acquisition of speech (prelingual onset), though a small number of hypomorphic missense mutations result in mild or moderate deafness of postlingual onset. We identified a novel GJB2 splice-site mutation, c. -22-2A>C, in three siblings with mild postlingual hearing impairment that were compound heterozygous for c. -22-2A>C and c.35delG. Reverse transcriptase-PCR experiments performed on total RNA extracted from saliva samples from one of these siblings confirmed that c. -22-2A>C abolished the acceptor splice site of the single GJB2 intron, resulting in the absence of normally processed transcripts from this allele. However, we did isolate transcripts from the c. -22-2A>C allele that keep an intact GJB2 coding region and that were generated by use of an alternative acceptor splice site previously unknown. The residual expression of wild-type connection-26 encoded by these transcripts probably underlies the mild severity and late onset of the hearing impairment of these subjects. PMID:24039984

  20. Risks of all-cause and site-specific fractures among hospitalized patients with COPD

    PubMed Central

    Liao, Kuang-Ming; Liang, Fu-Wen; Li, Chung-Yi

    2016-01-01

    Abstract Patients with chronic obstructive pulmonary disease (COPD) have a high prevalence of osteoporosis. The clinical sequel of osteoporosis is fracture. Patients with COPD who experience a fracture also have increased morbidity and mortality. Currently, the types of all-cause and site-specific fracture among patients with COPD are unknown. Thus, we elucidated the all-cause and site-specific fractures among patients with COPD. A retrospective, population-based, cohort study was conducted utilizing the Taiwan Longitudinal Health Insurance Database. Patients with COPD were defined as those who were hospitalized with an International Classification of Diseases, Ninth Revision, Clinical Modification code of 490 to 492 or 496 between 2001 and 2011. The index date was set as the date of discharge. The study patients were followed from the index date to the date when they sought care for any type of fracture, date of death, date of health insurance policy termination, or the last day of 2013. The types of fracture analyzed in this study included vertebral, rib, humeral, radial and ulnar/wrist, pelvic, femoral, and tibial and fibular fractures. The cohort consisted of 11,312 patients with COPD. Among these patients, 1944 experienced fractures. The most common site-specific fractures were vertebral, femoral, rib, and forearm fractures (radius, ulna, and wrist) at 32.4%, 31%, 12%, and 11.8%, respectively. The adjusted hazard ratios of fracture were 1.71 [95% confidence interval (95% CI) = 1.56–1.87] for female patient with COPD and 1.50 (95% CI = 1.39–1.52) for patients with osteoporosis after covariate adjustment. Vertebral and hip fractures are common among patients with COPD, especially among males with COPD. Many comorbidities contribute to the high risk of fracture among patients with COPD. PMID:27749576

  1. Safer Work Plan for CAUs 452, 454, 456, and 464 Closure of Historical UST Release Sites Nevada Test Site

    SciTech Connect

    Jerry Bonn

    1997-08-01

    This plan addresses characterization and closure of nine underground storage tank petroleum hydrocarbon release sites. The sites are located at the Nevada Test Site in Areas 2, 9, 12, 23, and 25. The underground storage tanks associated with the release sites and addressed by this plan were closed between 1990 and 1996 by the U. S. Department of Energy, Nevada Operations Office. One underground storage tank was closed in place (23-111-1) while the remaining eight were closed by removal. Hydrocarbon releases were identified at each of the sites based upon laboratory analytical data samples collected below the tank bottoms. The objective of this plan is to provide a method for implementing characterization and closure of historical underground storage tank hydrocarbon release sites.

  2. Identification of putative active site residues of ACAT enzymes.

    PubMed

    Das, Akash; Davis, Matthew A; Rudel, Lawrence L

    2008-08-01

    In this report, we sought to determine the putative active site residues of ACAT enzymes. For experimental purposes, a particular region of the C-terminal end of the ACAT protein was selected as the putative active site domain due to its high degree of sequence conservation from yeast to humans. Because ACAT enzymes have an intrinsic thioesterase activity, we hypothesized that by analogy with the thioesterase domain of fatty acid synthase, the active site of ACAT enzymes may comprise a catalytic triad of ser-his-asp (S-H-D) amino acid residues. Mutagenesis studies revealed that in ACAT1, S456, H460, and D400 were essential for activity. In ACAT2, H438 was required for enzymatic activity. However, mutation of D378 destabilized the enzyme. Surprisingly, we were unable to identify any S mutations of ACAT2 that abolished catalytic activity. Moreover, ACAT2 was insensitive to serine-modifying reagents, whereas ACAT1 was not. Further studies indicated that tyrosine residues may be important for ACAT activity. Mutational analysis showed that the tyrosine residue of the highly conserved FYXDWWN motif was important for ACAT activity. Furthermore, Y518 was necessary for ACAT1 activity, whereas the analogous residue in ACAT2, Y496, was not. The available data suggest that the amino acid requirement for ACAT activity may be different for the two ACAT isozymes.

  3. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site

    PubMed Central

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-01-01

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide. PMID:26908655

  4. Methanopyrus kandleri topoisomerase V contains three distinct AP lyase active sites in addition to the topoisomerase active site.

    PubMed

    Rajan, Rakhi; Osterman, Amy; Mondragón, Alfonso

    2016-04-20

    Topoisomerase V (Topo-V) is the only topoisomerase with both topoisomerase and DNA repair activities. The topoisomerase activity is conferred by a small alpha-helical domain, whereas the AP lyase activity is found in a region formed by 12 tandem helix-hairpin-helix ((HhH)2) domains. Although it was known that Topo-V has multiple repair sites, only one had been mapped. Here, we show that Topo-V has three AP lyase sites. The atomic structure and Small Angle X-ray Scattering studies of a 97 kDa fragment spanning the topoisomerase and 10 (HhH)2 domains reveal that the (HhH)2 domains extend away from the topoisomerase domain. A combination of biochemical and structural observations allow the mapping of the second repair site to the junction of the 9th and 10th (HhH)2 domains. The second site is structurally similar to the first one and to the sites found in other AP lyases. The 3rd AP lyase site is located in the 12th (HhH)2 domain. The results show that Topo-V is an unusual protein: it is the only known protein with more than one (HhH)2 domain, the only known topoisomerase with dual activities and is also unique by having three AP lyase repair sites in the same polypeptide.

  5. Local Activity Principle:. the Cause of Complexity and Symmetry Breaking

    NASA Astrophysics Data System (ADS)

    Mainzer, Klaus

    2013-01-01

    The principle of local activity is precisely the missing concept to explain the emergence of complex patterns in a homogeneous medium. Leon O. Chua discovered and defined this principle in the theory of nonlinear electronic circuits in a mathematically rigorous way. The local principle can be generalized and proven at least for the class of nonlinear reaction-diffusion systems in physics, chemistry, biology and brain research. Recently, it was realized by memristors for nanoelectronic device applications in technical brains. In general, the emergence of complex patterns and structures is explained by symmetry breaking in homogeneous media. The principle of local activity is the cause of symmetry breaking in homogeneous media. We argue that the principle of local activity is really fundamental in science and can even be identified in quantum cosmology as symmetry breaking of local gauge symmetries generating the complexity of matter and forces in our universe. Finally, we consider applications in economic, financial, and social systems with the emergence of equilibrium states, symmetry breaking at critical points of phase transitions and risky acting at the edge of chaos. In any case, the driving causes of symmetry breaking and the emergence of complexity are locally active elements, cells, units, or agents.

  6. A de novo splice site mutation in CASK causes FG syndrome-4 and congenital nystagmus.

    PubMed

    Dunn, P; Prigatano, G P; Szelinger, S; Roth, J; Siniard, A L; Claasen, A M; Richholt, R F; De Both, M; Corneveaux, J J; Moskowitz, A M; Balak, C; Piras, I S; Russell, M; Courtright, A L; Belnap, N; Rangasamy, S; Ramsey, K; Opitz, J M; Craig, D W; Narayanan, V; Huentelman, M J; Schrauwen, I

    2017-03-01

    Mutations in CASK cause X-linked intellectual disability, microcephaly with pontine and cerebellar hypoplasia, optic atrophy, nystagmus, feeding difficulties, GI hypomotility, and seizures. Here we present a patient with a de novo carboxyl-terminus splice site mutation in CASK (c.2521-2A>G) and clinical features of the rare FG syndrome-4 (FGS4). We provide further characterization of genotype-phenotype correlations in CASK mutations and the presentation of nystagmus and the FGS4 phenotype. There is considerable variability in clinical phenotype among patients with a CASK mutation, even among variants predicted to have similar functionality. Our patient presented with developmental delay, nystagmus, and severe gastrointestinal and gastroesophageal complications. From a cognitive and neuropsychological perspective, language skills and IQ are within normal range, although visual-motor, motor development, behavior, and working memory were impaired. The c.2521-2A>G splice mutation leads to skipping of exon 26 and a 9 base-pair deletion associated with a cryptic splice site, leading to a 28-AA and a 3-AA in-frame deletion, respectively (p.Ala841_Lys843del and p.Ala841_Glu868del). The predominant mutant transcripts contain an aberrant guanylate kinase domain and thus are predicted to degrade CASK's ability to interact with important neuronal and ocular development proteins, including FRMD7. Upregulation of CASK as well as dysregulation among a number of interactors is also evident by RNA-seq. This is the second CASK mutation known to us as cause of FGS4. © 2017 Wiley Periodicals, Inc.

  7. A Case of Surgical Site Infection Caused by Mycobacterium fortuitum, following Herniorrhaphy

    PubMed Central

    Malini, A; Sangma, Mima Maychet B

    2016-01-01

    Rapidly Growing Mycobacteria (RGM) are opportunistic pathogens found in the environment. Mycobacterium fortuitum, M. chelonae and M.abscessus are the important human pathogens of this group. They cause wound infections, disseminated cutaneous disease, pulmonary infection in patients with cystic fibrosis or bronchiectasis, bone and joint infections and keratitis. Infections due to these Non-Tuberculous Mycobacteria (NTM) are increasingly reported. Post laparoscopic wound infections, mesh site infections and other surgical site infections due to M. fortuitum and M. chelonae have been reported. Usually wound infections due to atypical mycobacteria have delayed onset and do not respond to conventional antibiotics. Identification of RGM can be done by a set of cumbersome biochemical tests, High Performance Liquid Chromatography (HPLC), molecular methods using DNA probes or by Polymerase Chain Reaction (PCR). We here report a case of post-herniorrhaphy wound infection due to M. fortuitum which was identified by molecular method (HAIN mycobacterial species system). This case report underscores the importance of examining Ziehl-Neelsen (ZN) stain of all exudates with sterile culture on day one for non fastidious bacteria. Timely identification can lead to prompt therapy of patients preventing further complications. PMID:28050369

  8. A Case of Surgical Site Infection Caused by Mycobacterium fortuitum, following Herniorrhaphy.

    PubMed

    Madhusudhan, N S; Malini, A; Sangma, Mima Maychet B

    2016-11-01

    Rapidly Growing Mycobacteria (RGM) are opportunistic pathogens found in the environment. Mycobacterium fortuitum, M. chelonae and M.abscessus are the important human pathogens of this group. They cause wound infections, disseminated cutaneous disease, pulmonary infection in patients with cystic fibrosis or bronchiectasis, bone and joint infections and keratitis. Infections due to these Non-Tuberculous Mycobacteria (NTM) are increasingly reported. Post laparoscopic wound infections, mesh site infections and other surgical site infections due to M. fortuitum and M. chelonae have been reported. Usually wound infections due to atypical mycobacteria have delayed onset and do not respond to conventional antibiotics. Identification of RGM can be done by a set of cumbersome biochemical tests, High Performance Liquid Chromatography (HPLC), molecular methods using DNA probes or by Polymerase Chain Reaction (PCR). We here report a case of post-herniorrhaphy wound infection due to M. fortuitum which was identified by molecular method (HAIN mycobacterial species system). This case report underscores the importance of examining Ziehl-Neelsen (ZN) stain of all exudates with sterile culture on day one for non fastidious bacteria. Timely identification can lead to prompt therapy of patients preventing further complications.

  9. Active chemisorption sites in functionalized ionic liquids for carbon capture.

    PubMed

    Cui, Guokai; Wang, Jianji; Zhang, Suojiang

    2016-07-25

    Development of novel technologies for the efficient and reversible capture of CO2 is highly desired. In the last decade, CO2 capture using ionic liquids has attracted intensive attention from both academia and industry, and has been recognized as a very promising technology. Recently, a new approach has been developed for highly efficient capture of CO2 by site-containing ionic liquids through chemical interaction. This perspective review focuses on the recent advances in the chemical absorption of CO2 using site-containing ionic liquids, such as amino-based ionic liquids, azolate ionic liquids, phenolate ionic liquids, dual-functionalized ionic liquids, pyridine-containing ionic liquids and so on. Other site-containing liquid absorbents such as amine-based solutions, switchable solvents, and functionalized ionic liquid-amine blends are also investigated. Strategies have been discussed for how to activate the existent reactive sites and develop novel reactive sites by physical and chemical methods to enhance CO2 absorption capacity and reduce absorption enthalpy. The carbon capture mechanisms of these site-containing liquid absorbents are also presented. Particular attention has been paid to the latest progress in CO2 capture in multiple-site interactions by amino-free anion-functionalized ionic liquids. In the last section, future directions and prospects for carbon capture by site-containing ionic liquids are outlined.

  10. Active Sites Environmental Monitoring Program: Mid-FY 1991 report

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1991-10-01

    This report summarizes the activities of the Active Sites Environmental Monitoring Program (ASEMP) from October 1990 through March 1991. The ASEMP was established in 1989 by Solid Waste Operations and the Environmental Sciences Division to provide early detection and performance monitoring at active low-level radioactive waste (LLW) disposal sites in Solid Waste Storage Area (SWSA) 6 and transuranic (TRU) waste storage sites in SWSA 5 as required by chapters II and III of US Department of Energy Order 5820.2A. Monitoring results continue to demonstrate the no LLW is being leached from the storage vaults on the tumulus pads. Loading of vaults on Tumulus II began during this reporting period and 115 vaults had been loaded by the end of March 1991.

  11. Active and regulatory sites of cytosolic 5'-nucleotidase.

    PubMed

    Pesi, Rossana; Allegrini, Simone; Careddu, Maria Giovanna; Filoni, Daniela Nicole; Camici, Marcella; Tozzi, Maria Grazia

    2010-12-01

    Cytosolic 5'-nucleotidase (cN-II), which acts preferentially on 6-hydroxypurine nucleotides, is essential for the survival of several cell types. cN-II catalyses both the hydrolysis of nucleotides and transfer of their phosphate moiety to a nucleoside acceptor through formation of a covalent phospho-intermediate. Both activities are regulated by a number of phosphorylated compounds, such as diadenosine tetraphosphate (Ap₄A), ADP, ATP, 2,3-bisphosphoglycerate (BPG) and phosphate. On the basis of a partial crystal structure of cN-II, we mutated two residues located in the active site, Y55 and T56. We ascertained that the ability to catalyse the transfer of phosphate depends on the presence of a bulky residue in the active site very close to the aspartate residue that forms the covalent phospho-intermediate. The molecular model indicates two possible sites at which adenylic compounds may interact. We mutated three residues that mediate interaction in the first activation site (R144, N154, I152) and three in the second (F127, M436 and H428), and found that Ap₄A and ADP interact with the same site, but the sites for ATP and BPG remain uncertain. The structural model indicates that cN-II is a homotetrameric protein that results from interaction through a specific interface B of two identical dimers that have arisen from interaction of two identical subunits through interface A. Point mutations in the two interfaces and gel-filtration experiments indicated that the dimer is the smallest active oligomerization state. Finally, gel-filtration and light-scattering experiments demonstrated that the native enzyme exists as a tetramer, and no further oligomerization is required for enzyme activation.

  12. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand

    SciTech Connect

    Parashar, Abhinav; Venkatachalam, Avanthika; Gideon, Daniel Andrew; Manoj, Kelath Murali

    2014-12-12

    Highlights: • Cyanide (CN) is a well-studied toxic principle, known to inhibit heme-enzymes. • Inhibition is supposed to result from CN binding at the active site as a ligand. • Diverse heme enzymes’ CN inhibition profiles challenge prevailing mechanism. • Poor binding efficiency of CN at low enzyme concentrations and ligand pressures. • CN-based diffusible radicals cause ‘non-productive electron transfers’ (inhibition). - Abstract: The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins’ active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.

  13. BAX Activation is Initiated at a Novel Interaction Site

    PubMed Central

    Gavathiotis, Evripidis; Suzuki, Motoshi; Davis, Marguerite L.; Pitter, Kenneth; Bird, Gregory H.; Katz, Samuel G.; Tu, Ho-Chou; Kim, Hyungjin; Cheng, Emily H.-Y.; Tjandra, Nico; Walensky, Loren D.

    2008-01-01

    BAX is a pro-apoptotic protein of the BCL-2 family stationed in the cytosol until activated by a diversity of stress stimuli to induce cell death. Anti-apoptotic proteins such as BCL-2 counteract BAX-mediated cell death. Although an interaction site that confers survival functionality has been defined for anti-apoptotic proteins, an activation site has not been identified for BAX, rendering its explicit trigger mechanism unknown. We previously developed Stabilized Alpha-Helix of BCL-2 domains (SAHBs) that directly initiate BAX-mediated mitochondrial apoptosis. Here we demonstrate by NMR analysis that BIM SAHB binds BAX at an interaction site that is distinct from the canonical binding groove characterized for anti-apoptotic proteins. The specificity of the BIM SAHB-BAX interaction is highlighted by point mutagenesis that abrogates functional activity, confirming that BAX activation is initiated at this novel structural location. Thus, we have now defined a BAX interaction site for direct activation, establishing a new target for therapeutic modulation of apoptosis. PMID:18948948

  14. Decline of a Rare Moth at Its Last Known English Site: Causes and Lessons for Conservation

    PubMed Central

    Baker, David; Barrett, Sinead; Beale, Colin M.; Crawford, Terry J.; Ellis, Sam; Gullett, Tallulah; Parsons, Mark S.; Relf, Penny; Robertson, Paul; Small, Julian; Wainwright, Dave

    2016-01-01

    The conditions required by rare species are often only approximately known. Monitoring such species over time can help refine management of their protected areas. We report population trends of a rare moth, the Dark Bordered Beauty Epione vespertaria (Linnaeus, 1767) (Lepidoptera: Geometridae) at its last known English site on a protected lowland heath, and those of its host-plant, Salix repens (L.) (Malpighiales: Salicaceae). Between 2007 and 2014, adult moth density reduced by an average of 30–35% annually over the monitored area, and its range over the monitored area contracted in concert. By comparing data from before this decline (2005) with data taken in 2013, we show that the density of host-plants over the monitored area reduced three-fold overall, and ten-fold in the areas of highest host-plant density. In addition, plants were significantly smaller in 2013. In 2005, moth larvae tended to be found on plants that were significantly larger than average at the time. By 2013, far fewer plants were of an equivalent size. This suggests that the rapid decline of the moth population coincides with, and is likely driven by, changes in the host-plant population. Why the host-plant population has changed remains less certain, but fire, frost damage and grazing damage have probably contributed. It is likely that a reduction in grazing pressure in parts of the site would aid host-plant recovery, although grazing remains an important site management activity. Our work confirms the value of constant monitoring of rare or priority insect species, of the risks posed to species with few populations even when their populations are large, of the potential conflict between bespoke management for species and generic management of habitats, and hence the value of refining our knowledge of rare species’ requirements so that their needs can be incorporated into the management of protected areas. PMID:27333285

  15. A single splice site mutation in human-specific ARHGAP11B causes basal progenitor amplification

    PubMed Central

    Florio, Marta; Namba, Takashi; Pääbo, Svante; Hiller, Michael; Huttner, Wieland B.

    2016-01-01

    The gene ARHGAP11B promotes basal progenitor amplification and is implicated in neocortex expansion. It arose on the human evolutionary lineage by partial duplication of ARHGAP11A, which encodes a Rho guanosine triphosphatase–activating protein (RhoGAP). However, a lack of 55 nucleotides in ARHGAP11B mRNA leads to loss of RhoGAP activity by GAP domain truncation and addition of a human-specific carboxy-terminal amino acid sequence. We show that these 55 nucleotides are deleted by mRNA splicing due to a single C→G substitution that creates a novel splice donor site. We reconstructed an ancestral ARHGAP11B complementary DNA without this substitution. Ancestral ARHGAP11B exhibits RhoGAP activity but has no ability to increase basal progenitors during neocortex development. Hence, a single nucleotide substitution underlies the specific properties of ARHGAP11B that likely contributed to the evolutionary expansion of the human neocortex. PMID:27957544

  16. Involvement of novel autophosphorylation sites in ATM activation.

    PubMed

    Kozlov, Sergei V; Graham, Mark E; Peng, Cheng; Chen, Philip; Robinson, Phillip J; Lavin, Martin F

    2006-08-09

    ATM kinase plays a central role in signaling DNA double-strand breaks to cell cycle checkpoints and to the DNA repair machinery. Although the exact mechanism of ATM activation remains unknown, efficient activation requires the Mre11 complex, autophosphorylation on S1981 and the involvement of protein phosphatases and acetylases. We report here the identification of several additional phosphorylation sites on ATM in response to DNA damage, including autophosphorylation on pS367 and pS1893. ATM autophosphorylates all these sites in vitro in response to DNA damage. Antibodies against phosphoserine 1893 revealed rapid and persistent phosphorylation at this site after in vivo activation of ATM kinase by ionizing radiation, paralleling that observed for S1981 phosphorylation. Phosphorylation was dependent on functional ATM and on the Mre11 complex. All three autophosphorylation sites are physiologically important parts of the DNA damage response, as phosphorylation site mutants (S367A, S1893A and S1981A) were each defective in ATM signaling in vivo and each failed to correct radiosensitivity, genome instability and cell cycle checkpoint defects in ataxia-telangiectasia cells. We conclude that there are at least three functionally important radiation-induced autophosphorylation events in ATM.

  17. Resonant active sites in catalytic ammonia synthesis: A structural model

    NASA Astrophysics Data System (ADS)

    Cholach, Alexander R.; Bryliakova, Anna A.; Matveev, Andrey V.; Bulgakov, Nikolai N.

    2016-03-01

    Adsorption sites Mn consisted of n adjacent atoms M, each bound to the adsorbed species, are considered within a realistic model. The sum of bonds Σ lost by atoms in a site in comparison with the bulk atoms was used for evaluation of the local surface imperfection, while the reaction enthalpy at that site was used as a measure of activity. The comparative study of Mn sites (n = 1-5) at basal planes of Pt, Rh, Ir, Fe, Re and Ru with respect to heat of N2 dissociative adsorption QN and heat of Nad + Had → NHad reaction QNH was performed using semi-empirical calculations. Linear QN(Σ) increase and QNH(Σ) decrease allowed to specify the resonant Σ for each surface in catalytic ammonia synthesis at equilibrium Nad coverage. Optimal Σ are realizable for Ru2, Re2 and Ir4 only, whereas other centers meet steric inhibition or unreal crystal structure. Relative activity of the most active sites in proportion 5.0 × 10- 5: 4.5 × 10- 3: 1: 2.5: 3.0: 1080: 2270 for a sequence of Pt4, Rh4, Fe4(fcc), Ir4, Fe2-5(bcc), Ru2, Re2, respectively, is in agreement with relevant experimental data. Similar approach can be applied to other adsorption or catalytic processes exhibiting structure sensitivity.

  18. COL1 C-propeptide Cleavage Site Mutations Cause High Bone Mass Osteogenesis Imperfecta

    PubMed Central

    Lindahl, Katarina; Barnes, Aileen M.; Fratzl-Zelman, Nadja; Whyte, Michael P.; Hefferan, Theresa E.; Makareeva, Elena; Brusel, Marina; Yaszemski, Michael J.; Rubin, Carl-Johan; Kindmark, Andreas; Roschger, Paul; Klaushofer, Klaus; McAlister, William H.; Mumm, Steven; Leikin, Sergey; Kessler, Efrat; Boskey, Adele L.; Ljunggren, Östen; Marini, Joan C.

    2011-01-01

    Osteogenesis imperfecta (OI) is most often caused by mutations in the type I procollagen genes (COL1A1/COL1A2). We identified two children with substitutions in the type I procollagen C-propeptide cleavage site, which disrupt a unique processing step in collagen maturation and define a novel phenotype within OI. The patients have mild OI caused by mutations in COL1A1 (Patient 1: p.Asp1219Asn) or COL1A2 (Patient 2: p.Ala1119Thr), respectively. Patient 1 L1-L4 DXA z-score was +3.9 and pQCT vBMD was +3.1; Patient 2 had L1-L4 DXA z-score of 0.0 and pQCT vBMD of −1.8. Patient BMD contrasts with radiographic osteopenia and histomorphometry without osteosclerosis. Mutant procollagen processing is impaired in pericellular and in vitro assays. Patient dermal collagen fibrils have irregular borders. Incorporation of pC-collagen into matrix leads to increased bone mineralization. FT-IR imaging confirms elevated mineral/matrix ratios in both patients, along with increased collagen maturation in trabecular bone, compared to normal or OI controls. Bone mineralization density distribution revealed a marked shift toward increased mineralization density for both patients. Patient 1 has areas of higher and lower bone mineralization than controls; Patient 2’s bone matrix has a mineral content exceeding even classical OI bone. These patients define a new phenotype of high BMD OI and demonstrate that procollagen C-propeptide cleavage is crucial to normal bone mineralization. PMID:21344539

  19. The three Mycobacterium tuberculosis antigen 85 isoforms have unique substrates and activities determined by non-active site regions.

    PubMed

    Backus, Keriann M; Dolan, Michael A; Barry, Conor S; Joe, Maju; McPhie, Peter; Boshoff, Helena I M; Lowary, Todd L; Davis, Benjamin G; Barry, Clifton E

    2014-09-05

    The three isoforms of antigen 85 (A, B, and C) are the most abundant secreted mycobacterial proteins and catalyze transesterification reactions that synthesize mycolated arabinogalactan, trehalose monomycolate (TMM), and trehalose dimycolate (TDM), important constituents of the outermost layer of the cellular envelope of Mycobacterium tuberculosis. These three enzymes are nearly identical at the active site and have therefore been postulated to exist to evade host immunity. Distal to the active site is a second putative carbohydrate-binding site of lower homology. Mutagenesis of the three isoforms at this second site affected both substrate selectivity and overall catalytic activity in vitro. Using synthetic and natural substrates, we show that these three enzymes exhibit unique selectivity; antigen 85A more efficiently mycolates TMM to form TDM, whereas C (and to a lesser extent B) has a higher rate of activity using free trehalose to form TMM. This difference in substrate selectivity extends to the hexasaccharide fragment of cell wall arabinan. Mutation of secondary site residues from the most active isoform (C) into those present in A or B partially interconverts this substrate selectivity. These experiments in combination with molecular dynamics simulations reveal that differences in the N-terminal helix α9, the adjacent Pro(216)-Phe(228) loop, and helix α5 are the likely cause of changes in activity and substrate selectivity. These differences explain the existence of three isoforms and will allow for future work in developing inhibitors.

  20. Chemical Modification of Papain and Subtilisin: An Active Site Comparison

    ERIC Educational Resources Information Center

    St-Vincent, Mireille; Dickman, Michael

    2004-01-01

    An experiment using methyle methanethiosulfonate (MMTS) and phenylmethylsulfonyl flouride (PMSF) to specifically modify the cysteine and serine residues in the active sites of papain and subtilism respectively is demonstrated. The covalent modification of these enzymes and subsequent rescue of papain shows the beginning biochemist that proteins…

  1. Energy transfer at the active sites of heme proteins

    SciTech Connect

    Dlott, D.D.; Hill, J.R.

    1995-12-31

    Experiments using a picosecond pump-probe apparatus at the Picosecond Free-electron Laser Center at Stanford University, were performed to investigate the relaxation of carbon monoxide bound to the active sites of heme proteins. The significance of these experiments is two-fold: (1) they provide detailed information about molecular dynamics occurring at the active sites of proteins; and (2) they provide insight into the nature of vibrational relaxation processes in condensed matter. Molecular engineering is used to construct various molecular systems which are studied with the FEL. We have studied native proteins, mainly myoglobin obtained from different species, mutant proteins produced by genetic engineering using recombinant DNA techniques, and a variety of model systems which mimic the structures of the active sites of native proteins, which are produced using molecular synthesis. Use of these different systems permits us to investigate how specific molecular structural changes affect dynamical processes occurring at the active sites. This research provides insight into the problems of how different species needs are fulfilled by heme proteins which have greatly different functionality, which is induced by rather small structural changes.

  2. What's Causing the Activity on Comet 67P?

    NASA Astrophysics Data System (ADS)

    Kohler, Susanna

    2015-09-01

    Comet 67P/ChuryumovGerasimenko made famous by the explorations of the Rosetta mission has been displaying puzzling activity as it hurtles toward the Sun. However, recent modeling of the comet by a group of scientists from the Cte dAzur University may now explain whats causing 67Ps activity.Shadowed ActivityA model of comet 67P, with the colors indicating the rate of change of the temperature on the comets surface. The most rapid temperature changes are seen at the comets neck, in the same locations as the early activity seen in the Rosetta images. [Al-Lagoa et al. 2015] Between June and September of 2014, Rosetta observed comet 67P displaying early activity in the form of jets of dust emitted from near the neck of the comet (its narrowest point). Such activity is usually driven by the sublimation of volatiles from the comets surface as a result of sun exposure. But the neck of the comet is frequently shadowed as the comet rotates, and it receives significantly less sunlight than the rest of the comet. So why would the early activity originate from the comets neck?The authors of a recent study, led by Victor Al-Lagoa, hypothesize that its precisely because the neck is receiving alternating sunlight/shadows that its displaying activity. They suggest that thermal cracking of the surface of the comet is happening faster in this region, due to the rapid changes in temperature that result from the shadows cast by the surrounding terrain. The cracking exposes subsurface ices in the neck faster than in other regions, and the ensuing sublimation of that ice is what creates the activity were seeing.Temperature Models: To test their hypothesis, the authors study the surface temperatures on comet 67P by means of a thermophysical model a model used to calculate the temperatures on an airless body, both on and below the surface. The model takes into account factors like thermal inertia (how quickly the bodys temperature responds to changes in the incident energy), shadowing, and

  3. Activation of phenylalanine hydroxylase by phenylalanine does not require binding in the active site.

    PubMed

    Roberts, Kenneth M; Khan, Crystal A; Hinck, Cynthia S; Fitzpatrick, Paul F

    2014-12-16

    Phenylalanine hydroxylase (PheH), a liver enzyme that catalyzes the hydroxylation of excess phenylalanine in the diet to tyrosine, is activated by phenylalanine. The lack of activity at low levels of phenylalanine has been attributed to the N-terminus of the protein's regulatory domain acting as an inhibitory peptide by blocking substrate access to the active site. The location of the site at which phenylalanine binds to activate the enzyme is unknown, and both the active site in the catalytic domain and a separate site in the N-terminal regulatory domain have been proposed. Binding of catecholamines to the active-site iron was used to probe the accessibility of the active site. Removal of the regulatory domain increases the rate constants for association of several catecholamines with the wild-type enzyme by ∼2-fold. Binding of phenylalanine in the active site is effectively abolished by mutating the active-site residue Arg270 to lysine. The k(cat)/K(phe) value is down 10⁴ for the mutant enzyme, and the K(m) value for phenylalanine for the mutant enzyme is >0.5 M. Incubation of the R270K enzyme with phenylalanine also results in a 2-fold increase in the rate constants for catecholamine binding. The change in the tryptophan fluorescence emission spectrum seen in the wild-type enzyme upon activation by phenylalanine is also seen with the R270K mutant enzyme in the presence of phenylalanine. Both results establish that activation of PheH by phenylalanine does not require binding of the amino acid in the active site. This is consistent with a separate allosteric site, likely in the regulatory domain.

  4. Changes in active site histidine hydrogen bonding trigger cryptochrome activation.

    PubMed

    Ganguly, Abir; Manahan, Craig C; Top, Deniz; Yee, Estella F; Lin, Changfan; Young, Michael W; Thiel, Walter; Crane, Brian R

    2016-09-06

    Cryptochrome (CRY) is the principal light sensor of the insect circadian clock. Photoreduction of the Drosophila CRY (dCRY) flavin cofactor to the anionic semiquinone (ASQ) restructures a C-terminal tail helix (CTT) that otherwise inhibits interactions with targets that include the clock protein Timeless (TIM). All-atom molecular dynamics (MD) simulations indicate that flavin reduction destabilizes the CTT, which undergoes large-scale conformational changes (the CTT release) on short (25 ns) timescales. The CTT release correlates with the conformation and protonation state of conserved His378, which resides between the CTT and the flavin cofactor. Poisson-Boltzmann calculations indicate that flavin reduction substantially increases the His378 pKa Consistent with coupling between ASQ formation and His378 protonation, dCRY displays reduced photoreduction rates with increasing pH; however, His378Asn/Arg variants show no such pH dependence. Replica-exchange MD simulations also support CTT release mediated by changes in His378 hydrogen bonding and verify other responsive regions of the protein previously identified by proteolytic sensitivity assays. His378 dCRY variants show varying abilities to light-activate TIM and undergo self-degradation in cellular assays. Surprisingly, His378Arg/Lys variants do not degrade in light despite maintaining reactivity toward TIM, thereby implicating different conformational responses in these two functions. Thus, the dCRY photosensory mechanism involves flavin photoreduction coupled to protonation of His378, whose perturbed hydrogen-bonding pattern alters the CTT and surrounding regions.

  5. Changes in active site histidine hydrogen bonding trigger cryptochrome activation

    PubMed Central

    Ganguly, Abir; Manahan, Craig C.; Top, Deniz; Yee, Estella F.; Lin, Changfan; Young, Michael W.; Thiel, Walter; Crane, Brian R.

    2016-01-01

    Cryptochrome (CRY) is the principal light sensor of the insect circadian clock. Photoreduction of the Drosophila CRY (dCRY) flavin cofactor to the anionic semiquinone (ASQ) restructures a C-terminal tail helix (CTT) that otherwise inhibits interactions with targets that include the clock protein Timeless (TIM). All-atom molecular dynamics (MD) simulations indicate that flavin reduction destabilizes the CTT, which undergoes large-scale conformational changes (the CTT release) on short (25 ns) timescales. The CTT release correlates with the conformation and protonation state of conserved His378, which resides between the CTT and the flavin cofactor. Poisson-Boltzmann calculations indicate that flavin reduction substantially increases the His378 pKa. Consistent with coupling between ASQ formation and His378 protonation, dCRY displays reduced photoreduction rates with increasing pH; however, His378Asn/Arg variants show no such pH dependence. Replica-exchange MD simulations also support CTT release mediated by changes in His378 hydrogen bonding and verify other responsive regions of the protein previously identified by proteolytic sensitivity assays. His378 dCRY variants show varying abilities to light-activate TIM and undergo self-degradation in cellular assays. Surprisingly, His378Arg/Lys variants do not degrade in light despite maintaining reactivity toward TIM, thereby implicating different conformational responses in these two functions. Thus, the dCRY photosensory mechanism involves flavin photoreduction coupled to protonation of His378, whose perturbed hydrogen-bonding pattern alters the CTT and surrounding regions. PMID:27551082

  6. Diagnosing causes of cloud parameterization deficiencies using ARM measurements over SGP site

    SciTech Connect

    Wu, W.; Liu, Y.; Betts, A. K.

    2010-03-15

    Decade-long continuous surface-based measurements at Great Southern Plains (SGP) collected by the US Department of Energy’s Atmospheric Radiation Measurement (ARM) Climate Research Facility are first used to evaluate the three major reanalyses (i.e., ERA-Interim, NCEP/NCAR Reanalysis I and NCEP/DOE Reanalysis II) to identify model biases in simulating surface shortwave cloud forcing and total cloud fraction. The results show large systematic lower biases in the modeled surface shortwave cloud forcing and cloud fraction from all the three reanalysis datasets. Then we focus on diagnosing the causes of these model biases using the Active Remote Sensing of Clouds (ARSCL) products (e.g., vertical distribution of cloud fraction, cloud-base and cloud-top heights, and cloud optical depth) and meteorological measurements (temperature, humidity and stability). Efforts are made to couple cloud properties with boundary processes in the diagnosis.

  7. Probing the promiscuous active site of myo-inositol dehydrogenase using synthetic substrates, homology modeling, and active site modification.

    PubMed

    Daniellou, Richard; Zheng, Hongyan; Langill, David M; Sanders, David A R; Palmer, David R J

    2007-06-26

    The active site of myo-inositol dehydrogenase (IDH, EC 1.1.1.18) from Bacillus subtilis recognizes a variety of mono- and disaccharides, as well as 1l-4-O-substituted inositol derivatives. It catalyzes the NAD+-dependent oxidation of the axial alcohol of these substrates with comparable kinetic constants. We have found that 4-O-p-toluenesulfonyl-myo-inositol does not act as a substrate for IDH, in contrast to structurally similar compounds such as those bearing substituted benzyl substituents in the same position. X-ray crystallographic analysis of 4-O-p-toluenesulfonyl-myo-inositol and 4-O-(2-naphthyl)methyl-myo-inositol, which is a substrate for IDH, shows a distinct difference in the preferred conformation of the aryl substituent. Conformational analysis of known substrates of IDH suggests that this conformational difference may account for the difference in reactivity of 4-O-p-toluenesulfonyl-myo-inositol in the presence of IDH. A sequence alignment of IDH with the homologous glucose-fructose oxidoreductase allowed the construction of an homology model of inositol dehydrogenase, to which NADH and 4-O-benzyl-scyllo-inosose were docked and the active site energy minimized. The active site model is consistent with all experimental results and suggests that a conserved tyrosine-glycine-tyrosine motif forms the hydrophobic pocket adjoining the site of inositol recognition. Y233F and Y235F retain activity, while Y233R and Y235R do not. A histidine-aspartate pair, H176 and D172, are proposed to act as a dyad in which H176 is the active site acid/base. The enzyme is inactivated by diethyl pyrocarbonate, and the mutants H176A and D172N show a marked loss of activity. Kinetic isotope effect experiments with D172N indicate that chemistry is rate-determining for this mutant.

  8. The active site structure and mechanism of phosphoenolpyruvate utilizing enzymes

    SciTech Connect

    Cheng, K.C.

    1989-01-01

    Arginine specific reagents showed irreversible inhibition of avian liver mitochondrial phosphoenolpyruvate carboxykinase. Potent protection against modification was elicited by CO{sub 2} or CO{sub 2} in the presence of other substrates. Labeling of enzyme with (7-{sup 14}C) phenylglyoxal showed that 1 or 2 arginines are involved in CO{sub 2} binding and activation. Peptide map studies showed this active site arginine residues is located at position 289. Histidine specific reagents showed pseudo first order inhibition of avian mitochondrial phosphoenolpyruvate carboxykinase activity. The best protection against modification was elicited by IDP or IDP and Mn{sup +2}. One histidine residue is at or near the phosphoenolpyruvate binding site as demonstrated in the increased absorbance at 240 nm and proton relaxation rate studies. Circular dichroism studies reveal that enzyme structure was perturbed by diethylpyrocarbonate modification. Metal binding studies suggest that this enzyme has only one metal binding site. The putative binding sites from several GTP and phosphoenolpyruvate utilizing enzymes are observed in P-enolpyruvate carboxykinase from different species.

  9. Novel splice-site mutation in TTLL5 causes cone dystrophy in a consanguineous family

    PubMed Central

    Dias, Miguel de Sousa; Hamel, Christian P.; Meunier, Isabelle; Varin, Juliette; Blanchard, Steven; Boyard, Fiona; Sahel, José-Alain

    2017-01-01

    Purpose To report the clinical and genetic findings of one family with autosomal recessive cone dystrophy (CD) and to identify the causative mutation. Methods An institutional study of three family members from two generations. The clinical examination included best-corrected Snellen visual acuity measurement, fundoscopy, the Farnsworth D-15 color vision test, a full-field electroretinogram (ERG) that incorporated the International Society for Clinical Electrophysiology of Vision standards and methodology, fundus autofluorescence (FAF) and infrared (IR), and spectral-domain optical coherence tomography (SD-OCT). Genetic findings were achieved with DNA analysis using whole exome sequencing (WES) and Sanger sequencing. Results The proband, a 9-year-old boy, presented with a condition that appeared to be congenital and stationary. The clinical presentation initially reflected incomplete congenital stationary night blindness (icCSNB) because of myopia, a decrease in visual acuity, abnormal oscillatory potentials, and reduced amplitudes on the 30 Hz flicker ERG but was atypical because there were no clear electronegative responses. However, no disease-causing mutations in the genes underlying icCSNB were identified. Following WES analysis of family members, a homozygous splice-site mutation in intron 3 of TTLL5 (c.182–3_182–1delinsAA) was found cosegregating within the phenotype in the family. Conclusions The distinction between icCSNB and CD phenotypes is not always straightforward in young patients. The patient was quite young, which most likely explains why the progression of the CD was not obvious. WES analysis provided prompt diagnosis for this family; thus, the use of this technique to refine the diagnosis is highlighted in this study. PMID:28356705

  10. Familial glucocorticoid resistance caused by a splice site deletion in the human glucocorticoid receptor gene

    SciTech Connect

    Karl, M.; Lamberts, S.W.J.; Detera-Wadleigh, S.D.; Encio, I.J.; Stratakis, C.A.; Hurley, D.M.; Accili, D.; Chrousos, G.P. Erasmus Univ. of Rotterdam )

    1993-03-01

    The clinical syndrome of generalized, compensated glucocorticoid resistance is characterized by increased cortisol secretion without clinical evidence of hyper- or hypocortisolism, and manifestations of androgen and/or mineralocorticoid excess. This condition results from partial failure of the glucocorticoid receptor (GR) to modulate transcription of its target genes. The authors studied the molecular mechanisms of this syndrome in a Dutch kindred, whose affected members had hypercortisolism and approximately half of normal GRs, and whose proband was a young woman with manifestations of hyperandrogenism. Using the polymerase chain reaction to amplify and sequence each of the nine exons of the GR gene [alpha], along with their 5[prime]- and 3[prime]-flanking regions, the authors identified a 4-base deletion at the 3[prime]-boundary of exon 6 in one GR allele ([Delta][sub 4]), which removed a donor splice site in all three affected members studied. In contrast, the sequence of exon 6 in the two unaffected siblings was normal. A single nucleotide substitution causing an amino acid substitution in the amino terminal domain of the GR (asparagine to serine, codon 363) was also discovered in exon 2 of the other allele (G[sub 1220]) in the proband, in one of her affected brothers and in her unaffected sister. This deletion in the glucocorticoid receptor gene was associated with the expression of only one allele and a decrease of GR protein by 50% in affected members of this glucocorticoid resistant family. The mutation identified in exon 2 did not segregate with the disease and appears to be of no functional significance. The presence of the null allele was apparently compensated for by increased cortisol production at the expense of concurrent hyperandrogenism. 40 refs., 3 figs.

  11. EXPOSURE TO ACROLEIN BY INHALATION CAUSES PLATELET ACTIVATION

    PubMed Central

    Sithu, Srinivas D; Srivastava, Sanjay; Siddiqui, Maqsood A; Vladykovskaya, Elena; Riggs, Daniel W; Conklin, Daniel J; Haberzettl, Petra; O’Toole, Timothy E; Bhatnagar, Aruni; D’Souza, Stanley E

    2010-01-01

    Acrolein is a common air pollutant that is present in high concentrations in wood, cotton, and tobacco smoke, automobile exhaust and industrial waste and emissions. Exposure to acrolein containing environmental pollutants such as tobacco smoke and automobile exhaust has been linked to the activation of the coagulation and hemostasis pathways and thereby to the predisposition of thrombotic events in human. To examine the effects of acrolein on platelets, adult male C57Bl/6 mice were subjected acute (5 ppm for 6 h) or sub-chronic (1 ppm, 6h/day for 4 days) acrolein inhalation exposures. The acute exposure to acrolein did not cause pulmonary inflammation and oxidative stress, dyslipidemia or induce liver damage or muscle injury. Platelet GSH levels in acrolein-exposed mice were comparable to controls, but acrolein-exposure increased the abundance of protein-acrolein adducts in platelets. Platelets isolated from mice, exposed to both acute and sub-chronic acrolein levels, showed increased ADP-induced platelet aggregation. Exposure to acrolein also led to an increase in the indices of platelet activation such as the formation of platelet-leukocyte aggregates in the blood, plasma PF4 levels, and increased platelet-fibrinogen binding. The bleeding time was decreased in acrolein exposed mice. Plasma levels of PF4 were also increased in mice exposed to environmental tobacco smoke. Similar to inhalation exposure, acrolein feeding to mice also increased platelet activation and established a pro-thrombotic state in mice. Together, our data suggest that acrolein is an important contributing factor to the pro-thrombotic risk in human exposure to pollutants such as tobacco smoke or automobile exhaust, or through dietary consumption. PMID:20678513

  12. Exposure to acrolein by inhalation causes platelet activation

    SciTech Connect

    Sithu, Srinivas D.; Srivastava, Sanjay; Siddiqui, Maqsood A.; Vladykovskaya, Elena; Riggs, Daniel W.; Conklin, Daniel J.; Haberzettl, Petra; O'Toole, Timothy E.; Bhatnagar, Aruni; D'Souza, Stanley E.

    2010-10-15

    Acrolein is a common air pollutant that is present in high concentrations in wood, cotton, and tobacco smoke, automobile exhaust and industrial waste and emissions. Exposure to acrolein containing environmental pollutants such as tobacco smoke and automobile exhaust has been linked to the activation of the coagulation and hemostasis pathways and thereby to the predisposition of thrombotic events in human. To examine the effects of acrolein on platelets, adult male C57Bl/6 mice were subjected acute (5 ppm for 6 h) or sub-chronic (1 ppm, 6 h/day for 4 days) acrolein inhalation exposures. The acute exposure to acrolein did not cause pulmonary inflammation and oxidative stress, dyslipidemia or induce liver damage or muscle injury. Platelet GSH levels in acrolein-exposed mice were comparable to controls, but acrolein-exposure increased the abundance of protein-acrolein adducts in platelets. Platelets isolated from mice, exposed to both acute and sub-chronic acrolein levels, showed increased ADP-induced platelet aggregation. Exposure to acrolein also led to an increase in the indices of platelet activation such as the formation of platelet-leukocyte aggregates in the blood, plasma PF4 levels, and increased platelet-fibrinogen binding. The bleeding time was decreased in acrolein exposed mice. Plasma levels of PF4 were also increased in mice exposed to environmental tobacco smoke. Similar to inhalation exposure, acrolein feeding to mice also increased platelet activation and established a pro-thrombotic state in mice. Together, our data suggest that acrolein is an important contributing factor to the pro-thrombotic risk in human exposure to pollutants such as tobacco smoke or automobile exhaust, or through dietary consumption.

  13. Exposure to acrolein by inhalation causes platelet activation.

    PubMed

    Sithu, Srinivas D; Srivastava, Sanjay; Siddiqui, Maqsood A; Vladykovskaya, Elena; Riggs, Daniel W; Conklin, Daniel J; Haberzettl, Petra; O'Toole, Timothy E; Bhatnagar, Aruni; D'Souza, Stanley E

    2010-10-15

    Acrolein is a common air pollutant that is present in high concentrations in wood, cotton, and tobacco smoke, automobile exhaust and industrial waste and emissions. Exposure to acrolein containing environmental pollutants such as tobacco smoke and automobile exhaust has been linked to the activation of the coagulation and hemostasis pathways and thereby to the predisposition of thrombotic events in human. To examine the effects of acrolein on platelets, adult male C57Bl/6 mice were subjected acute (5ppm for 6h) or sub-chronic (1ppm, 6h/day for 4days) acrolein inhalation exposures. The acute exposure to acrolein did not cause pulmonary inflammation and oxidative stress, dyslipidemia or induce liver damage or muscle injury. Platelet GSH levels in acrolein-exposed mice were comparable to controls, but acrolein-exposure increased the abundance of protein-acrolein adducts in platelets. Platelets isolated from mice, exposed to both acute and sub-chronic acrolein levels, showed increased ADP-induced platelet aggregation. Exposure to acrolein also led to an increase in the indices of platelet activation such as the formation of platelet-leukocyte aggregates in the blood, plasma PF4 levels, and increased platelet-fibrinogen binding. The bleeding time was decreased in acrolein exposed mice. Plasma levels of PF4 were also increased in mice exposed to environmental tobacco smoke. Similar to inhalation exposure, acrolein feeding to mice also increased platelet activation and established a pro-thrombotic state in mice. Together, our data suggest that acrolein is an important contributing factor to the pro-thrombotic risk in human exposure to pollutants such as tobacco smoke or automobile exhaust, or through dietary consumption.

  14. Lamellipodial actin mechanically links myosin activity with adhesion site formation

    PubMed Central

    Giannone, Gregory; Dubin-Thaler, Benjamin; Rossier, Olivier; Cai, Yunfei; Chaga, Oleg; Jiang, Guoying; Beaver, William; Döbereiner, Hans-Günther; Freund, Yoav; Borisy, Gary; Sheetz, Michael P.

    2013-01-01

    Summary Cell motility proceeds by cycles of edge protrusion, adhesion and retraction. Whether these functions are coordinated by biochemical or biomechanical processes is unknown. We find that myosin II pulls the rear of the lamellipodial actin network, causing upward bending, edge retraction and initiation of new adhesion sites. The network then separates from the edge and condenses over the myosin. Protrusion resumes as lamellipodial actin regenerates from the front and extends rearward until it reaches newly assembled myosin, initiating the next cycle. Upward bending, observed by evanescence and electron microscopy, results in ruffle formation when adhesion strength is low. Correlative fluorescence and electron microscopy shows that the regenerating lamellipodium forms a cohesive, separable layer of actin above the lamellum. Thus, actin polymerization periodically builds a mechanical link, the lamellipodium, connecting myosin motors with the initiation of adhesion sites, suggesting that the major functions driving motility are coordinated by a biomechanical process. PMID:17289574

  15. Identification of Ice Nucleation Active Sites on Silicate Dust Particles

    NASA Astrophysics Data System (ADS)

    Zolles, Tobias; Burkart, Julia; Häusler, Thomas; Pummer, Bernhard; Hitzenberger, Regina; Grothe, Hinrich

    2015-04-01

    Mineral dusts originating from Earth's crust are known to be important atmospheric ice nuclei. In agreement with earlier studies, feldspar was found as the most active of the tested natural mineral dusts [1-3]. Nevertheless, among those structures K-feldspar showed by far the highest ice nucleation activity. In this study, the reasons for its activity and the difference in the activity of the different feldspars were investigated in closer details. Conclusions are drawn from scanning electron microscopy, X-ray powder diffraction, infrared spectroscopy, and oil-immersion freezing experiments. We give a potential explanation of the increased ice nucleation activity of K-feldspar. The ice nucleating sites are very much dependent on the alkali ion present by altering the water structure and the feldspar surface. The higher activity of K-feldspar can be attributed to the presence of potassium ions on the surface and surface bilayer. The alkali-ions have different hydration shells and thus an influence on the ice nucleation activity of feldspar. Chaotropic behavior of Calcium and Sodium ions are lowering the ice nucleation potential of the other feldspars, while kosmotropic Potassium has a neutral or even positive effect. Furthermore we investigated the influence of milling onto the ice nucleation of quartz particles. The ice nucleation activity can be increased by mechanical milling, by introducing more molecular, nucleation active defects to the particle surface. This effect is larger than expected by plane surface increase. [1] Atkinson et al. The Importance of Feldspar for Ice Nucleation by Mineral Dust in Mixed-Phase Clouds. Nature 2013, 498, 355-358. [2] Yakobi-Hancock et al.. Feldspar Minerals as Efficient Deposition Ice Nuclei. Atmos. Chem. Phys. 2013, 13, 11175-11185. [3] Zolles et al. Identification of Ice Nucleation Active Sites on Feldspar Dust Particles. J. Phys. Chem. A 2015 accepted.

  16. An active site mutation increases the polymerase activity of the guinea pig-lethal Marburg virus.

    PubMed

    Koehler, Alexander; Kolesnikova, Larissa; Becker, Stephan

    2016-10-01

    Marburg virus (MARV) causes severe, often fatal, disease in humans and transient illness in rodents. Sequential passaging of MARV in guinea pigs resulted in selection of a lethal virus containing 4 aa changes. A D184N mutation in VP40 (VP40D184N), which leads to a species-specific gain of viral fitness, and three mutations in the active site of viral RNA-dependent RNA polymerase L, which were investigated in the present study for functional significance in human and guinea pig cells. The transcription/replication activity of L mutants was strongly enhanced by a substitution at position 741 (S741C), and inhibited by other substitutions (D758A and A759D) in both species. The polymerase activity of L carrying the S741C substitution was eightfold higher in guinea pig cells than in human cells upon co-expression with VP40D184N, suggesting that the additive effect of the two mutations provides MARV a replicative advantage in the new host.

  17. Face the Edges: Catalytic Active Sites of Nanomaterials

    PubMed Central

    Ni, Bing

    2015-01-01

    Edges are special sites in nanomaterials. The atoms residing on the edges have different environments compared to those in other parts of a nanomaterial and, therefore, they may have different properties. Here, recent progress in nanomaterial fields is summarized from the viewpoint of the edges. Typically, edge sites in MoS2 or metals, other than surface atoms, can perform as active centers for catalytic reactions, so the method to enhance performance lies in the optimization of the edge structures. The edges of multicomponent interfaces present even more possibilities to enhance the activities of nanomaterials. Nanoframes and ultrathin nanowires have similarities to conventional edges of nanoparticles, the application of which as catalysts can help to reduce the use of costly materials. Looking beyond this, the edge structures of graphene are also essential for their properties. In short, the edge structure can influence many properties of materials. PMID:27980960

  18. Mortality from external causes in Africa and Asia: evidence from INDEPTH Health and Demographic Surveillance System Sites

    PubMed Central

    Streatfield, P. Kim; Khan, Wasif A.; Bhuiya, Abbas; Hanifi, Syed M.A.; Alam, Nurul; Diboulo, Eric; Niamba, Louis; Sié, Ali; Lankoandé, Bruno; Millogo, Roch; Soura, Abdramane B.; Bonfoh, Bassirou; Kone, Siaka; Ngoran, Eliezer K.; Utzinger, Juerg; Ashebir, Yemane; Melaku, Yohannes A.; Weldearegawi, Berhe; Gomez, Pierre; Jasseh, Momodou; Azongo, Daniel; Oduro, Abraham; Wak, George; Wontuo, Peter; Attaa-Pomaa, Mary; Gyapong, Margaret; Manyeh, Alfred K.; Kant, Shashi; Misra, Puneet; Rai, Sanjay K.; Juvekar, Sanjay; Patil, Rutuja; Wahab, Abdul; Wilopo, Siswanto; Bauni, Evasius; Mochamah, George; Ndila, Carolyne; Williams, Thomas N.; Khaggayi, Christine; Nyaguara, Amek; Obor, David; Odhiambo, Frank O.; Ezeh, Alex; Oti, Samuel; Wamukoya, Marylene; Chihana, Menard; Crampin, Amelia; Collinson, Mark A.; Kabudula, Chodziwadziwa W.; Wagner, Ryan; Herbst, Kobus; Mossong, Joël; Emina, Jacques B.O.; Sankoh, Osman A.; Byass, Peter

    2014-01-01

    Background Mortality from external causes, of all kinds, is an important component of overall mortality on a global basis. However, these deaths, like others in Africa and Asia, are often not counted or documented on an individual basis. Overviews of the state of external cause mortality in Africa and Asia are therefore based on uncertain information. The INDEPTH Network maintains longitudinal surveillance, including cause of death, at population sites across Africa and Asia, which offers important opportunities to document external cause mortality at the population level across a range of settings. Objective To describe patterns of mortality from external causes at INDEPTH Network sites across Africa and Asia, according to the WHO 2012 verbal autopsy (VA) cause categories. Design All deaths at INDEPTH sites are routinely registered and followed up with VA interviews. For this study, VA archives were transformed into the WHO 2012 VA standard format and processed using the InterVA-4 model to assign cause of death. Routine surveillance data also provide person-time denominators for mortality rates. Results A total of 5,884 deaths due to external causes were documented over 11,828,253 person-years. Approximately one-quarter of those deaths were to children younger than 15 years. Causes of death were dominated by childhood drowning in Bangladesh, and by transport-related deaths and intentional injuries elsewhere. Detailed mortality rates are presented by cause of death, age group, and sex. Conclusions The patterns of external cause mortality found here generally corresponded with expectations and other sources of information, but they fill some important gaps in population-based mortality data. They provide an important source of information to inform potentially preventive intervention designs. PMID:25377327

  19. Active sites in char gasification: Final technical report

    SciTech Connect

    Wojtowicz, M.; Lilly, W.D.; Perkins, M.T.; Hradil, G.; Calo, J.M.; Suuberg, E.M.

    1987-09-01

    Among the key variables in the design of gasifiers and combustors is the reactivity of the chars which must be gasified or combusted. Significant loss of unburned char is unacceptable in virtually any process; the provision of sufficient residence time for complete conversion is essential. A very wide range of reactivities are observed, depending upon the nature of the char in a process. The current work focuses on furthering the understanding of gasification reactivities of chars. It has been well established that the reactivity of char to gasification generally depends upon three principal factors: (1) the concentration of ''active sites'' in the char; (2) mass transfer within the char; and (3) the type and concentration of catalytic impurities in the char. The present study primarily addresses the first factor. The subject of this research is the origin, nature, and fate of active sites in chars derived from parent hydrocarbons with coal-like structure. The nature and number of the active sites and their reactivity towards oxygen are examined in ''model'' chars derived from phenol-formaldehyde type resins. How the active sites are lost by the process of thermal annealing during heat treatment of chars are studied, and actual rate for the annealing process is derived. Since intrinsic char reactivities are of primary interest in the present study, a fair amount of attention was given to the model char synthesis and handling so that the effect of catalytic impurities and oxygen-containing functional groups in the chemical structure of the material were minimized, if not completely eliminated. The project would not be considered complete without comparing characteristic features of synthetic chars with kinetic behavior exhibited by natural chars, including coal chars.

  20. Testing the applicability of rapid on-site enzymatic activity detection for surface water monitoring

    NASA Astrophysics Data System (ADS)

    Stadler, Philipp; Vogl, Wolfgang; Juri, Koschelnik; Markus, Epp; Maximilian, Lackner; Markus, Oismüller; Monika, Kumpan; Peter, Strauss; Regina, Sommer; Gabriela, Ryzinska-Paier; Farnleitner Andreas, H.; Matthias, Zessner

    2015-04-01

    On-site detection of enzymatic activities has been suggested as a rapid surrogate for microbiological pollution monitoring of water resources (e.g. using glucuronidases, galactosidases, esterases). Due to the possible short measuring intervals enzymatic methods have high potential as near-real time water quality monitoring tools. This presentation describes results from a long termed field test. For twelve months, two ColiMinder devices (Vienna Water Monitoring, Austria) for on-site determination of enzymatic activity were tested for stream water monitoring at the experimental catchment HOAL (Hydrological Open Air Laboratory, Center for Water Resource Systems, Vienna University of Technology). The devices were overall able to follow and reflect the diverse hydrological and microbiological conditions of the monitored stream during the test period. Continuous data in high temporal resolution captured the course of enzymatic activity in stream water during diverse rainfall events. The method also proofed sensitive enough to determine diurnal fluctuations of enzymatic activity in stream water during dry periods. The method was able to capture a seasonal trend of enzymatic activity in stream water that matches the results gained from Colilert18 analysis for E. coli and coliform bacteria of monthly grab samples. Furthermore the comparison of ColiMinder data with measurements gained at the same test site with devices using the same method but having different construction design (BACTcontrol, microLAN) showed consistent measuring results. Comparative analysis showed significant differences between measured enzymatic activity (modified fishman units and pmol/min/100ml) and cultivation based analyses (most probable number, colony forming unit). Methods of enzymatic activity measures are capable to detect ideally the enzymatic activity caused by all active target bacteria members, including VBNC (viable but nonculturable) while cultivation based methods cannot detect VBNC

  1. Active site amino acid sequence of human factor D.

    PubMed

    Davis, A E

    1980-08-01

    Factor D was isolated from human plasma by chromatography on CM-Sephadex C50, Sephadex G-75, and hydroxylapatite. Digestion of reduced, S-carboxymethylated factor D with cyanogen bromide resulted in three peptides which were isolated by chromatography on Sephadex G-75 (superfine) equilibrated in 20% formic acid. NH2-Terminal sequences were determined by automated Edman degradation with a Beckman 890C sequencer using a 0.1 M Quadrol program. The smallest peptide (CNBr III) consisted of the NH2-terminal 14 amino acids. The other two peptides had molecular weights of 17,000 (CNBr I) and 7000 (CNBr II). Overlap of the NH2-terminal sequence of factor D with the NH2-terminal sequence of CNBr I established the order of the peptides. The NH2-terminal 53 residues of factor D are somewhat more homologous with the group-specific protease of rat intestine than with other serine proteases. The NH2-terminal sequence of CNBr II revealed the active site serine of factor D. The typical serine protease active site sequence (Gly-Asp-Ser-Gly-Gly-Pro was found at residues 12-17. The region surrounding the active site serine does not appear to be more highly homologous with any one of the other serine proteases. The structural data obtained point out the similarities between factor D and the other proteases. However, complete definition of the degree of relationship between factor D and other proteases will require determination of the remainder of the primary structure.

  2. Brownian aggregation rate of colloid particles with several active sites

    SciTech Connect

    Nekrasov, Vyacheslav M.; Yurkin, Maxim A.; Chernyshev, Andrei V.; Polshchitsin, Alexey A.; Yakovleva, Galina E.; Maltsev, Valeri P.

    2014-08-14

    We theoretically analyze the aggregation kinetics of colloid particles with several active sites. Such particles (so-called “patchy particles”) are well known as chemically anisotropic reactants, but the corresponding rate constant of their aggregation has not yet been established in a convenient analytical form. Using kinematic approximation for the diffusion problem, we derived an analytical formula for the diffusion-controlled reaction rate constant between two colloid particles (or clusters) with several small active sites under the following assumptions: the relative translational motion is Brownian diffusion, and the isotropic stochastic reorientation of each particle is Markovian and arbitrarily correlated. This formula was shown to produce accurate results in comparison with more sophisticated approaches. Also, to account for the case of a low number of active sites per particle we used Monte Carlo stochastic algorithm based on Gillespie method. Simulations showed that such discrete model is required when this number is less than 10. Finally, we applied the developed approach to the simulation of immunoagglutination, assuming that the formed clusters have fractal structure.

  3. [Mechanism of arginine deiminase activity by site-directed mutagenesis].

    PubMed

    Li, Lifeng; Ni, Ye; Sun, Zhihao

    2012-04-01

    Arginine deiminase (ADI) has been studied as a potential anti-cancer agent for inhibiting arginine-auxotrophic tumors (such as melanomas and hepatocellular carcinomas) in phase III clinical trials. In this work, we studied the molecular mechanism of arginine deiminase activity by site-directed mutagenesis. Three mutation sites, A128, H404 and 1410, were introduced into wild-type ADI gene by QuikChange site-directed mutagenesis method, and four ADI mutants M1 (A128T), M2 (H404R), M3 (I410L), and M4 (A128T, H404R) were obtained. The ADI mutants were individually expressed in Escherichia coli BL21 (DE3), and the enzymatic properties of the purified mutant proteins were determined. The results show that both A128T and H404R had enhanced optimum pH, higher activity and stability of ADI under physiological condition (pH 7.4), as well as reduced K(m) value. This study provides an insight into the molecular mechanism of the ADI activity, and also the experimental evidence for the rational protein evolution in the future.

  4. Potential sites of CFTR activation by tyrosine kinases

    PubMed Central

    Billet, Arnaud; Jia, Yanlin; Jensen, Timothy J.; Hou, Yue-Xian; Chang, Xiu-Bao; Riordan, John R.; Hanrahan, John W.

    2016-01-01

    ABSTRACT The CFTR chloride channel is tightly regulated by phosphorylation at multiple serine residues. Recently it has been proposed that its activity is also regulated by tyrosine kinases, however the tyrosine phosphorylation sites remain to be identified. In this study we examined 2 candidate tyrosine residues near the boundary between the first nucleotide binding domain and the R domain, a region which is important for channel function but devoid of PKA consensus sequences. Mutating tyrosines at positions 625 and 627 dramatically reduced responses to Src or Pyk2 without altering the activation by PKA, suggesting they may contribute to CFTR regulation. PMID:26645934

  5. A mutational analysis of the active site of human type II inosine 5'-monophosphate dehydrogenase.

    PubMed

    Futer, Olga; Sintchak, Michael D; Caron, Paul R; Nimmesgern, Elmar; DeCenzo, Maureen T; Livingston, David J; Raybuck, Scott A

    2002-01-31

    The oxidation of IMP to XMP is the rate-limiting step in the de novo synthesis of guanine ribonucleotides. This NAD-dependent reaction is catalyzed by the enzyme inosine monophosphate dehydrogenase (IMPDH). Based upon the recent structural determination of IMPDH complexed to oxidized IMP (XMP*) and the potent uncompetitive inhibitor mycophenolic acid (MPA), we have selected active site residues and prepared mutants of human type II IMPDH. The catalytic parameters of these mutants were determined. Mutations G326A, D364A, and the active site nucleophile C331A all abolish enzyme activity to less than 0.1% of wild type. These residues line the IMP binding pocket and are necessary for correct positioning of the substrate, Asp364 serving to anchor the ribose ring of the nucleotide. In the MPA/NAD binding site, significant loss of activity was seen by mutation of any residue of the triad Arg322, Asn303, Asp274 which form a hydrogen bonding network lining one side of this pocket. From a model of NAD bound to the active site consistent with the mutational data, we propose that these resides are important in binding the ribose ring of the nicotinamide substrate. Additionally, mutations in the pair Thr333, Gln441, which lies close to the xanthine ring, cause a significant drop in the catalytic activity of IMPDH. It is proposed that these residues serve to deliver the catalytic water molecule required for hydrolysis of the cysteine-bound XMP* intermediate formed after oxidation by NAD.

  6. MSK1 activity is controlled by multiple phosphorylation sites

    PubMed Central

    McCOY, Claire E.; Campbell, David G.; Deak, Maria; Bloomberg, Graham B.; Arthur, J. Simon C.

    2004-01-01

    MSK1 (mitogen- and stress-activated protein kinase) is a kinase activated in cells downstream of both the ERK1/2 (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) cascades. In the present study, we show that, in addition to being phosphorylated on Thr-581 and Ser-360 by ERK1/2 or p38, MSK1 can autophosphorylate on at least six sites: Ser-212, Ser-376, Ser-381, Ser-750, Ser-752 and Ser-758. Of these sites, the N-terminal T-loop residue Ser-212 and the ‘hydrophobic motif’ Ser-376 are phosphorylated by the C-terminal kinase domain of MSK1, and their phosphorylation is essential for the catalytic activity of the N-terminal kinase domain of MSK1 and therefore for the phosphorylation of MSK1 substrates in vitro. Ser-381 is also phosphorylated by the C-terminal kinase domain, and mutation of Ser-381 decreases MSK1 activity, probably through the inhibition of Ser-376 phosphorylation. Ser-750, Ser-752 and Ser-758 are phosphorylated by the N-terminal kinase domain; however, their function is not known. The activation of MSK1 in cells therefore requires the activation of the ERK1/2 or p38 MAPK cascades and does not appear to require additional signalling inputs. This is in contrast with the closely related RSK (p90 ribosomal S6 kinase) proteins, whose activity requires phosphorylation by PDK1 (3-phosphoinositide-dependent protein kinase 1) in addition to phosphorylation by ERK1/2. PMID:15568999

  7. USA300 genotype community-associated methicillin-resistant Staphylococcus aureus as a cause of surgical site infections.

    PubMed

    Patel, Mukesh; Kumar, Ritu A; Stamm, Alan M; Hoesley, Craig J; Moser, Stephen A; Waites, Ken B

    2007-10-01

    Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains are increasingly recovered from nosocomial settings. We conducted a retrospective study of surgical site infections (SSI) during 2004 and 2005 to determine the prevalence of CA-MRSA; 57% of MRSA strains tested belonged to the USA300 genotype. CA-MRSA has become a prominent cause of SSI at our institution.

  8. Current activities handbook: formerly utilized sites remedial action program

    SciTech Connect

    1981-02-27

    This volume is one of a series produced under contract with the DOE, by Politech Corporation to develop a legislative and regulatory data base to assist the FUSRAP management in addressing the institutional and socioeconomic issues involved in carrying out the Formerly Utilized Sites Remedial Action Program. This Information Handbook series contains information about all relevant government agencies at the Federal and state levels, the pertinent programs they administer, each affected state legislature, and current Federal and state legislative and regulatory initiatives. This volume is a compilation of information about the activities each of the thirteen state legislatures potentially affected by the Formerly Utilized Sites Remedial Action Program. It contains a description of the state legislative procedural rules and a schedule of each legislative session; a summary of pending relevant legislation; the name and telephone number of legislative and state agency contacts; and the full text of all bills identified.

  9. Vitamin K epoxide reductase: homology, active site and catalytic mechanism.

    PubMed

    Goodstadt, Leo; Ponting, Chris P

    2004-06-01

    Vitamin K epoxide reductase (VKOR) recycles reduced vitamin K, which is used subsequently as a co-factor in the gamma-carboxylation of glutamic acid residues in blood coagulation enzymes. VKORC1, a subunit of the VKOR complex, has recently been shown to possess this activity. Here, we show that VKORC1 is a member of a large family of predicted enzymes that are present in vertebrates, Drosophila, plants, bacteria and archaea. Four cysteine residues and one residue, which is either serine or threonine, are identified as likely active-site residues. In some plant and bacterial homologues the VKORC1 homologous domain is fused with domains of the thioredoxin family of oxidoreductases. These might reduce disulfide bonds of VKORC1-like enzymes as a prerequisite for their catalytic activities.

  10. Interaction of aspartic acid-104 and proline-287 with the active site of m-calpain.

    PubMed Central

    Arthur, J S; Elce, J S

    1996-01-01

    In an ongoing study of the mechanisms of calpain catalysis and Ca(2+)-induced activation, the effects of Asp-104-->Ser and Pro-287-->Ser large subunit mutations on m-calpain activity, the pH-activity profile, Ca(2+)-sensitivity, and autolysis were measured. The importance of these positions was suggested by sequence comparisons between the calpain and papain families of cysteine proteinases. Asp-104 is adjacent to the active-site Cys-105, and Pro-287 is adjacent to the active-site Asn-286 and probably to the active-site His-262; both Asp-104 and Pro-287 are absolutely conserved in the known calpains, but are replaced by highly conserved serine residues in the papains. The single mutants had approx. 10-15% of wild-type activity, due mainly to a decrease in kcat, since Km was only slightly increased. The Pro-287-->Ser mutation appeared to cause a local perturbation of the catalytic Cys-105/His-262 catalytic ion pair, reducing its efficiency without major effect on the conformation and stability of the enzyme. The Asp-104-->Ser mutation caused a marked narrowing of the pH-activity curve, a 9-fold increase in Ca2+ requirement, and an acceleration of autolysis, when compared with the wild-type enzyme. The results indicated that Asp-104 alters the nature of its interaction with the catalytic ion pair during Ca(2+)-induced conformational change in calpain. This interaction may be direct or indirect, but is important in activation of the enzyme. PMID:8912692

  11. Neuronal hyperactivity causes Na(+)/H(+) exchanger-induced extracellular acidification at active synapses.

    PubMed

    Chiacchiaretta, Martina; Latifi, Shahrzad; Bramini, Mattia; Fadda, Manuela; Fassio, Anna; Benfenati, Fabio; Cesca, Fabrizia

    2017-03-02

    Extracellular pH impacts on neuronal activity, which is in turn an important determinant of extracellular H(+) concentration. The aim of this study is to describe the spatio-temporal dynamics of extracellular pH at synaptic sites during neuronal hyperexcitability. To address this issue we created ex.E(2)GFP, a membrane-targeted extracellular ratiometric pH indicator exquisitely sensitive to acidic shifts. By monitoring ex.E(2)GFP fluorescence in real time in primary cortical neurons we were able to quantify pH fluctuations during network hyperexcitability induced by convulsant drugs or high frequency electrical stimulation. Sustained hyperactivity caused a pH decrease that was reversible upon silencing of neuronal activity and localized to active synapses. This acidic shift was not attributable to the outflow of synaptic vesicle protons into the cleft nor to the activity of membrane-exposed H(+)-vATPase, but rather to the activity of the Na(+)/H(+)-exchanger. Our data demonstrate that extracellular synaptic pH shifts take place during epileptic-like activity of neural cultures, underlying the strict links existing between synaptic activity and synaptic pH. This evidence may contribute to the understanding of the physio-pathological mechanisms associated with hyperexcitability in the epileptic brain.

  12. Identification of covalent active site inhibitors of dengue virus protease

    PubMed Central

    Koh-Stenta, Xiaoying; Joy, Joma; Wang, Si Fang; Kwek, Perlyn Zekui; Wee, John Liang Kuan; Wan, Kah Fei; Gayen, Shovanlal; Chen, Angela Shuyi; Kang, CongBao; Lee, May Ann; Poulsen, Anders; Vasudevan, Subhash G; Hill, Jeffrey; Nacro, Kassoum

    2015-01-01

    Dengue virus (DENV) protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described. PMID:26677315

  13. Human 15-LOX-1 active site mutations alter inhibitor binding and decrease potency.

    PubMed

    Armstrong, Michelle; van Hoorebeke, Christopher; Horn, Thomas; Deschamps, Joshua; Freedman, J Cody; Kalyanaraman, Chakrapani; Jacobson, Matthew P; Holman, Theodore

    2016-11-01

    Human 15-lipoxygenase-1 (h15-LOX-1 or h12/15-LOX) reacts with polyunsaturated fatty acids and produces bioactive lipid derivatives that are implicated in many important human diseases. One such disease is stroke, which is the fifth leading cause of death and the first leading cause of disability in America. The discovery of h15-LOX-1 inhibitors could potentially lead to novel therapeutics in the treatment of stroke, however, little is known about the inhibitor/active site interaction. This study utilizes site-directed mutagenesis, guided in part by molecular modeling, to gain a better structural understanding of inhibitor interactions within the active site. We have generated eight mutants (R402L, R404L, F414I, F414W, E356Q, Q547L, L407A, I417A) of h15-LOX-1 to determine whether these active site residues interact with two h15-LOX-1 inhibitors, ML351 and an ML094 derivative, compound 18. IC50 values and steady-state inhibition kinetics were determined for the eight mutants, with four of the mutants affecting inhibitor potency relative to wild type h15-LOX-1 (F414I, F414W, E356Q and L407A). The data indicate that ML351 and compound 18, bind in a similar manner in the active site to an aromatic pocket close to F414 but have subtle differences in their specific binding modes. This information establishes the binding mode for ML094 and ML351 and will be leveraged to develop next-generation inhibitors.

  14. Causes of variability in light absorption by particles in snow at sites in Idaho and Utah

    NASA Astrophysics Data System (ADS)

    Doherty, Sarah J.; Hegg, Dean A.; Johnson, James E.; Quinn, Patricia K.; Schwarz, Joshua P.; Dang, Cheng; Warren, Stephen G.

    2016-05-01

    A characterization of black carbon (BC) and other light-absorbing particles in snow is presented for three mountain valley sites in Idaho in early 2014 and for one site near Vernal, Utah, in early 2013 and 2014. The focus of the study was on constraining the magnitude and drivers of variations in particulate absorbers in midlatitude U.S. seasonal snow. Mass mixing ratios of BC in newly fallen snow were similar at all three Idaho sites, with a median of 4.7 ± 4.2 ng BC per gram of snow. The median total light-absorbing particulate mixing ratios in new snow, expressed as an equivalent mixing ratio of BC, was 18 ± 23 ng g-1. At the Utah site, which is near sources of both fossil fuel and dust, the mixing ratios of BC varied from 7 to 45 ng g-1 across seven new snowfall samples, and the BC-equivalent mixing ratios varied from 9 to 1500 ng g-1. At all sites, dry deposition and in-snow processes increase the mixing ratio of BC by up to an order of magnitude and increase the mixing ratio of all light-absorbing particulates by up to 2 orders of magnitude, highlighting the importance of capturing these processes for accurately representing snow albedo in climate models. Spatial variability at a range of scales is found to be considerably smaller than the temporal variations at a given site, with implications for the representativeness of field samples used in observation/model comparisons.

  15. Target-classification approach applied to active UXO sites

    NASA Astrophysics Data System (ADS)

    Shubitidze, F.; Fernández, J. P.; Shamatava, Irma; Barrowes, B. E.; O'Neill, K.

    2013-06-01

    This study is designed to illustrate the discrimination performance at two UXO active sites (Oklahoma's Fort Sill and the Massachusetts Military Reservation) of a set of advanced electromagnetic induction (EMI) inversion/discrimination models which include the orthonormalized volume magnetic source (ONVMS), joint diagonalization (JD), and differential evolution (DE) approaches and whose power and flexibility greatly exceed those of the simple dipole model. The Fort Sill site is highly contaminated by a mix of the following types of munitions: 37-mm target practice tracers, 60-mm illumination mortars, 75-mm and 4.5'' projectiles, 3.5'', 2.36'', and LAAW rockets, antitank mine fuzes with and without hex nuts, practice MK2 and M67 grenades, 2.5'' ballistic windshields, M2A1-mines with/without bases, M19-14 time fuzes, and 40-mm practice grenades with/without cartridges. The site at the MMR site contains targets of yet different sizes. In this work we apply our models to EMI data collected using the MetalMapper (MM) and 2 × 2 TEMTADS sensors. The data for each anomaly are inverted to extract estimates of the extrinsic and intrinsic parameters associated with each buried target. (The latter include the total volume magnetic source or NVMS, which relates to size, shape, and material properties; the former includes location, depth, and orientation). The estimated intrinsic parameters are then used for classification performed via library matching and the use of statistical classification algorithms; this process yielded prioritized dig-lists that were submitted to the Institute for Defense Analyses (IDA) for independent scoring. The models' classification performance is illustrated and assessed based on these independent evaluations.

  16. Identification of Phosphorylation Sites Altering Pollen Soluble Inorganic Pyrophosphatase Activity.

    PubMed

    Eaves, Deborah J; Haque, Tamanna; Tudor, Richard L; Barron, Yoshimi; Zampronio, Cleidiane G; Cotton, Nicholas P J; de Graaf, Barend H J; White, Scott A; Cooper, Helen J; Franklin, F Christopher H; Harper, Jeffery F; Franklin-Tong, Vernonica E

    2017-03-01

    Protein phosphorylation regulates numerous cellular processes. Identifying the substrates and protein kinases involved is vital to understand how these important posttranslational modifications modulate biological function in eukaryotic cells. Pyrophosphatases catalyze the hydrolysis of inorganic phosphate (PPi) to inorganic phosphate Pi, driving biosynthetic reactions; they are essential for low cytosolic inorganic phosphate. It was suggested recently that posttranslational regulation of Family I soluble inorganic pyrophosphatases (sPPases) may affect their activity. We previously demonstrated that two pollen-expressed sPPases, Pr-p26.1a and Pr-p26.1b, from the flowering plant Papaver rhoeas were inhibited by phosphorylation. Despite the potential significance, there is a paucity of data on sPPase phosphorylation and regulation. Here, we used liquid chromatographic tandem mass spectrometry to map phosphorylation sites to the otherwise divergent amino-terminal extensions on these pollen sPPases. Despite the absence of reports in the literature on mapping phosphorylation sites on sPPases, a database survey of various proteomes identified a number of examples, suggesting that phosphorylation may be a more widely used mechanism to regulate these enzymes. Phosphomimetic mutants of Pr-p26.1a/b significantly and differentially reduced PPase activities by up to 2.5-fold at pH 6.8 and 52% in the presence of Ca(2+) and hydrogen peroxide over unmodified proteins. This indicates that phosphoregulation of key sites can inhibit the catalytic responsiveness of these proteins in concert with key intracellular events. As sPPases are essential for many metabolic pathways in eukaryotic cells, our findings identify the phosphorylation of sPPases as a potential master regulatory mechanism that could be used to attenuate metabolism.

  17. Evidence for segmental mobility in the active site of pepsin

    SciTech Connect

    Pohl, J.; Strop, P.; Senn, H.; Foundling, S.; Kostka, V.

    1986-05-01

    The low hydrolytic activity (k/sub cat/ < 0.001 s/sup -1/) of chicken pepsin (CP) towards tri- and tetrapeptides is enhanced at least 100 times by modification of its single sulfhydryl group of Cys-115, with little effect on K/sub m/-values. Modification thus simulates the effect of secondary substrate binding on pepsin catalysis. The rate of Cys-115 modification is substantially decreased in the presence of some competitive inhibitors, suggesting its active site location. Experiments with CP alkylated at Cys-115 with Acrylodan as a fluorescent probe or with N-iodoacetyl-(4-fluoro)-aniline as a /sup 19/F-nmr probe suggest conformation change around Cys-115 to occur on substrate or substrate analog binding. The difference /sup 1/H-nmr spectra (500 MHz) of unmodified free and inhibitor-complexed CP reveal chemical shifts almost exclusively in the aromatic region. The effects of Cu/sup + +/ on /sup 19/F- and /sup 1/H-nmr spectra have been studied. Examination of a computer graphics model of CP based on E. parasitica pepsin-inhibitor complex X-ray coordinates suggests that Cys-115 is located near the S/sub 3//S/sub 5/ binding site. The results are interpreted in favor of segmental mobility of this region important for pepsin substrate binding and catalysis.

  18. First Principles Computational Study of the Active Site of Arginase

    SciTech Connect

    Ivanov, Ivaylo; Klien, Micheal

    2004-01-14

    Ab initio density functional theory (DFT) methods were used to investigate the structural features of the active site of the binuclear enzyme rat liver arginase. Special emphasis was placed on the crucial role of the second shell ligand interactions. These interactions were systematically studied by performing calculations on models of varying size. It was determined that a water molecule, and not hydroxide, is the bridging exogenous ligand. The carboxylate ligands facilitate the close approach of the Mn (II) ions by attenuating the metal-metal electrostatic repulsion. Of the two metals, MnA was shown to carry a larger positive charge. Analysis of the electronic properties of the active site revealed that orbitals involving the terminal Asp234 residue, as well as the flexible -1,1 bridging Asp232, lie at high energies, suggesting weaker coordination. This is reflected in certain structural variability present in our models and is also consistent with recent experimental findings. Finally, implications of our findings for the biological function of the enzyme are delineated.

  19. C-H Activation on Co,O Sites: Isolated Surface Sites versus Molecular Analogs.

    PubMed

    Estes, Deven P; Siddiqi, Georges; Allouche, Florian; Kovtunov, Kirill V; Safonova, Olga V; Trigub, Alexander L; Koptyug, Igor V; Copéret, Christophe

    2016-11-16

    The activation and conversion of hydrocarbons is one of the most important challenges in chemistry. Transition-metal ions (V, Cr, Fe, Co, etc.) isolated on silica surfaces are known to catalyze such processes. The mechanisms of these processes are currently unknown but are thought to involve C-H activation as the rate-determining step. Here, we synthesize well-defined Co(II) ions on a silica surface using a metal siloxide precursor followed by thermal treatment under vacuum at 500 °C. We show that these isolated Co(II) sites are catalysts for a number of hydrocarbon conversion reactions, such as the dehydrogenation of propane, the hydrogenation of propene, and the trimerization of terminal alkynes. We then investigate the mechanisms of these processes using kinetics, kinetic isotope effects, isotopic labeling experiments, parahydrogen induced polarization (PHIP) NMR, and comparison with a molecular analog. The data are consistent with all of these reactions occurring by a common mechanism, involving heterolytic C-H or H-H activation via a 1,2 addition across a Co-O bond.

  20. Polarizability of the active site of cytochrome c reduces the activation barrier for electron transfer

    PubMed Central

    Dinpajooh, Mohammadhasan; Martin, Daniel R.; Matyushov, Dmitry V.

    2016-01-01

    Enzymes in biology’s energy chains operate with low energy input distributed through multiple electron transfer steps between protein active sites. The general challenge of biological design is how to lower the activation barrier without sacrificing a large negative reaction free energy. We show that this goal is achieved through a large polarizability of the active site. It is polarized by allowing a large number of excited states, which are populated quantum mechanically by electrostatic fluctuations of the protein and hydration water shells. This perspective is achieved by extensive mixed quantum mechanical/molecular dynamics simulations of the half reaction of reduction of cytochrome c. The barrier for electron transfer is consistently lowered by increasing the number of excited states included in the Hamiltonian of the active site diagonalized along the classical trajectory. We suggest that molecular polarizability, in addition to much studied electrostatics of permanent charges, is a key parameter to consider in order to understand how enzymes work. PMID:27306204

  1. Postoperative interstitial hernia as a cause of obscure incisional wound site pain.

    PubMed

    Modrzejewski, Andrzej; Smietański, Maciej

    2012-03-01

    An interstitial hernia is one in which the hernia sac is located between the layers of the abdominal wall. The analysis of contemporary literature shows that interstitial hernias are most often seen in children as a type of inguinal hernia and often accompany undescended testis. The hernia sac is usually located between the external-oblique and internal-oblique muscles in a lateral-cephalic direction. The authors present 3 cases of interstitial hernia found during laparoscopic exploration of the front abdominal wall done due to incisional wound site pain. No previous diagnosis of hernia was considered in all the cases. Hernias were found as complications of appendectomy and wound healing after radiotherapy of uterine and cervical cancer. In conclusion, in obscure wound site pain, the presence of an interstitial postoperative hernia should be considered as a possible reason for the complaint. Laparoscopic examination of the anterior abdominal wall during adhesiolysis in patients with abdominal pain enables proper diagnosis and treatment.

  2. Active Sites Environmental Monitoring Program: Program plan. Revision 1

    SciTech Connect

    Ashwood, T.L.; Wickliff, D.S.; Morrissey, C.M.

    1992-02-01

    The Active Sites Environmental Monitoring Program (ASEMP), initiated in 1989, provides early detection and performance monitoring of transuranic (TRU) waste and active low-level waste (LLW) facilities at Oak Ridge National Laboratory (ORNL) in accordance with US Department of Energy (DOE) Order 5820.2A. Active LLW facilities in Solid Waste Storage Area (SWSA) 6 include Tumulus I and Tumulus II, the Interim Waste Management Facility (IWMF), LLW silos, high-range wells, asbestos silos, and fissile wells. The tumulus pads and IWMF are aboveground, high-strength concrete pads on which concrete vaults containing metal boxes of LLW are placed; the void space between the boxes and vaults is filled with grout. Eventually, these pads and vaults will be covered by an engineered multilayered cap. All other LLW facilities in SWSA 6 are below ground. In addition, this plan includes monitoring of the Hillcut Disposal Test Facility (HDTF) in SWSA 6, even though this facility was completed prior to the data of the DOE order. In SWSA 5 North, the TRU facilities include below-grade engineered caves, high-range wells, and unlined trenches. All samples from SWSA 6 are screened for alpha and beta activity, counted for gamma-emitting isotopes, and analyzed for tritium. In addition to these analytes, samples from SWSA 5 North are analyzed for specific transuranic elements.

  3. Conformational Change in the Active Site of Streptococcal Unsaturated Glucuronyl Hydrolase Through Site-Directed Mutagenesis at Asp-115.

    PubMed

    Nakamichi, Yusuke; Oiki, Sayoko; Mikami, Bunzo; Murata, Kousaku; Hashimoto, Wataru

    2016-08-01

    Bacterial unsaturated glucuronyl hydrolase (UGL) degrades unsaturated disaccharides generated from mammalian extracellular matrices, glycosaminoglycans, by polysaccharide lyases. Two Asp residues, Asp-115 and Asp-175 of Streptococcus agalactiae UGL (SagUGL), are completely conserved in other bacterial UGLs, one of which (Asp-175 of SagUGL) acts as a general acid and base catalyst. The other Asp (Asp-115 of SagUGL) also affects the enzyme activity, although its role in the enzyme reaction has not been well understood. Here, we show substitution of Asp-115 in SagUGL with Asn caused a conformational change in the active site. Tertiary structures of SagUGL mutants D115N and D115N/K370S with negligible enzyme activity were determined at 2.00 and 1.79 Å resolution, respectively, by X-ray crystallography. The side chain of Asn-115 is drastically shifted in both mutants owing to the interaction with several residues, including Asp-175, by formation of hydrogen bonds. This interaction between Asn-115 and Asp-175 probably prevents the mutants from triggering the enzyme reaction using Asp-175 as an acid catalyst.

  4. Active Site and Laminarin Binding in Glycoside Hydrolase Family 55*

    PubMed Central

    Bianchetti, Christopher M.; Takasuka, Taichi E.; Deutsch, Sam; Udell, Hannah S.; Yik, Eric J.; Bergeman, Lai F.; Fox, Brian G.

    2015-01-01

    The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β-1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium (Ishida, T., Fushinobu, S., Kawai, R., Kitaoka, M., Igarashi, K., and Samejima, M. (2009) Crystal structure of glycoside hydrolase family 55 β-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 284, 10100–10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ∼30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties. PMID:25752603

  5. Metal active site elasticity linked to activation of homocysteine in methionine synthases

    SciTech Connect

    Koutmos, Markos; Pejchal, Robert; Bomer, Theresa M.; Matthews, Rowena G.; Smith, Janet L.; Ludwig, Martha L.

    2008-04-02

    Enzymes possessing catalytic zinc centers perform a variety of fundamental processes in nature, including methyl transfer to thiols. Cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthases are two such enzyme families. Although they perform the same net reaction, transfer of a methyl group from methyltetrahydrofolate to homocysteine (Hcy) to form methionine, they display markedly different catalytic strategies, modular organization, and active site zinc centers. Here we report crystal structures of zinc-replete MetE and MetH, both in the presence and absence of Hcy. Structural investigation of the catalytic zinc sites of these two methyltransferases reveals an unexpected inversion of zinc geometry upon binding of Hcy and displacement of an endogenous ligand in both enzymes. In both cases a significant movement of the zinc relative to the protein scaffold accompanies inversion. These structures provide new information on the activation of thiols by zinc-containing enzymes and have led us to propose a paradigm for the mechanism of action of the catalytic zinc sites in these and related methyltransferases. Specifically, zinc is mobile in the active sites of MetE and MetH, and its dynamic nature helps facilitate the active site conformational changes necessary for thiol activation and methyl transfer.

  6. Site-specific profiles of estrogenic activity in agricultural areas of California's inland waters.

    PubMed

    Lavado, Ramon; Loyo-Rosales, Jorge E; Floyd, Emily; Kolodziej, Edward P; Snyder, Shane A; Sedlak, David L; Schlenk, Daniel

    2009-12-15

    To evaluate the occurrence and sources of compounds capable of feminizing fish in agriculturally impacted waterways of the Central Valley of California, water samples were extracted and subjected to chemical analyses as well as in vitro and in vivo measurements of vitellogenin in juvenile rainbow trout (Oncorhynchus mykiss). Among the 16 sites sampled, 6 locations frequently exhibited elevated concentrations of estrogenic substances with 17beta-estradiol equivalents up to 242 ng/L in vitro and 12 microg/kg in vivo. The patterns of activity varied among sites, with two sites showing elevated activity only in vitro, two showing elevated activity only in vivo, and two showing elevated activity in both assays. Sequential elution of solid-phase extraction (SPE) disks followed by bioassay-guided fractionation was used to characterize water samples from the two locations where activity was observed in both bioassays. The highest estrogenic activity was observed in the most nonpolar fractions (80-100% methanol eluent) from the Napa River, while most of the activity in the Sacramento River Delta eluted in the 60% methanol eluent. Quantitative analyses of SPE extracts and additional HPLC fractionation of the SPE extracts by GC-MS/MS and LC-MS/MS indicated concentrations of steroid hormones, alkylphenol polyethoxylates, and herbicides that were at least 1-3 orders of magnitude below bioassay 17beta-estradiol equivalent calculations. Given the different patterns of activity and chemical properties of the estrogenic compounds, it appears that estrogenic activity in these agriculturally impacted surface waters is attributable to multiple compounds. Further investigation is needed to identify the compounds causing the estrogenic activity and to determine the potential impacts of these compounds on feral fish.

  7. An Active Site Water Network in the Plasminogen Activator Pla from Yersinia pestis

    SciTech Connect

    Eren, Elif; Murphy, Megan; Goguen, Jon; van den Berg, Bert

    2010-08-13

    The plasminogen activator Pla from Yersinia pestis is an outer membrane protease (omptin) that is important for the virulence of plague. Here, we present the high-resolution crystal structure of wild-type, enzymatically active Pla at 1.9 {angstrom}. The structure shows a water molecule located between active site residues D84 and H208, which likely corresponds to the nucleophilic water. A number of other water molecules are present in the active site, linking residues important for enzymatic activity. The R211 sidechain in loop L4 is close to the nucleophilic water and possibly involved in the stabilization of the oxyanion intermediate. Subtle conformational changes of H208 result from the binding of lipopolysaccharide to the outside of the barrel, explaining the unusual dependence of omptins on lipopolysaccharide for activity. The Pla structure suggests a model for the interaction with plasminogen substrate and provides a more detailed understanding of the catalytic mechanism of omptin proteases.

  8. An active site water network in the plasminogen activator pla from Yersinia pestis.

    PubMed

    Eren, Elif; Murphy, Megan; Goguen, Jon; van den Berg, Bert

    2010-07-14

    The plasminogen activator Pla from Yersinia pestis is an outer membrane protease (omptin) that is important for the virulence of plague. Here, we present the high-resolution crystal structure of wild-type, enzymatically active Pla at 1.9 A. The structure shows a water molecule located between active site residues D84 and H208, which likely corresponds to the nucleophilic water. A number of other water molecules are present in the active site, linking residues important for enzymatic activity. The R211 sidechain in loop L4 is close to the nucleophilic water and possibly involved in the stabilization of the oxyanion intermediate. Subtle conformational changes of H208 result from the binding of lipopolysaccharide to the outside of the barrel, explaining the unusual dependence of omptins on lipopolysaccharide for activity. The Pla structure suggests a model for the interaction with plasminogen substrate and provides a more detailed understanding of the catalytic mechanism of omptin proteases.

  9. Site-specific range uncertainties caused by dose calculation algorithms for proton therapy

    NASA Astrophysics Data System (ADS)

    Schuemann, J.; Dowdell, S.; Grassberger, C.; Min, C. H.; Paganetti, H.

    2014-08-01

    The purpose of this study was to assess the possibility of introducing site-specific range margins to replace current generic margins in proton therapy. Further, the goal was to study the potential of reducing margins with current analytical dose calculations methods. For this purpose we investigate the impact of complex patient geometries on the capability of analytical dose calculation algorithms to accurately predict the range of proton fields. Dose distributions predicted by an analytical pencil-beam algorithm were compared with those obtained using Monte Carlo (MC) simulations (TOPAS). A total of 508 passively scattered treatment fields were analyzed for seven disease sites (liver, prostate, breast, medulloblastoma-spine, medulloblastoma-whole brain, lung and head and neck). Voxel-by-voxel comparisons were performed on two-dimensional distal dose surfaces calculated by pencil-beam and MC algorithms to obtain the average range differences and root mean square deviation for each field for the distal position of the 90% dose level (R90) and the 50% dose level (R50). The average dose degradation of the distal falloff region, defined as the distance between the distal position of the 80% and 20% dose levels (R80-R20), was also analyzed. All ranges were calculated in water-equivalent distances. Considering total range uncertainties and uncertainties from dose calculation alone, we were able to deduce site-specific estimations. For liver, prostate and whole brain fields our results demonstrate that a reduction of currently used uncertainty margins is feasible even without introducing MC dose calculations. We recommend range margins of 2.8% + 1.2 mm for liver and prostate treatments and 3.1% + 1.2 mm for whole brain treatments, respectively. On the other hand, current margins seem to be insufficient for some breast, lung and head and neck patients, at least if used generically. If no case specific adjustments are applied, a generic margin of 6.3% + 1.2 mm would be

  10. Resistance to Linezolid Caused by Modifications at Its Binding Site on the Ribosome

    PubMed Central

    Long, Katherine S.

    2012-01-01

    Linezolid is an oxazolidinone antibiotic in clinical use for the treatment of serious infections of resistant Gram-positive bacteria. It inhibits protein synthesis by binding to the peptidyl transferase center on the ribosome. Almost all known resistance mechanisms involve small alterations to the linezolid binding site, so this review will therefore focus on the various changes that can adversely affect drug binding and confer resistance. High-resolution structures of linezolid bound to the 50S ribosomal subunit show that it binds in a deep cleft that is surrounded by 23S rRNA nucleotides. Mutation of 23S rRNA has for some time been established as a linezolid resistance mechanism. Although ribosomal proteins L3 and L4 are located further away from the bound drug, mutations in specific regions of these proteins are increasingly being associated with linezolid resistance. However, very little evidence has been presented to confirm this. Furthermore, recent findings on the Cfr methyltransferase underscore the modification of 23S rRNA as a highly effective and transferable form of linezolid resistance. On a positive note, detailed knowledge of the linezolid binding site has facilitated the design of a new generation of oxazolidinones that show improved properties against the known resistance mechanisms. PMID:22143525

  11. Characterization of the active site of ADP-ribosyl cyclase.

    PubMed

    Munshi, C; Thiel, D J; Mathews, I I; Aarhus, R; Walseth, T F; Lee, H C

    1999-10-22

    ADP-ribosyl cyclase synthesizes two Ca(2+) messengers by cyclizing NAD to produce cyclic ADP-ribose and exchanging nicotinic acid with the nicotinamide group of NADP to produce nicotinic acid adenine dinucleotide phosphate. Recombinant Aplysia cyclase was expressed in yeast and co-crystallized with a substrate, nicotinamide. x-ray crystallography showed that the nicotinamide was bound in a pocket formed in part by a conserved segment and was near the central cleft of the cyclase. Glu(98), Asn(107) and Trp(140) were within 3.5 A of the bound nicotinamide and appeared to coordinate it. Substituting Glu(98) with either Gln, Gly, Leu, or Asn reduced the cyclase activity by 16-222-fold, depending on the substitution. The mutant N107G exhibited only a 2-fold decrease in activity, while the activity of W140G was essentially eliminated. The base exchange activity of all mutants followed a similar pattern of reduction, suggesting that both reactions occur at the same active site. In addition to NAD, the wild-type cyclase also cyclizes nicotinamide guanine dinucleotide to cyclic GDP-ribose. All mutant enzymes had at least half of the GDP-ribosyl cyclase activity of the wild type, some even 2-3-fold higher, indicating that the three coordinating amino acids are responsible for positioning of the substrate but not absolutely critical for catalysis. To search for the catalytic residues, other amino acids in the binding pocket were mutagenized. E179G was totally devoid of GDP-ribosyl cyclase activity, and both its ADP-ribosyl cyclase and the base exchange activities were reduced by 10,000- and 18,000-fold, respectively. Substituting Glu(179) with either Asn, Leu, Asp, or Gln produced similar inactive enzymes, and so was the conversion of Trp(77) to Gly. However, both E179G and the double mutant E179G/W77G retained NAD-binding ability as shown by photoaffinity labeling with [(32)P]8-azido-NAD. These results indicate that both Glu(179) and Trp(77) are crucial for catalysis and

  12. Mutations inducing an active-site aperture in Rhizobium sp. sucrose isomerase confer hydrolytic activity.

    PubMed

    Lipski, Alexandra; Watzlawick, Hildegard; Ravaud, Stéphanie; Robert, Xavier; Rhimi, Moez; Haser, Richard; Mattes, Ralf; Aghajari, Nushin

    2013-02-01

    Sucrose isomerase is an enzyme that catalyzes the production of sucrose isomers of high biotechnological and pharmaceutical interest. Owing to the complexity of the chemical synthesis of these isomers, isomaltulose and trehalulose, enzymatic conversion remains the preferred method for obtaining these products. Depending on the microbial source, the ratio of the sucrose-isomer products varies significantly. In studies aimed at understanding and explaining the underlying molecular mechanisms of these reactions, mutations obtained using a random-mutagenesis approach displayed a major hydrolytic activity. Two of these variants, R284C and F164L, of sucrose isomerase from Rhizobium sp. were therefore crystallized and their crystal structures were determined. The three-dimensional structures of these mutants allowed the identification of the molecular determinants that favour hydrolytic activity compared with transferase activity. Substantial conformational changes resulting in an active-site opening were observed, as were changes in the pattern of water molecules bordering the active-site region.

  13. Molecular dynamics simulations of Zika virus NS3 helicase: Insights into RNA binding site activity.

    PubMed

    Mottin, Melina; Braga, Rodolpho C; da Silva, Roosevelt A; Silva, Joao H Martins da; Perryman, Alexander L; Ekins, Sean; Andrade, Carolina Horta

    2017-03-21

    America is still suffering with the outbreak of Zika virus (ZIKV) infection. Congenital ZIKV syndrome has already caused a public health emergency of international concern. However, there are still no vaccines to prevent or drugs to treat the infection caused by ZIKV. The ZIKV NS3 helicase (NS3h) protein is a promising target for drug discovery due to its essential role in viral genome replication. NS3h unwinds the viral RNA to enable the replication of the viral genome by the NS5 protein. NS3h contains two important binding sites: the NTPase binding site and the RNA binding site. Here, we used molecular dynamics (MD) simulations to study the molecular behavior of ZIKV NS3h in the presence and absence of ssRNA and the potential implications for NS3h activity and inhibition. Although there is conformational variability and poor electron densities of the RNA binding loop in various apo flaviviruses NS3h crystallographic structures, the MD trajectories of NS3h-ssRNA demonstrated that the RNA binding loop becomes more stable when NS3h is occupied by RNA. Our results suggest that the presence of RNA generates important interactions with the RNA binding loop, and these interactions stabilize the loop sufficiently that it remains in a closed conformation. This closed conformation likely keeps the ssRNA bound to the protein for a sufficient duration to enable the unwinding/replication activities of NS3h to occur. In addition, conformational changes of this RNA binding loop can change the nature and location of the optimal ligand binding site, according to ligand binding site prediction results. These are important findings to help guide the design and discovery of new inhibitors of NS3h as promising compounds to treat the ZIKV infection.

  14. Periodontitis-activated monocytes/macrophages cause aortic inflammation.

    PubMed

    Miyajima, Shin-ichi; Naruse, Keiko; Kobayashi, Yasuko; Nakamura, Nobuhisa; Nishikawa, Toru; Adachi, Kei; Suzuki, Yuki; Kikuchi, Takeshi; Mitani, Akio; Mizutani, Makoto; Ohno, Norikazu; Noguchi, Toshihide; Matsubara, Tatsuaki

    2014-06-04

    A relationship between periodontal disease and atherosclerosis has been suggested by epidemiological studies. Ligature-induced experimental periodontitis is an adequate model for clinical periodontitis, which starts from plaque accumulation, followed by inflammation in the periodontal tissue. Here we have demonstrated using a ligature-induced periodontitis model that periodontitis activates monocytes/macrophages, which subsequently circulate in the blood and adhere to vascular endothelial cells without altering the serum TNF-α concentration. Adherent monocytes/macrophages induced NF-κB activation and VCAM-1 expression in the endothelium and increased the expression of the TNF-α signaling cascade in the aorta. Peripheral blood-derived mononuclear cells from rats with experimental periodontitis showed enhanced adhesion and increased NF-κB/VCAM-1 in cultured vascular endothelial cells. Our results suggest that periodontitis triggers the initial pathogenesis of atherosclerosis, inflammation of the vasculature, through activating monocytes/macrophages.

  15. Tumor hypoxia causes DNA hypermethylation by reducing TET activity

    PubMed Central

    Kuchnio, Anna; Ploumakis, Athanasios; Ghesquière, Bart; Van Dyck, Laurien; Boeckx, Bram; Schoonjans, Luc; Hermans, Els; Amant, Frederic; Kristensen, Vessela N.; Peng Koh, Kian; Mazzone, Massimiliano; Coleman, Mathew; Carell, Thomas; Carmeliet, Peter; Lambrechts, Diether

    2016-01-01

    Summary Hypermethylation of tumor suppressor gene (TSG) promoters confers growth advantages to cancer cells, but how these changes arise is poorly understood. Here, we report that tumor hypoxia reduces the activity of oxygen-dependent TET enzymes, which catalyze DNA de-methylation through 5-methylcytosine oxidation. This occurs independently of hypoxia-associated alterations in TET expression, proliferation, metabolism, HIF activity or reactive oxygen, but directly depends on oxygen shortage. Hypoxia-induced loss of TET activity increases hypermethylation at gene promoters in vitro. Also in patients, TSG promoters are markedly more methylated in hypoxic tumors, independently of proliferation, stromal cell infiltration and tumor characteristics. Our data suggest cellular selection of hypermethylation events, with almost half of them being ascribable to hypoxia across tumor types. Accordingly, increased hypoxia after vessel pruning in murine breast tumors increases hypermethylation, while restored tumor oxygenation by vessel normalization abrogates this effect. Tumor hypoxia thus acts as a novel regulator underlying DNA methylation. PMID:27533040

  16. Causes of daily cycle variability of atmospheric pollutants in a western Mediterranean urban site (DAURE campaign)

    NASA Astrophysics Data System (ADS)

    Reche, Cristina; Moreno, Teresa; Viana, Mar; Querol, Xavier; Alastuey, Andrés.; Jimenez, Jose L.; Pandolfi, Marco; Amato, Fulvio; Pérez, Noemí; Moreno, Natalia

    2010-05-01

    The 2009 DAURE Aerosol Campaign (23-February-2009 to 27-March-2009 and 1-July to 31-July) (see Presentation: Pandolfi et al., section AS3.2) had the objective of characterising the main sources and chemical processes controlling atmospheric pollution due to particulate matter in the Mediterranean site of Barcelona (Spain). An urban and a rural background site were selected in order to describe both kinds of pollution setting. Several parameters were taken into consideration, including the variability of mass concentration in the coarse and fine fractions, particle number, amount of black carbon and the concentration of gaseous pollutants (SO2, H2S, NO, NO2, CO, O3) present. Comparisons between the chemical composition of ambient atmospheric particles during day versus night were made using twelve-hour PM samples. The data shown here are focused on results obtained for the urban site where two main atmospheric settings were distinguishable in winter, namely Atlantic advection versus local air mass recirculation. During the warmer months Saharan dust intrusions added a third important influence on PM background. The data demonstrate that superimposed upon these background influences on city air quality are important local contributions from road traffic, construction-demolition works and shipping. There is also a major local contribution of secondary aerosols, with elevated number of particles occurring at midday (and especially in summer) when nucleation processes are favoured by photochemistry. Concentrations of SO2 peak at different times to the other gaseous pollutants due to regular daytime onshore south-easterly breezes bringing harbour emissions into the city. Road traffic in Barcelona also has a great impact on air quality, as demonstrated by daily and weekly cycles of gaseous pollutants, black carbon and the finer fraction of PM, with peaks being coincident with traffic rush-hours (8-10h and 20-22h), levels of pollution increasing from Monday to Friday, and

  17. Permanent Neonatal Diabetes Caused by Creation of an Ectopic Splice Site within the INS Gene

    PubMed Central

    Gastaldo, Elena; Harries, Lorna W.; Rubio-Cabezas, Oscar; Castaño, Luis

    2012-01-01

    Background The aim of this study was to characterize the genetic etiology in a patient who presented with permanent neonatal diabetes at 2 months of age. Methodology/Principal Findings Regulatory elements and coding exons 2 and 3 of the INS gene were amplified and sequenced from genomic and complementary DNA samples. A novel heterozygous INS mutation within the terminal intron of the gene was identified in the proband and her affected father. This mutation introduces an ectopic splice site leading to the insertion of 29 nucleotides from the intronic sequence into the mature mRNA, which results in a longer and abnormal transcript. Conclusions/Significance This study highlights the importance of routinely sequencing the exon-intron boundaries and the need to carry out additional studies to confirm the pathogenicity of any identified intronic genetic variants. PMID:22235272

  18. Identification of a novel K311 ubiquitination site critical for androgen receptor transcriptional activity.

    PubMed

    McClurg, Urszula L; Cork, David M W; Darby, Steven; Ryan-Munden, Claudia A; Nakjang, Sirintra; Mendes Côrtes, Leticia; Treumann, Achim; Gaughan, Luke; Robson, Craig N

    2016-11-29

    The androgen receptor (AR) is the main driver of prostate cancer (PC) development and progression, and the primary therapeutic target in PC. To date, two functional ubiquitination sites have been identified on AR, both located in its C-terminal ligand binding domain (LBD). Recent reports highlight the emergence of AR splice variants lacking the LBD that can arise during disease progression and contribute to castrate resistance. Here, we report a novel N-terminal ubiquitination site at lysine 311. Ubiquitination of this site plays a role in AR stability and is critical for its transcriptional activity. Inactivation of this site causes AR to accumulate on chromatin and inactivates its transcriptional function as a consequence of inability to bind to p300. Additionally, mutation at lysine 311 affects cellular transcriptome altering the expression of genes involved in chromatin organization, signaling, adhesion, motility, development and metabolism. Even though this site is present in clinically relevant AR-variants it can only be ubiquitinated in cells when AR retains LBD suggesting a role for AR C-terminus in E2/E3 substrate recognition. We report that as a consequence AR variants lacking the LBD cannot be ubiquitinated in the cellular environment and their protein turnover must be regulated via an alternate pathway.

  19. Site-specific PEGylation of lidamycin and its antitumor activity

    PubMed Central

    Li, Liang; Shang, Boyang; Hu, Lei; Shao, Rongguang; Zhen, Yongsu

    2015-01-01

    In this study, N-terminal site-specific mono-PEGylation of the recombinant lidamycin apoprotein (rLDP) of lidamycin (LDM) was prepared using a polyethyleneglycol (PEG) derivative (Mw 20 kDa) through a reactive terminal aldehyde group under weak acidic conditions (pH 5.5). The biochemical properties of mPEG-rLDP-AE, an enediyne-integrated conjugate, were analyzed by SDS-PAGE, RP-HPLC, SEC-HPLC and MALDI-TOF. Meanwhile, in vitro and in vivo antitumor activity of mPEG-rLDP-AE was evaluated by MTT assays and in xenograft model. The results indicated that mPEG-rLDP-AE showed significant antitumor activity both in vitro and in vivo. After PEGylation, mPEG-rLDP still retained the binding capability to the enediyne AE and presented the physicochemical characteristics similar to that of native LDP. It is of interest that the PEGylation did not diminish the antitumor efficacy of LDM, implying the possibility that this derivative may function as a payload to deliver novel tumor-targeted drugs. PMID:26579455

  20. Allosteric site-mediated active site inhibition of PBP2a using Quercetin 3-O-rutinoside and its combination.

    PubMed

    Rani, Nidhi; Vijayakumar, Saravanan; P T V, Lakshmi; Arunachalam, Annamalai

    2016-08-01

    Recent crystallographic study revealed the involvement of allosteric site in active site inhibition of penicillin binding protein (PBP2a), where one molecule of Ceftaroline (Cef) binds to the allosteric site of PBP2a and paved way for the other molecule (Cef) to bind at the active site. Though Cef has the potency to inhibit the PBP2a, its adverse side effects are of major concern. Previous studies have reported the antibacterial property of Quercetin derivatives, a group of natural compounds. Hence, the present study aims to evaluate the effect of Quercetin 3-o-rutinoside (Rut) in allosteric site-mediated active site inhibition of PBP2a. The molecular docking studies between allosteric site and ligands (Rut, Que, and Cef) revealed a better binding efficiency (G-score) of Rut (-7.790318) and Cef (-6.194946) with respect to Que (-5.079284). Molecular dynamic (MD) simulation studies showed significant changes at the active site in the presence of ligands (Rut and Cef) at allosteric site. Four different combinations of Rut and Cef were docked and their G-scores ranged between -6.320 and -8.623. MD studies revealed the stability of the key residue (Ser403) with Rut being at both sites, compared to other complexes. Morphological analysis through electron microscopy confirmed that combination of Rut and Cefixime was able to disturb the bacterial cell membrane in a similar fashion to that of Rut and Cefixime alone. The results of this study indicate that the affinity of Rut at both sites were equally good, with further validations Rut could be considered as an alternative for inhibiting MRSA growth.

  1. Calcific tendinitis of biceps femoris: an unusual site and cause for lateral knee pain.

    PubMed

    Chan, Warwick; Chase, Helen Emily; Cahir, John G; Walton, Neil Patrick

    2016-07-29

    A 37-year-old man presented to the acute knee and sports medicine clinic with atraumatic lateral knee pain. He had point tenderness over the lateral aspect of his knee which had not settled with anti-inflammatory medications. Imaging revealed a large opaque lesion lateral to the knee and although there was no clear mechanism, injury to the posterolateral corner was considered. An MRI subsequently revealed a rare case of calcific tendinitis to the biceps femoris tendon insertion. This condition was self-limiting and did not require interventions such as steroid injections. This is the first reported case of calcific tendinitis of biceps femoris as a cause of acute knee pain.

  2. Epileptic activity in Alzheimer's disease: causes and clinical relevance.

    PubMed

    Vossel, Keith A; Tartaglia, Maria C; Nygaard, Haakon B; Zeman, Adam Z; Miller, Bruce L

    2017-04-01

    Epileptic activity is frequently associated with Alzheimer's disease; this association has therapeutic implications, because epileptic activity can occur at early disease stages and might contribute to pathogenesis. In clinical practice, seizures in patients with Alzheimer's disease can easily go unrecognised because they usually present as non-motor seizures, and can overlap with other symptoms of the disease. In patients with Alzheimer's disease, seizures can hasten cognitive decline, highlighting the clinical relevance of early recognition and treatment. Some evidence indicates that subclinical epileptiform activity in patients with Alzheimer's disease, detected by extended neurophysiological monitoring, can also lead to accelerated cognitive decline. Treatment of clinical seizures in patients with Alzheimer's disease with select antiepileptic drugs (AEDs), in low doses, is usually well tolerated and efficacious. Moreover, studies in mouse models of Alzheimer's disease suggest that certain classes of AEDs that reduce network hyperexcitability have disease-modifying properties. These AEDs target mechanisms of epileptogenesis involving amyloid β and tau. Clinical trials targeting network hyperexcitability in patients with Alzheimer's disease will identify whether AEDs or related strategies could improve their cognitive symptoms or slow decline.

  3. Extensive site-directed mutagenesis reveals interconnected functional units in the alkaline phosphatase active site

    PubMed Central

    Sunden, Fanny; Peck, Ariana; Salzman, Julia; Ressl, Susanne; Herschlag, Daniel

    2015-01-01

    Enzymes enable life by accelerating reaction rates to biological timescales. Conventional studies have focused on identifying the residues that have a direct involvement in an enzymatic reaction, but these so-called ‘catalytic residues’ are embedded in extensive interaction networks. Although fundamental to our understanding of enzyme function, evolution, and engineering, the properties of these networks have yet to be quantitatively and systematically explored. We dissected an interaction network of five residues in the active site of Escherichia coli alkaline phosphatase. Analysis of the complex catalytic interdependence of specific residues identified three energetically independent but structurally interconnected functional units with distinct modes of cooperativity. From an evolutionary perspective, this network is orders of magnitude more probable to arise than a fully cooperative network. From a functional perspective, new catalytic insights emerge. Further, such comprehensive energetic characterization will be necessary to benchmark the algorithms required to rationally engineer highly efficient enzymes. DOI: http://dx.doi.org/10.7554/eLife.06181.001 PMID:25902402

  4. A split active site couples cap recognition by Dcp2 to activation

    PubMed Central

    Floor, Stephen N.; Jones, Brittnee N.; Hernandez, Gail A.; Gross, John D.

    2010-01-01

    Decapping by Dcp2 is an essential step in 5′-3′ mRNA decay. In yeast, decapping requires an open-to-closed transition in Dcp2, though the link between closure and catalysis remains elusive. Here we show using NMR that cap binds conserved residues on both the catalytic and regulatory domains of Dcp2. Lesions in the cap-binding site on the regulatory domain reduce the catalytic step two orders of magnitude and block formation of the closed state whereas Dcp1 enhances the catalytic step by a factor of ten and promotes closure. We conclude that closure occurs during the rate-limiting catalytic step of decapping, juxtaposing the cap-binding region of each domain to form a composite active site. This work suggests a model for regulation of decapping, where coactivators trigger decapping by stabilizing a labile composite active site. PMID:20711189

  5. Damage to lens fiber cells causes TRPV4-dependent Src family kinase activation in the epithelium.

    PubMed

    Shahidullah, M; Mandal, A; Delamere, N A

    2015-11-01

    The bulk of the lens consists of tightly packed fiber cells. Because mature lens fibers lack mitochondria and other organelles, lens homeostasis relies on a monolayer of epithelial cells at the anterior surface. The detection of various signaling pathways in lens epithelial cells suggests they respond to stimuli that influence lens function. Focusing on Src Family Kinases (SFKs) and Transient Receptor Potential Vanilloid 4 (TRPV4), we tested whether the epithelium can sense and respond to an event that occurs in fiber mass. The pig lens was subjected to localized freeze-thaw (FT) damage to fibers at posterior pole then the lens was incubated for 1-10 min in Krebs solution at 37 °C. Transient SFK activation in the epithelium was detectable at 1 min. Using a western blot approach, the ion channel TRPV4 was detected in the epithelium but was sparse or absent in fiber cells. Even though TRPV4 expression appears low at the actual site of FT damage to the fibers, SFK activation in the epithelium was suppressed in lenses subjected to FT damage then incubated with the TRPV4 antagonist HC067047 (10 μM). Na,K-ATPase activity was examined because previous studies report changes of Na,K-ATPase activity associated with SFK activation. Na,K-ATPase activity doubled in the epithelium removed from FT-damaged lenses and the response was prevented by HC067047 or the SFK inhibitor PP2 (10 μM). Similar changes were observed in response to fiber damage caused by injection of 5 μl hyperosmotic NaCl or mannitol solution beneath the surface of the posterior pole. The findings point to a TRPV4-dependent mechanism that enables the epithelial cells to detect remote damage in the fiber mass and respond within minutes by activating SFK and increasing Na,K-ATPase activity. Because TRPV4 channels are mechanosensitive, we speculate they may be stimulated by swelling of the lens structure caused by damage to the fibers. Increased Na,K-ATPase activity gives the lens greater capacity to

  6. Chronic immune activation and inflammation as the cause of malignancy

    PubMed Central

    O'Byrne, K J; Dalgleish, A G

    2001-01-01

    Several chronic infections known to be associated with malignancy have established oncogenic properties. However the existence of chronic inflammatory conditions that do not have an established infective cause and are associated with the development of tumours strongly suggests that the inflammatory process itself provides the prerequisite environment for the development of malignancy. This environment includes upregulation of mediators of the inflammatory response such as cyclo-oxygenase (COX)-2 leading to the production of inflammatory cytokines and prostaglandins which themselves may suppress cell mediated immune responses and promote angiogenesis. These factors may also impact on cell growth and survival signalling pathways resulting in induction of cell proliferation and inhibition of apoptosis. Furthermore, chronic inflammation may lead to the production of reactive oxygen species and metabolites such as malondialdehyde within the affected cells that may in turn induce DNA damage and mutations and, as a result, be carcinogenic. Here it is proposed that the conditions provided by a chronic inflammatory environment are so essential for the progression of the neoplastic process that therapeutic intervention aimed at inhibiting inflammation, reducing angiogenesis and stimulating cell mediated immune responses may have a major role in reducing the incidence of common cancers. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11506482

  7. A sediment mesocosm experiment to determine if the remediation of a shoreline waste disposal site in Antarctica caused further environmental impacts.

    PubMed

    Stark, Jonathan S; Johnstone, Glenn J; Riddle, Martin J

    2014-12-15

    A shoreline waste disposal site at Casey Station, Antarctica was removed because it was causing impacts in the adjacent marine environment (Brown Bay). We conducted a field experiment to determine whether the excavation created further impacts. Trays of clean, defaunated sediment were deployed at two locations within Brown Bay and two control locations, two years prior to remediation. Trays were sampled one year before, 1month before, 1month after and two years after the excavation. An increase in metals was found at Brown Bay two years after the remediation. However there was little evidence of impacts on sediment assemblages. Communities at each location were different, but differences from before to after the remediation were comparable, indicating there were unlikely to have been further impacts. We demonstrate that abandoned waste disposal sites in hydrologically active places in Antarctica can be removed without creating greater adverse impacts to ecosystems downstream.

  8. Effect of prolonged exposure to organic solvents on the active site environment of subtilisin Carlsberg.

    PubMed

    Bansal, Vibha; Delgado, Yamixa; Fasoli, Ezio; Ferrer, Amaris; Griebenow, Kai; Secundo, Francesco; Barletta, Gabriel L

    2010-06-01

    The potential of enzyme catalysis as a tool for organic synthesis is nowadays indisputable, as is the fact that organic solvents affect an enzyme's activity, selectivity and stability. Moreover, it was recently realized that an enzyme's initial activity is substantially decreased after prolonged exposure to organic media, an effect that further hampers their potential as catalysts for organic synthesis. Regrettably, the mechanistic reasons for these effects are still debatable. In the present study we have made an attempt to explain the reasons behind the partial loss of enzyme activity on prolonged exposure to organic solvents. Fluorescence spectroscopic studies of the serine protease subtilisin Carlsberg chemically modified with polyethylene glycol (PEG-SC) and inhibited with a Dancyl fluorophore, and dissolved in two organic solvents (acetonitrile and 1,4-dioxane) indicate that when the enzyme is initially introduced into these solvents, the active site environment is similar to that in water; however prolonged exposure to the organic medium causes this environment to resemble that of the solvent in which the enzyme is dissolved. Furthermore, kinetic studies show a reduction on both V(max) and K(M) as a result of prolonged exposure to the solvents. One interpretation of these results is that during this prolonged exposure to organic solvents the active-site fluorescent label inhibitor adopts a different binding conformation. Extrapolating this to an enzymatic reaction we argue that substrates bind in a less catalytically favorable conformation after the enzyme has been exposed to organic media for several hours.

  9. Stacking transition in bilayer graphene caused by thermally activated rotation

    NASA Astrophysics Data System (ADS)

    Zhu, Mengjian; Ghazaryan, Davit; Son, Seok-Kyun; Woods, Colin R.; Misra, Abhishek; He, Lin; Taniguchi, Takashi; Watanabe, Kenji; Novoselov, Kostya S.; Cao, Yang; Mishchenko, Artem

    2017-03-01

    Crystallographic alignment between two-dimensional crystals in van der Waals heterostructures brought a number of profound physical phenomena, including observation of Hofstadter butterfly and topological currents, and promising novel applications, such as resonant tunnelling transistors. Here, by probing the electronic density of states in graphene using graphene-hexagonal boron nitride-graphene tunnelling transistors, we demonstrate a structural transition of bilayer graphene from incommensurate twisted stacking state into a commensurate AB stacking due to a macroscopic graphene self-rotation. This structural transition is accompanied by a topological transition in the reciprocal space and by pseudospin texturing. The stacking transition is driven by van der Waals interaction energy of the two graphene layers and is thermally activated by unpinning the microscopic chemical adsorbents which are then removed by the self-cleaning of graphene.

  10. Photonic crystals cause active colour change in chameleons.

    PubMed

    Teyssier, Jérémie; Saenko, Suzanne V; van der Marel, Dirk; Milinkovitch, Michel C

    2015-03-10

    Many chameleons, and panther chameleons in particular, have the remarkable ability to exhibit complex and rapid colour changes during social interactions such as male contests or courtship. It is generally interpreted that these changes are due to dispersion/aggregation of pigment-containing organelles within dermal chromatophores. Here, combining microscopy, photometric videography and photonic band-gap modelling, we show that chameleons shift colour through active tuning of a lattice of guanine nanocrystals within a superficial thick layer of dermal iridophores. In addition, we show that a deeper population of iridophores with larger crystals reflects a substantial proportion of sunlight especially in the near-infrared range. The organization of iridophores into two superposed layers constitutes an evolutionary novelty for chameleons, which allows some species to combine efficient camouflage with spectacular display, while potentially providing passive thermal protection.

  11. Photonic crystals cause active colour change in chameleons

    PubMed Central

    Teyssier, Jérémie; Saenko, Suzanne V.; van der Marel, Dirk; Milinkovitch, Michel C.

    2015-01-01

    Many chameleons, and panther chameleons in particular, have the remarkable ability to exhibit complex and rapid colour changes during social interactions such as male contests or courtship. It is generally interpreted that these changes are due to dispersion/aggregation of pigment-containing organelles within dermal chromatophores. Here, combining microscopy, photometric videography and photonic band-gap modelling, we show that chameleons shift colour through active tuning of a lattice of guanine nanocrystals within a superficial thick layer of dermal iridophores. In addition, we show that a deeper population of iridophores with larger crystals reflects a substantial proportion of sunlight especially in the near-infrared range. The organization of iridophores into two superposed layers constitutes an evolutionary novelty for chameleons, which allows some species to combine efficient camouflage with spectacular display, while potentially providing passive thermal protection. PMID:25757068

  12. Laboratory activities involving transmissible spongiform encephalopathy causing agents

    PubMed Central

    Leunda, Amaya; Van Vaerenbergh, Bernadette; Baldo, Aline; Roels, Stefan; Herman, Philippe

    2013-01-01

    Since the appearance in 1986 of epidemic of bovine spongiform encephalopathy (BSE), a new form of neurological disease in cattle which also affected human beings, many diagnostic and research activities have been performed to develop detection and therapeutic tools. A lot of progress was made in better identifying, understanding and controlling the spread of the disease by appropriate monitoring and control programs in European countries. This paper reviews the recent knowledge on pathogenesis, transmission and persistence outside the host of prion, the causative agent of transmissible spongiform encephalopathies (TSE) in mammals with a particular focus on risk (re)assessment and management of biosafety measures to be implemented in diagnostic and research laboratories in Belgium. Also, in response to the need of an increasing number of European diagnostic laboratories stopping TSE diagnosis due to a decreasing number of TSE cases reported in the last years, decontamination procedures and a protocol for decommissioning TSE diagnostic laboratories is proposed. PMID:24055928

  13. Characterization of Active Site Residues of Nitroalkane Oxidase†

    PubMed Central

    Valley, Michael P.; Fenny, Nana S.; Ali, Shah R.; Fitzpatrick, Paul F.

    2010-01-01

    The flavoenzyme nitroalkane oxidase catalyzes the oxidation of primary and secondary nitrolkanes to the corresponding aldehydes and ketones plus nitrite. The structure of the enzyme shows that Serl71 forms a hydrogen bond to the flavin N5, suggesting that it plays a role in catalysis. Cys397 and Tyr398 were previously identified by chemical modification as potential active site residues. To more directly probe the roles of these residues, the S171A, S171V, S171T, C397S, and Y398F enzymes have been characterized with nitroethane as substrate. The C397S and Y398 enzymes were less stable than the wild-type enzyme, and the C397S enzyme routinely contained a substoichiometric amount of FAD. Analysis of the steady-state kinetic parameters for the mutant enzymes, including deuterium isotope effects, establishes that all of the mutations result in decreases in the rate constants for removal of the substrate proton by ~5-fold and decreases in the rate constant for product release of ~2-fold. Only the S171V and S171T mutations alter the rate constant for flavin oxidation. These results establish that these residues are not involved in catalysis, but rather are required for maintaining the protein structure. PMID:20056514

  14. Detection limit for activation measurements in ultralow background sites

    NASA Astrophysics Data System (ADS)

    Trache, Livius; Chesneanu, D.; Margineanu, R.; Pantelica, A.; Ghita, D. G.; Burducea, I.; Straticiuc, M.; Tang, X. D.

    2014-09-01

    We used 12C +13C fusion at the beam energies E = 6, 7 and 8 MeV to determine the sensitivity and the limits of activation method measurements in ultralow background sites. A 13C beam of 0.5 μA from the 3 MV Tandem accelerator of the Horia Hulubei National Institute of Physics and Nuclear Engineering - IFIN HH impinged on thick graphite targets. After about 24 hrs of irradiation targets were measured in two different laboratories: one with a heavy shielded Ge detector in the institute (at the surface) and one located underground in the microBequerel laboratory, in the salt mine of Slanic-Prahova, Romania. The 1369- and 2754 keV peaks from 24Na deactivation were clearly observed in the γ-ray spectra obtained for acquisitions lasting a few hours, or a few days. Determination of the detection limit in evaluating the cross sections for the target irradiated at Ec . m = 3 MeV indicates the fact that it is possible to measure gamma spectrum in underground laboratory down to Ec . m = 2 . 6 MeV. Cleaning the spectra with beta-gamma coincidences and increasing beam intensity 20 times will take as further down. The measurements are motivated by the study of the 12 C +12 C reaction at astrophysical energies.

  15. Disturbance opens recruitment sites for bacterial colonization in activated sludge.

    PubMed

    Vuono, David C; Munakata-Marr, Junko; Spear, John R; Drewes, Jörg E

    2016-01-01

    Little is known about the role of immigration in shaping bacterial communities or the factors that may dictate success or failure of colonization by bacteria from regional species pools. To address these knowledge gaps, the influence of bacterial colonization into an ecosystem (activated sludge bioreactor) was measured through a disturbance gradient (successive decreases in the parameter solids retention time) relative to stable operational conditions. Through a DNA sequencing approach, we show that the most abundant bacteria within the immigrant community have a greater probability of colonizing the receiving ecosystem, but mostly as low abundance community members. Only during the disturbance do some of these bacterial populations significantly increase in abundance beyond background levels and in few cases become dominant community members post-disturbance. Two mechanisms facilitate the enhanced enrichment of immigrant populations during disturbance: (i) the availability of resources left unconsumed by established species and (ii) the increased availability of niche space for colonizers to establish and displace resident populations. Thus, as a disturbance decreases local diversity, recruitment sites become available to promote colonization. This work advances our understanding of microbial resource management and diversity maintenance in complex ecosystems.

  16. Zfx facilitates tumorigenesis caused by activation of the Hedgehog pathway

    PubMed Central

    Palmer, Colin J.; Galan-Caridad, Jose M.; Weisberg, Stuart P.; Lei, Liang; Esquilin, Jose M.; Croft, Gist F.; Wainwright, Brandon; Canoll, Peter; Owens, David M.; Reizis, Boris

    2014-01-01

    The Hedgehog (Hh) signaling pathway regulates normal development and cell proliferation in metazoan organisms, but its aberrant activation can promote tumorigenesis. Hh-induced tumors arise from various tissues and they may be indolent or aggressive, as is the case with skin basal cell carcinoma (BCC) or cerebellar medulloblastoma (MB), respectively. Little is known about common cell-intrinsic factors that control the development of such diverse Hh-dependent tumors. Transcription factor Zfx is required for the self-renewal of hematopoietic and embryonic stem cells, as well as for the propagation of acute myeloid and T-lymphoblastic leukemias. We report here that Zfx facilitates the development of experimental BCC and MB in mice initiated by deletion of the Hh inhibitory receptor Ptch1. Simultaneous deletion of Zfx along with Ptch1 prevented BCC formation and delayed MB development. In contrast, Zfx was dispensable for tumorigenesis in a mouse model of glioblastoma. We used genome-wide expression and chromatin binding analysis in a human MB cell line to characterize direct, evolutionarily conserved targets of Zfx, identifying Dis3L and Ube2j1 as two targets required for the growth of the human MB cells. Our results establish Zfx as a common cell-intrinsic regulator of diverse Hh-induced tumors, with implications for the definition of new therapeutic targets in these malignancies. PMID:25164012

  17. Biological activities caused by far-infrared radiation

    NASA Astrophysics Data System (ADS)

    Inoué, Shojiro; Kabaya, Morihiro

    1989-09-01

    Contrary to previous presumption, accumulated evidence indicates that far-infrared rays are biologically active. A small ceramic disk that emist far-infrared rays (4 16 μm) has commonly been applied to a local spot or a whole part of the body for exposure. Pioneering attempts to experimentally analyze an effect of acute and chronic radiation of far-infrared rays on living organisms have detected a growth-promoting effect in growing rats, a sleep-modulatory effect in freely behaving rats and an insomiac patient, and a blood circulation-enhancing effect in human skin. Question-paires to 542 users of far-infrared radiator disks embedded in bedelothes revealed that the majority of the users subjectively evaluated an improvement of their health. These effects on living organisms appear to be non-specifically triggered by an exposure to far-infrared rays, which eventually induce an increase in temperature of the body tissues or, more basically, an elevated motility of body fluids due to decrease in size of water clusters.

  18. Identification of a novel splice-site mutation in the Lebercilin (LCA5) gene causing Leber congenital amaurosis

    PubMed Central

    Ramprasad, Vedam Lakshmi; Soumittra, Nagasamy; Nancarrow, Derek; Sen, Parveen; McKibbin, Martin; Williams, Grange A; Arokiasamy, Tharigopala; Lakshmipathy, Praveena; Inglehearn, Chris F

    2008-01-01

    Purpose Leber congenital amaurosis (LCA) is one of the most common causes of hereditary blindness in infants. To date, mutations in 13 known genes and at two other loci have been implicated in LCA causation. An examination of the known genes highlights several processes which, when defective, cause LCA, including photoreceptor development and maintenance, phototransduction, vitamin A metabolism, and protein trafficking. In addition, it has been known for some time that defects in sensory cilia can cause syndromes involving hereditary blindness. More recently evidence has come to light that non-syndromic LCA can also be a “ciliopathy.” Methods Here we present a homozygosity mapping analysis in a consanguineous sibship that led to the identification of a mutation in the recently discovered LCA5 gene. Homozygosity mapping was done using Affymetrix 10K Xba I Gene Chip and a 24.5cM region on chromosome 6 (6q12- q16.3) was identified to be significantly homozygous. The LCA5 gene on this region was sequenced and cDNA sequencing also done to characterize the mutation. Results A c.955G>A missense mutation in the last base of exon 6 causing disruption of the splice donor site was identified in both the affected sibs. Since there is a second consensus splice donor sequence 5 bp into the adjacent intron, this mutation results in a transcript with a 5 bp insertion of intronic sequence, leading to a frameshift and premature truncation. Conclusions We report a missense mutation functionally altering the splice donor site and leading to a truncated protein. This is the second report of LCA5 mutations causing LCA. It may also be significant that one affected child died at eleven months of age due to asphyxia during sleep. To date the only phenotype unambiguously associated with mutations in this gene is LCA. However the LCA5 gene is known to be expressed in nasopharynx, trachea and lungs and was originally identified in the proteome of bronchial epithelium ciliary axonemes. The

  19. Mosaic Activating Mutations in FGFR1 Cause Encephalocraniocutaneous Lipomatosis

    PubMed Central

    Bennett, James T.; Tan, Tiong Yang; Alcantara, Diana; Tétrault, Martine; Timms, Andrew E.; Jensen, Dana; Collins, Sarah; Nowaczyk, Malgorzata J.M.; Lindhurst, Marjorie J.; Christensen, Katherine M.; Braddock, Stephen R.; Brandling-Bennett, Heather; Hennekam, Raoul C.M.; Chung, Brian; Lehman, Anna; Su, John; Ng, SuYuen; Amor, David J.; Majewski, Jacek; Biesecker, Les G.; Boycott, Kym M.; Dobyns, William B.; O’Driscoll, Mark; Moog, Ute; McDonell, Laura M.

    2016-01-01

    Encephalocraniocutaneous lipomatosis (ECCL) is a sporadic condition characterized by ocular, cutaneous, and central nervous system anomalies. Key clinical features include a well-demarcated hairless fatty nevus on the scalp, benign ocular tumors, and central nervous system lipomas. Seizures, spasticity, and intellectual disability can be present, although affected individuals without seizures and with normal intellect have also been reported. Given the patchy and asymmetric nature of the malformations, ECCL has been hypothesized to be due to a post-zygotic, mosaic mutation. Despite phenotypic overlap with several other disorders associated with mutations in the RAS-MAPK and PI3K-AKT pathways, the molecular etiology of ECCL remains unknown. Using exome sequencing of DNA from multiple affected tissues from five unrelated individuals with ECCL, we identified two mosaic mutations, c.1638C>A (p.Asn546Lys) and c.1966A>G (p.Lys656Glu) within the tyrosine kinase domain of FGFR1, in two affected individuals each. These two residues are the most commonly mutated residues in FGFR1 in human cancers and are associated primarily with CNS tumors. Targeted resequencing of FGFR1 in multiple tissues from an independent cohort of individuals with ECCL identified one additional individual with a c.1638C>A (p.Asn546Lys) mutation in FGFR1. Functional studies of ECCL fibroblast cell lines show increased levels of phosphorylated FGFRs and phosphorylated FRS2, a direct substrate of FGFR1, as well as constitutive activation of RAS-MAPK signaling. In addition to identifying the molecular etiology of ECCL, our results support the emerging overlap between mosaic developmental disorders and tumorigenesis. PMID:26942290

  20. Active Site Structures in Nitrogen-Doped Carbon-Supported Cobalt Catalysts for the Oxygen Reduction Reaction.

    PubMed

    Qian, Yingdan; Liu, Zheng; Zhang, Hui; Wu, Ping; Cai, Chenxin

    2016-12-07

    The catalytic mechanism and the nature of active sites are revealed for the oxygen reduction reaction (ORR) with new non-noble-metal nitrogen-doped carbon-supported transition-metal catalysts (metal-N-C catalyst). Specifically, new nitrogen-doped carbon-supported cobalt catalysts (Co-N-C catalysts) are made by pyrolyzing various ratios of the nitrogen-atom rich heterocycle compound, 1-ethyl-3-methyl imidazolium dicyanamide (EMIM-dca) and cobalt salt (Co(NO3)2). The ORR activity (JK at 0.8 V vs RHE, in 0.1 M KOH solution) of a typical catalyst in this family, Co15-N-C800, is 8.25 mA/mg, which is much higher than the ORR activity values of N-C catalysts (0.41 mA/mg). The active site in the catalyst is found to be the Co-N species, which is most likely in the form of Co2N. Metallic cobalt (Co) particles, Co3C species, and N-C species are not catalytically active sites, nor do these moieties interact with the Co-N active sites during the catalysis of the ORR. Increasing the Co salt content during the synthesis favors the formation of Co-N active sites in the final catalyst. Higher pyrolysis temperatures (e.g., a temperature higher than 800 °C) do not favor the formation of the Co-N active sites, but cause the formed Co-N active sites to decompose, which, therefore, leads to a lower catalytic activity. This reveals that the control of the parameters that affect the final structure is critical to catalyst performance and, therefore, the effective development of high-performance heteroatom-doped non-noble-metal ORR catalysts.

  1. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 9 2012-07-01 2012-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing...

  2. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 9 2014-07-01 2014-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an active... visible emissions to the outside air from any active waste disposal site where asbestos-containing...

  3. Hydroxynonenal inactivates cathepsin B by forming Michael adducts with active site residues.

    PubMed

    Crabb, John W; O'Neil, June; Miyagi, Masaru; West, Karen; Hoff, Henry F

    2002-04-01

    Oxidation of plasma low-density lipoprotein (oxLDL) generates the lipid peroxidation product 4-hydroxy-2 nonenal (HNE) and also reduces proteolytic degradation of oxLDL and other proteins internalized by mouse peritoneal macrophages in culture. This leads to accumulation of undegraded material in lysosomes and formation of ceroid, a component of foam cells in atherosclerotic lesions. To explore the possibility that HNE contributes directly to the inactivation of proteases, structure-function studies of the lysosomal protease cathepsin B have been pursued. We found that treatment of mouse macrophages with HNE reduces degradation of internalized maleyl bovine serine albumin and cathepsin B activity. Purified bovine cathepsin B treated briefly with 15 microM HNE lost approximately 76% of its protease activity and also developed immunoreactivity with antibodies to HNE adducts in Western blot analysis. After stabilization of the potential Michael adducts by sodium borohydride reduction, modified amino acids were localized within the bovine cathepsin B protein structure by mass spectrometric analysis of tryptic peptides. Michael adducts were identified by tandem mass spectrometry at cathepsin B active site residues Cys 29 (mature A chain) and His 150 (mature B chain). Thus, covalent interaction between HNE and critical active site residues inactivates cathepsin B. These results support the hypothesis that the accumulation of undegraded macromolecules in lysosomes after oxidative damage are caused in part by direct protease inactivation by adduct formation with lipid peroxidation products such as HNE.

  4. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  5. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  6. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  7. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  8. 10 CFR 63.16 - Review of site characterization activities. 2

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... IN A GEOLOGIC REPOSITORY AT YUCCA MOUNTAIN, NEVADA Licenses Preapplication Review § 63.16 Review of... conduct of site characterization activities at the Yucca Mountain site, DOE shall report the nature and... activities at the Yucca Mountain site, NRC staff shall be permitted to visit and inspect the locations...

  9. Robotics and Automation Activities at the Savannah River Site: A Site Report for SUBWOG 39F

    SciTech Connect

    Teese, G.D.

    1995-09-28

    The Savannah River Site has successfully used robots, teleoperators, and remote video to reduce exposure to ionizing radiation, improve worker safety, and improve the quality of operations. Previous reports have described the use of mobile teleoperators in coping with a high level liquid waste spill, the removal of highly contaminated equipment, and the inspection of nuclear reactor vessels. This report will cover recent applications at the Savannah River, as well as systems which SRS has delivered to other DOE site customers.

  10. GAS HYDRATES AT TWO SITES OF AN ACTIVE CONTINENTAL MARGIN.

    USGS Publications Warehouse

    Kvenvolden, K.A.

    1985-01-01

    Sediment containing gas hydrates from two distant Deep Sea Drilling Project sites (565 and 568), located about 670 km apart on the landward flank of the Middle America Trench, was studied to determine the geochemical conditions that characterize the occurrence of gas hydrates. Site 565 was located in the Pacific Ocean offshore the Nicoya Peninsula of Costa Rica in 3,111 m of water. The depth of the hole at this site was 328 m, and gas hydrates were recovered from 285 and 319 m. Site 568 was located about 670 km to the northwest offshore Guatemala in 2,031 m of water. At this site the hole penetrated to 418 m, and gas hydrates were encountered at 404 m.

  11. Lidar research activities and observations at NARL site, Gadanki, India

    NASA Astrophysics Data System (ADS)

    Yellapragada, Bhavani Kumar

    2016-05-01

    The National Atmospheric Research Laboratory (NARL), a unit of Department of Space (DOS), located at Gadanki village (13.5°N, 79.2°E, 370 m AMSL) in India, is involved in the development of lidar remote sensing technologies for atmospheric research. Several advanced lidar technologies employing micropulse, polarization, Raman and scanning have been developed at this site and demonstrated for atmospheric studies during the period between 2008 and 2015. The technology of micropulse lidar, operates at 532 nm wavelength, was successfully transferred to an industry and the commercial version has been identified for Indian Lidar network (I-LINK) programme. Under this lidar network activity, several lidar units were installed at different locations in India to study tropospheric aerosols and clouds. The polarization sensitive lidar technology was realized using a set of mini photomultiplier tube (PMT) units and has the capability to operate during day and night without a pause. The lidar technology uses a compact flashlamp pumped Qswitched laser and employs biaxial configuration between the transmitter and receiver units. The lidar technology has been utilized for understanding the polarization characteristics of boundary layer aerosols during the mixed layer development. The demonstrated Raman lidar technology, uses the third harmonic wavelength of Nd:YAG laser, provides the altitude profiles of aerosol backscattering, extinction and water vapor covering the boundary layer range and allows operation during nocturnal periods. The Raman lidar derived height profiles of aerosol backscattering and extinction coefficient, lidar ratio, and watervapor mixing ratio inform the tropical boundary layer aerosol characteristics. The scanning lidar technology uses a near infrared laser wavelength for probing the lower atmosphere and has been utilized for high resolution cloud profiling during convective periods. The lidar technology is also used for rain rate measurement during

  12. Dynamically achieved active site precision in enzyme catalysis.

    PubMed

    Klinman, Judith P

    2015-02-17

    CONSPECTUS: The grand challenge in enzymology is to define and understand all of the parameters that contribute to enzymes' enormous rate accelerations. The property of hydrogen tunneling in enzyme reactions has moved the focus of research away from an exclusive focus on transition state stabilization toward the importance of the motions of the heavy atoms of the protein, a role for reduced barrier width in catalysis, and the sampling of a protein conformational landscape to achieve a family of protein substates that optimize enzyme-substrate interactions and beyond. This Account focuses on a thermophilic alcohol dehydrogenase for which the chemical step of hydride transfer is rate determining across a wide range of experimental conditions. The properties of the chemical coordinate have been probed using kinetic isotope effects, indicating a transition in behavior below 30 °C that distinguishes nonoptimal from optimal C-H activation. Further, the introduction of single site mutants has the impact of either enhancing or eliminating the temperature dependent transition in catalysis. Biophysical probes, which include time dependent hydrogen/deuterium exchange and fluorescent lifetimes and Stokes shifts, have also been pursued. These studies allow the correlation of spatially resolved transitions in protein motions with catalysis. It is now possible to define a long-range network of protein motions in ht-ADH that extends from a dimer interface to the substrate binding domain across to the cofactor binding domain, over a distance of ca. 30 Å. The ongoing challenge to obtaining spatial and temporal resolution of catalysis-linked protein motions is discussed.

  13. Characterization of the interactions between the active site of a protein tyrosine kinase and a divalent metal activator

    PubMed Central

    Lin, Xiaofeng; Ayrapetov, Marina K; Sun, Gongqin

    2005-01-01

    Background Protein tyrosine kinases are important enzymes for cell signalling and key targets for anticancer drug discovery. The catalytic mechanisms of protein tyrosine kinase-catalysed phosphorylation are not fully understood. Protein tyrosine kinase Csk requires two Mg2+ cations for activity: one (M1) binds to ATP, and the other (M2) acts as an essential activator. Results Experiments in this communication characterize the interaction between M2 and Csk. Csk activity is sensitive to pH in the range of 6 to 7. Kinetic characterization indicates that the sensitivity is not due to altered substrate binding, but caused by the sensitivity of M2 binding to pH. Several residues in the active site with potential of binding M2 are mutated and the effect on metal activation studied. An active mutant of Asn319 is generated, and this mutation does not alter the metal binding characteristics. Mutations of Glu236 or Asp332 abolish the kinase activity, precluding a positive or negative conclusion on their role in M2 coordination. Finally, the ability of divalent metal cations to activate Csk correlates to a combination of ionic radius and the coordination number. Conclusion These studies demonstrate that M2 binding to Csk is sensitive to pH, which is mainly responsible for Csk activity change in the acidic arm of the pH response curve. They also demonstrate critical differences in the metal activator coordination sphere in protein tyrosine kinase Csk and a protein Ser/Thr kinase, the cAMP-dependent protein kinase. They shed light on the physical interactions between a protein tyrosine kinase and a divalent metal activator. PMID:16305747

  14. Active-site zinc ligands and activated H2O of zinc enzymes.

    PubMed Central

    Vallee, B L; Auld, D S

    1990-01-01

    The x-ray crystallographic structures of 12 zinc enzymes have been chosen as standards of reference to identify the ligands to the catalytic and structural zinc atoms of other members of their respective enzyme families. Universally, H2O is a ligand and critical component of the catalytically active zinc sites. In addition, three protein side chains bind to the catalytic zinc atom, whereas four protein ligands bind to the structural zinc atom. The geometry and coordination number of zinc can vary greatly to accommodate particular ligands. Zinc forms complexes with nitrogen and oxygen just as readily as with sulfur, and this is reflected in catalytic zinc sites having a binding frequency of His much greater than Glu greater than Asp = Cys, three of which bind to the metal atom. The systematic spacing between the ligands is striking. For all catalytic zinc sites except the coenzyme-dependent alcohol dehydrogenase, the first two ligands are separated by a "short-spacer" consisting of 1 to 3 amino acids. These ligands are separated from the third ligand by a "long spacer" of approximately 20 to approximately 120 amino acids. The spacer enables formation of a primary bidentate zinc complex, whereas the long spacer contributes flexibility to the coordination sphere, which can poise the zinc for catalysis as well as bring other catalytic and substrate binding groups into apposition with the active site. The H2O is activated by ionization, polarization, or poised for displacement. Collectively, the data imply that the preferred mechanistic pathway for activating the water--e.g., zinc hydroxide or Lewis acid catalysis--will be determined by the identity of the other three ligands and their spacing. Images PMID:2104979

  15. Lethal Factor Active-Site Mutations Affect Catalytic Activity In Vitro

    PubMed Central

    Hammond, S. E.; Hanna, P. C.

    1998-01-01

    The lethal factor (LF) protein of Bacillus anthracis lethal toxin contains the thermolysin-like active-site and zinc-binding consensus motif HEXXH (K. R. Klimpel, N. Arora, and S. H. Leppla, Mol. Microbiol. 13:1093–1100, 1994). LF is hypothesized to act as a Zn2+ metalloprotease in the cytoplasm of macrophages, but no proteolytic activities have been previously shown on any target substrate. Here, synthetic peptides are hydrolyzed by LF in vitro. Mass spectroscopy and peptide sequencing of isolated cleavage products separated by reverse-phase high-pressure liquid chromatography indicate that LF seems to prefer proline-containing substrates. Substitution mutations within the consensus active-site residues completely abolish all in vitro catalytic functions, as does addition of 1,10-phenanthroline, EDTA, and certain amino acid hydroxamates, including the novel zinc metalloprotease inhibitor ZINCOV. In contrast, the protease inhibitors bestatin and lysine CMK, previously shown to block LF activity on macrophages, did not block LF activity in vitro. These data provide the first direct evidence that LF may act as an endopeptidase. PMID:9573135

  16. The yeast regulator of transcription protein Rtr1 lacks an active site and phosphatase activity.

    PubMed

    Xiang, Kehui; Manley, James L; Tong, Liang

    2012-07-10

    The activity of RNA polymerase II (Pol II) is controlled in part by the phosphorylation state of the C-terminal domain (CTD) of its largest subunit. Recent reports have suggested that yeast regulator of transcription protein, Rtr1, and its human homologue RPAP2, possess Pol II CTD Ser5 phosphatase activity. Here we report the crystal structure of Kluyveromyces lactis Rtr1, which reveals a new type of zinc finger protein and does not have any close structural homologues. Importantly, the structure does not show evidence of an active site, and extensive experiments to demonstrate its CTD phosphatase activity have been unsuccessful, suggesting that Rtr1 has a non-catalytic role in CTD dephosphorylation.

  17. Sustained mitogen-activated protein kinase activation with Aggregatibacter actinomycetemcomitans causes inflammatory bone loss.

    PubMed

    Dunmyer, J; Herbert, B; Li, Q; Zinna, R; Martin, K; Yu, H; Kirkwood, K L

    2012-10-01

    Aggregatibacter actinomycetemcomitans is a gram-negative facultative capnophile involved in pathogenesis of aggressive forms of periodontal disease. In the present study, we interrogated the ability of A. actinomycetemcomitans to stimulate innate immune signaling and cytokine production and established that A. actinomycetemcomitans causes bone loss in a novel rat calvarial model. In vitro studies indicated that A. actinomycetemcomitans stimulated considerable production of soluble cytokines, tumor necrosis factor-α, interleukin-6 and interleukin-10 in both primary bone marrow-derived macrophages and NR8383 macrophages. Immunoblot analysis indicated that A. actinomycetemcomitans exhibits sustained activation of all major mitogen-activated protein kinase (MAPK) pathways, as well as the negative regulator of MAPK signaling, MAPK phosphatase-1 (MKP-1), for at least 8 h. In a rat calvarial model of inflammatory bone loss, high and low doses of formalin-fixed A. actinomycetemcomitans were microinjected into the supraperiosteal calvarial space for 1-2 weeks. Histological staining and micro-computed tomography of rat calvariae revealed a significant increase of inflammatory and fibroblast infiltrate and increased bone resorption as measured by total lacunar pit formation. From these data, we provide new evidence that fixed whole cell A. actinomycetemcomitans stimulation elicits a pro-inflammatory host response through sustained MAPK signaling, leading to enhanced bone resorption within the rat calvarial bone.

  18. The active site of oxidative phosphorylation and the origin of hyperhomocysteinemia in aging and dementia.

    PubMed

    McCully, Kilmer S

    2015-01-01

    The active site of oxidative phosphorylation and adenosine triphosphate (ATP) synthesis in mitochondria is proposed to consist of two molecules of thioretinamide bound to cobalamin, forming thioretinaco, complexed with ozone, oxygen, nicotinamide adenine dinucleotide. and inorganic phosphate, TR2CoO3O2NAD(+)H2PO4(-). Reduction of the pyridinium nitrogen of the nicotinamide group by an electron from electron transport complexes initiates polymerization of phosphate with adenosine diphosphate, yielding nicotinamide riboside and ATP bound to thioretinaco ozonide oxygen. A second electron reduces oxygen to hydroperoxyl radical, releasing ATP from the active site. A proton gradient is created within F1F0 ATPase complexes of mitochondria by reaction of protons with reduced nicotinamide riboside and with hydroperoxyl radical, yielding reduced nicotinamide riboside and hydroperoxide. The hyperhomocysteinemia of aging and dementia is attributed to decreased synthesis of adenosyl methionine by thioretinaco ozonide and ATP, causing decreased allosteric activation of cystathionine synthase and decreased allosteric inhibition of methylenetetrahydrofolate reductase and resulting in dysregulation of methionine metabolism.

  19. Site-specific phosphorylation and microtubule dynamics control Pyrin inflammasome activation

    PubMed Central

    Gao, Wenqing; Yang, Jieling; Liu, Wang; Wang, Yupeng; Shao, Feng

    2016-01-01

    Pyrin, encoded by the MEFV gene, is best known for its gain-of-function mutations causing familial Mediterranean fever (FMF), an autoinflammatory disease. Pyrin forms a caspase-1–activating inflammasome in response to inactivating modifications of Rho GTPases by various bacterial toxins or effectors. Pyrin-mediated innate immunity is unique in that it senses bacterial virulence rather than microbial molecules, but its mechanism of activation is unknown. Here we show that Pyrin was phosphorylated in bone marrow-derived macrophages and dendritic cells. We identified Ser-205 and Ser-241 in mouse Pyrin whose phosphorylation resulted in inhibitory binding by cellular 14-3-3 proteins. The two serines underwent dephosphorylation upon toxin stimulation or bacterial infection, triggering 14-3-3 dissociation, which correlated with Pyrin inflammasome activation. We developed antibodies specific for phosphorylated Ser-205 and Ser-241, which confirmed the stimuli-induced dephosphorylation of endogenous Pyrin. Mutational analyses indicated that both phosphorylation and signal-induced dephosphorylation of Ser-205/241 are important for Pyrin activation. Moreover, microtubule drugs, including colchicine, commonly used to treat FMF, effectively blocked activation of the Pyrin inflammasome. These drugs did not affect Pyrin dephosphorylation and 14-3-3 dissociation but inhibited Pyrin-mediated apoptosis-associated Speck-like protein containing CARD (ASC) aggregation. Our study reveals that site-specific (de)phosphorylation and microtubule dynamics critically control Pyrin inflammasome activation, illustrating a fine and complex mechanism in cytosolic immunity. PMID:27482109

  20. Outline structure of the human L1 cell adhesion molecule and the sites where mutations cause neurological disorders.

    PubMed Central

    Bateman, A; Jouet, M; MacFarlane, J; Du, J S; Kenwrick, S; Chothia, C

    1996-01-01

    The L1 cell adhesion molecule has six domains homologous to members of the immunoglobulin superfamily and five homologous to fibronectin type III domains. We determined the outline structure of the L1 domains by showing that they have, at the key sites that determine conformation, residues similar to those in proteins of known structure. The outline structure describes the relative positions of residues, the major secondary structures and residue solvent accessibility. We use the outline structure to investigate the likely effects of 22 mutations that cause neurological diseases. The mutations are not randomly distributed but cluster in a few regions of the structure. They can be divided into those that act mainly by changing conformation or denaturing their domain and those that alter its surface properties. Images PMID:8947027

  1. Prediction of P53 Mutants (Multiple Sites) Transcriptional Activity Based on Structural (2D&3D) Properties

    PubMed Central

    Geetha Ramani, R.; Jacob, Shomona Gracia

    2013-01-01

    Prediction of secondary site mutations that reinstate mutated p53 to normalcy has been the focus of intense research in the recent past owing to the fact that p53 mutants have been implicated in more than half of all human cancers and restoration of p53 causes tumor regression. However laboratory investigations are more often laborious and resource intensive but computational techniques could well surmount these drawbacks. In view of this, we formulated a novel approach utilizing computational techniques to predict the transcriptional activity of multiple site (one-site to five-site) p53 mutants. The optimal MCC obtained by the proposed approach on prediction of one-site, two-site, three-site, four-site and five-site mutants were 0.775,0.341,0.784,0.916 and 0.655 respectively, the highest reported thus far in literature. We have also demonstrated that 2D and 3D features generate higher prediction accuracy of p53 activity and our findings revealed the optimal results for prediction of p53 status, reported till date. We believe detection of the secondary site mutations that suppress tumor growth may facilitate better understanding of the relationship between p53 structure and function and further knowledge on the molecular mechanisms and biological activity of p53, a targeted source for cancer therapy. We expect that our prediction methods and reported results may provide useful insights on p53 functional mechanisms and generate more avenues for utilizing computational techniques in biological data analysis. PMID:23468845

  2. [Sop proteins can cause transcriptional silencing of genes located close to the centromere sites of linear plasmid N15].

    PubMed

    Mardanov, A V; Lane, D; Ravin, N V

    2010-01-01

    Stable inheritance of bacterial chromosomes and low copy number plasmids is ensured by accurate partitioning of replicated molecules between the daughter cells at division. Partitioning of the prophage of the temperate bacteriophage N15, which exists as a linear plasmid molecule with covalently closed ends, depends on the sop locus, comprising genes sopA and sopB, as well as four centromere sites located in different regions of the N15 genome essential for replication and the control of lysogeny. We found that binding of SopB to the centromere can silence centromere-proximal promoters, presumably due to subsequent polymerizing of SopB along the DNA. Close to the IR4 centromere site we identified a promoter, P59, able to drive expression of phage late genes encoding the structural proteins of virion. We found that following binding to IR4 the N15 Sop proteins can cause repression of this promoter. The repression depends on SopB and became stronger in the presence of SopA. Sop-dependent silencing of centromere-proximal promoters control gene expression in phage N15, particularly preventing undesired expression of late genes in the N15 prophage. Thus, the phage N15 sop system not only ensures plasmid partitioning but is also involved in the genetic network controlling prophage replication and the maintenance of lysogeny.

  3. Nuclear Site Security in the Event of Terrorist Activity

    SciTech Connect

    Thomson, M.L.; Sims, J.

    2008-07-01

    This paper, presented as a poster, identifies why ballistic protection should now be considered at nuclear sites to counter terrorist threats. A proven and flexible form of multi purpose protection is described in detail with identification of trial results that show its suitability for this role. (authors)

  4. Preliminary siting activities for new waste handling facilities at the Idaho National Engineering Laboratory

    SciTech Connect

    Taylor, D.D.; Hoskinson, R.L.; Kingsford, C.O.; Ball, L.W.

    1994-09-01

    The Idaho Waste Processing Facility, the Mixed and Low-Level Waste Treatment Facility, and the Mixed and Low-Level Waste Disposal Facility are new waste treatment, storage, and disposal facilities that have been proposed at the Idaho National Engineering Laboratory (INEL). A prime consideration in planning for such facilities is the selection of a site. Since spring of 1992, waste management personnel at the INEL have been involved in activities directed to this end. These activities have resulted in the (a) identification of generic siting criteria, considered applicable to either treatment or disposal facilities for the purpose of preliminary site evaluations and comparisons, (b) selection of six candidate locations for siting,and (c) site-specific characterization of candidate sites relative to selected siting criteria. This report describes the information gathered in the above three categories for the six candidate sites. However, a single, preferred site has not yet been identified. Such a determination requires an overall, composite ranking of the candidate sites, which accounts for the fact that the sites under consideration have different advantages and disadvantages, that no single site is superior to all the others in all the siting criteria, and that the criteria should be assigned different weighing factors depending on whether a site is to host a treatment or a disposal facility. Stakeholder input should now be solicited to help guide the final selection. This input will include (a) siting issues not already identified in the siting, work to date, and (b) relative importances of the individual siting criteria. Final site selection will not be completed until stakeholder input (from the State of Idaho, regulatory agencies, the public, etc.) in the above areas has been obtained and a strategy has been developed to make a composite ranking of all candidate sites that accounts for all the siting criteria.

  5. Revealing the nature of the active site on the carbon catalyst for C-H bond activation.

    PubMed

    Sun, XiaoYing; Li, Bo; Su, Dangsheng

    2014-09-28

    A reactivity descriptor for the C-H bond activation on the nanostructured carbon catalyst is proposed. Furthermore the calculations reveal that the single ketone group can be an active site in ODH reaction.

  6. Cellular Active N-Hydroxyurea FEN1 Inhibitors Block Substrate Entry to the Active Site

    PubMed Central

    Exell, Jack C.; Thompson, Mark J.; Finger, L. David; Shaw, Steven J.; Debreczeni, Judit; Ward, Thomas A.; McWhirter, Claire; Siöberg, Catrine L. B.; Martinez Molina, Daniel; Mark Abbott, W.; Jones, Clifford D.; Nissink, J. Willem M.; Durant, Stephen T.; Grasby, Jane A.

    2016-01-01

    The structure-specific nuclease human flap endonuclease-1 (hFEN1) plays a key role in DNA replication and repair and may be of interest as an oncology target. We present the first crystal structure of inhibitor-bound hFEN1 and show a cyclic N-hydroxyurea bound in the active site coordinated to two magnesium ions. Three such compounds had similar IC50 values but differed subtly in mode of action. One had comparable affinity for protein and protein–substrate complex and prevented reaction by binding to active site catalytic metal ions, blocking the unpairing of substrate DNA necessary for reaction. Other compounds were more competitive with substrate. Cellular thermal shift data showed engagement of both inhibitor types with hFEN1 in cells with activation of the DNA damage response evident upon treatment. However, cellular EC50s were significantly higher than in vitro inhibition constants and the implications of this for exploitation of hFEN1 as a drug target are discussed. PMID:27526030

  7. Active Layer and Moisture Measurements for Intensive Site 0 and 1, Barrow, Alaska

    DOE Data Explorer

    John Peterson

    2015-04-17

    These are measurements of Active Layer Thickness collected along several lines beginning in September, 2011 to the present. The data were collected at several time periods along the Site0 L2 Line, the Site1 AB Line, and an ERT Monitoring Line near Area A in Site1.

  8. Identification of promiscuous ene-reductase activity by mining structural databases using active site constellations

    PubMed Central

    Steinkellner, Georg; Gruber, Christian C.; Pavkov-Keller, Tea; Binter, Alexandra; Steiner, Kerstin; Winkler, Christoph; Łyskowski, Andrzej; Schwamberger, Orsolya; Oberer, Monika; Schwab, Helmut; Faber, Kurt; Macheroux, Peter; Gruber, Karl

    2014-01-01

    The exploitation of catalytic promiscuity and the application of de novo design have recently opened the access to novel, non-natural enzymatic activities. Here we describe a structural bioinformatic method for predicting catalytic activities of enzymes based on three-dimensional constellations of functional groups in active sites (‘catalophores’). As a proof-of-concept we identify two enzymes with predicted promiscuous ene-reductase activity (reduction of activated C–C double bonds) and compare them with known ene-reductases, that is, members of the Old Yellow Enzyme family. Despite completely different amino acid sequences, overall structures and protein folds, high-resolution crystal structures reveal equivalent binding modes of typical Old Yellow Enzyme substrates and ligands. Biochemical and biocatalytic data show that the two enzymes indeed possess ene-reductase activity and reveal an inverted stereopreference compared with Old Yellow Enzymes for some substrates. This method could thus be a tool for the identification of viable starting points for the development and engineering of novel biocatalysts. PMID:24954722

  9. Fungal diversity of Norway spruce litter: effects of site conditions and premature leaf fall caused by bark beetle outbreak.

    PubMed

    Przybył, K; Karolewski, P; Oleksyn, J; Labedzki, A; Reich, P B

    2008-08-01

    Fungi play an important role in leaf litter decomposition due to their ability to break down the lignocellulose matrix, which other organisms are unable to digest. However, little is known regarding the factors affecting components of fungal diversity. Here, we quantified richness of internal fungi in relation to litter nutrient and phenolic concentrations, sampling season (spring or fall), and premature leaf shedding due to low precipitation and infestation of bark beetles (mainly Ips typographus and Ips duplicatus). The study was conducted in 37-year-old Norway spruce [Picea abies (L.) Karst.] stands, with three plots each in mixed forest (MF) and coniferous forest (CF) site conditions in south-central Poland. Fifty-four species of sporulating fungi were identified in 2,330 freshly fallen needles sampled during 2003-2005, including 45 species in MF and 31 in CF. The significantly higher number of species in MF was likely related to moister conditions at that site. Among isolated fungi, 22% (12 species) were identified as endophytes of Norway spruce in prior studies. During spring of 2005, we found less than half the number of isolates and fungal species at each forest site as compared to fall for the two prior years. This pattern was observed in typical soil fungi (e.g., Penicillium daleae, Penicillium purpurogenum) and endophytes/epiphytes (e.g., Aureobasidium pullulans, Alternaria alternata, Cladosporium spp., and Lophodermium piceae). Premature shedding of needles was the most likely cause of this decline because it shortened the time period for fungi to infect green needles while on the tree. For all sites and sampling periods, richness of internal fungi was strongly and positively related to the age of freshly fallen litter (assessed using needle Ca concentration as a needle age tracer) and was also negatively related to litter phenolic concentration. Richness of internal fungi in freshly fallen litter may be adversely affected by low soil moisture status

  10. Are nest sites actively chosen? Testing a common assumption for three non-resource limited birds

    NASA Astrophysics Data System (ADS)

    Goodenough, A. E.; Elliot, S. L.; Hart, A. G.

    2009-09-01

    Many widely-accepted ecological concepts are simplified assumptions about complex situations that remain largely untested. One example is the assumption that nest-building species choose nest sites actively when they are not resource limited. This assumption has seen little direct empirical testing: most studies on nest-site selection simply assume that sites are chosen actively (and seek explanations for such behaviour) without considering that sites may be selected randomly. We used 15 years of data from a nestbox scheme in the UK to test the assumption of active nest-site choice in three cavity-nesting bird species that differ in breeding and migratory strategy: blue tit ( Cyanistes caeruleus), great tit ( Parus major) and pied flycatcher ( Ficedula hypoleuca). Nest-site selection was non-random (implying active nest-site choice) for blue and great tits, but not for pied flycatchers. We also considered the relative importance of year-specific and site-specific factors in determining occupation of nest sites. Site-specific factors were more important than year-specific factors for the tit species, while the reverse was true for pied flycatchers. Our results show that nest-site selection, in birds at least, is not always the result of active choice, such that choice should not be assumed automatically in studies of nesting behaviour. We use this example to highlight the need to test key ecological assumptions empirically, and the importance of doing so across taxa rather than for single "model" species.

  11. Early Site Permit Demonstration Program: Recommendations for communication activities and public participation in the Early Site Permit Demonstration Program

    SciTech Connect

    Not Available

    1993-01-27

    On October 24, 1992, President Bush signed into law the National Energy Policy Act of 1992. The bill is a sweeping, comprehensive overhaul of the Nation`s energy laws, the first in more than a decade. Among other provisions, the National Energy Policy Act reforms the licensing process for new nuclear power plants by adopting a new approach developed by the US Nuclear Regulatory Commission (NRC) in 1989, and upheld in court in 1992. The NRC 10 CFR Part 52 rule is a three-step process that guarantees public participation at each step. The steps are: early site permit approval; standard design certifications; and, combined construction/operating licenses for nuclear power reactors. Licensing reform increases an organization`s ability to respond to future baseload electricity generation needs with less financial risk for ratepayers and the organization. Costly delays can be avoided because design, safety and siting issues will be resolved before a company starts to build a plant. Specifically, early site permit approval allows for site suitability and acceptability issues to be addressed prior to an organization`s commitment to build a plant. Responsibility for site-specific activities, including communications and public participation, rests with those organizations selected to try out early site approval. This plan has been prepared to assist those companies (referred to as sponsoring organizations) in planning their communications and public involvement programs. It provides research findings, information and recommendations to be used by organizations as a resource and starting point in developing their own plans.

  12. Structural characterization of single nucleotide variants at ligand binding sites and enzyme active sites of human proteins

    PubMed Central

    Yamada, Kazunori D.; Nishi, Hafumi; Nakata, Junichi; Kinoshita, Kengo

    2016-01-01

    Functional sites on proteins play an important role in various molecular interactions and reactions between proteins and other molecules. Thus, mutations in functional sites can severely affect the overall phenotype. Progress of genome sequencing projects has yielded a wealth of information on single nucleotide variants (SNVs), especially those with less than 1% minor allele frequency (rare variants). To understand the functional influence of genetic variants at a protein level, we investigated the relationship between SNVs and protein functional sites in terms of minor allele frequency and the structural position of variants. As a result, we observed that SNVs were less abundant at ligand binding sites, which is consistent with a previous study on SNVs and protein interaction sites. Additionally, we found that non-rare variants tended to be located slightly apart from enzyme active sites. Examination of non-rare variants revealed that most of the mutations resulted in moderate changes of the physico-chemical properties of amino acids, suggesting the existence of functional constraints. In conclusion, this study shows that the mapping of genetic variants on protein structures could be a powerful approach to evaluate the functional impact of rare genetic variations. PMID:27924270

  13. Stereospecific suppression of active site mutants by methylphosphonate substituted substrates reveals the stereochemical course of site-specific DNA recombination.

    PubMed

    Rowley, Paul A; Kachroo, Aashiq H; Ma, Chien-Hui; Maciaszek, Anna D; Guga, Piotr; Jayaram, Makkuni

    2015-07-13

    Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The SP preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or RP-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5' to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions.

  14. Active-Site Hydration and Water Diffusion in Cytochrome P450cam: A Highly Dynamic Process

    SciTech Connect

    Miao, Yinglong; Baudry, Jerome Y

    2011-01-01

    Long-timescale molecular dynamics simulations (300 ns) are performed on both the apo- (i.e., camphor-free) and camphor-bound cytochrome P450cam (CYP101). Water diffusion into and out of the protein active site is observed without biased sampling methods. During the course of the molecular dynamics simulation, an average of 6.4 water molecules is observed in the camphor-binding site of the apo form, compared to zero water molecules in the binding site of the substrate-bound form, in agreement with the number of water molecules observed in crystal structures of the same species. However, as many as 12 water molecules can be present at a given time in the camphor-binding region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydration of the protein active site, with water molecules exchanging rapidly with the bulk solvent. Water molecules are also found to exchange locations frequently inside the active site, preferentially clustering in regions surrounding the water molecules observed in the crystal structure. Potential-of-mean-force calculations identify thermodynamically favored trans-protein pathways for the diffusion of water molecules between the protein active site and the bulk solvent. Binding of camphor in the active site modifies the free-energy landscape of P450cam channels toward favoring the diffusion of water molecules out of the protein active site.

  15. Structural mechanism of RuBisCO activation by carbamylation of the active site lysine.

    PubMed

    Stec, Boguslaw

    2012-11-13

    Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is a crucial enzyme in carbon fixation and the most abundant protein on earth. It has been studied extensively by biochemical and structural methods; however, the most essential activation step has not yet been described. Here, we describe the mechanistic details of Lys carbamylation that leads to RuBisCO activation by atmospheric CO(2). We report two crystal structures of nitrosylated RuBisCO from the red algae Galdieria sulphuraria with O(2) and CO(2) bound at the active site. G. sulphuraria RuBisCO is inhibited by cysteine nitrosylation that results in trapping of these gaseous ligands. The structure with CO(2) defines an elusive, preactivation complex that contains a metal cation Mg(2+) surrounded by three H(2)O/OH molecules. Both structures suggest the mechanism for discriminating gaseous ligands by their quadrupole electric moments. We describe conformational changes that allow for intermittent binding of the metal ion required for activation. On the basis of these structures we propose the individual steps of the activation mechanism. Knowledge of all these elements is indispensable for engineering RuBisCO into a more efficient enzyme for crop enhancement or as a remedy to global warming.

  16. Using Unnatural Amino Acids to Probe the Energetics of Oxyanion Hole Hydrogen Bonds in the Ketosteroid Isomerase Active Site

    PubMed Central

    2015-01-01

    Hydrogen bonds are ubiquitous in enzyme active sites, providing binding interactions and stabilizing charge rearrangements on substrate groups over the course of a reaction. But understanding the origin and magnitude of their catalytic contributions relative to hydrogen bonds made in aqueous solution remains difficult, in part because of complexities encountered in energetic interpretation of traditional site-directed mutagenesis experiments. It has been proposed for ketosteroid isomerase and other enzymes that active site hydrogen bonding groups provide energetic stabilization via “short, strong” or “low-barrier” hydrogen bonds that are formed due to matching of their pKa or proton affinity to that of the transition state. It has also been proposed that the ketosteroid isomerase and other enzyme active sites provide electrostatic environments that result in larger energetic responses (i.e., greater “sensitivity”) to ground-state to transition-state charge rearrangement, relative to aqueous solution, thereby providing catalysis relative to the corresponding reaction in water. To test these models, we substituted tyrosine with fluorotyrosines (F-Tyr’s) in the ketosteroid isomerase (KSI) oxyanion hole to systematically vary the proton affinity of an active site hydrogen bond donor while minimizing steric or structural effects. We found that a 40-fold increase in intrinsic F-Tyr acidity caused no significant change in activity for reactions with three different substrates. F-Tyr substitution did not change the solvent or primary kinetic isotope effect for proton abstraction, consistent with no change in mechanism arising from these substitutions. The observed shallow dependence of activity on the pKa of the substituted Tyr residues suggests that the KSI oxyanion hole does not provide catalysis by forming an energetically exceptional pKa-matched hydrogen bond. In addition, the shallow dependence provides no indication of an active site electrostatic

  17. Silver-Coated Nylon Dressing Plus Active DC Microcurrent for Healing of Autogenous Skin Donor Sites

    DTIC Science & Technology

    2013-08-01

    Silver-Coated Nylon Dressing Plus Active DC Microcurrent for Healing of Autogenous Skin Donor Sites Edward W. Malin, MD, Chaya M. Galin, BSN, RN... microcurrent in comparison to silver-coated dressing with sham microcurrent on wound-closure time for autogenous skin donor sites. Methods: Four...hundred five patients were screened for treatment of their donor sites using a silver-coated nylon dressing with either sham or active microcurrent

  18. Identification of Ice Nucleation Active Sites on Feldspar Dust Particles

    PubMed Central

    2015-01-01

    Mineral dusts originating from Earth’s crust are known to be important atmospheric ice nuclei. In agreement with earlier studies, feldspar was found as the most active of the tested natural mineral dusts. Here we investigated in closer detail the reasons for its activity and the difference in the activity of the different feldspars. Conclusions are drawn from scanning electron microscopy, X-ray powder diffraction, infrared spectroscopy, and oil-immersion freezing experiments. K-feldspar showed by far the highest ice nucleation activity. Finally, we give a potential explanation of this effect, finding alkali-metal ions having different hydration shells and thus an influence on the ice nucleation activity of feldspar surfaces. PMID:25584435

  19. 76 FR 30696 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-26

    ... Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites AGENCY: Department of... eligible active uranium and thorium processing site licensees for reimbursement under Title X of the Energy... requires DOE to reimburse eligible uranium and thorium licensees for certain costs of...

  20. 76 FR 24871 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-03

    ... Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites AGENCY: Department of... from eligible active uranium and thorium processing site licensees for reimbursement under Title X of...). Title X requires DOE to reimburse eligible uranium and thorium licensees for certain costs...

  1. Methane activation by platinum: critical role of edge and corner sites of metal nanoparticles.

    PubMed

    Viñes, Francesc; Lykhach, Yaroslava; Staudt, Thorsten; Lorenz, Michael P A; Papp, Christian; Steinrück, Hans-Peter; Libuda, Jörg; Neyman, Konstantin M; Görling, Andreas

    2010-06-11

    Complete dehydrogenation of methane is studied on model Pt catalysts by means of state-of-the-art DFT methods and by a combination of supersonic molecular beams with high-resolution photoelectron spectroscopy. The DFT results predict that intermediate species like CH(3) and CH(2) are specially stabilized at sites located at particles edges and corners by an amount of 50-80 kJ mol(-1). This stabilization is caused by an enhanced activity of low-coordinated sites accompanied by their special flexibility to accommodate adsorbates. The kinetics of the complete dehydrogenation of methane is substantially modified according to the reaction energy profiles when switching from Pt(111) extended surfaces to Pt nanoparticles. The CH(3) and CH(2) formation steps are endothermic on Pt(111) but markedly exothermic on Pt(79). An important decrease of the reaction barriers is observed in the latter case with values of approximately 60 kJ mol(-1) for first C-H bond scission and 40 kJ mol(-1) for methyl decomposition. DFT predictions are experimentally confirmed by methane decomposition on Pt nanoparticles supported on an ordered CeO(2) film on Cu(111). It is shown that CH(3) generated on the Pt nanoparticles undergoes spontaneous dehydrogenation at 100 K. This is in sharp contrast to previous results on Pt single-crystal surfaces in which CH(3) was stable up to much higher temperatures. This result underlines the critical role of particle edge sites in methane activation and dehydrogenation.

  2. A model of the rabies virus glycoprotein active site.

    PubMed

    Rustici, M; Bracci, L; Lozzi, L; Neri, P; Santucci, A; Soldani, P; Spreafico, A; Niccolai, N

    1993-06-01

    The glycoprotein from the neurotropic rabies virus shows a significant homology with the alpha neurotoxin that binds to the nicotinic acetylcholine receptor. The crystal structure of the alpha neurotoxins suggests that the Arg 37 guanidinium group and the Asp 31 side-chain carboxylate of the erabutoxin have stereochemical features resembling those of acetylcholine. Conformational studies on the Asn194-Ser195-Arg196-Gly197 tetrapeptide, an essential part of the binding site of the rabies virus glycoprotein, indicate that the side chains of Asn and Arg could also mimic the acetylcholine structure. This observation is consistent with the recently proposed mechanism of the viral infection.

  3. Functional properties and active-site topographies of factor X Gla- and prothrombin Gla-domain chimeras of activated protein C.

    PubMed

    Qureshi, Shabir H; Yang, Likui; Manithody, Chandrashekhara; Bae, Jong-Sup; Rezaie, Alireza R

    2008-09-01

    Substitution of the Gla-domain of activated protein C (APC) with the Gla-domain of prothrombin (APC-PTGla) improves the anticoagulant activity of APC independent of protein S. Previous FRET studies showed that this substitution alters the active-site topography of this mutant, rendering it identical to the active site of the APC-protein S complex. In this study, we characterized the functional properties and the active-site topography of another APC chimera containing the Gla-domain of factor X (APC-FXGla). We discovered that the anticoagulant activity of this mutant was similarly improved independent of protein S. The average distance of the closest approach (L) between the donor dye fluorescein attached to the active site of APC derivatives and the acceptor dye octadecylrhodamine incorporated into PC/PS vesicles was determined to be 99 A for APC and 84-86 A for both APC-PTGla and APC-FXGla. Protein S minimally influenced the L values of the APC chimeras, however, it lowered this value to 87 A for wild-type APC. Further studies revealed that neither chimera elicits a protective signaling response in the TNF-alpha-activated endothelial cells. These results suggest that unique structural features within the Gla-domain of APC enable the protease to interact with endothelial protein C receptor in the antiinflammatory pathway, while the same features also cause an inherently lower specific activity for APC in the anticoagulant pathway. This adaptation has made APC a cofactor-dependent protease, requiring the cofactor function of protein S for its optimal anticoagulant function, which appears to involve the alteration of the active-site topography of APC above the membrane surface.

  4. Active Site Hydrophobicity and the Convergent Evolution of Paraoxonase Activity in Structurally Divergent Enzymes: The Case of Serum Paraoxonase 1

    PubMed Central

    2016-01-01

    Serum paraoxonase 1 (PON1) is a native lactonase capable of promiscuously hydrolyzing a broad range of substrates, including organophosphates, esters, and carbonates. Structurally, PON1 is a six-bladed β-propeller with a flexible loop (residues 70–81) covering the active site. This loop contains a functionally critical Tyr at position 71. We have performed detailed experimental and computational analyses of the role of selected Y71 variants in the active site stability and catalytic activity in order to probe the role of Y71 in PON1’s lactonase and organophosphatase activities. We demonstrate that the impact of Y71 substitutions on PON1’s lactonase activity is minimal, whereas the kcat for the paraoxonase activity is negatively perturbed by up to 100-fold, suggesting greater mutational robustness of the native activity. Additionally, while these substitutions modulate PON1’s active site shape, volume, and loop flexibility, their largest effect is in altering the solvent accessibility of the active site by expanding the active site volume, allowing additional water molecules to enter. This effect is markedly more pronounced in the organophosphatase activity than the lactonase activity. Finally, a detailed comparison of PON1 to other organophosphatases demonstrates that either a similar “gating loop” or a highly buried solvent-excluding active site is a common feature of these enzymes. We therefore posit that modulating the active site hydrophobicity is a key element in facilitating the evolution of organophosphatase activity. This provides a concrete feature that can be utilized in the rational design of next-generation organophosphate hydrolases that are capable of selecting a specific reaction from a pool of viable substrates. PMID:28026940

  5. Proteome-wide analysis of nonsynonymous single-nucleotide variations in active sites of human proteins.

    PubMed

    Dingerdissen, Hayley; Motwani, Mona; Karagiannis, Konstantinos; Simonyan, Vahan; Mazumder, Raja

    2013-03-01

    An enzyme's active site is essential to normal protein activity such that any disruptions at this site may lead to dysfunction and disease. Nonsynonymous single-nucleotide variations (nsSNVs), which alter the amino acid sequence, are one type of disruption that can alter the active site. When this occurs, it is assumed that enzyme activity will vary because of the criticality of the site to normal protein function. We integrate nsSNV data and active site annotations from curated resources to identify all active-site-impacting nsSNVs in the human genome and search for all pathways observed to be associated with this data set to assess the likely consequences. We find that there are 934 unique nsSNVs that occur at the active sites of 559 proteins. Analysis of the nsSNV data shows an over-representation of arginine and an under-representation of cysteine, phenylalanine and tyrosine when comparing the list of nsSNV-impacted active site residues with the list of all possible proteomic active site residues, implying a potential bias for or against variation of these residues at the active site. Clustering analysis shows an abundance of hydrolases and transferases. Pathway and functional analysis shows several pathways over- or under-represented in the data set, with the most significantly affected pathways involved in carbohydrate metabolism. We provide a table of 32 variation-substrate/product pairs that can be used in targeted metabolomics experiments to assay the effects of specific variations. In addition, we report the significant prevalence of aspartic acid to histidine variation in eight proteins associated with nine diseases including glycogen storage diseases, lacrimo-auriculo-dento-digital syndrome, Parkinson's disease and several cancers.

  6. Sec3 Mutations Are Synthetically Lethal with Profilin Mutations and Cause Defects in Diploid-Specific Bud-Site Selection

    PubMed Central

    Haarer, B. K.; Corbett, A.; Kweon, Y.; Petzold, A. S.; Silver, P.; Brown, S. S.

    1996-01-01

    Replacement of the wild-type yeast profilin gene (PFY1) with a mutated form (pfy1-111) that has codon 72 changed to encode glutamate rather than arginine results in defects similar to, but less severe than, those that result from complete deletion of the profilin gene. We have used a colony color-sectoring assay to identify mutations that cause pfy1-111, but not wild-type, cells to be inviable. These profilin synthetic lethal (psl) mutations result in various degrees of abnormal growth, morphology, and temperature sensitivity in PFY1 cells. We have examined psl1 strains in the most detail. Interestingly, these strains display a diploid-specific defect in bud-site selection; haploid strains bud normally, while homozygous diploid strains show a dramatic increase in random budding. We discovered that PSL1 is the late secretory gene, SEC3, and have found that mutations in several other late secretory genes are also synthetically lethal with pfy1-111. Our results are likely to reflect an interdependence between the actin cytoskeleton and secretory processes in directing cell polarity and growth. Moreover, they indicate that the secretory pathway is especially crucial for maintaining budding polarity in diploids. PMID:8889515

  7. Exon 10 skipping in ACAT1 caused by a novel c.949G>A mutation located at an exonic splice enhancer site.

    PubMed

    Otsuka, Hiroki; Sasai, Hideo; Nakama, Mina; Aoyama, Yuka; Abdelkreem, Elsayed; Ohnishi, Hidenori; Konstantopoulou, Vassiliki; Sass, Jörn Oliver; Fukao, Toshiyuki

    2016-11-01

    Beta-ketothiolase deficiency, also known as mitochondrial acetoacetyl-CoA thiolase (T2) deficiency, is an autosomal recessive disease caused by mutations in the acetyl‑CoA acetyltransferase 1 (ACAT1) gene. A German T2‑deficient patient that developed a severe ketoacidotic episode at the age of 11 months, was revealed to be a compound heterozygote of a previously reported null mutation, c.472A>G (p.N158D) and a novel mutation, c.949G>A (p.D317N), in ACAT1. The c.949G>A mutation was suspected to cause aberrant splicing as it is located within an exonic splicing enhancer sequence (c. 947CTGACGC) that is a potential binding site for serine/arginine‑rich splicing factor 1. A mutation in this sequence, c.951C>T, results in exon 10 skipping. A minigene construct was synthesized that included exon 9‑truncated intron 9‑exon 10‑truncated intron 10‑exon 11, and the splicing of this minigene revealed that the c.949G>A mutant construct caused exon 10 skipping in a proportion of the transcripts. Furthermore, additional substitution of G for C at the first nucleotide of exon 10 (c.941G>C) abolished the effect of the c.949G>A mutation. Transient expression analysis of the c.949G>A mutant cDNA revealed no residual T2 activity in the mutated D317N enzyme. Therefore, c.949G>A (D317N) is a pathogenic missense mutation, and diminishes the effect of an exonic splicing enhancer and causes exon 10 skipping. The present study demonstrates that a missense mutation, or even a synonymous substitution, may disrupt enzyme function by interference with splicing.

  8. Assessment of activation products in the Savannah River Site environment

    SciTech Connect

    Carlton, W.H.; Denham, M.

    1996-07-01

    This document assesses the impact of radioactive activation products released from SRS facilities since the first reactor became operational late in 1953. The isotopes reported here are those whose release resulted in the highest dose to people living near SRS: {sup 32}P, {sup 51}Cr, {sup 60}C, and {sup 65}Zn. Release pathways, emission control features, and annual releases to the aqueous and atmospheric environments are discussed. No single incident has resulted in a major acute release of activation products to the environment. The releases were the result of normal operations of the reactors and separations facilities. Releases declined over the years as better controls were established and production was reduced. The overall radiological impact of SRS activation product atmospheric releases from 1954 through 1994 on the offsite maximally exposed individual can be characterized by a total dose of 0.76 mrem. During the same period, such an individual received a total dose of 14,400 mrem from non-SRS sources of ionizing radiation present in the environment. SRS activation product aqueous releases between 1954 and 1994 resulted in a total dose of 54 mrem to the offsite maximally exposed individual. The impact of SRS activation product releases on offsite populations also has been evaluated.

  9. All the catalytic active sites of MoS2 for hydrogen evolution

    DOE PAGES

    Li, Guoqing; Zhang, Du; Qiao, Qiao; ...

    2016-11-29

    MoS2 presents a promising low-cost catalyst for the hydrogen evolution reaction (HER), but the understanding about its active sites has remained limited. Here we present an unambiguous study of the catalytic activities of all possible reaction sites of MoS2, including edge sites, sulfur vacancies, and grain boundaries. We demonstrate that, in addition to the well-known catalytically active edge sites, sulfur vacancies provide another major active site for the HER, while the catalytic activity of grain boundaries is much weaker. Here, the intrinsic turnover frequencies (Tafel slopes) of the edge sites, sulfur vacancies, and grain boundaries are estimated to be 7.5more » s–1 (65–75 mV/dec), 3.2 s–1 (65–85 mV/dec), and 0.1 s–1 (120–160 mV/dec), respectively. We also demonstrate that the catalytic activity of sulfur vacancies strongly depends on the density of the vacancies and the local crystalline structure in proximity to the vacancies. Unlike edge sites, whose catalytic activity linearly depends on the length, sulfur vacancies show optimal catalytic activities when the vacancy density is in the range of 7–10%, and the number of sulfur vacancies in high crystalline quality MoS2 is higher than that in low crystalline quality MoS2, which may be related with the proximity of different local crystalline structures to the vacancies.« less

  10. Quantifying the density and utilization of active sites in non-precious metal oxygen electroreduction catalysts

    PubMed Central

    Sahraie, Nastaran Ranjbar; Kramm, Ulrike I.; Steinberg, Julian; Zhang, Yuanjian; Thomas, Arne; Reier, Tobias; Paraknowitsch, Jens-Peter; Strasser, Peter

    2015-01-01

    Carbon materials doped with transition metal and nitrogen are highly active, non-precious metal catalysts for the electrochemical conversion of molecular oxygen in fuel cells, metal air batteries, and electrolytic processes. However, accurate measurement of their intrinsic turn-over frequency and active-site density based on metal centres in bulk and surface has remained difficult to date, which has hampered a more rational catalyst design. Here we report a successful quantification of bulk and surface-based active-site density and associated turn-over frequency values of mono- and bimetallic Fe/N-doped carbons using a combination of chemisorption, desorption and 57Fe Mössbauer spectroscopy techniques. Our general approach yields an experimental descriptor for the intrinsic activity and the active-site utilization, aiding in the catalyst development process and enabling a previously unachieved level of understanding of reactivity trends owing to a deconvolution of site density and intrinsic activity. PMID:26486465

  11. [Increased fibrinolytic activity during cardiopulmonary bypass is caused by activated protein C system].

    PubMed

    Gando, S; Tedo, I; Masio, H; Goda, Y; Kawahigashi, H

    1994-06-01

    To examine the hypothesis that activated protein C system during cardiopulmonary bypass surgery may increase fibrinolytic activity during cardiopulmonary bypass, protein C activity, protein C antigen and thrombomodulin of sixteen patients undergoing elective cardiopulmonary bypass surgery were investigated after induction of anesthesia, before and after cardiopulmonary bypass, and at the end of operation. Protein C activity decreased and thrombomodulin increased significantly after the cardiopulmonary bypass. There were no significant correlations of thrombomodulin with protein C activity and protein C antigen. In conclusion, we have demonstrated that protein C system is activated and circulating thrombomodulin appears in the systemic circulation during cardiopulmonary bypass surgery and this enhanced activation of protein C system is possibly related to the reported increase of fibrinolytic activity during cardiopulmonary bypass.

  12. The influence of small-mammal burrowing activity on water storage at the Hanford Site

    SciTech Connect

    Landeen, D.S.

    1994-12-31

    This paper summarizes the activities that were conducted in support of the long-term surface barrier development program by Westinghouse Hanford Company to determine the degree that small-mammal burrow systems affect the loss or retention of water in the soils at the Hanford Site in Washington state. An animal intrusion lysimeter facility was constructed, consisting of two outer boxes buried at grade, which served as receptacles for six animal intrusion lysimeters. Small burrowing animals common the Hanford Site were introduced over a 3- to 4-month period. Supplemental precipitation was added monthly to three of the lysimeters with a rainfall simulator (rainulator). Information collected from the five tests indicated that (1) during summer months, water was lost in all the lysimeters, including the supplemental precipitation added with the rainulator; and (2) during winter months, all lysimeters gained water. The data indicate little difference in the amount of water stored between control and animal lysimeters. The overall water loss was attributed to surface evaporation, a process that occurred equally in control and treatment lysimeters. Other causes of water loss are a result of (1) constant soil turnover and subsequent drying, and (2) burrow ventilation effects. This suggests that burrow systems will not contribute to any significant water storage at depth and, in fact, may enhance the removal of water from the soil.

  13. Marine Biology Field Trip Sites. Ocean Related Curriculum Activities.

    ERIC Educational Resources Information Center

    Pauls, John

    The ocean affects all of our lives. Therefore, awareness of and information about the interconnections between humans and oceans are prerequisites to making sound decisions for the future. Project ORCA (Ocean Related Curriculum Activities) has developed interdisciplinary curriculum materials designed to meet the needs of students and teachers…

  14. Encroachment of Human Activity on Sea Turtle Nesting Sites

    NASA Astrophysics Data System (ADS)

    Ziskin, D.; Aubrecht, C.; Elvidge, C.; Tuttle, B.; Baugh, K.; Ghosh, T.

    2008-12-01

    The encroachment of anthropogenic lighting on sea turtle nesting sites poses a serious threat to the survival of these animals [Nicholas, 2001]. This danger is quantified by combining two established data sets. The first is the Nighttime Lights data produced by the NOAA National Geophysical Data Center [Elvidge et al., 1997]. The second is the Marine Turtle Database produced by the World Conservation Monitoring Centre (WCMC). The technique used to quantify the threat of encroachment is an adaptation of the method described in Aubrecht et al. [2008], which analyzes the stress on coral reef systems by proximity to nighttime lights near the shore. Nighttime lights near beaches have both a direct impact on turtle reproductive success since they disorient hatchlings when they mistake land-based lights for the sky-lit surf [Lorne and Salmon, 2007] and the lights are also a proxy for other anthropogenic threats. The identification of turtle nesting sites with high rates of encroachment will hopefully steer conservation efforts to mitigate their effects [Witherington, 1999]. Aubrecht, C, CD Elvidge, T Longcore, C Rich, J Safran, A Strong, M Eakin, KE Baugh, BT Tuttle, AT Howard, EH Erwin, 2008, A global inventory of coral reef stressors based on satellite observed nighttime lights, Geocarto International, London, England: Taylor and Francis. In press. Elvidge, CD, KE Baugh, EA Kihn, HW Kroehl, ER Davis, 1997, Mapping City Lights with Nighttime Data from the DMSP Operational Linescan System, Photogrammatic Engineering and Remote Sensing, 63:6, pp. 727-734. Lorne, JK, M Salmon, 2007, Effects of exposure to artificial lighting on orientation of hatchling sea turtles on the beach and in the ocean, Endangered Species Research, Vol. 3: 23-30. Nicholas, M, 2001, Light Pollution and Marine Turtle Hatchlings: The Straw that Breaks the Camel's Back?, George Wright Forum, 18:4, p77-82. Witherington, BE, 1999, Reducing Threats To Nesting Habitat, Research and Management Techniques for

  15. Molecular Basis for Enzymatic Sulfite Oxidation -- HOW THREE CONSERVED ACTIVE SITE RESIDUES SHAPE ENZYME ACTIVITY

    SciTech Connect

    Bailey, Susan; Rapson, Trevor; Johnson-Winters, Kayunta; Astashkin, Andrei; Enemark, John; Kappler, Ulrike

    2008-11-10

    Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased Km(sulfite)(app) by 2-3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDHWT and SDHY236F, the catalytic turnover rates of SDHR55M and SDHH57A are relatively insensitive to pH (~;;60 and 200 s-1, respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDHH57A and SDHY236F) or absence of Arg-55 (SDHR55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDHR55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDHR55M. Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH.

  16. Identification of inhibitors against the potential ligandable sites in the active cholera toxin.

    PubMed

    Gangopadhyay, Aditi; Datta, Abhijit

    2015-04-01

    The active cholera toxin responsible for the massive loss of water and ions in cholera patients via its ADP ribosylation activity is a heterodimer of the A1 subunit of the bacterial holotoxin and the human cytosolic ARF6 (ADP Ribosylation Factor 6). The active toxin is a potential target for the design of inhibitors against cholera. In this study we identified the potential ligandable sites of the active cholera toxin which can serve as binding sites for drug-like molecules. By employing an energy-based approach to identify ligand binding sites, and comparison with the results of computational solvent mapping, we identified two potential ligandable sites in the active toxin which can be targeted during structure-based drug design against cholera. Based on the probe affinities of the identified ligandable regions, docking-based virtual screening was employed to identify probable inhibitors against these sites. Several indole-based alkaloids and phosphates showed strong interactions to the important residues of the ligandable region at the A1 active site. On the other hand, 26 top scoring hits were identified against the ligandable region at the A1 ARF6 interface which showed strong hydrogen bonding interactions, including guanidines, phosphates, Leucopterin and Aristolochic acid VIa. This study has important implications in the application of hybrid structure-based and ligand-based methods against the identified ligandable sites using the identified inhibitors as reference ligands, for drug design against the active cholera toxin.

  17. Barium ions selectively activate BK channels via the Ca2+-bowl site.

    PubMed

    Zhou, Yu; Zeng, Xu-Hui; Lingle, Christopher J

    2012-07-10

    Activation of Ca(2+)-dependent BK channels is increased via binding of micromolar Ca(2+) to two distinct high-affinity sites per BK α-subunit. One site, termed the Ca(2+) bowl, is embedded within the second RCK domain (RCK2; regulator of conductance for potassium) of each α-subunit, while oxygen-containing residues in the first RCK domain (RCK1) have been linked to a separate Ca(2+) ligation site. Although both sites are activated by Ca(2+) and Sr(2+), Cd(2+) selectively favors activation via the RCK1 site. Divalent cations of larger ionic radius than Sr(2+) are thought to be ineffective at activating BK channels. Here we show that Ba(2+), better known as a blocker of K(+) channels, activates BK channels and that this effect arises exclusively from binding at the Ca(2+)-bowl site. Compared with previous estimates for Ca(2+) bowl-mediated activation by Ca(2+), the affinity of Ba(2+) to the Ca(2+) bowl is reduced about fivefold, and coupling of binding to activation is reduced from ∼3.6 for Ca(2+) to about ∼2.8 for Ba(2+). These results support the idea that ionic radius is an important determinant of selectivity differences among different divalent cations observed for each Ca(2+)-binding site.

  18. Activation of brown adipose tissue mitochondrial GDP binding sites

    SciTech Connect

    Swick, A.G.

    1987-01-01

    The primary function of brown adipose tissue (BAT) is heat production. This ability is attributed to the existence of a unique inner mitochondrial membrane protein termed the uncoupling protein or thermogenin. This protein is permeable to H+ and thus allows respiration (and therefore thermogenesis) to proceed at a rapid rate, independent of ADP phosphorylation. Proton conductance can be inhibited by the binding of purine nucleotides to the uncoupling protein. The binding of (/sup 3/H)-GDP to BAT mitochondria is frequently used as a measure of BAT thermogenic activity. Rats fed a diet that was low but adequate in protein exhibited a decrease in feed efficiency. In addition, BAT thermogenesis was activated as indicated by an elevation in the level of GDP binding to BAT mitochondria. This phenomena occurred in older rats and persisted over time.

  19. Site-Directed Mutagenesis from Arg195 to His of a Microalgal Putatively Chloroplastidial Glycerol-3-Phosphate Acyltransferase Causes an Increase in Phospholipid Levels in Yeast

    PubMed Central

    Ouyang, Long-Ling; Li, Hui; Yan, Xiao-Jun; Xu, Ji-Lin; Zhou, Zhi-Gang

    2016-01-01

    To analyze the contribution of glycerol-3-phosphate acyltransferase (GPAT) to the first acylation of glycerol-3-phosphate (G-3-P), the present study focused on a functional analysis of the GPAT gene from Lobosphaera incisa (designated as LiGPAT). A full-length cDNA of LiGPAT consisting of a 1,305-bp ORF, a 1,652-bp 5′-UTR, and a 354-bp 3′-UTR, was cloned. The ORF encoded a 434-amino acid peptide, of which 63 residues at the N-terminus defined a chloroplast transit peptide. Multiple sequence alignment and phylogeny analysis of GPAT homologs provided the convincible bioinformatics evidence that LiGPAT was localized to chloroplasts. Considering the conservation of His among the G-3-P binding sites from chloroplastidial GPATs and the substitution of His by Arg at position 195 in the LiGPAT mature protein (designated mLiGPAT), we established the heterologous expression of either mLiGPAT or its mutant (Arg195His) (sdmLiGPAT) in the GPAT-deficient yeast mutant gat1Δ. Lipid profile analyses of these transgenic yeasts not only validated the acylation function of LiGPAT but also indicated that the site-directed mutagenesis from Arg195 to His led to an increase in the phospholipid level in yeast. Semi-quantitative analysis of mLiGPAT and sdmLiGPAT, together with the structural superimposition of their G-3-P binding sites, indicated that the increased enzymatic activity was caused by the enlarged accessible surface of the phosphate group binding pocket when Arg195 was mutated to His. Thus, the potential of genetic manipulation of GPAT to increase the glycerolipid level in L. incisa and other microalgae would be of great interest. PMID:27014309

  20. Structure and Reactivity of the Phosphotriesterase Active Site

    DTIC Science & Technology

    2002-01-01

    characterize different catalytic conformations for chorismate mutase . Preliminary evidence for water binding in phosphotriesterase suggests that activity in...MD/QM study of the chorismate mutase catalyzed Claisen rearrangement reaction. 2001.subm. J.Phys.Chem.B 22.Day, P.N.J., J.H.; Gordon,M.S.; Webb,S.P...Claisen rearrangement of an unusual substrate in chorismate mutase . 2001.subm. J.Phys.Chem.B 38.Stevens, W.J., Basch,H., Krauss,M., Compact effective

  1. Comparison of soil heavy metal pollution caused by e-waste recycling activities and traditional industrial operations.

    PubMed

    He, Kailing; Sun, Zehang; Hu, Yuanan; Zeng, Xiangying; Yu, Zhiqiang; Cheng, Hefa

    2017-02-23

    The traditional industrial operations are well recognized as an important source of heavy metal pollution, while that caused by the e-waste recycling activities, which have sprouted in some developing countries, is often overlooked. This study was carried out to compare the status of soil heavy metal pollution caused by the traditional industrial operations and the e-waste recycling activities in the Pearl River Delta, and assess whether greater attention should be paid to control the pollution arising from e-waste recycling activities. Both the total contents and the chemical fractionation of major heavy metals (As, Cr, Cd, Ni, Pb, Cu, and Zn) in 50 surface soil samples collected from the e-waste recycling areas and 20 soil samples from the traditional industrial zones were determined. The results show that the soils in the e-waste recycling areas were mainly polluted by Cu, Zn, As, and Cd, while Cu, Zn, As, Cd, and Pb were the major heavy metals in the soils from the traditional industrial zones. Statistical analyses consistently show that Cu, Cd, Pb, and Zn in the surface soils from both types of sites were contributed mostly by human activities, while As, Cr, and Ni in the soils were dominated by natural background. No clear distinction was found on the pollution characteristic of heavy metals in the surface soils between the e-waste recycling areas and traditional industrial zones. The potential ecological risk posed by heavy metals in the surface soils from both types of sites, which was dominated by that from Cd, ranged from low to moderate. Given the much shorter development history of e-waste recycling and its largely unregulated nature, significant efforts should be made to crack down on illegal e-waste recycling and strengthen pollution control for related activities.

  2. Active urea transport by the skin of Bufo viridis: Amiloride- and phloretin-sensitive transport sites

    SciTech Connect

    Rapoport, J.; Abuful, A.; Chaimovitz, C.; Noeh, Z.; Hays, R.M. Albert Einstein College of Medicine, New York, NY )

    1988-09-01

    Urea is actively transported inwardly (J{sub i}) across the skin of the green toad Bufo viridis. J{sub i} is markedly enhanced in toads adapted to hypertonic saline. The authors studied urea transport across the skin of Bufo viridis under a variety of experimental conditions, including treatment with amiloride and phloretin, agents that inhibit urea permeability in the bladder of Bufo marinus. Amiloride (10{sup {minus}4} M) significantly inhibited J{sub i} in both adapted and unadapted animals and was unaffected by removal of sodium from the external medium. Phloretin (10{sup {minus}4} M) significantly inhibited J{sub i} in adapted animals by 23-46%; there was also a reduction in J{sub i} in unadapted toads at 10{sup {minus}4} and 5 {times} 10{sup {minus}4} M phloretin. A dose-response study revealed that the concentration of phloretin causing half-maximal inhibition (K{sub {1/2}}) was 5 {times} 10{sup {minus}4} M for adapted animals. J{sub i} was unaffected by the substitution of sucrose for Ringer solution or by ouabain. They conclude (1) the process of adaptation appears to involve an increase in the number of amiloride- and phloretin-inhibitable urea transport sites in the skin, with a possible increase in the affinity of the sites for phloretin; (2) the adapted skin resembles the Bufo marinus urinary bladder with respect to amiloride and phloretin-inhibitable sites; (3) they confirm earlier observations that J{sub i} is independent of sodium transport.

  3. Active site proton delivery and the lyase activity of human CYP17A1

    SciTech Connect

    Khatri, Yogan; Gregory, Michael C.; Grinkova, Yelena V.; Denisov, Ilia G.; Sligar, Stephen G.

    2014-01-03

    equivalents and protons are funneled into non-productive pathways. This is similar to previous work with other P450 catalyzed hydroxylation. However, catalysis of carbon–carbon bond scission by the T306A mutant was largely unimpeded by disruption of the CYP17A1 acid-alcohol pair. The unique response of CYP17A1 lyase activity to mutation of Thr306 is consistent with a reactive intermediate formed independently of proton delivery in the active site, and supports involvement of a nucleophilic peroxo-anion rather than the traditional Compound I in catalysis.

  4. Pathways of H2 toward the Active Site of [NiFe]-Hydrogenase

    PubMed Central

    Teixeira, Vitor H.; Baptista, António M.; Soares, Cláudio M.

    2006-01-01

    Hydrogenases catalyze the reversible oxidation of molecular hydrogen (H2), but little is known about the diffusion of H2 toward the active site. Here we analyze pathways for H2 permeation using molecular dynamics (MD) simulations in explicit solvent. Various MD simulation replicates were done, to improve the sampling of the system states. H2 easily permeates hydrogenase in every simulation and it moves preferentially in channels. All H2 molecules that reach the active site made their approach from the side of the Ni ion. H2 is able to reach distances of <4 Å from the active site, although after 6 Å permeation is difficult. In this region we mutated Val-67 into alanine and perform new MD simulations. These simulations show an increase of H2 inside the protein and at lower distances from the active site. This valine can be a control point in the H2 access to the active center. PMID:16731562

  5. Identification of a putative binding site critical for general anesthetic activation of TRPA1.

    PubMed

    Ton, Hoai T; Phan, Thieu X; Abramyan, Ara M; Shi, Lei; Ahern, Gerard P

    2017-04-04

    General anesthetics suppress CNS activity by modulating the function of membrane ion channels, in particular, by enhancing activity of GABAA receptors. In contrast, several volatile (isoflurane, desflurane) and i.v. (propofol) general anesthetics excite peripheral sensory nerves to cause pain and irritation upon administration. These noxious anesthetics activate transient receptor potential ankyrin repeat 1 (TRPA1), a major nociceptive ion channel, but the underlying mechanisms and site of action are unknown. Here we exploit the observation that pungent anesthetics activate mammalian but not Drosophila TRPA1. Analysis of chimeric Drosophila and mouse TRPA1 channels reveal a critical role for the fifth transmembrane domain (S5) in sensing anesthetics. Interestingly, we show that anesthetics share with the antagonist A-967079 a potential binding pocket lined by residues in the S5, S6, and the first pore helix; isoflurane competitively disrupts A-967079 antagonism, and introducing these mammalian TRPA1 residues into dTRPA1 recapitulates anesthetic agonism. Furthermore, molecular modeling predicts that isoflurane and propofol bind to this pocket by forming H-bond and halogen-bond interactions with Ser-876, Met-915, and Met-956. Mutagenizing Met-915 or Met-956 selectively abolishes activation by isoflurane and propofol without affecting actions of A-967079 or the agonist, menthol. Thus, our combined experimental and computational results reveal the potential binding mode of noxious general anesthetics at TRPA1. These data may provide a structural basis for designing drugs to counter the noxious and vasorelaxant properties of general anesthetics and may prove useful in understanding effects of anesthetics on related ion channels.

  6. Multiple-site mutations of phage Bp7 endolysin improves its activities against target bacteria.

    PubMed

    Zhang, Can; Wang, Yuanchao; Sun, Huzhi; Ren, Huiying

    2015-10-01

    The widespread use of antibiotics has caused serious drug resistance. Bacteria that were once easily treatable are now extremely difficult to treat. Endolysin can be used as an alternative to antibiotics for the treatment of drug-resistant bacteria. To analyze the antibacterial activity of the endolysin of phage Bp7 (Bp7e), a 489-bp DNA fragment of endolysin Bp7e was PCR-amplified from a phage Bp7 genome and cloned, and then a pET28a-Bp7e prokaryotic expression vector was constructed. Two amino acids were mutated (L99A, M102E) to construct pET28a-Bp7Δe, with pET28a-Bp7e as a template. Phylogenetic analysis suggested that BP7e belongs to a T4-like phage endolysin group. Bp7e and its mutant Bp7Δe were expressed in Escherichia coli BL21(DE3) as soluble proteins. They were purified by affinity chromatography, and then their antibacterial activities were analyzed. The results demonstrated that the recombinant proteins Bp7e and Bp7Δe showed obvious antibacterial activity against Micrococcus lysodeikticus but no activity against Staphylococcus aureus. In the presence of malic acid, Bp7e and Bp7Δe exhibited an effect on most E. coli strains which could be lysed by phage Bp7, but no effect on Salmonella paratyphi or Pseudomonas aeruginosa. Moreover, Bp7Δe with double-site mutations showed stronger antibacterial activity and a broader lysis range than Bp7e.

  7. Maintenance of plastid RNA editing activities independently of their target sites.

    PubMed

    Tillich, Michael; Poltnigg, Peter; Kushnir, Sergei; Schmitz-Linneweber, Christian

    2006-03-01

    RNA editing in plant organelles is mediated by site-specific, nuclear-encoded factors. Previous data suggested that the maintenance of these factors depends on the presence of their rapidly evolving cognate sites. The surprising ability of allotetraploid Nicotiana tabacum (tobacco) to edit a foreign site in the chloroplast ndhA messenger RNA was thought to be inherited from its diploid male ancestor, Nicotiana tomentosiformis. Here, we show that the same ndhA editing activity is also present in Nicotiana sylvestris, which is the female diploid progenitor of tobacco and which lacks the ndhA site. Hence, heterologous editing is not simply a result of tobacco's allopolyploid genome organization. Analyses of other editing sites after sexual or somatic transfer between land plants showed that heterologous editing occurs at a surprisingly high frequency. This suggests that the corresponding editing activities are conserved despite the absence of their target sites, potentially because they serve other functions in the plant cell.

  8. Stimulation, Inhibition, or Stabilization of Na,K-ATPase Caused by Specific Lipid Interactions at Distinct Sites

    PubMed Central

    Habeck, Michael; Haviv, Haim; Katz, Adriana; Kapri-Pardes, Einat; Ayciriex, Sophie; Shevchenko, Andrej; Ogawa, Haruo; Toyoshima, Chikashi; Karlish, Steven J. D.

    2015-01-01

    The activity of membrane proteins such as Na,K-ATPase depends strongly on the surrounding lipid environment. Interactions can be annular, depending on the physical properties of the membrane, or specific with lipids bound in pockets between transmembrane domains. This paper describes three specific lipid-protein interactions using purified recombinant Na,K-ATPase. (a) Thermal stability of the Na,K-ATPase depends crucially on a specific interaction with 18:0/18:1 phosphatidylserine (1-stearoyl-2-oleoyl-sn-glycero-3-phospho-l-serine; SOPS) and cholesterol, which strongly amplifies stabilization. We show here that cholesterol associates with SOPS, FXYD1, and the α subunit between trans-membrane segments αTM8 and -10 to stabilize the protein. (b) Polyunsaturated neutral lipids stimulate Na,K-ATPase turnover by >60%. A screen of the lipid specificity showed that 18:0/20:4 and 18:0/22:6 phosphatidylethanolamine (PE) are the optimal phospholipids for this effect. (c) Saturated phosphatidylcholine and sphingomyelin, but not saturated phosphatidylserine or PE, inhibit Na,K-ATPase activity by 70–80%. This effect depends strongly on the presence of cholesterol. Analysis of the Na,K-ATPase activity and E1-E2 conformational transitions reveals the kinetic mechanisms of these effects. Both stimulatory and inhibitory lipids poise the conformational equilibrium toward E2, but their detailed mechanisms of action are different. PE accelerates the rate of E1 → E2P but does not affect E2(2K)ATP → E13NaATP, whereas sphingomyelin inhibits the rate of E2(2K)ATP → E13NaATP, with very little effect on E1 → E2P. We discuss these lipid effects in relation to recent crystal structures of Na,K-ATPase and propose that there are three separate sites for the specific lipid interactions, with potential physiological roles to regulate activity and stability of the pump. PMID:25533463

  9. Stimulation, inhibition, or stabilization of Na,K-ATPase caused by specific lipid interactions at distinct sites.

    PubMed

    Habeck, Michael; Haviv, Haim; Katz, Adriana; Kapri-Pardes, Einat; Ayciriex, Sophie; Shevchenko, Andrej; Ogawa, Haruo; Toyoshima, Chikashi; Karlish, Steven J D

    2015-02-20

    The activity of membrane proteins such as Na,K-ATPase depends strongly on the surrounding lipid environment. Interactions can be annular, depending on the physical properties of the membrane, or specific with lipids bound in pockets between transmembrane domains. This paper describes three specific lipid-protein interactions using purified recombinant Na,K-ATPase. (a) Thermal stability of the Na,K-ATPase depends crucially on a specific interaction with 18:0/18:1 phosphatidylserine (1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine; SOPS) and cholesterol, which strongly amplifies stabilization. We show here that cholesterol associates with SOPS, FXYD1, and the α subunit between trans-membrane segments αTM8 and -10 to stabilize the protein. (b) Polyunsaturated neutral lipids stimulate Na,K-ATPase turnover by >60%. A screen of the lipid specificity showed that 18:0/20:4 and 18:0/22:6 phosphatidylethanolamine (PE) are the optimal phospholipids for this effect. (c) Saturated phosphatidylcholine and sphingomyelin, but not saturated phosphatidylserine or PE, inhibit Na,K-ATPase activity by 70-80%. This effect depends strongly on the presence of cholesterol. Analysis of the Na,K-ATPase activity and E1-E2 conformational transitions reveals the kinetic mechanisms of these effects. Both stimulatory and inhibitory lipids poise the conformational equilibrium toward E2, but their detailed mechanisms of action are different. PE accelerates the rate of E1 → E2P but does not affect E2(2K)ATP → E13NaATP, whereas sphingomyelin inhibits the rate of E2(2K)ATP → E13NaATP, with very little effect on E1 → E2P. We discuss these lipid effects in relation to recent crystal structures of Na,K-ATPase and propose that there are three separate sites for the specific lipid interactions, with potential physiological roles to regulate activity and stability of the pump.

  10. A Processive Carbohydrate Polymerase That Mediates Bifunctional Catalysis Using a Single Active Site

    PubMed Central

    May, John F.; Levengood, Matthew R.; Splain, Rebecca A.; Brown, Christopher D.; Kiessling, Laura L.

    2012-01-01

    Even in the absence of a template, glycosyltransferases can catalyze the synthesis of carbohydrate polymers of specific sequence. The paradigm has been that one enzyme catalyzes the formation of one type of glycosidic linkage, yet certain glycosyltransferases generate polysaccharide sequences composed of two distinct linkage types. In principle, bifunctional glycosyltransferases can possess separate active sites for each catalytic activity or one active site with dual activities. We encountered the fundamental question of one or two distinct active sites in our investigation of the galactosyltransferase GlfT2. GlfT2 catalyzes the formation of mycobacterial galactan, a critical cell-wall polymer composed of galactofuranose residues connected with alternating, regioisomeric linkages. We found that GlfT2 mediates galactan polymerization using only one active site that manifests dual regioselectivity. Structural modeling of the bifunctional glycosyltransferases hyaluronan synthase and cellulose synthase suggests that these enzymes also generate multiple glycosidic linkages using a single active site. These results highlight the versatility of glycosyltransferases for generating polysaccharides of specific sequence. We postulate that a hallmark of processive elongation of a carbohydrate polymer by a bifunctional enzyme is that one active site can give rise to two separate types of glycosidic bonds. PMID:22217153

  11. Catalytically Active Guanylyl Cyclase B Requires Endoplasmic Reticulum-mediated Glycosylation, and Mutations That Inhibit This Process Cause Dwarfism.

    PubMed

    Dickey, Deborah M; Edmund, Aaron B; Otto, Neil M; Chaffee, Thomas S; Robinson, Jerid W; Potter, Lincoln R

    2016-05-20

    C-type natriuretic peptide activation of guanylyl cyclase B (GC-B), also known as natriuretic peptide receptor B or NPR2, stimulates long bone growth, and missense mutations in GC-B cause dwarfism. Four such mutants (L658F, Y708C, R776W, and G959A) bound (125)I-C-type natriuretic peptide on the surface of cells but failed to synthesize cGMP in membrane GC assays. Immunofluorescence microscopy also indicated that the mutant receptors were on the cell surface. All mutant proteins were dephosphorylated and incompletely glycosylated, but dephosphorylation did not explain the inactivation because the mutations inactivated a "constitutively phosphorylated" enzyme. Tunicamycin inhibition of glycosylation in the endoplasmic reticulum or mutation of the Asn-24 glycosylation site decreased GC activity, but neither inhibition of glycosylation in the Golgi by N-acetylglucosaminyltransferase I gene inactivation nor PNGase F deglycosylation of fully processed GC-B reduced GC activity. We conclude that endoplasmic reticulum-mediated glycosylation is required for the formation of an active catalytic, but not ligand-binding domain, and that mutations that inhibit this process cause dwarfism.

  12. An Accessory Agonist Binding Site Promotes Activation of α4β2* Nicotinic Acetylcholine Receptors*

    PubMed Central

    Wang, Jingyi; Kuryatov, Alexander; Sriram, Aarati; Jin, Zhuang; Kamenecka, Theodore M.; Kenny, Paul J.; Lindstrom, Jon

    2015-01-01

    Neuronal nicotinic acetylcholine receptors containing α4, β2, and sometimes other subunits (α4β2* nAChRs) regulate addictive and other behavioral effects of nicotine. These nAChRs exist in several stoichiometries, typically with two high affinity acetylcholine (ACh) binding sites at the interface of α4 and β2 subunits and a fifth accessory subunit. A third low affinity ACh binding site is formed when this accessory subunit is α4 but not if it is β2. Agonists selective for the accessory ACh site, such as 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283), cannot alone activate a nAChR but can facilitate more efficient activation in combination with agonists at the canonical α4β2 sites. We therefore suggest categorizing agonists according to their site selectivity. NS9283 binds to the accessory ACh binding site; thus it is termed an accessory site-selective agonist. We expressed (α4β2)2 concatamers in Xenopus oocytes with free accessory subunits to obtain defined nAChR stoichiometries and α4/accessory subunit interfaces. We show that α2, α3, α4, and α6 accessory subunits can form binding sites for ACh and NS9283 at interfaces with α4 subunits, but β2 and β4 accessory subunits cannot. To permit selective blockage of the accessory site, α4 threonine 126 located on the minus side of α4 that contributes to the accessory site, but not the α4β2 sites, was mutated to cysteine. Alkylation of this cysteine with a thioreactive reagent blocked activity of ACh and NS9283 at the accessory site. Accessory agonist binding sites are promising drug targets. PMID:25869137

  13. Mutagenesis and crystallographic studies of the catalytic residues of the papain family protease bleomycin hydrolase: new insights into active-site structure

    PubMed Central

    O'Farrell, Paul A.; Joshua-Tor, Leemor

    2006-01-01

    Bleomycin hydrolase (BH) is a hexameric papain family cysteine protease which is involved in preparing peptides for antigen presentation and has been implicated in tumour cell resistance to bleomycin chemotherapy. Structures of active-site mutants of yeast BH yielded unexpected results. Replacement of the active-site asparagine with alanine, valine or leucine results in the destabilization of the histidine side chain, demonstrating unambiguously the role of the asparagine residue in correctly positioning the histidine for catalysis. Replacement of the histidine with alanine or leucine destabilizes the asparagine position, indicating a delicate arrangement of the active-site residues. In all of the mutants, the C-terminus of the protein, which lies in the active site, protrudes further into the active site. All mutants were compromised in their catalytic activity. The structures also revealed the importance of a tightly bound water molecule which stabilizes a loop near the active site and which is conserved throughout the papain family. It is displaced in a number of the mutants, causing destabilization of this loop and a nearby loop, resulting in a large movement of the active-site cysteine. The results imply that this water molecule plays a key structural role in this family of enzymes. PMID:17007609

  14. Extending the Diffuse Layer Model of Surface Acidity Behavior: III. Estimating Bound Site Activity Coefficients

    EPA Science Inventory

    Although detailed thermodynamic analyses of the 2-pK diffuse layer surface complexation model generally specify bound site activity coefficients for the purpose of accounting for those non-ideal excess free energies contributing to bound site electrochemical potentials, in applic...

  15. 75 FR 71677 - Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-11-24

    ... Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites AGENCY: Department of... uranium and thorium processing site licensees for reimbursement under Title X of the Energy Policy Act of... requires DOE to reimburse eligible uranium and thorium licensees for certain costs of...

  16. Exploring functionally related enzymes using radially distributed properties of active sites around the reacting points of bound ligands

    PubMed Central

    2012-01-01

    Background Structural genomics approaches, particularly those solving the 3D structures of many proteins with unknown functions, have increased the desire for structure-based function predictions. However, prediction of enzyme function is difficult because one member of a superfamily may catalyze a different reaction than other members, whereas members of different superfamilies can catalyze the same reaction. In addition, conformational changes, mutations or the absence of a particular catalytic residue can prevent inference of the mechanism by which catalytic residues stabilize and promote the elementary reaction. A major hurdle for alignment-based methods for prediction of function is the absence (despite its importance) of a measure of similarity of the physicochemical properties of catalytic sites. To solve this problem, the physicochemical features radially distributed around catalytic sites should be considered in addition to structural and sequence similarities. Results We showed that radial distribution functions (RDFs), which are associated with the local structural and physicochemical properties of catalytic active sites, are capable of clustering oxidoreductases and transferases by function. The catalytic sites of these enzymes were also characterized using the RDFs. The RDFs provided a measure of the similarity among the catalytic sites, detecting conformational changes caused by mutation of catalytic residues. Furthermore, the RDFs reinforced the classification of enzyme functions based on conventional sequence and structural alignments. Conclusions Our results demonstrate that the application of RDFs provides advantages in the functional classification of enzymes by providing information about catalytic sites. PMID:22536854

  17. 40 CFR 61.154 - Standard for active waste disposal sites.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...). (e) For all asbestos-containing waste material received, the owner or operator of the active waste... 40 Protection of Environment 9 2013-07-01 2013-07-01 false Standard for active waste disposal... for Asbestos § 61.154 Standard for active waste disposal sites. Each owner or operator of an...

  18. Unmasking tandem site interaction in human acetylcholinesterase. Substrate activation with a cationic acetanilide substrate.

    PubMed

    Johnson, Joseph L; Cusack, Bernadette; Davies, Matthew P; Fauq, Abdul; Rosenberry, Terrone L

    2003-05-13

    Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge, and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. A conformational interaction between the A- and P-sites has recently been found to modulate ligand affinities. We now demonstrate that this interaction is of functional importance by showing that the acetylation rate constant of a substrate bound to the A-site is increased by a factor a when a second molecule of substrate binds to the P-site. This demonstration became feasible through the introduction of a new acetanilide substrate analogue of acetylcholine, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), for which a = 4. This substrate has a low acetylation rate constant and equilibrates with the catalytic site, allowing a tractable algebraic solution to the rate equation for substrate hydrolysis. ATMA affinities for the A- and P-sites deduced from the kinetic analysis were confirmed by fluorescence titration with thioflavin T as a reporter ligand. Values of a >1 give rise to a hydrolysis profile called substrate activation, and the AChE site-specific mutant W86F, and to a lesser extent wild-type human AChE itself, showed substrate activation with acetylthiocholine as the substrate. Substrate activation was incorporated into a previous catalytic scheme for AChE in which a bound P-site ligand can also block product dissociation from the A-site, and two additional features of the AChE catalytic pathway were revealed. First, the ability of a bound P-site ligand to increase the substrate acetylation rate constant varied with the structure of the ligand: thioflavin T accelerated ATMA acetylation by a factor a(2) of 1.3, while propidium failed to accelerate. Second, catalytic rate

  19. Structure and nuclearity of active sites in Fe-zeolites: comparison with iron sites in enzymes and homogeneous catalysts.

    PubMed

    Zecchina, Adriano; Rivallan, Mickaël; Berlier, Gloria; Lamberti, Carlo; Ricchiardi, Gabriele

    2007-07-21

    Fe-ZSM-5 and Fe-silicalite zeolites efficiently catalyse several oxidation reactions which find close analogues in the oxidation reactions catalyzed by homogeneous and enzymatic compounds. The iron centres are highly dispersed in the crystalline matrix and on highly diluted samples, mononuclear and dinuclear structures are expected to become predominant. The crystalline and robust character of the MFI framework has allowed to hypothesize that the catalytic sites are located in well defined crystallographic positions. For this reason these catalysts have been considered as the closest and best defined heterogeneous counterparts of heme and non heme iron complexes and of Fenton type Fe(2+) homogeneous counterparts. On this basis, an analogy with the methane monooxygenase has been advanced several times. In this review we have examined the abundant literature on the subject and summarized the most widely accepted views on the structure, nuclearity and catalytic activity of the iron species. By comparing the results obtained with the various characterization techniques, we conclude that Fe-ZSM-5 and Fe-silicalite are not the ideal samples conceived before and that many types of species are present, some active and some other silent from adsorptive and catalytic point of view. The relative concentration of these species changes with thermal treatments, preparation procedures and loading. Only at lowest loadings the catalytically active species become the dominant fraction of the iron species. On the basis of the spectroscopic titration of the active sites by using NO as a probe, we conclude that the active species on very diluted samples are isolated and highly coordinatively unsaturated Fe(2+) grafted to the crystalline matrix. Indication of the constant presence of a smaller fraction of Fe(2+) presumably located on small clusters is also obtained. The nitrosyl species formed upon dosing NO from the gas phase on activated Fe-ZSM-5 and Fe-silicalite, have been analyzed

  20. Nest success, cause-specific nest failure, and hatchability of aquatic birds at selenium-contaminated Kesterson Reservoir and a reference site

    USGS Publications Warehouse

    Ohlendorf, Harry M.; Hothem, Roger L.; Welsh, Daniel

    1989-01-01

    During 1983-1985, we studied the reproductive success of several species of aquatic birds (coots, ducks, shorebirds, and grebes) nesting at two sites in Merced County, California: a selenium-contaminated site (Kesterson Reservoir) and a nearby reference site (Volta Wildlife Area). We used a computer program (MICROMORT) developed for the analysis of radiotelemetry data (Heisey and Fuller 1985) to estimate nest success and cause-specific failure rates, and then compared these parameters and hatchability between sites and among years. Nest success and causes of failure varied by species, site, and year. The most important causes of nest failure were usually predation, desertion, and water-level changes. However, embryotoxicosis (mortality, deformity, and lack of embryonic development) was the most important cause of nest failure in Eared Grebes (Podiceps nigricollis) at Kesterson Reservoir. Embryotoxicosis also reduced the hatchability of eggs of all other species at Kesterson in one or more years; embryonic mortality occurred rarely at Volta, and abnormalities were not observed.

  1. Monocopper active site for partial methane oxidation in Cu-exchanged 8MR zeolites

    SciTech Connect

    Kulkarni, Ambarish R.; Zhao, Zhi -Jian; Siahrostami, Samira; Nørskov, Jens K.; Studt, Felix

    2016-08-17

    Direct conversion of methane to methanol using oxygen is experiencing renewed interest owing to the availability of new natural gas resources. Copper-exchanged zeolites such as mordenite and ZSM-5 have shown encouraging results, and di- and tri-copper species have been suggested as active sites. Recently, small eight-membered ring (8MR) zeolites including SSZ-13, -16, and -39 have been shown to be active for methane oxidation, but the active sites and reaction mechanisms in these 8MR zeolites are not known. In this work, we use density functional theory (DFT) calculations to systematically evaluate monocopper species as active sites for the partial methane oxidation reaction in Cu-exchanged SSZ-13. On the basis of kinetic and thermodynamic arguments, we suggest that [CuIIOH]+ species in the 8MR are responsible for the experimentally observed activity. Furthermore, our results successfully explain the available spectroscopic data and experimental observations including (i) the necessity of water for methanol extraction and (ii) the effect of Si/Al ratio on the catalyst activity. Monocopper species have not yet been suggested as an active site for the partial methane oxidation reaction, and our results suggest that [CuIIOH]+ active site may provide complementary routes for methane activation in zeolites in addition to the known [Cu–O–Cu]2+ and Cu3O3 motifs.

  2. Monocopper active site for partial methane oxidation in Cu-exchanged 8MR zeolites

    DOE PAGES

    Kulkarni, Ambarish R.; Zhao, Zhi -Jian; Siahrostami, Samira; ...

    2016-08-17

    Direct conversion of methane to methanol using oxygen is experiencing renewed interest owing to the availability of new natural gas resources. Copper-exchanged zeolites such as mordenite and ZSM-5 have shown encouraging results, and di- and tri-copper species have been suggested as active sites. Recently, small eight-membered ring (8MR) zeolites including SSZ-13, -16, and -39 have been shown to be active for methane oxidation, but the active sites and reaction mechanisms in these 8MR zeolites are not known. In this work, we use density functional theory (DFT) calculations to systematically evaluate monocopper species as active sites for the partial methane oxidationmore » reaction in Cu-exchanged SSZ-13. On the basis of kinetic and thermodynamic arguments, we suggest that [CuIIOH]+ species in the 8MR are responsible for the experimentally observed activity. Furthermore, our results successfully explain the available spectroscopic data and experimental observations including (i) the necessity of water for methanol extraction and (ii) the effect of Si/Al ratio on the catalyst activity. Monocopper species have not yet been suggested as an active site for the partial methane oxidation reaction, and our results suggest that [CuIIOH]+ active site may provide complementary routes for methane activation in zeolites in addition to the known [Cu–O–Cu]2+ and Cu3O3 motifs.« less

  3. 1993 annual report of hazardous waste activities for the Oak Ridge K-25 site

    SciTech Connect

    Not Available

    1994-02-01

    This report is a detailed listing of all of the Hazardous Waste activities occurring at Martin Marietta`s K-25 site. Contained herein are hazardous waste notification forms, waste stream reports, generator fee forms and various TSDR reports.

  4. Structural Insights into the Protease-like Antigen Plasmodium falciparum SERA5 and Its Noncanonical Active-Site Serine

    SciTech Connect

    Hodder, Anthony N.; Malby, Robyn L.; Clarke, Oliver B.; Fairlie, W. Douglas; Colman, Peter M.; Crabb, Brendan S.; Smith, Brian J.

    2009-08-28

    The sera genes of the malaria-causing parasite Plasmodium encode a family of unique proteins that are maximally expressed at the time of egress of parasites from infected red blood cells. These multi-domain proteins are unique, containing a central papain-like cysteine-protease fragment enclosed between the disulfide-linked N- and C-terminal domains. However, the central fragment of several members of this family, including serine repeat antigen 5 (SERA5), contains a serine (S596) in place of the active-site cysteine. Here we report the crystal structure of the central protease-like domain of Plasmodium falciparum SERA5, revealing a number of anomalies in addition to the putative nucleophilic serine: (1) the structure of the putative active site is not conducive to binding substrate in the canonical cysteine-protease manner; (2) the side chain of D594 restricts access of substrate to the putative active site; and (3) the S{sub 2} specificity pocket is occupied by the side chain of Y735, reducing this site to a small depression on the protein surface. Attempts to determine the structure in complex with known inhibitors were not successful. Thus, despite having revealed its structure, the function of the catalytic domain of SERA5 remains an enigma.

  5. AMPK Causes Cell Cycle Arrest in LKB1-deficient Cells via Activation of CAMKK2

    PubMed Central

    Fogarty, Sarah; Ross, Fiona A.; Ciruelos, Diana Vara; Gray, Alexander; Gowans, Graeme J.; Hardie, D. Grahame

    2017-01-01

    The AMP-activated protein kinase (AMPK) is activated by phosphorylation at Thr172, either by the tumor suppressor kinase LKB1 or by an alternate pathway involving the Ca2+/calmodulin-dependent kinase, CAMKK2. Increases in AMP:ATP and ADP:ATP ratios, signifying energy deficit, promote allosteric activation and net Thr172 phosphorylation mediated by LKB1, so that the LKB1-AMPK pathway acts as an energy sensor. Many tumor cells carry loss-of-function mutations in the STK11 gene encoding LKB1, but LKB1 re-expression in these cells causes cell cycle arrest. Therefore, it was investigated as to whether arrest by LKB1 is caused by activation of AMPK or of one of the AMPK-related kinases, which are also dependent on LKB1 but are not activated by CAMKK2. In three LKB1-null tumor cell lines, treatment with the Ca2+ ionophore A23187 caused a G1-arrest that correlated with AMPK activation and Thr172 phosphorylation. In G361 cells, expression of a truncated, CAMKK2 mutant also caused G1-arrest similar to that caused by expression of LKB1, while expression of a dominant negative AMPK mutant, or a double knockout of both AMPK-α subunits, also prevented the cell cycle arrest caused by A23187. These mechanistic findings confirm that AMPK activation triggers cell cycle arrest, and also suggest that the rapid proliferation of LKB1-null tumor cells is due to lack of the restraining influence of AMPK. However, cell cycle arrest can be restored by re-expressing LKB1 or a constitutively active CAMKK2, or by pharmacological agents that increase intracellular Ca2+ and thus activate endogenous CAMKK2. Implications Evidence here reveals that the rapid growth and proliferation of cancer cells lacking the tumor suppressor LKB1 is due to reduced activity of AMPK, and suggests a therapeutic approach by which this block might be circumvented. PMID:27141100

  6. The surface chemistry of heterogeneous catalysis: mechanisms, selectivity, and active sites.

    PubMed

    Zaera, Francisco

    2005-01-01

    The role of chemical kinetics in defining the requirements for the active sites of heterogeneous catalysts is discussed. A personal view is presented, with specific examples from our laboratory to illustrate the role of the chemical composition, structure, and electronic properties of specific surface sites in determining reaction activity and selectivity. Manipulation of catalytic behavior via the addition of chemical modifiers and by tuning of the reaction conditions is also introduced.

  7. The Impact of Level of Solar Activity on Mortality by Cause in Longtime Period

    NASA Astrophysics Data System (ADS)

    Podolska, Katerina

    2014-05-01

    The aim of this presentation is to show the dependence of the intensity of mortality in the Czech Republic, according to the chosen causes of death according to ICD-10, on the solar activity during the increasing and decreasing phase of the solar cycle No.23 in the period 1994-2011. We use the methods of multivariate statistical analysis. The typology of time profiles for the causes of death is identified with the help of cluster analysis using time. The solar activity is represented by the indices R, Kp, F10.7 and Dst, and also by the height of the F2 layer and TEC for the Czech Republic. There are investigated groups of causes of death according to ICD-10 II. Neoplasms, VI. Diseases of the nervous system, XII. Diseases of the skin and subcutaneous tissue and XVII. Congenital malformations, deformations and chromosomal abnormalities. The correlation between the intensity of mortality from cardiovascular disease e.g. I21 (acute myocardial infarction) and I64 (stroke) and birth defect e.g. Q91 (Edwards' and Pataus' syndrom) and the solar activity parameters is discovered, as well as a stronger dependence on the height of the F2 layer and TEC. We also explored the influence of the above parameters on mortality by causes on degenerative diseases. Typology of time profiles for these causes of death are identified by cluster analysis using time and have found large differences between diagnoses.

  8. Nuclear waste: Status of DOE`s nuclear waste site characterization activities

    SciTech Connect

    1987-12-31

    Three potential nuclear waste repository sites have been selected to carry out characterization activities-the detailed geological testing to determine the suitability of each site as a repository. The sites are Hanford in south-central Washington State, Yucca Mountain in southern Nevada, and Deaf Smith in the Texas Panhandle. Two key issues affecting the total program are the estimations of the site characterization completion data and costs and DOE`s relationship with the Nuclear Regulatory Commission which has been limited and its relations with affected states and Indian tribes which continue to be difficult.

  9. SUMMARY REPORT: THE CAUSES AND CONTROL OF ACTIVATED SLUDGE BULKING AND FOAMING

    EPA Science Inventory

    This 92-page Technology Transfer Summary Report provides reference material on the causes and controls of sludge bulking and foaming in activated sludge treatment that can be readily understood, and it includes sufficient detail to help plant operators control their systems. The ...

  10. Number and locations of agonist binding sites required to activate homomeric Cys-loop receptors.

    PubMed

    Rayes, Diego; De Rosa, María José; Sine, Steven M; Bouzat, Cecilia

    2009-05-06

    Homo-pentameric Cys-loop receptors contain five identical agonist binding sites, each formed at a subunit interface. To determine the number and locations of binding sites required to generate a stable active state, we constructed a receptor subunit with a mutation that disables the agonist binding site and a reporter mutation that alters unitary conductance and coexpressed mutant and nonmutant subunits. Although receptors with a range of different subunit compositions are produced, patch-clamp recordings reveal that the amplitude of each single-channel opening event reports the number and, for certain subunit combinations, the locations of subunits with intact binding sites. We find that receptors with three binding sites at nonconsecutive subunit interfaces exhibit maximal mean channel open time, receptors with binding sites at three consecutive or two nonconsecutive interfaces exhibit intermediate open time, and receptors with binding sites at two consecutive or one interface exhibit brief open time. Macroscopic recordings after rapid application of agonist reveal that channel activation slows and the extent of desensitization decreases as the number of binding sites per receptor decreases. The overall results provide a framework for defining mechanisms of activation and drug modulation for homo-pentameric Cys-loop receptors.

  11. Molecular dynamics explorations of active site structure in designed and evolved enzymes.

    PubMed

    Osuna, Sílvia; Jiménez-Osés, Gonzalo; Noey, Elizabeth L; Houk, K N

    2015-04-21

    This Account describes the use of molecular dynamics (MD) simulations to reveal how mutations alter the structure and organization of enzyme active sites. As proposed by Pauling about 70 years ago and elaborated by many others since then, biocatalysis is efficient when functional groups in the active site of an enzyme are in optimal positions for transition state stabilization. Changes in mechanism and covalent interactions are often critical parts of enzyme catalysis. We describe our explorations of the dynamical preorganization of active sites using MD, studying the fluctuations between active and inactive conformations normally concealed to static crystallography. MD shows how the various arrangements of active site residues influence the free energy of the transition state and relates the populations of the catalytic conformational ensemble to the enzyme activity. This Account is organized around three case studies from our laboratory. We first describe the importance of dynamics in evaluating a series of computationally designed and experimentally evolved enzymes for the Kemp elimination, a popular subject in the enzyme design field. We find that the dynamics of the active site is influenced not only by the original sequence design and subsequent mutations but also by the nature of the ligand present in the active site. In the second example, we show how microsecond MD has been used to uncover the role of remote mutations in the active site dynamics and catalysis of a transesterase, LovD. This enzyme was evolved by Tang at UCLA and Codexis, Inc., and is a useful commercial catalyst for the production of the drug simvastatin. X-ray analysis of inactive and active mutants did not reveal differences in the active sites, but relatively long time scale MD in solution showed that the active site of the wild-type enzyme preorganizes only upon binding of the acyl carrier protein (ACP) that delivers the natural acyl group to the active site. In the absence of bound ACP

  12. Correlated structural kinetics and retarded solvent dynamics at the metalloprotease active site

    SciTech Connect

    Grossman, Moran; Born, Benjamin; Heyden, Matthias; Tworowski, Dmitry; Fields, Gregg B.; Sagi, Irit; Havenith, Martina

    2011-09-18

    Solvent dynamics can play a major role in enzyme activity, but obtaining an accurate, quantitative picture of solvent activity during catalysis is quite challenging. Here, we combine terahertz spectroscopy and X-ray absorption analyses to measure changes in the coupled water-protein motions during peptide hydrolysis by a zinc-dependent human metalloprotease. These changes were tightly correlated with rearrangements at the active site during the formation of productive enzyme-substrate intermediates and were different from those in an enzyme–inhibitor complex. Molecular dynamics simulations showed a steep gradient of fast-to-slow coupled protein-water motions around the protein, active site and substrate. Our results show that water retardation occurs before formation of the functional Michaelis complex. We propose that the observed gradient of coupled protein-water motions may assist enzyme-substrate interactions through water-polarizing mechanisms that are remotely mediated by the catalytic metal ion and the enzyme active site.

  13. Two interacting binding sites for quinacrine derivatives in the active site of trypanothione reductase – a template for drug design

    PubMed Central

    Saravanamuthu, Ahilan; Vickers, Tim J.; Bond, Charles S.; Peterson, Mark R.; Hunter, William N.; Fairlamb, Alan H.

    2012-01-01

    SUMMARY Trypanothione reductase is a key enzyme in the trypanothione-based redox metabolism of pathogenic trypanosomes. Since this system is absent in humans, being replaced with glutathione and glutathione reductase, it offers a target for selective inhibition. The rational design of potent inhibitors requires accurate structures of enzyme-inhibitor complexes, but this is lacking for trypanothione reductase. We therefore used quinacrine mustard, an alkylating derivative of the competitive inhibitor quinacrine, to probe the active site of this dimeric flavoprotein. Quinacrine mustard irreversibly inactivates Trypanosoma cruzi trypanothione reductase, but not human glutathione reductase, in a time-dependent manner with a stoichiometry of two inhibitors bound per monomer. The rate of inactivation is dependent upon the oxidation state of trypanothione reductase, with the NADPH-reduced form being inactivated significantly faster than the oxidised form. Inactivation is slowed by clomipramine and a melarsen oxide-trypanothione adduct (both are competitive inhibitors) but accelerated by quinacrine. The structure of the trypanothione reductase-quinacrine mustard adduct was determined to 2.7 Å, revealing two molecules of inhibitor bound in the trypanothione-binding site. The acridine moieties interact with each other through π-stacking effects, and one acridine interacts in a similar fashion with a tryptophan residue. These interactions provide a molecular explanation for the differing effects of clomipramine and quinacrine on inactivation by quinacrine mustard. Synergism with quinacrine occurs as a result of these planar acridines being able to stack together in the active site cleft, thereby gaining an increased number of binding interactions, whereas antagonism occurs with non-planar molecules, such as clomipramine, where stacking is not possible. PMID:15102853

  14. Low-order chaos in sympathetic nerve activity causes 1/f fluctuation of heartbeat intervals

    NASA Astrophysics Data System (ADS)

    Osaka, Motohisa; Kumagai, Hiroo; Sakata, Katsufumi; Onami, Toshiko; Chon, Ki H.; Watanabe, Mari A.; Saruta, Takao

    2004-04-01

    The mechanism of 1/f scaling of heartbeat intervals remains unknown. We recorded heartbeat intervals, sympathetic nerve activity, and blood pressure in conscious rats with normal or high blood pressure. Using nonlinear analyses, we demonstrate that the dynamics of this system of 3 variables is low-order chaos, and that sympathetic nerve activity leads to heartbeat interval and blood pressure changes. It is suggested that 1/f scaling of heartbeat intervals results from the low-order chaos of these variables and that impaired regulation of blood pressure by sympathetic nerve activity is likely to cause experimentally observable steeper scaling of heartbeat intervals in hypertensive (high blood pressure) rats.

  15. Joint associations of alcohol consumption and physical activity with all-cause and cardiovascular mortality.

    PubMed

    Soedamah-Muthu, Sabita S; De Neve, Melissa; Shelton, Nicola J; Tielemans, Susanne M A J; Stamatakis, Emmanuel

    2013-08-01

    Individual associations of alcohol consumption and physical activity with cardiovascular disease are relatively established, but the joint associations are not clear. Therefore, the aim of this study was to examine prospectively the joint associations between alcohol consumption and physical activity with cardiovascular mortality (CVM) and all-cause mortality. Four population-based studies in the United Kingdom were included, the 1997 and 1998 Health Surveys for England and the 1998 and 2003 Scottish Health Surveys. In men and women, respectively, low physical activity was defined as 0.1 to 5 and 0.1 to 4 MET-hours/week and high physical activity as ≥5 and ≥4 MET-hours/week. Moderate or moderately high alcohol intake was defined as >0 to 35 and >0 to 21 units/week and high levels of alcohol intake as >35 and >21 units/week. In total, there were 17,410 adults without prevalent cardiovascular diseases and complete data on alcohol and physical activity (43% men, median age 55 years). During a median follow-up period of 9.7 years, 2,204 adults (12.7%) died, 638 (3.7%) with CVM. Cox proportional-hazards models were adjusted for potential confounders such as marital status, social class, education, ethnicity, and longstanding illness. In the joint associations analysis, low activity combined with high levels of alcohol (CVM: hazard ratio [HR] 1.95, 95% confidence interval [CI] 1.28 to 2.96, p = 0.002; all-cause mortality: HR 1.64, 95% CI 1.32 to 2.03, p <0.001) and low activity combined with no alcohol (CVM: HR 1.93, 95% CI 1.35 to 2.76, p <0.001; all-cause mortality: HR 1.50, 95% CI 1.24 to 1.81, p <0.001) were linked to the highest risk, compared with moderate drinking and higher levels of physical activity. Within each given alcohol group, low activity was linked to increased CVM risk (e.g., HR 1.48, 95% CI 1.08 to 2.03, p = 0.014, for the moderate drinking group), but in the presence of high physical activity, high alcohol intake was not linked to increased CVM

  16. Fragment-based identification of determinants of conformational and spectroscopic change at the ricin active site

    SciTech Connect

    Carra,J.; McHugh, C.; Mulligan, S.; Machiesky, L.; Soares, A.; Millard, C.

    2007-01-01

    We found that amide ligands can bind weakly but specifically to the ricin active site, producing significant shifts in positions of the critical active site residues Arg180 and Tyr80. These results indicate that fragment-based drug discovery methods are capable of identifying minimal bonding determinants of active-site side-chain rearrangements and the mechanistic origins of spectroscopic shifts. Our results suggest that tryptophan fluorescence provides a sensitive probe for the geometric relationship of arginine-tryptophan pairs, which often have significant roles in protein function. Using the unusual characteristics of the RTA system, we measured the still controversial thermodynamic changes of site-specific urea binding to a protein, results that are relevant to understanding the physical mechanisms of protein denaturation.

  17. Asparagine deprivation mediated by Salmonella asparaginase causes suppression of activation-induced T cell metabolic reprogramming.

    PubMed

    Torres, AnnMarie; Luke, Joanna D; Kullas, Amy L; Kapilashrami, Kanishk; Botbol, Yair; Koller, Antonius; Tonge, Peter J; Chen, Emily I; Macian, Fernando; van der Velden, Adrianus W M

    2016-02-01

    Salmonellae are pathogenic bacteria that induce immunosuppression by mechanisms that remain largely unknown. Previously, we showed that a putative type II l-asparaginase produced by Salmonella Typhimurium inhibits T cell responses and mediates virulence in a murine model of infection. Here, we report that this putative L-asparaginase exhibits L-asparagine hydrolase activity required for Salmonella Typhimurium to inhibit T cells. We show that L-asparagine is a nutrient important for T cell activation and that L-asparagine deprivation, such as that mediated by the Salmonella Typhimurium L-asparaginase, causes suppression of activation-induced mammalian target of rapamycin signaling, autophagy, Myc expression, and L-lactate secretion. We also show that L-asparagine deprivation mediated by the Salmonella Typhimurium L-asparaginase causes suppression of cellular processes and pathways involved in protein synthesis, metabolism, and immune response. Our results advance knowledge of a mechanism used by Salmonella Typhimurium to inhibit T cell responses and mediate virulence, and provide new insights into the prerequisites of T cell activation. We propose a model in which l-asparagine deprivation inhibits T cell exit from quiescence by causing suppression of activation-induced metabolic reprogramming.

  18. First aid for dental trauma caused by sports activities: state of knowledge, treatment and prevention.

    PubMed

    Emerich, Katarzyna; Kaczmarek, Jan

    2010-05-01

    In view of the widespread lack of knowledge of first aid procedures in cases of dental trauma, this article describes the current state of knowledge and highlights the need for education of those likely to witness or be victims of dental trauma while practising sports. Dental and oral injuries, the commonest type of orofacial injuries, are often sustained by athletes playing contact sports; indeed, they represent the most frequent type of sporting injury. Studies of a large group of children and adults have shown that as many as 31% of all orofacial injuries are caused by sporting activities. Furthermore, current literature on the subject emphasizes that awareness of appropriate triage procedures following dental trauma is unsatisfactory. Delay in treatment is the single most influential factor affecting prognosis. What should we know and, more importantly, what should we do? Immediate replantation of an avulsed tooth is the best treatment option at the site of the accident. If replantation is impossible, milk is the preferred transport medium for the avulsed tooth. There is a general low level of awareness about the need for prompt triage of traumatic dental injuries sustained in sports, despite their relative frequency. When a cohort of Swiss basketball players was interviewed, only half were aware that an avulsed tooth could be replanted. Cheap, commercially available tooth storage devices containing an isotonic transport medium (so-called 'Save-a-Tooth boxes'), can maintain the viability of an avulsed tooth for up to 72 hours, prior to replantation. More readily available storage media such as milk, sterile saline or even saliva may be used, but knowledge of this information is rare among sports participants. For example, just 6.6% of the Swiss basketball players interviewed were aware of the 'Tooth Rescue box' products. Sporting organizations seem to offer very little information about sports-related risks or preventive strategies for orodental trauma. Having

  19. Structural and Kinetic Analyses of Macrophage Migration Inhibitory Factor Active Site Interactions

    SciTech Connect

    Crichlow, G.; Lubetsky, J; Leng, L; Bucala, R; Lolis, E

    2009-01-01

    Macrophage migration inhibitory factor (MIF) is a secreted protein expressed in numerous cell types that counters the antiinflammatory effects of glucocorticoids and has been implicated in sepsis, cancer, and certain autoimmune diseases. Interestingly, the structure of MIF contains a catalytic site resembling the tautomerase/isomerase sites of microbial enzymes. While bona fide physiological substrates remain unknown, model substrates have been identified. Selected compounds that bind in the tautomerase active site also inhibit biological functions of MIF. It had previously been shown that the acetaminophen metabolite, N-acetyl-p-benzoquinone imine (NAPQI), covalently binds to the active site of MIF. In this study, kinetic data indicate that NAPQI inhibits MIF both covalently and noncovalently. The structure of MIF cocrystallized with NAPQI reveals that the NAPQI has undergone a chemical alteration forming an acetaminophen dimer (bi-APAP) and binds noncovalently to MIF at the mouth of the active site. We also find that the commonly used protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), forms a covalent complex with MIF and inhibits the tautomerase activity. Crystallographic analysis reveals the formation of a stable, novel covalent bond for PMSF between the catalytic nitrogen of the N-terminal proline and the sulfur of PMSF with complete, well-defined electron density in all three active sites of the MIF homotrimer. Conclusions are drawn from the structures of these two MIF-inhibitor complexes regarding the design of novel compounds that may provide more potent reversible and irreversible inhibition of MIF.

  20. All the catalytic active sites of MoS2 for hydrogen evolution

    SciTech Connect

    Li, Guoqing; Zhang, Du; Qiao, Qiao; Yu, Yifei; Peterson, David; Zafar, Abdullah; Kumar, Raj; Curtarolo, Stefano; Hunte, Frank; Shannon, Steve; Zhu, Yimei; Yang, Weitao; Cao, Linyou

    2016-11-29

    MoS2 presents a promising low-cost catalyst for the hydrogen evolution reaction (HER), but the understanding about its active sites has remained limited. Here we present an unambiguous study of the catalytic activities of all possible reaction sites of MoS2, including edge sites, sulfur vacancies, and grain boundaries. We demonstrate that, in addition to the well-known catalytically active edge sites, sulfur vacancies provide another major active site for the HER, while the catalytic activity of grain boundaries is much weaker. Here, the intrinsic turnover frequencies (Tafel slopes) of the edge sites, sulfur vacancies, and grain boundaries are estimated to be 7.5 s–1 (65–75 mV/dec), 3.2 s–1 (65–85 mV/dec), and 0.1 s–1 (120–160 mV/dec), respectively. We also demonstrate that the catalytic activity of sulfur vacancies strongly depends on the density of the vacancies and the local crystalline structure in proximity to the vacancies. Unlike edge sites, whose catalytic activity linearly depends on the length, sulfur vacancies show optimal catalytic activities when the vacancy density is in the range of 7–10%, and the number of sulfur vacancies in high crystalline quality MoS2 is higher than that in low crystalline quality MoS2, which may be related with the proximity of different local crystalline structures to the vacancies.

  1. Causes of Activation and Deactivation of Modified Nanogold Catalysts during Prolonged Storage and Redox Treatments.

    PubMed

    Kolobova, Ekaterina; Kotolevich, Yulia; Pakrieva, Ekaterina; Mamontov, Grigory; Farías, Mario H; Bogdanchikova, Nina; Cortés Corberán, Vicente; Pestryakov, Alexey

    2016-04-13

    The catalytic properties of modified Au/TiO₂ catalysts for low-temperature CO oxidation are affected by deactivation and reactivation after long-term storage and by redox treatments. The effect of these phenomena on the catalysts was studied by HRTEM, BET, SEM, FTIR CO, XPS and H₂ TPR methods. The main cause for the deactivation and reactivation of catalytic properties is the variation in the electronic state of the supported gold, mainly, the proportion of singly charged ions Au⁺. The most active samples are those with the highest proportion of singly charged gold ions, while catalysts with a high content of trivalent gold ions are inactive at low-temperatures. Active states of gold, resistant to changes caused by the reaction process and storage conditions, can be stabilized by modification of the titanium oxide support with transition metals oxides. The catalyst modified with lanthanum oxide shows the highest stability and activity.

  2. A novel TMPRSS6 mutation that prevents protease auto-activation causes IRIDA

    PubMed Central

    Altamura, Sandro; D'Alessio, Flavia; Selle, Barbara; Muckenthaler, Martina U.

    2010-01-01

    IRIDA (iron-refractory iron-deficiency anaemia) is a rare autosomal-recessive disorder hallmarked by hypochromic microcytic anaemia, low transferrin saturation and high levels of the iron-regulated hormone hepcidin. The disease is caused by mutations in the transmembrane serine protease TMPRSS6 (transmembrane protease serine 6) that prevent inactivation of HJV (haemojuvelin), an activator of hepcidin transcription. In the present paper, we describe a patient with IRIDA who carries a novel mutation (Y141C) in the SEA domain of the TMPRSS6 gene. Functional characterization of the TMPRSS6(Y141C) mutant protein in cultured cells showed that it localizes to similar subcellular compartments as wild-type TMPRSS6 and binds HJV, but fails to auto-catalytically activate itself. As a consequence, hepcidin mRNA expression is increased, causing the clinical symptoms observed in this IRIDA patient. The present study provides important mechanistic insight into how TMPRSS6 is activated. PMID:20704562

  3. A Mutational Analysis of the Active Site Loop Residues in cis-3-Chloroacrylic Acid Dehalogenase

    PubMed Central

    Schroeder, Gottfried K.; Huddleston, Jamison P.; Johnson, William H.; Whitman, Christian P.

    2013-01-01

    cis -3-Chloroacrylic acid dehalogenase (cis-CaaD) from Pseudomonas pavonaceae 170 and a homologue from Corynebacterium glutamicum designated Cg10062 share 34% sequence identity (54% similarity). The former catalyzes a key step in a bacterial catabolic pathway for the nematocide 1,3-dichloropropene, whereas the latter has no known biological activity. Although Cg10062 has the six active site residues (Pro-1, His-28, Arg-70, Arg-73, Tyr-103, Glu-114) that are critical for cis-CaaD activity, it shows only a low level cis-CaaD activity and lacks the specificity of cis-CaaD: Cg10062 processes both isomers of 3-chloroacrylate with a preference for the cis-isomer. Although the basis for these differences is unknown, a comparison of the crystal structures of the enzymes covalently modified by an adduct resulting from their incubation with the same inhibitor offers a possible explanation. A 6-residue active site loop in cis-CaaD shows a strikingly different conformation from that observed in Cg10062: the loop closes down on the active site of cis-CaaD, but not on that of Cg10062. In order to examine what this loop might contribute to cis-CaaD catalysis and specificity, the residues were changed individually to those found in Cg10062. Subsequent kinetic and mechanistic analysis suggests that the T34A mutant of cis-CaaD is more Cg10062-like. The mutant enzyme shows a 4-fold increase in Km (using cis-3-bromoacrylate), but not to the degree observed for Cg10062 (687-fold). The mutation also causes a 4-fold decrease in the burst rate (compared to the wild type cis-CaaD), whereas Cg10062 shows no burst rate. More telling is the reaction of the T34A mutant of cis-CaaD with the alternate substrate, 2,3-butadienoate. In the presence of NaBH4 and the allene, cis-CaaD is completely inactivated after one turnover due to the covalent modification of Pro-1. The same experiment with Cg10062 does not result in the covalent modification of Pro-1. The different outcomes are attributed to

  4. Comparison between active sensor and radiosonde cloud boundaries over the ARM Southern Great Plains site

    NASA Astrophysics Data System (ADS)

    Naud, Catherine M.; Muller, Jan-Peter; Clothiaux, Eugene E.

    2003-02-01

    In order to test the strengths and limitations of cloud boundary retrievals from radiosonde profiles, 4 years of radar, lidar, and ceilometer data collected at the Atmospheric Radiation Measurements Southern Great Plains site from November 1996 through October 2000 are used to assess the retrievals of [1995] and [1996]. The lidar and ceilometer data yield lowest-level cloud base heights that are, on average, within approximately 125 m of each other when both systems detect a cloud. These quantities are used to assess the accuracy of coincident cloud base heights obtained from radar and the two radiosonde-based methods applied to 200 m resolution profiles obtained at the same site. The lidar/ceilometer and radar cloud base heights agree by 0.156 ± 0.423 km for 85.27% of the observations, while the agreement between the lidar/ceilometer and radiosonde-derived heights is at best -0.044 ± 0.559 km for 74.60% of all cases. Agreement between radar- and radiosonde-derived cloud boundaries is better for cloud base height than for cloud top height, being at best 0.018 ± 0.641 km for 70.91% of the cloud base heights and 0.348 ± 0.729 km for 68.27% of the cloud top heights. The disagreements between radar- and radiosonde-derived boundaries are mainly caused by broken cloud situations when it is difficult to verify that drifting radiosondes and fixed active sensors are observing the same clouds. In the case of the radar the presence of clutter (e.g., vegetal particles or insects) can affect the measurements from the surface up to approximately 3-5 km, preventing comparisons with radiosonde-derived boundaries. Overall, [1995] tend to classify moist layers that are not clouds as clouds and both radiosonde techniques report high cloud top heights that are higher than the corresponding heights from radar.

  5. Depletion of WRN protein causes RACK1 to activate several protein kinase C isoforms.

    PubMed

    Massip, L; Garand, C; Labbé, A; Perreault, E; Turaga, R V N; Bohr, V A; Lebel, M

    2010-03-11

    Werner's syndrome (WS) is a rare autosomal disease characterized by the premature onset of several age-associated pathologies. The protein defective in patients with WS (WRN) is a helicase/exonuclease involved in DNA repair, replication, transcription and telomere maintenance. In this study, we show that a knock down of the WRN protein in normal human fibroblasts induces phosphorylation and activation of several protein kinase C (PKC) enzymes. Using a tandem affinity purification strategy, we found that WRN physically and functionally interacts with receptor for activated C-kinase 1 (RACK1), a highly conserved anchoring protein involved in various biological processes, such as cell growth and proliferation. RACK1 binds strongly to the RQC domain of WRN and weakly to its acidic repeat region. Purified RACK1 has no impact on the helicase activity of WRN, but selectively inhibits WRN exonuclease activity in vitro. Interestingly, knocking down RACK1 increased the cellular frequency of DNA breaks. Depletion of the WRN protein in return caused a fraction of nuclear RACK1 to translocate out of the nucleus to bind and activate PKCdelta and PKCbetaII in the membrane fraction of cells. In contrast, different DNA-damaging treatments known to activate PKCs did not induce RACK1/PKCs association in cells. Overall, our results indicate that a depletion of the WRN protein in normal fibroblasts causes the activation of several PKCs through translocation and association of RACK1 with such kinases.

  6. Depletion of WRN protein causes RACK1 to activate several protein kinase C isoforms

    PubMed Central

    Massip, L; Garand, C; Labbé, A; Perreault, È; Turaga, RVN; Bohr, VA; Lebel, M

    2015-01-01

    Werner’s syndrome (WS) is a rare autosomal disease characterized by the premature onset of several age-associated pathologies. The protein defective in patients with WS (WRN) is a helicase/exonuclease involved in DNA repair, replication, transcription and telomere maintenance. In this study, we show that a knock down of the WRN protein in normal human fibroblasts induces phosphorylation and activation of several protein kinase C (PKC) enzymes. Using a tandem affinity purification strategy, we found that WRN physically and functionally interacts with receptor for activated C-kinase 1 (RACK1), a highly conserved anchoring protein involved in various biological processes, such as cell growth and proliferation. RACK1 binds strongly to the RQC domain of WRN and weakly to its acidic repeat region. Purified RACK1 has no impact on the helicase activity of WRN, but selectively inhibits WRN exonuclease activity in vitro. Interestingly, knocking down RACK1 increased the cellular frequency of DNA breaks. Depletion of the WRN protein in return caused a fraction of nuclear RACK1 to translocate out of the nucleus to bind and activate PKCδ and PKCβII in the membrane fraction of cells. In contrast, different DNA-damaging treatments known to activate PKCs did not induce RACK1/PKCs association in cells. Overall, our results indicate that a depletion of the WRN protein in normal fibroblasts causes the activation of several PKCs through translocation and association of RACK1 with such kinases. PMID:19966859

  7. Controls on interannual variability in lightning-caused fire activity in the western US

    NASA Astrophysics Data System (ADS)

    Abatzoglou, John T.; Kolden, Crystal A.; Balch, Jennifer K.; Bradley, Bethany A.

    2016-04-01

    Lightning-caused wildfires account for a majority of burned area across the western United States (US), yet lightning remains among the more unpredictable spatiotemporal aspects of the fire environment and a challenge for both modeling and managing fire activity. A data synthesis of cloud-to-ground lightning strikes, climate and fire data across the western US from 1992 to 2013 was conducted to better understand geographic variability in lightning-caused wildfire and the factors that influence interannual variability in lightning-caused wildfire at regional scales. Distinct geographic variability occurred in the proportion of fires and area burned attributed to lightning, with a majority of fires in the interior western US attributed to lightning. Lightning ignition efficiency was highest across the western portion of the region due to the concomitance of peak lightning frequency and annual nadir in fuel moisture in mid-to-late summer. For most regions the number of total and dry lightning strikes exhibited strong interannual correlation with the number of lightning-caused fires, yet were a poor predictor of area burned at regional scales. Commonality in climate-fire relationships for regional annual area burned by lightning- versus human-ignited fires suggests climate conditions, rather than lightning activity, are the predominant control of interannual variability in area burned by lightning-caused fire across much of the western US.

  8. Association between domains of physical activity and all-cause, cardiovascular and cancer mortality.

    PubMed

    Autenrieth, Christine S; Baumert, Jens; Baumeister, Sebastian E; Fischer, Beate; Peters, Annette; Döring, Angela; Thorand, Barbara

    2011-02-01

    Few studies have investigated the independent effects of domain-specific physical activity on mortality. We sought to investigate the association of physical activity performed in different domains of daily living on all-cause, cardiovascular (CVD) and cancer mortality. Using a prospective cohort design, 4,672 men and women, aged 25-74 years, who participated in the baseline examination of the MONICA/KORA Augsburg Survey 1989/1990 were classified according to their activity level (no, light, moderate, vigorous). Domains of self-reported physical activity (work, transportation, household, leisure time) and total activity were assessed by the validated MOSPA (MONICA Optional Study on Physical Activity) questionnaire. After a median follow-up of 17.8 years, a total of 995 deaths occurred, with 452 from CVD and 326 from cancer. For all-cause mortality, hazard ratios and 95% confidence interval (HR, 95% CI) of the highly active versus the inactive reference group were 0.69 (0.48-1.00) for work, 0.48 (0.36-0.65) for leisure time, and 0.73 (0.59-0.90) for total activity after multivariable adjustments. Reduced risks of CVD mortality were observed for high levels of work (0.54, 0.31-0.93), household (0.80, 0.54-1.19), leisure time (0.50, 0.31-0.79) and total activity (0.75, 0.55-1.03). Leisure time (0.36, 0.23-0.59) and total activity (0.62, 0.43-0.88) were associated with reduced risks of cancer mortality. Light household activity was related to lower all-cause (0.82, 0.71-0.95) and CVD (0.72, 0.58-0.89) mortality. No clear effects were found for transportation activities. Our findings suggest that work, household, leisure time and total physical activity, but not transportation activity, may protect from premature mortality.

  9. Identification of a novel phosphorylation site, Ser-170, as a regulator of bad pro-apoptotic activity.

    PubMed

    Dramsi, Shaynoor; Scheid, Michael P; Maiti, Arpita; Hojabrpour, Payman; Chen, Xianming; Schubert, Kathryn; Goodlett, David R; Aebersold, Ruedi; Duronio, Vincent

    2002-02-22

    Bad is a pro-apoptotic member of the Bcl-2 family of proteins that is thought to exert a death-promoting effect by heterodimerization with Bcl-X(L), nullifying its anti-apoptotic activity. Growth factors may promote cell survival at least partially through phosphorylation of Bad at one or more of Ser-112, -136, or -155. Our previous work showed that Bad is also phosphorylated in response to cytokines at another site, which we now identify as Ser-170. The functional role of this novel phosphorylation site was assessed by site-directed mutagenesis and analysis of the pro-apoptotic function of Bad in transiently transfected HEK293 and COS-7 cells or by stable expression in the cytokine-dependent cell line, MC/9. In general, mutation of Ser-170 to Ala results in a protein with increased ability to induce apoptosis, similar to the S112A mutant. Mutation of Ser-170 to Asp, mimicking a constitutively phosphorylated site, results in a protein that is virtually unable to induce apoptosis. Similarly, the S112A/S170D double mutant does not cause apoptosis in HEK293 and MC/9 cell lines. These data strongly suggest that phosphorylation of Bad at Ser-170 is a critical event in blocking the pro-apoptotic activity of Bad.

  10. Nature of Catalytic Active Sites Present on the Surface of Advanced Bulk Tantalum Mixed Oxide Photocatalysts

    SciTech Connect

    Phivilay, Somphonh; Puretzky, Alexander A; Domen, Kazunari Domen; Wachs, Israel

    2013-01-01

    The most active photocatalyst system for water splitting under UV irradiation (270 nm) is the promoted 0.2%NiO/NaTaO3:2%La photocatalyst with optimized photonic efficiency (P.E.) of 56%, but fundamental issues about the nature of the surface catalytic active sites and their involvement in the photocatalytic process still need to be clarified. This is the first study to apply cutting edge surface spectroscopic analyses to determine the surface nature of tantalum mixed oxide photocatalysts. Surface analysis with HR-XPS (1-3nm) and HS-LEIS (0.3nm) spectroscopy indicates that the NiO and La2O3 promoters are concentrated in the surface region of the bulk NaTaO3 phase. The La2O3 is concentrated on the NaTaO3 outermost surface layers while NiO is distributed throughout the NaTaO3 surface region (1-3nm). Raman and UV-vis spectroscopy revealed that the bulk molecular and electronic structures, respectively, of NaTaO3 were not modified by the addition of the La2O3 and NiO promoters, with La2O3 resulting in a slightly more ordered structure. Photoluminescence (PL) spectroscopy reveals that the addition of La2O3 and NiO produces a greater number of electron traps resulting in the suppression of the recombination of excited electrons/holes. In contrast to earlier reports, the La2O3 is only a textural promoter (increasing the BET surface area ~7x by stabilizing smaller NaTaO3 particles), but causes a ~3x decrease in the specific photocatalytic TORs ( mol H2/m2/h) rate because surface La2O3 blocks exposed catalytic active NaTaO3 sites. The NiO promoter was found to be a potent electronic promoter that enhances the NaTaO3 surface normalized TORs by a factor of ~10-50 and TOF by a factor of ~10. The level of NiO promotion is the same in the absence and presence of La2O3 demonstrating that there is no promotional synergistic interaction between the NiO and La2O3 promoters. This study demonstrates the important contributions of the photocatalyst surface properties to the fundamental

  11. Effect of acidic amino acids engineered into the active site cleft of Thermopolyspora flexuosa GH11 xylanase.

    PubMed

    Li, He; Turunen, Ossi

    2015-01-01

    Thermopolyspora flexuosa GH11 xylanase (XYN11A) shows optimal activity at pH 6-7 and 75-80 °C. We studied how mutation to aspartic acid (N46D and V48D) in the vicinity of the catalytic acid/base affects the pH activity of highly thermophilic GH11 xylanase. Both mutations shifted the pH activity profile toward acidic pH. In general, the Km values were lower at pH 4-5 than at pH 6, and in line with this, the rate of hydrolysis of xylotetraose was slightly faster at pH 4 than at pH 6. The N46D mutation and also lower pH in XYN11A increased the hydrolysis of xylotriose. The Km value increased remarkably (from 2.5 to 11.6 mg/mL) because of V48D, which indicates the weakening of binding affinity of the substrate to the active site. Xylotetraose functioned well as a substrate for other enzymes, but with lowered reaction rate for V48D. Both N46D and V48D increased the enzyme inactivation by ionic liquid [emim]OAc. In conclusion, the pH activity profile could be shifted to acidic pH due to an effect from two different directions, but the tightly packed GH11 active site can cause steric problems for the mutations.

  12. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

    PubMed Central

    Wilkens, Casper; Dilokpimol, Adiphol; Nakai, Hiroyuki; Lewińska, Anna; Abou Hachem, Maher; Svensson, Birte

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical for the activity of their cognate enzyme, though they are not readily detected in the sequence of a protein, but normally require a crystal structure of a complex for their identification. A variety of methods, including affinity electrophoresis (AE), insoluble polysaccharide pulldown (IPP) and surface plasmon resonance (SPR) have been used to study auxiliary binding sites. These techniques are complementary as AE allows monitoring of binding to soluble polysaccharides, IPP to insoluble polysaccharides and SPR to oligosaccharides. Here we show that these methods are useful not only for analyzing known binding sites, but also for identifying new ones, even without structural data available. We further verify the chosen assays discriminate between known SBS/CBM containing enzymes and negative controls. Altogether 35 enzymes are screened for the presence of SBSs or CBMs and several novel binding sites are identified, including the first SBS ever reported in a cellulase. This work demonstrates that combinations of these methods can be used as a part of routine enzyme characterization to identify new binding sites and advance the study of SBSs and CBMs, allowing them to be detected in the absence of structural data. PMID:27504624

  13. Active site of bimetallic heterogeneous catalyst by atomic resolution aberration-corrected STEM

    NASA Astrophysics Data System (ADS)

    Hsiao, Chien-Nan; Lin, Chun-Ting

    2015-11-01

    The localized defect of Au-Pd bimetallic heterogeneous nanoparticles catalyst was investigated using HRTEM and aberration-corrected HRSTEM. The phase plates were calculated from the aberration coefficients of the measured probe tableau for various outer tilt angle of the optical axis and the accuracy required for the compensation of the various residual aberration coefficients in order to achieve sub-angstrom resolution with the electron optics system was evaluated up to the fifth order aberrations. It is found that the interplanar spacing of the Au-Pd nanoparticle (1 1 1) planes observed along the [1 1 0] zone axis was approximately 0.24 nm measured by HRTEM. In addition, the HRSTEM HAADF image demonstrated that the twin boundaries on the surfaces of heterogeneous nanoparticles catalysts at atomic scale. These defects might be introduced during the growth to alleviate the internal stress caused by the 4.6% lattice mismatch of Au-Pd bimetallic system. Current research could be applied to the study of active sites in nanocatalysts.

  14. Beta zero thalassemia caused by a base substitution that creates an alternative splice acceptor site in an intron.

    PubMed Central

    Metherall, J E; Collins, F S; Pan, J; Weissman, S M; Forget, B G

    1986-01-01

    A thalassemic beta-globin gene cloned from a haplotype I chromosome contains a T to G transversion at position 116 of IVS1 which results in the generation of an abnormal alternative acceptor splice site. Transient expression studies revealed a 4-fold decrease in the amount of RNA produced with greater than 99% of it being abnormally spliced despite preservation of the normal acceptor splice site at position 130. These results suggest that the mutation at IVS1 position 116 results in beta zero thalassemia. A closely related mutation at position 110 of IVS1 also generates a novel acceptor site and results in a similar decrease in total mRNA produced, but approximately 20% of the mRNA produced is normally spliced and thus the phenotype is that of beta + thalassemia. These observations suggest that short range position effects may play a dramatic role in the choice of potential splice acceptor sites. We demonstrate the presence of abnormally spliced mRNA in reticulocytes of affected individuals and show the mutation at IVS1 position 116 segregating from the mutation at IVS1 position 110 in a three generation pedigree. The mutation results in the creation of a MaeI restriction site, as do a number of other thalassemic mutations, and we demonstrate some difficulties that may arise in the differential diagnosis of these mutations. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:3780671

  15. Synergistic effect between defect sites and functional groups on the hydrolysis of cellulose over activated carbon.

    PubMed

    Foo, Guo Shiou; Sievers, Carsten

    2015-02-01

    The chemical oxidation of activated carbon by H2 O2 and H2 SO4 is investigated, structural and chemical modifications are characterized, and the materials are used as catalysts for the hydrolysis of cellulose. Treatment with H2 O2 enlarges the pore size and imparts functional groups such as phenols, lactones, and carboxylic acids. H2 SO4 treatment targets the edges of carbon sheets primarily, and this effect is more pronounced with a higher temperature. Adsorption isotherms demonstrate that the adsorption of oligomers on functionalized carbon is dominated by van der Waals forces. The materials treated chemically are active for the hydrolysis of cellulose despite the relative weakness of most of their acid sites. It is proposed that a synergistic effect between defect sites and functional groups enhances the activity by inducing a conformational change in the glucan chains if they are adsorbed at defect sites. This activates the glycosidic bonds for hydrolysis by in-plane functional groups.

  16. Denaturation studies of active-site labeled papain using electron paramagnetic resonance and fluorescence spectroscopy.

    PubMed Central

    Ping, Z A; Butterfiel, D A

    1991-01-01

    A spin-labeled p-chloromercuribenzoate (SL-PMB) and a fluorescence probe, 6-acryloyl-2-dimethylaminonaphthalene (Acrylodan), both of which bind to the single SH group located in the active site of papain, were used to investigate the interaction of papain (EC 3.4.22.2) with two protein denaturants. It was found that the active site of papain was highly stable in urea solution, but underwent a large conformational change in guanidine hydrochloride solution. Electron paramagnetic resonance and fluorescence results were in agreement and both paralleled enzymatic activity of papain with respect to both the variation in pH and denaturation. These results strongly suggest that SL-PMB and Acrylodan labels can be used to characterize the physical state of the active site of the enzyme. PMID:1657229

  17. Intracellular alkalization causes pain sensation through activation of TRPA1 in mice

    PubMed Central

    Fujita, Fumitaka; Uchida, Kunitoshi; Moriyama, Tomoko; Shima, Asako; Shibasaki, Koji; Inada, Hitoshi; Sokabe, Takaaki; Tominaga, Makoto

    2008-01-01

    Vertebrate cells require a very narrow pH range for survival. Cells accordingly possess sensory and defense mechanisms for situations where the pH deviates from the viable range. Although the monitoring of acidic pH by sensory neurons has been attributed to several ion channels, including transient receptor potential vanilloid 1 channel (TRPV1) and acid-sensing ion channels (ASICs), the mechanisms by which these cells detect alkaline pH are not well understood. Here, using Ca2+ imaging and patch-clamp recording, we showed that alkaline pH activated transient receptor potential cation channel, subfamily A, member 1 (TRPA1) and that activation of this ion channel was involved in nociception. In addition, intracellular alkalization activated TRPA1 at the whole-cell level, and single-channel openings were observed in the inside-out configuration, indicating that alkaline pH activated TRPA1 from the inside. Analyses of mutants suggested that the two N-terminal cysteine residues in TRPA1 were involved in activation by intracellular alkalization. Furthermore, intraplantar injection of ammonium chloride into the mouse hind paw caused pain-related behaviors that were not observed in TRPA1-deficient mice. These results suggest that alkaline pH causes pain sensation through activation of TRPA1 and may provide a molecular explanation for some of the human alkaline pH–related sensory disorders whose mechanisms are largely unknown. PMID:19033673

  18. Enhancement of Polymerase Activity of the Large Fragment in DNA Polymerase I from Geobacillus stearothermophilus by Site-Directed Mutagenesis at the Active Site

    PubMed Central

    Ma, Yi; Zhang, Beilei; Wang, Meng; Ou, Yanghui

    2016-01-01

    The large fragment of DNA polymerase I from Geobacillus stearothermophilus GIM1.543 (Bst DNA polymerase) with 5′-3′ DNA polymerase activity while in absence of 5′-3′ exonuclease activity possesses high thermal stability and polymerase activity. Bst DNA polymerase was employed in isothermal multiple self-matching initiated amplification (IMSA) which amplified the interest sequence with high selectivity and was widely applied in the rapid detection of human epidemic diseases. However, the detailed information of commercial Bst DNA polymerase is unpublished and well protected by patents, which makes the high price of commercial kits. In this study, wild-type Bst DNA polymerase (WT) and substitution mutations for improving the efficiency of DNA polymerization were expressed and purified in E. coli. Site-directed substitutions of four conserved residues (Gly310, Arg412, Lys416, and Asp540) in the activity site of Bst DNA polymerase influenced efficiency of polymerizing dNTPs. The substitution of residue Gly310 by alanine or leucine and residue Asp540 by glutamic acid increased the efficiency of polymerase activity. All mutants with higher polymerizing efficiency were employed to complete the rapid detection of EV71-associated hand, foot, and mouth disease (HFMD) by IMSA approach with relatively shorter period which is suitable for the primary diagnostics setting in rural and underdeveloped areas. PMID:27981047

  19. [Evaluation of beta-glucuronidase activity for the isolation of diarrhea-causing Escherichia coli].

    PubMed

    Muto, T; Arai, K; Miyai, M

    1991-06-01

    To compare the isolating efficiency of diarrhea-causing Escherichia coli between Fluorocult agar plates, which reveal the beta-glucuronidase activity of E. coli, and a combination of SS and DHL agar plates, a total of 330 fecal specimens collected from outpatients were examined. Diarrhea-causing E. coli, identified by serological and toxigenic characters, were demonstrated in 52 samples. Among these specimens, 35 samples tested were positive on the Fluorocult agar plates, and 26 samples on the combination of SS and DHL agar plates. However, only 10 samples were positive on both the Fluorocult agar plates and the combination of SS and DHL agar plates. Thus, using Fluorocult agar plates for the isolation of diarrhea-causing E. coli in addition to the conventional SS and DHL agar plates will improve isolating efficacy.

  20. Plasma membrane overgrowth causes fibrotic collagen accumulation and immune activation in Drosophila adipocytes

    PubMed Central

    Zang, Yiran; Wan, Ming; Liu, Min; Ke, Hongmei; Ma, Shuangchun; Liu, Lu-Ping; Ni, Jian-Quan; Carlos Pastor-Pareja, José

    2015-01-01

    Many chronic diseases are associated with fibrotic deposition of Collagen and other matrix proteins. Little is known about the factors that determine preferential onset of fibrosis in particular tissues. Here we show that plasma membrane (PM) overgrowth causes pericellular Collagen accumulation in Drosophila adipocytes. We found that loss of Dynamin and other endocytic components causes pericellular trapping of outgoing Collagen IV due to dramatic cortex expansion when endocytic removal of PM is prevented. Deposits also form in the absence of negative Toll immune regulator Cactus, excess PM being caused in this case by increased secretion. Finally, we show that trimeric Collagen accumulation, downstream of Toll or endocytic defects, activates a tissue damage response. Our work indicates that traffic imbalances and PM topology may contribute to fibrosis. It also places fibrotic deposits both downstream and upstream of immune signaling, consistent with the chronic character of fibrotic diseases. DOI: http://dx.doi.org/10.7554/eLife.07187.001 PMID:26090908

  1. Fighting the stigma caused by mental disorders: past perspectives, present activities, and future directions

    PubMed Central

    STUART, HEATHER

    2008-01-01

    People who live with mental illnesses are among the most stigmatized groups in society. In 1996, in recognition of the particularly harsh burden caused by the stigma associated with schizophrenia, the WPA initiated a global anti-stigma program, Open-the-Doors. In 2005, a WPA Section on Stigma and Mental Health was created, with a broader mandate to reduce stigma and discrimination caused by mental disabilities in general. In light of these impor-tant developments, and the growing public health interest in stigma reduction, this paper reflects on the past perspectives that have led us to our current position, reviews present activities and accomplishments, and identifies challenges that the Section members will face in their future efforts to reduce the stigma caused by mental disorders. PMID:18836546

  2. In silico analysis of Pycnoporus cinnabarinus laccase active site with toxic industrial dyes.

    PubMed

    Prasad, Nirmal K; Vindal, Vaibhav; Narayana, Siva Lakshmi; Ramakrishna, V; Kunal, Swaraj Priyaranjan; Srinivas, M

    2012-05-01

    Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in various industrial oxidative processes like production of fine chemicals to bioremediation of contaminated soil and water. In order to understand the mechanisms of substrate binding and interaction between substrates and Pycnoporus cinnabarinus laccase, a homology model was generated. The resulted model was further validated and used for docking studies with toxic industrial dyes- acid blue 74, reactive black 5 and reactive blue 19. Interactions of chemical mediators with the laccase was also examined. The docking analysis showed that the active site always cannot accommodate the dye molecules, due to constricted nature of the active site pocket and steric hindrance of the residues whereas mediators are relatively small and can easily be accommodated into the active site pocket, which, thereafter leads to the productive binding. The binding properties of these compounds along with identification of critical active site residues can be used for further site-directed mutagenesis experiments in order to identify their role in activity and substrate specificity, ultimately leading to improved mutants for degradation of these toxic compounds.

  3. AutoDock-GIST: Incorporating Thermodynamics of Active-Site Water into Scoring Function for Accurate Protein-Ligand Docking.

    PubMed

    Uehara, Shota; Tanaka, Shigenori

    2016-11-23

    Water plays a significant role in the binding process between protein and ligand. However, the thermodynamics of water molecules are often underestimated, or even ignored, in protein-ligand docking. Usually, the free energies of active-site water molecules are substantially different from those of waters in the bulk region. The binding of a ligand to a protein causes a displacement of these waters from an active site to bulk, and this displacement process substantially contributes to the free energy change of protein-ligand binding. The free energy of active-site water molecules can be calculated by grid inhomogeneous solvation theory (GIST), using molecular dynamics (MD) and the trajectory of a target protein and water molecules. Here, we show a case study of the combination of GIST and a docking program and discuss the effectiveness of the displacing gain of unfavorable water in protein-ligand docking. We combined the GIST-based desolvation function with the scoring function of AutoDock4, which is called AutoDock-GIST. The proposed scoring function was assessed employing 51 ligands of coagulation factor Xa (FXa), and results showed that both scoring accuracy and docking success rate were improved. We also evaluated virtual screening performance of AutoDock-GIST using FXa ligands in the directory of useful decoys-enhanced (DUD-E), thus finding that the displacing gain of unfavorable water is effective for a successful docking campaign.

  4. Substrate shuttling between active sites of uroporphyrinogen decarboxylase is not required to generate coproporphyrinogen

    PubMed Central

    Phillips, John D.; Warby, Christy A.; Whitby, Frank G.; Kushner, James P.; Hill, Christopher P.

    2009-01-01

    Summary Uroporphyrinogen Decarboxylase (URO-D; EC 4.1.1.37), the fifth enzyme of the heme biosynthetic pathway, is required for the production of heme, vitamin B12, siroheme, and chlorophyll precursors. URO-D catalyzes the sequential decarboxylation of the four acetate side chains on the pyrrole groups of uroporphyrinogen to produce coproporphyrinogen. URO-D is a stable homodimer with the active site clefts of the two subunits adjacent to each other. It has been hypothesized that the two catalytic centers interact functionally, perhaps by shuttling of reaction intermediates between subunits. We tested this hypothesis by construction of a single chain protein (scURO-D) in which the two subunits were connected by a flexible linker. The crystal structure of this protein was shown to be superimposible with wild-type activity and have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of scURO-D resulted in approximately half of wild-type activity. The distribution of reaction intermediates was the same for mutant and wild-type sequences, and was unaltered in a competition experiment using the I and III isomer substrates. These observations indicate that communication between active sites is not required for enzyme function, and suggest that the dimeric structure of URO-D is required to achieve conformational stability and create a large active site cleft. PMID:19362562

  5. Substrate Shuttling Between Active Sites of Uroporphyrinogen Decarboxylase in Not Required to Generate Coproporphyrinogen

    SciTech Connect

    Phillips, J.; Warby, C; Whitby, F; Kushner, J; Hill, C

    2009-01-01

    Uroporphyrinogen decarboxylase (URO-D; EC 4.1.1.37), the fifth enzyme of the heme biosynthetic pathway, is required for the production of heme, vitamin B12, siroheme, and chlorophyll precursors. URO-D catalyzes the sequential decarboxylation of four acetate side chains in the pyrrole groups of uroporphyrinogen to produce coproporphyrinogen. URO-D is a stable homodimer, with the active-site clefts of the two subunits adjacent to each other. It has been hypothesized that the two catalytic centers interact functionally, perhaps by shuttling of reaction intermediates between subunits. We tested this hypothesis by construction of a single-chain protein (single-chain URO-D) in which the two subunits were connected by a flexible linker. The crystal structure of this protein was shown to be superimposable with wild-type activity and to have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of single-chain URO-D resulted in approximately half of wild-type activity. The distributions of reaction intermediates were the same for mutant and wild-type sequences and were unaltered in a competition experiment using I and III isomer substrates. These observations indicate that communication between active sites is not required for enzyme function and suggest that the dimeric structure of URO-D is required to achieve conformational stability and to create a large active-site cleft.

  6. Crystal structure of an avian influenza polymerase PA[subscript N] reveals an endonuclease active site

    SciTech Connect

    Yuan, Puwei; Bartlam, Mark; Lou, Zhiyong; Chen, Shoudeng; Zhou, Jie; He, Xiaojing; Lv, Zongyang; Ge, Ruowen; Li, Xuemei; Deng, Tao; Fodor, Ervin; Rao, Zihe; Liu, Yingfang

    2009-11-10

    The heterotrimeric influenza virus polymerase, containing the PA, PB1 and PB2 proteins, catalyses viral RNA replication and transcription in the nucleus of infected cells. PB1 holds the polymerase active site and reportedly harbours endonuclease activity, whereas PB2 is responsible for cap binding. The PA amino terminus is understood to be the major functional part of the PA protein and has been implicated in several roles, including endonuclease and protease activities as well as viral RNA/complementary RNA promoter binding. Here we report the 2.2 angstrom (A) crystal structure of the N-terminal 197 residues of PA, termed PA(N), from an avian influenza H5N1 virus. The PA(N) structure has an alpha/beta architecture and reveals a bound magnesium ion coordinated by a motif similar to the (P)DX(N)(D/E)XK motif characteristic of many endonucleases. Structural comparisons and mutagenesis analysis of the motif identified in PA(N) provide further evidence that PA(N) holds an endonuclease active site. Furthermore, functional analysis with in vivo ribonucleoprotein reconstitution and direct in vitro endonuclease assays strongly suggest that PA(N) holds the endonuclease active site and has critical roles in endonuclease activity of the influenza virus polymerase, rather than PB1. The high conservation of this endonuclease active site among influenza strains indicates that PA(N) is an important target for the design of new anti-influenza therapeutics.

  7. Transcriptional activation by LR1 at the Eµ enhancer and switch region sites

    PubMed Central

    Hanakahi, L. A.; Maizels, Nancy

    2000-01-01

    LR1 is a B cell-specific, sequence-specific duplex DNA binding activity which is induced in B cells carrying out class switch recombination. Here we identify several properties of LR1 which enable it to function in transcriptional regulation. We show that LR1 contributes to transcriptional activation by the Eµ immunoglobulin heavy chain intron enhancer by binding to a site within the enhancer core. We further show that LR1 bends DNA upon binding. In addition, we show that LR1 is itself a bona fide transcriptional activator, as multimerized LR1 sites produce an element which can enhance transcription from a minimal promoter. In order for class switch recombination to occur, an activating signal must be transmitted via the Eµ core, and both S regions targeted for recombination must be actively transcribed. The properties of LR1 that we have identified suggest distinct potential functions of LR1 duplex DNA binding activity in class switch recombination. First, LR1 may contribute to recombinational activation by the Eµ core. Second, there are multiple potential LR1 duplex binding sites in each of the G-rich switch regions, and LR1 bound at contiguous sites may enhance recombination by stimulating transcription of the S regions. PMID:10908319

  8. Cyclooxygenase activity contributes to the monoaminergic damage caused by serial exposure to stress and methamphetamine.

    PubMed

    Northrop, Nicole A; Yamamoto, Bryan K

    2013-09-01

    Methamphetamine (Meth) is a widely abused psychostimulant that causes long-term dopamine (DA) and serotonin (5-HT) depletions. Stress and Meth abuse are comorbid events in society and stress exacerbates Meth-induced monoaminergic terminal damage. Stress is also known to produce neuroinflammation. This study examined the role of the neuroinflammatory mediator, cyclooxygenase (COX), in the depletions of monoamines caused by serial exposure to chronic unpredictable stress (CUS) and Meth. CUS produced an increase in COX-2 protein expression and enhanced Meth-induced monoaminergic depletions in the striatum and hippocampus. The enhanced DA and 5-HT depletions in the striatum, but not the hippocampus, were prevented by pretreatment with COX inhibitor, ketoprofen, during stress or during Meth; however, ketoprofen did not attenuate the monoaminergic damage caused by Meth alone. The COX-dependent enhancement by stress of Meth-induced monoaminergic depletions was independent of hyperthermia, as ketoprofen did not attenuate Meth-induced hyperthermia. In addition, the EP1 receptor antagonist, SC-51089, did not attenuate DA or 5-HT depletions caused by stress and Meth. These findings illustrate that COX activity, but not activation of the EP1 receptor, is responsible for the potentiation of Meth-induced damage to striatal monoamine terminals by stress and suggests the use of anti-inflammatory drugs for mitigating the neurotoxic effects associated with the combination of stress and Meth.

  9. Regulation of Dpp activity by tissue-specific cleavage of an upstream site within the prodomain

    PubMed Central

    Sopory, Shailaja; Kwon, Sunjong; Wehrli, Marcel; Christian, Jan L.

    2010-01-01

    BMP4 is synthesized as an inactive precursor that is cleaved at two sites during maturation: initially at a site (S1) adjacent to the ligand domain, and then at an upstream site (S2) within the prodomain. Cleavage at the second site regulates the stability of mature BMP4 and this in turn influences its signaling intensity and range of action. The Drosophila ortholog of BMP4, Dpp, functions as a long- or short-range signaling molecule in the wing disc or embryonic midgut, respectively but mechanisms that differentially regulate its bioactivity in these tissues have not been explored. In the current studies we demonstrate, by dpp mutant rescue, that cleavage at the S2 site of proDpp is required for development of the wing and leg imaginal discs, whereas cleavage at the S1 site is sufficient to rescue Dpp function in the midgut. Both the S1 and S2 site of proDpp are cleaved in the wing disc, and S2-cleavage is essential to generate sufficient ligand to exceed the threshold for pMAD activation at both short- and long-range in most cells. By contrast, proDpp is cleaved at the S1 site alone in the embryonic mesoderm and this generates sufficient ligand to activate physiological target genes in neighboring cells. These studies provide the first biochemical and genetic evidence that that selective cleavage of the S2 site of proDPP provides a tissue-specific mechanism for regulating Dpp activity, and that differential cleavage can contribute to, but is not an absolute determinant of signaling range. PMID:20659445

  10. Multiple active site residues are important for photochemical efficiency in the light-activated enzyme protochlorophyllide oxidoreductase (POR).

    PubMed

    Menon, Binuraj R K; Hardman, Samantha J O; Scrutton, Nigel S; Heyes, Derren J

    2016-08-01

    Protochlorophyllide oxidoreductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide), an essential, regulatory step in chlorophyll biosynthesis. The unique requirement of the enzyme for light has provided the opportunity to investigate how light energy can be harnessed to power biological catalysis and enzyme dynamics. Excited state interactions between the Pchlide molecule and the protein are known to drive the subsequent reaction chemistry. However, the structural features of POR and active site residues that are important for photochemistry and catalysis are currently unknown, because there is no crystal structure for POR. Here, we have used static and time-resolved spectroscopic measurements of a number of active site variants to study the role of a number of residues, which are located in the proposed NADPH/Pchlide binding site based on previous homology models, in the reaction mechanism of POR. Our findings, which are interpreted in the context of a new improved structural model, have identified several residues that are predicted to interact with the coenzyme or substrate. Several of the POR variants have a profound effect on the photochemistry, suggesting that multiple residues are important in stabilizing the excited state required for catalysis. Our work offers insight into how the POR active site geometry is finely tuned by multiple active site residues to support enzyme-mediated photochemistry and reduction of Pchlide, both of which are crucial to the existence of life on Earth.

  11. Multiparity causes uncoordinated activity of pelvic- and perineal-striated muscles and urodynamic changes in rabbits.

    PubMed

    Martínez-Gómez, Margarita; Mendoza-Martínez, Germán; Corona-Quintanilla, Dora Luz; Fajardo, Víctor; Rodríguez-Antolín, Jorge; Castelán, Francisco

    2011-12-01

    Temporal and coordinated activation of pelvic- (pubococcygeous) and perineal- (bulbospongiosus and ischiocavernosus) striated muscles occurs during micturition in female rabbits. We have hypothesized that the coordinated activation of pelvic and perineal muscles is modified during the micturition of young multiparous rabbits. Young virgin and multiparous female chinchilla rabbits were used to simultaneously record cystometrograms and electromyograms of the pubococcygeous, ischocavernosus, and bulbospongiosus muscles. Bladder function was assessed using standard urodynamic variables. The temporal coordination of pelvic- and perineal-striated muscle activity was changed in multiparous rabbits. The cystometrogram recordings were different than those obtained from virgin rabbits, as seen in alterations of the threshold volume, the residual volume, the voiding duration, and the maximum pressure. In rabbits, we find that multiparity causes uncoordinated activity of pubococcygeous, ischiocavernosus, and bulbospongiosus muscles and modifies the urodynamics.

  12. An Electromagnetic Interference Study of Potential Transmitter Sites for the HF Active Auroral Research Program (HAARP)

    DTIC Science & Technology

    1993-07-19

    heating . The measurements described in this report were conducted at a number of candidate HAARP transmitter sites in the vicinity of Fairbanks...employ the High Power Auroral Stimulation (HIPAS) RF heating facility [1], located in the Chena River valley area near Fairbanks. HAARP will be an...Potential Transmitter Sites for the HF Active Auroral Research Program ( HAARP ) JOSEP11 A. GOLDSTEIN EDWARD 1. KENNEDY ADRIAN S. ELEY 4 IMICHlAEL A. RuPAR C

  13. [Combination of two signals of danger--a principal cause of activation of chronic inflammatory processes].

    PubMed

    Lebedev, K A

    2012-01-01

    Chronic inflammatory processes have long current during which there is a change of remission by an aggravation of disease. Until recently was considered, that occurrence and activation of chronic inflammatory process is caused by one signal of danger. Most often it are served with microorganisms. The sum of our end literary data shows that activation is connected with accumulation in the center of an inflammation of macrophages and their hyperactivation as a result of action of two signals of danger--microorganisms and xenobiotics.

  14. Probing the catalytic mechanism of bovine CD38/NAD+ glycohydrolase by site directed mutagenesis of key active site residues.

    PubMed

    Kuhn, Isabelle; Kellenberger, Esther; Cakir-Kiefer, Céline; Muller-Steffner, Hélène; Schuber, Francis

    2014-07-01

    Bovine CD38/NAD(+) glycohydrolase catalyzes the hydrolysis of NAD(+) to nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose via a stepwise reaction mechanism. Our recent crystallographic study of its Michaelis complex and covalently-trapped intermediates provided insights into the modalities of substrate binding and the molecular mechanism of bCD38. The aim of the present work was to determine the precise role of key conserved active site residues (Trp118, Glu138, Asp147, Trp181 and Glu218) by focusing mainly on the cleavage of the nicotinamide-ribosyl bond. We analyzed the kinetic parameters of mutants of these residues which reside within the bCD38 subdomain in the vicinity of the scissile bond of bound NAD(+). To address the reaction mechanism we also performed chemical rescue experiments with neutral (methanol) and ionic (azide, formate) nucleophiles. The crucial role of Glu218, which orients the substrate for cleavage by interacting with the N-ribosyl 2'-OH group of NAD(+), was highlighted. This contribution to catalysis accounts for almost half of the reaction energy barrier. Other contributions can be ascribed notably to Glu138 and Asp147 via ground-state destabilization and desolvation in the vicinity of the scissile bond. Key interactions with Trp118 and Trp181 were also proven to stabilize the ribooxocarbenium ion-like transition state. Altogether we propose that, as an alternative to a covalent acylal reaction intermediate with Glu218, catalysis by bCD38 proceeds through the formation of a discrete and transient ribooxocarbenium intermediate which is stabilized within the active site mostly by electrostatic interactions.

  15. Activation of Nrf2 in keratinocytes causes chloracne (MADISH)-like skin disease in mice

    PubMed Central

    Schäfer, Matthias; Willrodt, Ann-Helen; Kurinna, Svitlana; Link, Andrea S; Farwanah, Hany; Geusau, Alexandra; Gruber, Florian; Sorg, Olivier; Huebner, Aaron J; Roop, Dennis R; Sandhoff, Konrad; Saurat, Jean-Hilaire; Tschachler, Erwin; Schneider, Marlon R; Langbein, Lutz; Bloch, Wilhelm; Beer, Hans-Dietmar; Werner, Sabine

    2014-01-01

    The transcription factor Nrf2 is a key regulator of the cellular stress response, and pharmacological Nrf2 activation is a promising strategy for skin protection and cancer prevention. We show here that prolonged Nrf2 activation in keratinocytes causes sebaceous gland enlargement and seborrhea in mice due to upregulation of the growth factor epigen, which we identified as a novel Nrf2 target. This was accompanied by thickening and hyperkeratosis of hair follicle infundibula. These abnormalities caused dilatation of infundibula, hair loss, and cyst development upon aging. Upregulation of epigen, secretory leukocyte peptidase inhibitor (Slpi), and small proline-rich protein 2d (Sprr2d) in hair follicles was identified as the likely cause of infundibular acanthosis, hyperkeratosis, and cyst formation. These alterations were highly reminiscent to the phenotype of chloracne/“metabolizing acquired dioxin-induced skin hamartomas” (MADISH) patients. Indeed, SLPI, SPRR2, and epigen were strongly expressed in cysts of MADISH patients and upregulated by dioxin in human keratinocytes in an NRF2-dependent manner. These results identify novel Nrf2 activities in the pilosebaceous unit and point to a role of NRF2 in MADISH pathogenesis. PMID:24503019

  16. Evolution of anatase surface active sites probed by in situ sum-frequency phonon spectroscopy.

    PubMed

    Cao, Yue; Chen, Shiyou; Li, Yadong; Gao, Yi; Yang, Deheng; Shen, Yuen Ron; Liu, Wei-Tao

    2016-09-01

    Surface active sites of crystals often govern their relevant surface chemistry, yet to monitor them in situ in real atmosphere remains a challenge. Using surface-specific sum-frequency spectroscopy, we identified the surface phonon mode associated with the active sites of undercoordinated titanium ions and conjoint oxygen vacancies, and used it to monitor them on anatase (TiO2) (101) under ambient conditions. In conjunction with theory, we determined related surface structure around the active sites and tracked the evolution of oxygen vacancies under ultraviolet irradiation. We further found that unlike in vacuum, the surface oxygen vacancies, which dominate the surface reactivity, are strongly regulated by ambient gas molecules, including methanol and water, as well as weakly associated species, such as nitrogen and hydrogen. The result revealed a rich interplay between prevailing ambient species and surface reactivity, which can be omnipresent in environmental and catalytic applications of titanium dioxides.

  17. Non-canonical active site architecture of the radical SAM thiamin pyrimidine synthase

    SciTech Connect

    Fenwick, Michael K.; Mehta, Angad P.; Zhang, Yang; Abdelwahed, Sameh H.; Begley, Tadhg P.; Ealick, Steven E.

    2015-03-27

    Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5'-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a β-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the β-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active site metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.

  18. Kinetics of nucleotide entry into RNA polymerase active site provides mechanism for efficiency and fidelity.

    PubMed

    Wang, Beibei; Sexton, Rachel E; Feig, Michael

    2017-04-01

    During transcription, RNA polymerase II elongates RNA by adding nucleotide triphosphates (NTPs) complementary to a DNA template. Structural studies have suggested that NTPs enter and exit the active site via the narrow secondary pore but details have remained unclear. A kinetic model is presented that integrates molecular dynamics simulations with experimental data. Previous simulations of trigger loop dynamics and the dynamics of matched and mismatched NTPs in and near the active site were combined with new simulations describing NTP exit from the active site via the secondary pore. Markov state analysis was applied to identify major states and estimate kinetic rates for transitions between those states. The kinetic model predicts elongation and misincorporation rates in close agreement with experiment and provides mechanistic hypotheses for how NTP entry and exit via the secondary pore is feasible and a key feature for achieving high elongation and low misincorporation rates during RNA elongation.

  19. Evolution of anatase surface active sites probed by in situ sum-frequency phonon spectroscopy

    PubMed Central

    Cao, Yue; Chen, Shiyou; Li, Yadong; Gao, Yi; Yang, Deheng; Shen, Yuen Ron; Liu, Wei-Tao

    2016-01-01

    Surface active sites of crystals often govern their relevant surface chemistry, yet to monitor them in situ in real atmosphere remains a challenge. Using surface-specific sum-frequency spectroscopy, we identified the surface phonon mode associated with the active sites of undercoordinated titanium ions and conjoint oxygen vacancies, and used it to monitor them on anatase (TiO2) (101) under ambient conditions. In conjunction with theory, we determined related surface structure around the active sites and tracked the evolution of oxygen vacancies under ultraviolet irradiation. We further found that unlike in vacuum, the surface oxygen vacancies, which dominate the surface reactivity, are strongly regulated by ambient gas molecules, including methanol and water, as well as weakly associated species, such as nitrogen and hydrogen. The result revealed a rich interplay between prevailing ambient species and surface reactivity, which can be omnipresent in environmental and catalytic applications of titanium dioxides. PMID:27704049

  20. Gamma exposure rates due to neutron activation of soil: site of Hood detonation, Operation Plumbbob

    SciTech Connect

    Auxier, J.A.; Ohnesorge, W.F.

    1980-06-01

    This paper is the result of some recent discussions of exposure rates within the first few hours of the Hood detonation of the Plumbbob series due to neutron activation of soil. We estimated the exposure rates from 1/2 to 3 h after the detonation from ground zero to 1000 yards from ground zero. The area was assumed to be uncontaminated by fallout. Soil samples from the area of the Nevada Test Site at which the Hood device was detonated were sent to ORNL by Dr. John Malik of Los Alamos and by Mr. Gordon Jacks of the Nevada Test Site. These samples were irradiated at the DOSAR facility and the resulting activity analyzed. Calculations of exposure rates were then made based on the analyzed activity and the measured thermal neutron fluences at DOSAR and at the Hood Site.

  1. Systematic mutagenesis of the active site omega loop of TEM-1 beta-lactamase.

    PubMed Central

    Petrosino, J F; Palzkill, T

    1996-01-01

    Beta-Lactamase is a bacterial protein that provides resistance against beta-lactam antibiotics. TEM-1 beta-lactamase is the most prevalent plasmid-mediated beta-lactamase in gram-negative bacteria. Normally, this enzyme has high levels of hydrolytic activity for penicillins, but mutant beta-lactamases have evolved with activity toward a variety of beta-lactam antibiotics. It has been shown that active site substitutions are responsible for changes in the substrate specificity. Since mutant beta-lactamases pose a serious threat to antimicrobial therapy, the mechanisms by which mutations can alter the substrate specificity of TEM-1 beta-lactamase are of interest. Previously, screens of random libraries encompassing 31 of 55 active site amino acid positions enabled the identification of the residues responsible for maintaining the substrate specificity of TEM-1 beta-lactamase. In addition to substitutions found in clinical isolates, many other specificity-altering mutations were also identified. Interestingly, many nonspecific substitutions in the N-terminal half of the active site omega loop were found to increase ceftazidime hydrolytic activity and decrease ampicillin hydrolytic activity. To complete the active sight study, eight additional random libraries were constructed and screened for specificity-altering mutations. All additional substitutions found to alter the substrate specificity were located in the C-terminal half of the active site loop. These mutants, much like the N-terminal omega loop mutants, appear to be less stable than the wild-type enzyme. Further analysis of a 165-YYG-167 triple mutant, selected for high levels of ceftazidime hydrolytic activity, provides an example of the correlation which exists between enzyme instability and increased ceftazidime hydrolytic activity in the ceftazidime-selected omega loop mutants. PMID:8606154

  2. Improving the neutral phytase activity from Bacillus amyloliquefaciens DSM 1061 by site-directed mutagenesis.

    PubMed

    Xu, Wei; Shao, Rong; Wang, Zupeng; Yan, Xiuhua

    2015-03-01

    Neutral phytase is used as a feed additive for degradation of anti-nutritional phytate in aquatic feed industry. Site-directed mutagenesis of Bacillus amyloliquefaciens DSM 1061 phytase was performed with an aim to increase its activity. Mutation residues were chosen based on multiple sequence alignments and structure analysis of neutral phytsaes from different microorganisms. The mutation sites on surface (D148E, S197E and N156E) and around the active site (D52E) of phytase were selected. Analysis of the phytase variants showed that the specific activities of mutants D148E and S197E remarkably increased by about 35 and 13% over a temperature range of 40-75 °C at pH 7.0, respectively. The k cat of mutants D148E and S197E were 1.50 and 1.25 times than that of the wild-type phytase, respectively. Both D148E and S197E showed much higher thermostability than that of the wild-type phytase. However, mutants N156E and D52E led to significant loss of specific activity of the enzyme. Structural analysis revealed that these mutations may affect conformation of the active site of phytase. The present mutant phytases D148E and S197E with increased activities and thermostabilities have application potential as additives in aquaculture feed.

  3. Threatened and endangered wildlife species of the Hanford Site related to CERCLA characterization activities

    SciTech Connect

    Fitzner, R.E.; Weiss, S.G.; Stegen, J.A.

    1994-06-01

    The US Department of Energy`s (DOE) Hanford Site has been placed on the National Priorities List, which requires that it be remediated under the Comprehensive Environmental Response, Compensation, and Liability Act (CERCLA) or Superfund. Potentially contaminated areas of the Hanford Site were grouped into operable units, and detailed characterization and investigation plans were formulated. The DOE Richland Operations Office requested Westinghouse Hanford Company (WHC) to conduct a biological assessment of the potential impact of these characterization activities on the threatened, endangered, and sensitive wildlife species of the Hanford Site. Additional direction for WHC compliances with wildlife protection can be found in the Environmental Compliance Manual. This document is intended to meet these requirements, in part, for the CERCLA characterization activities, as well as for other work comparable in scope. This report documents the biological assessment and describes the pertinent components of the Hanford Site as well as the planned characterization activities. Also provided are accounts of endangered, threatened, and federal candidate wildlife species on the Hanford Site and information as to how human disturbances can affect these species. Potential effects of the characterization activities are described with recommendations for mitigation measures.

  4. A Variable Active Site Residue Influences the Kinetics of Response Regulator Phosphorylation and Dephosphorylation.

    PubMed

    Immormino, Robert M; Silversmith, Ruth E; Bourret, Robert B

    2016-10-04

    Two-component regulatory systems, minimally composed of a sensor kinase and a response regulator protein, are common mediators of signal transduction in microorganisms. All response regulators contain a receiver domain with conserved active site residues that catalyze the signal activating and deactivating phosphorylation and dephosphorylation reactions. We explored the impact of variable active site position T+1 (one residue C-terminal to the conserved Thr/Ser) on reaction kinetics and signaling fidelity, using wild type and mutant Escherichia coli CheY, CheB, and NarL to represent the three major sequence classes observed across response regulators: Ala/Gly, Ser/Thr, and Val/Ile/Met, respectively, at T+1. Biochemical and structural data together suggested that different amino acids at T+1 impacted reaction kinetics by altering access to the active site while not perturbing overall protein structure. A given amino acid at position T+1 had similar effects on autodephosphorylation in each protein background tested, likely by modulating access of the attacking water molecule to the active site. Similarly, rate constants for CheY autophosphorylation with three different small molecule phosphodonors were consistent with the steric constraints on access to the phosphorylation site arising from combination of specific phosphodonors with particular amino acids at T+1. Because other variable active site residues also influence response regulator phosphorylation biochemistry, we began to explore how context (here, the amino acid at T+2) affected the influence of position T+1 on CheY autocatalytic reactions. Finally, position T+1 affected the fidelity and kinetics of phosphotransfer between sensor kinases and response regulators but was not a primary determinant of their interaction.

  5. The active site of low-temperature methane hydroxylation in iron-containing zeolites

    NASA Astrophysics Data System (ADS)

    Snyder, Benjamin E. R.; Vanelderen, Pieter; Bols, Max L.; Hallaert, Simon D.; Böttger, Lars H.; Ungur, Liviu; Pierloot, Kristine; Schoonheydt, Robert A.; Sels, Bert F.; Solomon, Edward I.

    2016-08-01

    An efficient catalytic process for converting methane into methanol could have far-reaching economic implications. Iron-containing zeolites (microporous aluminosilicate minerals) are noteworthy in this regard, having an outstanding ability to hydroxylate methane rapidly at room temperature to form methanol. Reactivity occurs at an extra-lattice active site called α-Fe(II), which is activated by nitrous oxide to form the reactive intermediate α-O; however, despite nearly three decades of research, the nature of the active site and the factors determining its exceptional reactivity are unclear. The main difficulty is that the reactive species—α-Fe(II) and α-O—are challenging to probe spectroscopically: data from bulk techniques such as X-ray absorption spectroscopy and magnetic susceptibility are complicated by contributions from inactive ‘spectator’ iron. Here we show that a site-selective spectroscopic method regularly used in bioinorganic chemistry can overcome this problem. Magnetic circular dichroism reveals α-Fe(II) to be a mononuclear, high-spin, square planar Fe(II) site, while the reactive intermediate, α-O, is a mononuclear, high-spin Fe(IV)=O species, whose exceptional reactivity derives from a constrained coordination geometry enforced by the zeolite lattice. These findings illustrate the value of our approach to exploring active sites in heterogeneous systems. The results also suggest that using matrix constraints to activate metal sites for function—producing what is known in the context of metalloenzymes as an ‘entatic’ state—might be a useful way to tune the activity of heterogeneous catalysts.

  6. Reduced sodium/proton exchanger NHE3 activity causes congenital sodium diarrhea.

    PubMed

    Janecke, Andreas R; Heinz-Erian, Peter; Yin, Jianyi; Petersen, Britt-Sabina; Franke, Andre; Lechner, Silvia; Fuchs, Irene; Melancon, Serge; Uhlig, Holm H; Travis, Simon; Marinier, Evelyne; Perisic, Vojislav; Ristic, Nina; Gerner, Patrick; Booth, Ian W; Wedenoja, Satu; Baumgartner, Nadja; Vodopiutz, Julia; Frechette-Duval, Marie-Christine; De Lafollie, Jan; Persad, Rabindranath; Warner, Neil; Tse, C Ming; Sud, Karan; Zachos, Nicholas C; Sarker, Rafiquel; Zhu, Xinjun; Muise, Aleixo M; Zimmer, Klaus-Peter; Witt, Heiko; Zoller, Heinz; Donowitz, Mark; Müller, Thomas

    2015-12-01

    Congenital sodium diarrhea (CSD) refers to an intractable diarrhea of intrauterine onset with high fecal sodium loss. CSD is clinically and genetically heterogeneous. Syndromic CSD is caused by SPINT2 mutations. While we recently described four cases of the non-syndromic form of CSD that were caused by dominant activating mutations in intestinal receptor guanylate cyclase C (GC-C), the genetic cause for the majority of CSD is still unknown. Therefore, we aimed to determine the genetic cause for non-GC-C non-syndromic CSD in 18 patients from 16 unrelated families applying whole-exome sequencing and/or chromosomal microarray analyses and/or direct Sanger sequencing. SLC9A3 missense, splicing and truncation mutations, including an instance of uniparental disomy, and whole-gene deletion were identified in nine patients from eight families with CSD. Two of these nine patients developed inflammatory bowel disease (IBD) at 4 and 16 years of age. SLC9A3 encodes Na(+)/H(+) antiporter 3 (NHE3), which is the major intestinal brush-border Na(+)/H(+) exchanger. All mutations were in the NHE3 N-terminal transport domain, and all missense mutations were in the putative membrane-spanning domains. Identified SLC9A3 missense mutations were functionally characterized in plasma membrane NHE null fibroblasts. SLC9A3 missense mutations compromised NHE3 activity by reducing basal surface expression and/or loss of basal transport function of NHE3 molecules, whereas acute regulation was normal. This study identifies recessive mutations in NHE3, a downstream target of GC-C, as a cause of CSD and implies primary basal NHE3 malfunction as a predisposition for IBD in a subset of patients.

  7. Transcriptional pausing and stalling causes multiple clustered mutations by human activation-induced deaminase

    PubMed Central

    Canugovi, Chandrika; Samaranayake, Mala; Bhagwat, Ashok S.

    2009-01-01

    Transcription of the rearranged immunoglobulin gene and expression of the enzyme activation-induced deaminase (AID) are essential for somatic hypermutations of this gene during antibody maturation. While AID acts as a single-strand DNA-cytosine deaminase creating U · G mispairs that lead to mutations, the role played by transcription in this process is less clear. We have used in vitro transcription of the kan gene by the T7 RNA polymerase (RNAP) in the presence of AID and a genetic reversion assay for kanamycin-resistance to investigate the causes of multiple clustered mutations (MCMs) during somatic hypermutations. We find that, depending on transcription conditions, AID can cause single-base substitutions or MCMs. When wild-type RNAP is used for transcription at physiologically relevant concentrations of ribonucleoside triphosphates (NTPs), few MCMs are found. In contrast, slowing the rate of elongation by reducing the NTP concentration or using a mutant RNAP increases several-fold the percent of revertants containing MCMs. Arresting the elongation complexes by a quick removal of NTPs leads to formation of RNA-DNA hybrids (R-loops). Treatment of these structures with AID results in a high percentage of KanR revertants with MCMs. Furthermore, selecting for transcription elongation complexes stalled near the codon that suffers mutations during acquisition of kanamycin-resistance results in an overwhelming majority of revertants with MCMs. These results show that if RNAP II pauses or stalls during transcription of immunoglobulin gene, AID is likely to promote MCMs. As changes in physiological conditions such as occurrence of certain DNA primary or secondary structures or DNA adducts are known to cause transcriptional pausing and stalling in mammalian cells, this process may cause MCMs during somatic hypermutation.—Canugovi, C., Samaranayake, M., Bhagwat, A. S. Transcriptional pausing and stalling causes multiple clustered mutations by human activation

  8. Dynamics of the Active Sites of Dimeric Seryl tRNA Synthetase from Methanopyrus kandleri.

    PubMed

    Dutta, Saheb; Nandi, Nilashis

    2015-08-27

    Aminoacyl tRNA synthetases (aaRSs) carry out the first step of protein biosynthesis. Several aaRSs are multimeric, and coordination between the dynamics of active sites present in each monomer is a prerequisite for the fast and accurate aminoacylation. However, important lacunae of understanding exist concerning the conformational dynamics of multimeric aaRSs. Questions remained unanswered pertaining to the dynamics of the active site. Little is known concerning the conformational dynamics of the active sites in response to the substrate binding, reorganization of the catalytic residues around reactants, time-dependent changes at the reaction center, which are essential for facilitating the nucleophilic attack, and interactions at the interface of neighboring monomers. In the present work, we carried out all-atom molecular dynamics simulation of dimeric (mk)SerRS from Methanopyrus kandleri bound with tRNA using an explicit solvent system. Two dimeric states of seryl tRNA synthetase (open, substrate bound, and adenylate bound) and two monomeric states (open and substrate bound) are simulated with bound tRNA. The aim is to understand the conformational dynamics of (mk)SerRS during its reaction cycle. While the present results provide a clear dynamical perspective of the active sites of (mk)SerRS, they corroborate with the results from the time-averaged experimental data such as crystallographic and mutation analysis of methanogenic SerRS from M. kandleri and M. barkeri. It is observed from the present simulation that the motif 2 loop gates the active site and its Glu351 and Arg360 stabilizes ATP in a bent state favorable for nucleophilic attack. The flexibility of the walls of the active site gradually reduces near reaction center, which is a more organized region compared to the lid region. The motif 2 loop anchors Ser and ATP using Arg349 in a hydrogen bonded geometry crucial for nucleophilic attack and favorably influences the electrostatic potential at the

  9. Counting Active Sites on Titanium Oxide-Silica Catalysts for Hydrogen Peroxide Activation through In Situ Poisoning with Phenylphosphonic Acid

    SciTech Connect

    Eaton, Todd R.; Boston, Andrew M.; Thompson, Anthony B.; Gray, Kimberly A.; Notestein, Justin M.

    2015-06-04

    Quantifying specific active sites in supported catalysts improves our understanding and assists in rational design. Supported oxides can undergo significant structural changes as surface densities increase from site-isolated cations to monolayers and crystallites, which changes the number of kinetically relevant sites. Herein, TiOx domains are titrated on TiOx–SiO2 selectively with phenylphosphonic acid (PPA). An ex situ method quantifies all fluid-accessible TiOx, whereas an in situ titration during cis-cyclooctene epoxidation provides previously unavailable values for the number of tetrahedral Ti sites on which H2O2 activation occurs. We use this method to determine the active site densities of 22 different catalysts with different synthesis methods, loadings, and characteristic spectra and find a single intrinsic turnover frequency for cis-cyclooctene epoxidation of (40±7) h-1. This simple method gives molecular-level insight into catalyst structure that is otherwise hidden when bulk techniques are used.

  10. METHAMPHETAMINE TREATMENT CAUSES DELAYED DECREASE IN NOVELTY-INDUCED LOCOMOTOR ACTIVITY IN MICE

    PubMed Central

    Krasnova, Irina N.; Hodges, Amber B.; Ladenheim, Bruce; Rhoades, Raina; Phillip, Crystal G.; Ceseňa, Angela; Ivanova, Ekaterina; Hohmann, Christine F.; Cadet, Jean Lud

    2009-01-01

    Methamphetamine (METH) is a psychostimulant that causes damage to dopamine (DA) axons and to non-monoaminergic neurons in the brain. The aim of the present study was to investigate short- and long-term effects of neurotoxic METH treatment on novelty-induced locomotor activity in mice. Male BALB/c mice, 12–14 weeks old, were injected with saline or METH (i.p., 7.5 mg/kg × 4 times, every 2 hours). Behavior and neurotoxic effects were assessed at 10 days, 3 and 5 months following drug treatment. METH administration caused marked decreases in DA levels in the mouse striatum and cortex at 10 days post-drug. However, METH did not induce any changes in novelty-induced locomotor activity. At 3 and 5 months after treatment METH-exposed mice showed significant recovery of DA levels in the striatum and cortex. In contrast, these animals demonstrated significant decreases in locomotor activity at 5 months in comparison to aged-matched control mice. Further assessment of METH toxicity using TUNEL staining showed that the drug induced increased cell death in the striatum and cortex at 3 days after administration. Taken together, these data suggest that delayed deficits in novelty-induced locomotor activity observed in METH exposed animals are not due to neurodegeneration of DA terminals but to combined effects of METH and age-dependent dysfunction of non-DA intrinsic striatal and/or corticostriatal neurons. PMID:19559060

  11. Activated microglia cause reversible apoptosis of pheochromocytoma cells, inducing their cell death by phagocytosis

    PubMed Central

    Hornik, Tamara C.; Vilalta, Anna; Brown, Guy C.

    2016-01-01

    ABSTRACT Some apoptotic processes, such as phosphatidylserine exposure, are potentially reversible and do not necessarily lead to cell death. However, phosphatidylserine exposure can induce phagocytosis of a cell, resulting in cell death by phagocytosis: phagoptosis. Phagoptosis of neurons by microglia might contribute to neuropathology, whereas phagoptosis of tumour cells by macrophages might limit cancer. Here, we examined the mechanisms by which BV-2 microglia killed co-cultured pheochromocytoma (PC12) cells that were either undifferentiated or differentiated into neuronal cells. We found that microglia activated by lipopolysaccharide rapidly phagocytosed PC12 cells. Activated microglia caused reversible phosphatidylserine exposure on and reversible caspase activation in PC12 cells, and caspase inhibition prevented phosphatidylserine exposur and decreased subsequent phagocytosis. Nitric oxide was necessary and sufficient to induce the reversible phosphatidylserine exposure and phagocytosis. The PC12 cells were not dead at the time they were phagocytised, and inhibition of their phagocytosis left viable cells. Cell loss was inhibited by blocking phagocytosis mediated by phosphatidylserine, MFG-E8, vitronectin receptors or P2Y6 receptors. Thus, activated microglia can induce reversible apoptosis of target cells, which is insufficient to cause apoptotic cell death, but sufficient to induce their phagocytosis and therefore cell death by phagoptosis. PMID:26567213

  12. Activated microglia cause reversible apoptosis of pheochromocytoma cells, inducing their cell death by phagocytosis.

    PubMed

    Hornik, Tamara C; Vilalta, Anna; Brown, Guy C

    2016-01-01

    Some apoptotic processes, such as phosphatidylserine exposure, are potentially reversible and do not necessarily lead to cell death. However, phosphatidylserine exposure can induce phagocytosis of a cell, resulting in cell death by phagocytosis: phagoptosis. Phagoptosis of neurons by microglia might contribute to neuropathology, whereas phagoptosis of tumour cells by macrophages might limit cancer. Here, we examined the mechanisms by which BV-2 microglia killed co-cultured pheochromocytoma (PC12) cells that were either undifferentiated or differentiated into neuronal cells. We found that microglia activated by lipopolysaccharide rapidly phagocytosed PC12 cells. Activated microglia caused reversible phosphatidylserine exposure on and reversible caspase activation in PC12 cells, and caspase inhibition prevented phosphatidylserine exposur and decreased subsequent phagocytosis. Nitric oxide was necessary and sufficient to induce the reversible phosphatidylserine exposure and phagocytosis. The PC12 cells were not dead at the time they were phagocytised, and inhibition of their phagocytosis left viable cells. Cell loss was inhibited by blocking phagocytosis mediated by phosphatidylserine, MFG-E8, vitronectin receptors or P2Y6 receptors. Thus, activated microglia can induce reversible apoptosis of target cells, which is insufficient to cause apoptotic cell death, but sufficient to induce their phagocytosis and therefore cell death by phagoptosis.

  13. Functional biomimetic models for the active site in the respiratory enzyme cytochrome c oxidase.

    PubMed

    Collman, James P; Decréau, Richard A

    2008-11-07

    A functional analog of the active site in the respiratory enzyme, cytochrome c oxidase (CcO) reproduces every feature in CcO's active site: a myoglobin-like heme (heme a3), a distal tridentate imidazole copper complex (Cu(B)), a phenol (Tyr244), and a proximal imidazole. When covalently attached to a liquid-crystalline SAM film on an Au electrode, this functional model continuously catalyzes the selective four-electron reduction of dioxygen at physiological potential and pH, under rate-limiting electron flux (as occurs in CcO).

  14. New active site oriented glyoxyl-agarose derivatives of Escherichia coli penicillin G acylase

    PubMed Central

    Cecchini, Davide A; Serra, Immacolata; Ubiali, Daniela; Terreni, Marco; Albertini, Alessandra M

    2007-01-01

    Background Immobilized Penicillin G Acylase (PGA) derivatives are biocatalysts that are industrially used for the hydrolysis of Penicillin G by fermentation and for the kinetically controlled synthesis of semi-synthetic β-lactam antibiotics. One of the most used supports for immobilization is glyoxyl-activated agarose, which binds the protein by reacting through its superficial Lys residues. Since in E. coli PGA Lys are also present near the active site, an immobilization that occurs through these residues may negatively affect the performance of the biocatalyst due to the difficult diffusion of the substrate into the active site. A preferential orientation of the enzyme with the active site far from the support surface would be desirable to avoid this problem. Results Here we report how it is possible to induce a preferential orientation of the protein during the binding process on aldehyde activated supports. A superficial region of PGA, which is located on the opposite side of the active site, is enriched in its Lys content. The binding of the enzyme onto the support is consequently forced through the Lys rich region, thus leaving the active site fully accessible to the substrate. Different mutants with an increasing number of Lys have been designed and, when active, immobilized onto glyoxyl agarose. The synthetic performances of these new catalysts were compared with those of the immobilized wild-type (wt) PGA. Our results show that, while the synthetic performance of the wt PGA sensitively decreases after immobilization, the Lys enriched mutants have similar performances to the free enzyme even after immobilization. We also report the observations made with other mutants which were unable to undergo a successful maturation process for the production of active enzymes or which resulted toxic for the host cell. Conclusion The desired orientation of immobilized PGA with the active site freely accessible can be obtained by increasing the density of Lys residues

  15. Physiological Brain Activity Causes DNA Double Strand Breaks in Neurons — Exacerbation by Amyloid-β

    PubMed Central

    Suberbielle, Elsa; Sanchez, Pascal E.; Kravitz, Alexxai V.; Wang, Xin; Ho, Kaitlyn; Eilertson, Kirsten; Devidze, Nino; Kreitzer, Anatol C.; Mucke, Lennart

    2013-01-01

    We show that a natural behavior, exploration of a novel environment, causes DNA double-strand breaks (DSBs) in neurons of young adult wildtype mice. DSBs occurred in multiple brain regions, were most abundant in the dentate gyrus, which is involved in spatial learning and memory, and were repaired within 24 hours. Increasing neuronal activity by sensory or optogenetic stimulation increased neuronal DSBs in relevant but not irrelevant networks. Human amyloid precursor protein (hAPP) transgenic mice, which simulate key aspects of Alzheimer's disease, had increased neuronal DSBs at baseline and more severe and prolonged DSBs after exploration. Interventions that suppress aberrant neuronal activity and improve memory in hAPP mice normalized their levels of DSBs. Blocking extrasynaptic NMDA-type glutamate receptors prevented amyloid-β (Aβ)-induced DSBs in neuronal cultures. Thus, transient increases in neuronal DSBs occur as a result of physiological brain activity and Aβ exacerbates DNA damage, most likely by eliciting synaptic dysfunction. PMID:23525040

  16. Pathogenesis of hypertrophic cardiomyopathy caused by myozenin 2 mutations is independent of calcineurin activity

    PubMed Central

    Ruggiero, Alessandra; Chen, Suet Nee; Lombardi, Raffaella; Rodriguez, Gabriela; Marian, Ali J.

    2013-01-01

    Aims The role of calcineurin protein phosphatase 2B (PP2B) in the pathogenesis of human hypertrophic cardiomyopathy (HCM) remains unsettled. We determined potential involvement of calcineurin in the pathogenesis of HCM caused by mutations in myozenin 2 (MYOZ2), an inhibitor of calcineurin. Methods and results We generated multiple lines of transgenic mice expressing either Flag-tagged wild-type (WT) (MYOZ2WT) or mutant MYOZ2S48P and MYOZ2I246M, identified in families with HCM, in the heart. To mimic the human genotype, we generated bigenic mice expressing WT and mutant MYOZ2 in the background of hemizygous endogenous MYOZ2 (Myoz2+/−). Transgene proteins constituted 15–48% of the total MYOZ2 protein in the heart. Mutant MYOZ2 mice showed molecular, cellular, and gross cardiac hypertrophy, preserved systolic function, and interstitial fibrosis. Immunofluorescence staining showed co-localization of WT and mutant MYOZ2 proteins with α-actinin at the Z disks. Electron microscopy showed disrupted and mal-aligned Z disks in the mutant mice. Cardiac calcineurin activity, determined by quantifying Rcan1.4 mRNA and protein levels, luciferase activity in triple transgenic Myoz2+/−:NFATc-Luc:MYOZ2I246M and Myoz2+/−:NFATc-Luc:MYOZ2WT mice, and NFATc transcriptional activity assay, was unchanged in the mutant transgenic mice. However, levels of phospho-ERK1/2 and JNK54/46 were altered in the transgenic mice. Likewise, lentiviral-mediated expression of the MYOZ2I246M did not affect RCAN1.4 and calcineurin (PPP3CB) protein levels. Conclusions Thus, the cardiac phenotype in HCM caused by MYOZ2 mutations might be independent of calcineurin activity in the heart. Z disk abnormalities might provide the stimulus for the induction of cardiac hypertrophy caused by MYOZ2 mutations. PMID:22987565

  17. Wobble Pairs of the HDV Ribozyme Play Specific Roles in Stabilization of Active Site Dynamics

    PubMed Central

    Sripathi, Kamali N.; Banáš, Pavel; Reblova, Kamila; Šponer, Jiři; Otyepka, Michal

    2015-01-01

    The hepatitis delta virus (HDV) is the only known human pathogen whose genome contains a catalytic RNA motif (ribozyme). The overall architecture of the HDV ribozyme is that of a double-nested pseudoknot, with two GU pairs flanking the active site. Although extensive studies have shown that mutation of either wobble results in decreased catalytic activity, little work has focused on linking these mutations to specific structural effects on catalytic fitness. Here we use molecular dynamics simulations based on an activated structure to probe the active site dynamics as a result of wobble pair mutations. In both wild-type and mutant ribozymes, the in-line fitness of the active site (as a measure of catalytic proficiency) strongly depends on the presence of a C75(N3H3+)N1(O5′) hydrogen bond, which positions C75 as the general acid for the reaction. Our mutational analyses show that each GU wobble supports catalytically fit conformations in distinct ways; the reverse G25U20 wobble promotes high in-line fitness, high occupancy of the C75(N3H3+)G1(O5′) general-acid hydrogen bond and stabilization of the G1U37 wobble, while the G1U37 wobble acts more locally by stabilizing high in-line fitness and the C75(N3H3+)G1(O5′) hydrogen bond. We also find that stable type I A-minor and P1.1 hydrogen bonding above and below the active site, respectively, prevent local structural disorder from spreading and disrupting global conformation. Taken together, our results define specific, often redundant architectural roles for several structural motifs of the HDV ribozyme active site, expanding the known roles of these motifs within all HDV-like ribozymes and other structured RNAs. PMID:25631765

  18. Conserved phosphorylation sites in the activation loop of the Arabidopsis phytosulfokine receptor PSKR1 differentially affect kinase and receptor activity

    PubMed Central

    Hartmann, Jens; Linke, Dennis; Bönniger, Christine; Tholey, Andreas; Sauter, Margret

    2015-01-01

    PSK (phytosulfokine) is a plant peptide hormone perceived by a leucine-rich repeat receptor kinase. Phosphosite mapping of epitope-tagged PSKR1 (phytosulfokine receptor 1) from Arabidopsis thaliana plants identified Ser696 and Ser698 in the JM (juxtamembrane) region and probably Ser886 and/or Ser893 in the AL (activation loop) as in planta phosphorylation sites. In vitro-expressed kinase was autophosphorylated at Ser717 in the JM, and at Ser733, Thr752, Ser783, Ser864, Ser911, Ser958 and Thr998 in the kinase domain. The LC–ESI–MS/MS spectra provided support that up to three sites (Thr890, Ser893 and Thr894) in the AL were likely to be phosphorylated in vitro. These sites are evolutionarily highly conserved in PSK receptors, indicative of a conserved function. Site-directed mutagenesis of the four conserved residues in the activation segment, Thr890, Ser893, Thr894 and Thr899, differentially altered kinase activity in vitro and growth-promoting activity in planta. The T899A and the quadruple-mutated TSTT-A (T890A/S893A/T894A/T899A) mutants were both kinase-inactive, but PSKR1(T899A) retained growth-promoting activity. The T890A and S893A/T894A substitutions diminished kinase activity and growth promotion. We hypothesize that phosphorylation within the AL activates kinase activity and receptor function in a gradual and distinctive manner that may be a means to modulate the PSK response. PMID:26472115

  19. Human activities in Natura 2000 sites: a highly diversified conservation network.

    PubMed

    Tsiafouli, Maria A; Apostolopoulou, Evangelia; Mazaris, Antonios D; Kallimanis, Athanasios S; Drakou, Evangelia G; Pantis, John D

    2013-05-01

    The Natura 2000 network was established across the European Union's (EU) Member States with the aim to conserve biodiversity, while ensuring the sustainability of human activities. However, to what kind and to what extent Natura 2000 sites are subject to human activities and how this varies across Member States remains unspecified. Here, we analyzed 111,269 human activity records from 14,727 protected sites in 20 Member States. The frequency of occurrence of activities differs among countries, with more than 86 % of all sites being subjected to agriculture or forestry. Activities like hunting, fishing, urbanization, transportation, and tourism are more frequently recorded in south European sites than in northern or eastern ones. The observed variations indicate that Natura 2000 networks are highly heterogeneous among EU Member States. Our analysis highlights the importance of agriculture in European landscapes and indicates possible targets for policy interventions at national, European, or "sub-European" level. The strong human presence in the Natura 2000 network throughout Member States, shows that conservation initiatives could succeed only by combining social and ecological sustainability and by ensuring the integration of policies affecting biodiversity.

  20. Kv3 channel assembly, trafficking and activity are regulated by zinc through different binding sites.

    PubMed

    Gu, Yuanzheng; Barry, Joshua; Gu, Chen

    2013-05-15

    Zinc, a divalent heavy metal ion and an essential mineral for life, regulates synaptic transmission and neuronal excitability via ion channels. However, its binding sites and regulatory mechanisms are poorly understood. Here, we report that Kv3 channel assembly, localization and activity are regulated by zinc through different binding sites. Local perfusion of zinc reversibly reduced spiking frequency of cultured neurons most likely by suppressing Kv3 channels. Indeed, zinc inhibited Kv3.1 channel activity and slowed activation kinetics, independent of its site in the N-terminal T1 domain. Biochemical assays surprisingly identified a novel zinc-binding site in the Kv3.1 C-terminus, critical for channel activity and axonal targeting, but not for the zinc inhibition. Finally, mutagenesis revealed an important role of the junction between the first transmembrane (TM) segment and the first extracellular loop in sensing zinc. Its mutant enabled fast spiking with relative resistance to the zinc inhibition. Therefore, our studies provide novel mechanistic insights into the multifaceted regulation of Kv3 channel activity and localization by divalent heavy metal ions.

  1. Active-Site Monovalent Cations Revealed in a 1.55 Å Resolution Hammerhead Ribozyme Structure

    PubMed Central

    Anderson, Michael; Schultz, Eric P.; Martick, Monika; Scott, William G.

    2013-01-01

    We have obtained a 1.55 Å crystal structure of a hammerhead ribozyme derived from Schistosoma mansoni in conditions that permit detailed observations of Na+ ion binding in the ribozyme's active site. At least two such Na+ ions are observed. The first Na+ ion binds to the N7 of G10.1 and the adjacent A9 phosphate in a manner identical to that previously observed for divalent cations. A second Na+ ion binds to the Hoogsteen face of G12, the general base in the hammerhead cleavage reaction, thereby potentially dissipating the negative charge of the catalytically active enolate form of the nucleotide base. A potential but more ambiguous third site bridges the A9 and scissile phosphates in a manner consistent with previous predictions. Hammerhead ribozymes have been observed to be active in the presence of high concentrations of monovalent cations, including Na+, but the mechanism by which monovalent cations substitute for divalent cations in hammerhead catalysis remains unclear. Our results enable us to suggest that Na+ directly and specifically substitutes for divalent cations in the hammerhead active site. The detailed geometry of the pre-catalytic active site complex is also revealed with a new level of precision, thanks to the quality of the electron density maps obtained from what is currently the highest resolution ribozyme structure in the protein data bank. PMID:23711504

  2. Tuned by metals: the TET peptidase activity is controlled by 3 metal binding sites.

    PubMed

    Colombo, Matteo; Girard, Eric; Franzetti, Bruno

    2016-02-08

    TET aminopeptidases are dodecameric particles shared in the three life domains involved in various biological processes, from carbon source provider in archaea to eye-pressure regulation in humans. Each subunit contains a dinuclear metal site (M1 and M2) responsible for the enzyme catalytic activity. However, the role of each metal ion is still uncharacterized. Noteworthy, while mesophilic TETs are activated by Mn(2+), hyperthermophilic TETs prefers Co(2+). Here, by means of anomalous x-ray crystallography and enzyme kinetics measurements of the TET3 aminopeptidase from the hyperthermophilic organism Pyrococcus furiosus (PfTET3), we show that M2 hosts the catalytic activity of the enzyme, while M1 stabilizes the TET3 quaternary structure and controls the active site flexibility in a temperature dependent manner. A new third metal site (M3) was found in the substrate binding pocket, modulating the PfTET3 substrate preferences. These data show that TET activity is tuned by the molecular interplay among three metal sites.

  3. Triclosan causes spontaneous abortion accompanied by decline of estrogen sulfotransferase activity in humans and mice

    PubMed Central

    Wang, Xiaoli; Chen, Xiaojiao; Feng, Xuejiao; Chang, Fei; Chen, Minjian; Xia, Yankai; Chen, Ling

    2015-01-01

    Triclosan (TCS), an antibacterial agent, is identified in serum and urine of humans. Here, we show that the level of urinary TCS in 28.3% patients who had spontaneous abortion in mid-gestation were increased by 11.3-fold (high-TCS) compared with normal pregnancies. Oral administration of TCS (10 mg/kg/day) in mice (TCS mice) caused an equivalent urinary TCS level as those in the high-TCS abortion patients. The TCS-exposure from gestation day (GD) 5.5 caused dose-dependently fetal death during GD12.5–16.5 with decline of live fetal weight. GD15.5 TCS mice appeared placental thrombus and tissue necrosis with enhancement of platelet aggregation. The levels of placenta and plasma estrogen sulfotransferase (EST) mRNA and protein in TCS mice or high-TCS abortion patients were not altered, but their EST activities were significantly reduced compared to controls. Although the levels of serum estrogen (E2) in TCS mice and high-TCS abortion patients had no difference from controls, their ratio of sulfo-conjugated E2 and unconjugated E2 was reduced. The estrogen receptor antagonist ICI-182,780 prevented the enhanced platelet aggregation and placental thrombosis and attenuated the fetal death in TCS mice. The findings indicate that TCS-exposure might cause spontaneous abortion probably through inhibition of EST activity to produce placental thrombosis. PMID:26666354

  4. ATM kinase inhibition in glial cells activates the innate immune response and causes neurodegeneration in Drosophila.

    PubMed

    Petersen, Andrew J; Rimkus, Stacey A; Wassarman, David A

    2012-03-13

    To investigate the mechanistic basis for central nervous system (CNS) neurodegeneration in the disease ataxia-telangiectasia (A-T), we analyzed flies mutant for the causative gene A-T mutated (ATM). ATM encodes a protein kinase that functions to monitor the genomic integrity of cells and control cell cycle, DNA repair, and apoptosis programs. Mutation of the C-terminal amino acid in Drosophila ATM inhibited the kinase activity and caused neuron and glial cell death in the adult brain and a reduction in mobility and longevity. These data indicate that reduced ATM kinase activity is sufficient to cause neurodegeneration in A-T. ATM kinase mutant flies also had elevated expression of innate immune response genes in glial cells. ATM knockdown in glial cells, but not neurons, was sufficient to cause neuron and glial cell death, a reduction in mobility and longevity, and elevated expression of innate immune response genes in glial cells, indicating that a non-cell-autonomous mechanism contributes to neurodegeneration in A-T. Taken together, these data suggest that early-onset CNS neurodegeneration in A-T is similar to late-onset CNS neurodegeneration in diseases such as Alzheimer's in which uncontrolled inflammatory response mediated by glial cells drives neurodegeneration.

  5. Activation of mTORC1 in collecting ducts causes hyperkalemia.

    PubMed

    Chen, Zhenguo; Dong, Heling; Jia, Chunhong; Song, Qiancheng; Chen, Juan; Zhang, Yue; Lai, Pinglin; Fan, Xiaorong; Zhou, Xuan; Liu, Miao; Lin, Jun; Yang, Cuilan; Li, Ming; Gao, Tianming; Bai, Xiaochun

    2014-03-01

    Mutation of TSC (encoding tuberous sclerosis complex protein) and activation of mammalian target of rapamycin (mTOR) have been implicated in the pathogenesis of several renal diseases, such as diabetic nephropathy and polycystic kidney disease. However, the role of mTOR in renal potassium excretion and hyperkalemia is not known. We showed that mice with collecting-duct (CD)-specific ablation of TSC1 (CDTsc1KO) had greater mTOR complex 1 (mTORC1) activation in the CD and demonstrated features of pseudohypoaldosteronism, including hyperkalemia, hyperaldosteronism, and metabolic acidosis. mTORC1 activation caused endoplasmic reticulum stress, columnar cell lesions, and dedifferentiation of CD cells with loss of aquaporin-2 and epithelial-mesenchymal transition-like phenotypes. Of note, mTORC1 activation also reduced the expression of serum- and glucocorticoid-inducible kinase 1, a crucial regulator of potassium homeostasis in the kidney, and decreased the expression and/or activity of epithelial sodium channel-α, renal outer medullary potassium channel, and Na(+), K(+)-ATPase in the CD, which probably contributed to the aldosterone resistance and hyperkalemia in these mice. Rapamycin restored these phenotypic changes. Overall, this study identifies a novel function of mTORC1 in regulating potassium homeostasis and demonstrates that loss of TSC1 and activation of mTORC1 results in dedifferentiation and dysfunction of the CD and causes hyperkalemia. The CDTsc1KO mice provide a novel model for hyperkalemia induced exclusively by dysfunction of the CD.

  6. Activity of Cu{sup 2+} ions on the tetrahedral and octahedral sites of spinel oxide catalysts for CO oxidation

    SciTech Connect

    Ghose, J.; Murthy, K.S.R.C.

    1996-09-01

    In studies of CO oxidation on substituted copper chromite spinel oxide catalyst decreases as the Cu{sup 2+} content of the catalyst decreases, either by substitution with a divalent ion, i.e., Cu{sub 1-x} Mg{sub x} [Cr{sub 2}]O{sub 4}, or by reduction of Cu{sup 2+} to Cu{sup 1+}. Crystallographic studies have shown that Cu[Cr{sub 2}]O{sub 4} changes from normal to partially inverse when Cr{sup 3+} is replaced by Al{sup 3+}. Thus, in aluminum-substituted copper chromite catalysts, copper is present on both tetrahedral and octahedral sites of the spinel lattice, i.e., Cu{sub 1-x}Al{sub x} [Cu{sub x}Cr{sub 2-(x+y)}Al{sub y}]O{sub 4}. ESCA studies have shown that upon Al substitution some of the tetrahedral Cu{sup 2+} ions are reduced to Cu{sup 1+} and this causes a reduction in the catalytic activity of the catalysts. The present work was taken up to compare the activity of Cu{sup 2+} on tetrahedral sites with that on octahedral sites of the spinel oxide catalysts. For this, CO oxidation studies were carried out on the inverse spinel CuFe{sub 2}O{sub 4} and on the normal spinel CuRh{sub 2}O{sub 4} catalysts. 7 refs., 1 fig.

  7. Two novel splicing mutations in the SLC45A2 gene cause Oculocutaneous Albinism Type IV by unmasking cryptic splice sites.

    PubMed

    Straniero, Letizia; Rimoldi, Valeria; Soldà, Giulia; Mauri, Lucia; Manfredini, Emanuela; Andreucci, Elena; Bargiacchi, Sara; Penco, Silvana; Gesu, Giovanni P; Del Longo, Alessandra; Piozzi, Elena; Asselta, Rosanna; Primignani, Paola

    2015-09-01

    Oculocutaneous albinism (OCA) is characterized by hypopigmentation of the skin, hair and eye, and by ophthalmologic abnormalities caused by a deficiency in melanin biosynthesis. OCA type IV (OCA4) is one of the four commonly recognized forms of albinism, and is determined by mutation in the SLC45A2 gene. Here, we investigated the genetic basis of OCA4 in an Italian child. The mutational screening of the SLC45A2 gene identified two novel potentially pathogenic splicing mutations: a synonymous transition (c.888G>A) involving the last nucleotide of exon 3 and a single-nucleotide insertion (c.1156+2dupT) within the consensus sequence of the donor splice site of intron 5. As computer-assisted analysis for mutant splice-site prediction was not conclusive, we investigated the effects on pre-mRNA splicing of these two variants by using an in vitro minigene approach. Production of mutant transcripts in HeLa cells demonstrated that both mutations cause the almost complete abolishment of the physiologic donor splice site, with the concomitant unmasking of cryptic donor splice sites. To our knowledge, this work represents the first in-depth molecular characterization of splicing defects in a OCA4 patient.

  8. Ferruginous hawk nesting on the US DOE Hanford Site: Case history of a recent invasion caused by transmission lines

    SciTech Connect

    Fitzner, R.E.; Newell, R.L.

    1989-04-01

    Ferruginous hawks were an uncommon nesting raptor on the US DOE Hanford Site prior to 1987, with only one or two pairs nesting each year. In 1987, four pair were found nesting on transmission towers. In 1988, seven pairs were observed nesting on the towers. This recent increase in nesting pairs may correspond to an acceptance phenomena related to age of the transmission towers. The increase may be due to an overall increase in the population of ferruginous hawks in Washington. 12 refs., 4 figs.

  9. Vanillin causes the activation of Yap1 and mitochondrial fragmentation in Saccharomyces cerevisiae.

    PubMed

    Nguyen, Trinh Thi My; Iwaki, Aya; Ohya, Yoshikazu; Izawa, Shingo

    2014-01-01

    Vanillin and furfural are derived from lignocellulosic biomass and inhibit yeast growth and fermentation as biomass conversion inhibitors. Furfural has been shown to induce oxidative stress in Saccharomyces cerevisiae. Since there has been no report on the relationship between vanillin and oxidative stress, we investigated whether vanillin caused oxidative stress in yeast cells. We showed that vanillin caused the nuclear accumulation of Yap1, an oxidative stress responsive transcription factor, and subsequent transcriptional activation of Yap1-target genes. The growth of the null mutant of the YAP1 gene (yap1Δ) was delayed in the presence of vanillin, which indicated that Yap1 plays a role in the acquisition of tolerance to vanillin. We also demonstrated that vanillin facilitated the fragmentation of mitochondria. These findings suggest that the toxicity of vanillin involves damage induced by oxidative stress.

  10. Complete androgen insensitivity syndrome caused by a deep intronic pseudoexon-activating mutation in the androgen receptor gene

    PubMed Central

    Känsäkoski, Johanna; Jääskeläinen, Jarmo; Jääskeläinen, Tiina; Tommiska, Johanna; Saarinen, Lilli; Lehtonen, Rainer; Hautaniemi, Sampsa; Frilander, Mikko J.; Palvimo, Jorma J.; Toppari, Jorma; Raivio, Taneli

    2016-01-01

    Mutations in the X-linked androgen receptor (AR) gene underlie complete androgen insensitivity syndrome (CAIS), the most common cause of 46,XY sex reversal. Molecular genetic diagnosis of CAIS, however, remains uncertain in patients who show normal coding region of AR. Here, we describe a novel mechanism of AR disruption leading to CAIS in two 46,XY sisters. We analyzed whole-genome sequencing data of the patients for pathogenic variants outside the AR coding region. Patient fibroblasts from the genital area were used for AR cDNA analysis and protein quantification. Analysis of the cDNA revealed aberrant splicing of the mRNA caused by a deep intronic mutation (c.2450-118A>G) in the intron 6 of AR. The mutation creates a de novo 5′ splice site and a putative exonic splicing enhancer motif, which leads to the preferential formation of two aberrantly spliced mRNAs (predicted to include a premature stop codon). Patient fibroblasts contained no detectable AR protein. Our results show that patients with CAIS and normal AR coding region need to be examined for deep intronic mutations that can lead to pseudoexon activation. PMID:27609317

  11. Norepinephrine causes epigenetic repression of PKCε gene in rodent hearts by activating Nox1-dependent reactive oxygen species production.

    PubMed

    Xiong, Fuxia; Xiao, Daliao; Zhang, Lubo

    2012-07-01

    Heart disease is the leading cause of death in the United States. Recent studies demonstrate that fetal programming of PKCε gene repression results in ischemia-sensitive phenotype in the heart. The present study tests the hypothesis that increased norepinephrine causes epigenetic repression of PKCε gene in the heart via Nox1-dependent reactive oxygen species (ROS) production. Prolonged norepinephrine treatment increased ROS production in fetal rat hearts and embryonic ventricular myocyte H9c2 cells via a selective increase in Nox1 expression. Norepinephrine-induced ROS resulted in an increase in PKCε promoter methylation at Egr-1 and Sp-1 binding sites, leading to PKCε gene repression. N-acetylcysteine, diphenyleneiodonium, and apocynin blocked norepinephrine-induced ROS production and the promoter methylation, and also restored PKCε mRNA and protein to control levels in vivo in fetal hearts and in vitro in embryonic myocyte cells. Accordingly, norepinephrine-induced ROS production, promoter methylation, and PKCε gene repression were completely abrogated by knockdown of Nox1 in cardiomyocytes. These findings provide evidence of a novel interaction between elevated norepinephrine and epigenetic repression of PKCε gene in the heart mediated by Nox1-dependent oxidative stress and suggest new insights of molecular mechanisms linking the heightened sympathetic activity to aberrant cardioprotection and increased ischemic vulnerability in the heart.

  12. Transcainide causes two modes of open-channel block with different voltage sensitivities in batrachotoxin-activated sodium channels.

    PubMed Central

    Zamponi, G W; French, R J

    1994-01-01

    Transcainide, a complex derivative of lidocaine, blocks the open state of BTX-activated sodium channels from bovine heart and rat skeletal muscle in two distinct ways. When applied to either side of the membrane, transcainide caused discrete blocking events a few hundred milliseconds in duration (slow block), and a concomitant reduction in apparent single-channel amplitude, presumably because of rapid block beyond the temporal resolution of our recordings (fast block). We quantitatively analyzed block from the cytoplasmic side. Both modes of block occurred via binding of the drug to the open channel, approximately followed 1:1 stoichiometry, and were similar for both channel subtypes. For slow block, the blocking rate increased, and the unblocking rate decreased with depolarization, yielding an overall enhancement of block at positive potentials, and suggesting a blocking site at an apparent electrical distance about 45% of the way from the cytoplasmic end of the channel (z delta approximately 0.45). In contrast, the fast blocking mode was only slightly enhanced by depolarization (z delta approximately 0.15). Phenomenologically, the bulky and complex transcainide molecule combines the almost voltage-insensitive blocking action of phenylhydrazine (Zamponi and French, 1994a (companion paper)) with a slow open-channel blocking action that shows a voltage dependence typical of simpler amines. Only the slower blocking mode was sensitive to the removal of external sodium ions, suggesting that the two types of block occur at distinct sites. Dose-response relations were also consistent with independent binding of transcainide to two separate sites on the channel. PMID:7811913

  13. Docking and molecular dynamics studies at trypanothione reductase and glutathione reductase active sites.

    PubMed

    Iribarne, Federico; Paulino, Margot; Aguilera, Sara; Murphy, Miguel; Tapia, Orlando

    2002-05-01

    A theoretical docking study on the active sites of trypanothione reductase (TR) and glutathione reductase (GR) with the corresponding natural substrates, trypanothione disulfide (T[S]2) and glutathione disulfide (GSSG), is reported. Molecular dynamics simulations were carried out in order to check the robustness of the docking results. The energetic results are in agreement with previous experimental findings and show the crossed complexes have lower stabilization energies than the natural ones. To test DOCK3.5, four nitro furanic compounds, previously designed as potentially active anti-chagasic molecules, were docked at the GR and TR active sites with the DOCK3.5 procedure. A good correlation was found between differential inhibitory activity and relative interaction energy (affinity). The results provide a validation test for the use of DOCK3.5 in connection with the design of anti-chagasic drugs.

  14. Acidophilic actinobacteria synthesised silver nanoparticles showed remarkable activity against fungi-causing superficial mycoses in humans.

    PubMed

    Anasane, N; Golińska, P; Wypij, M; Rathod, D; Dahm, H; Rai, M

    2016-03-01

    Superficial mycoses are limited to the most external part of the skin and hair and caused by Malassezia sp., Trichophyton sp. and Candida sp. We report extracellular biosynthesis of silver nanoparticles (AgNPs) by acidophilic actinobacteria (SF23, C9) and its in vitro antifungal activity against fungi-causing superficial mycoses. The phylogenetic analysis based on the 16S rRNA gene sequence of strains SF23 and C9 showed that they are most closely related to Pilimelia columellifera subsp. pallida GU269552(T). The detection of AgNPs was confirmed by visual observation of colour changes from colourless to brown, and UV-vis spectrophotometer analysis, which showed peaks at 432 and 427 nm, respectively. These AgNPs were further characterised by nanoparticle tracking analysis (NTA), Zeta potential, Fourier-transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). The FTIR analysis exhibited the presence of proteins as capping agents. The TEM analysis revealed the formation of spherical and polydispersed nanoparticles in the size range of 4-36 nm and 8-60 nm, respectively. The biosynthesised AgNPs were screened against fungi-causing superficial mycoses viz., Malassezia furfur, Trichophyton rubrum, Candida albicans and C. tropicalis. The highest antifungal activity of AgNPs from SF23 and C9 against T. rubrum and the least against M. furfur and C. albicans was observed as compared to other tested fungi. The biosynthesised AgNPs were found to be potential anti-antifungal agent against fungi-causing superficial mycoses.

  15. A Carboxyl Ester Lipase (CEL) Mutant Causes Chronic Pancreatitis by Forming Intracellular Aggregates That Activate Apoptosis.

    PubMed

    Xiao, Xunjun; Jones, Gabrielle; Sevilla, Wednesday A; Stolz, Donna B; Magee, Kelsey E; Haughney, Margaret; Mukherjee, Amitava; Wang, Yan; Lowe, Mark E

    2016-10-28

    Patients with chronic pancreatitis (CP) frequently have genetic risk factors for disease. Many of the identified genes have been connected to trypsinogen activation or trypsin inactivation. The description of CP in patients with mutations in the variable number of tandem repeat (VNTR) domain of carboxyl ester lipase (CEL) presents an opportunity to study the pathogenesis of CP independently of trypsin pathways. We tested the hypothesis that a deletion and frameshift mutation (C563fsX673) in the CEL VNTR causes CP through proteotoxic gain-of-function activation of maladaptive cell signaling pathways including cell death pathways. HEK293 or AR42J cells were transfected with constructs expressing CEL with 14 repeats in the VNTR (CEL14R) or C563fsX673 CEL (CEL maturity onset diabetes of youth with a deletion mutation in the VNTR (MODY)). In both cell types, CEL MODY formed intracellular aggregates. Secretion of CEL MODY was decreased compared with that of CEL14R. Expression of CEL MODY increased endoplasmic reticulum stress, activated the unfolded protein response, and caused cell death by apoptosis. Our results demonstrate that disorders of protein homeostasis can lead to CP and suggest that novel therapies to decrease the intracellular accumulation of misfolded protein may be successful in some patients with CP.

  16. Effect of Monophasic Pulsed Current on Heel Pain and Functional Activities caused by Plantar Fasciitis

    PubMed Central

    Alotaibi, Abdullah K.; Petrofsky, Jerrold S.; Daher, Noha S.; Lohman, Everett; Laymon, Michael; Syed, Hasan M.

    2015-01-01

    Background Plantar fasciitis (PF) is a soft tissue disorder considered to be one of the most common causes of inferior heel pain. The aim of this study was to investigate the effect of monophasic pulsed current (MPC) and MPC coupled with plantar fascia-specific stretching exercises (SE) on the treatment of PF. Material/Methods Forty-four participants (22 women and 22 men, with a mean age of 49 years) diagnosed with PF were randomly assigned to receive MPC (n=22) or MPC coupled with plantar fascia-specific SE (n=22). Prior to and after 4 weeks of treatment, participants underwent baseline evaluation; heel pain was evaluated using a visual analogue scale (VAS), heel tenderness threshold was quantified using a handheld pressure algometer (PA), and functional activities level was assessed using the Activities of Daily Living subscale of the Foot and Ankle Ability Measure (ADL/FAAM). Results Heel pain scores showed a significant reduction in both groups compared to baseline VAS scores (P<0.001). Heel tenderness improved significantly in both groups compared with baseline PA scores (P<0.001). Functional activity level improved significantly in both groups compared with baseline (ADL/FAAM) scores (P<0.001). However, no significant differences existed between the 2 treatment groups in all post-intervention outcome measures. Conclusions This trial showed that MPC is useful in treating inferior heel symptoms caused by PF. PMID:25791231

  17. Pyrene is a Novel Constitutive Androstane Receptor (CAR) Activator and Causes Hepatotoxicity by CAR.

    PubMed

    Zhang, Xiao-Jie; Shi, Zhe; Lyv, Jing-Xi; He, Xuyan; Englert, Neal A; Zhang, Shu-Yun

    2015-10-01

    Polycyclic aromatic hydrocarbons (PAHs) are a class of ubiquitous persistent environmental pollutants which are primarily formed from the incomplete combustion of organic materials. Many potential sources of human exposure to PAHs exist, including daily exposures from the ambient environment or occupational settings. PAHs have been found to cause harmful effects on human health. Here, we evaluated the adverse effects of pyrene, a common PAH, on the liver. The present study demonstrates that pyrene is able to activate mouse constitutive androstane receptor (CAR). CAR protein, as measured by Western blot analysis, was observed to translocate into the nucleus from the cytoplasm in mouse liver after exposure to pyrene. Utilizing CAR null mice, we identified that CAR mediates pyrene-induced hepatotoxicity. Increased relative liver weight, hepatocellular hypertrophy, and elevated serum alanine aminotransferase levels were found in wild-type but not CAR null mice after orally administered pyrene. We further show that pyrene induced the expression of mouse liver metabolism enzymes including CYP2B10, CYP3A11, GSTm1, GSTm3, and SULT1A1, and caused hepatic glutathione depletion in wild-type but not CAR null mice. Moreover, by luciferase reporter assay and quantitative real-time PCR analysis, pyrene was found to be a potential inducer of CYP2B6 expression via activation of human CAR in HepG2 cells and human primary hepatocytes. Our observations suggest that pyrene is a novel CAR activator and that CAR is essential for mediating pyrene-induced liver injury.

  18. Activation of cyclic GMP-AMP synthase by self-DNA causes autoimmune diseases.

    PubMed

    Gao, Daxing; Li, Tuo; Li, Xiao-Dong; Chen, Xiang; Li, Quan-Zhen; Wight-Carter, Mary; Chen, Zhijian J

    2015-10-20

    TREX1 is an exonuclease that digests DNA in the cytoplasm. Loss-of-function mutations of TREX1 are linked to Aicardi-Goutieres Syndrome (AGS) and systemic lupus erythematosus (SLE) in humans. Trex1(-/-) mice exhibit autoimmune and inflammatory phenotypes that are associated with elevated expression of interferon (IFN)-induced genes (ISGs). Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that activates the IFN pathway. Upon binding to DNA, cGAS is activated to catalyze the synthesis of cGAMP, which functions as a second messenger that binds and activates the adaptor protein STING to induce IFNs and other cytokines. Here we show that genetic ablation of cGas in Trex1(-/-) mice eliminated all detectable pathological and molecular phenotypes, including ISG induction, autoantibody production, aberrant T-cell activation, and lethality. Even deletion of just one allele of cGas largely rescued the phenotypes of Trex1(-/-) mice. Similarly, deletion of cGas in mice lacking DNaseII, a lysosomal enzyme that digests DNA, rescued the lethal autoimmune phenotypes of the DNaseII(-/-) mice. Through quantitative mass spectrometry, we found that cGAMP accumulated in mouse tissues deficient in Trex1 or DNaseII and that this accumulation was dependent on cGAS. These results demonstrate that cGAS activation causes the autoimmune diseases in Trex1(-/-) and DNaseII(-/-) mice and suggest that inhibition of cGAS may lead to prevention and treatment of some human autoimmune diseases caused by self-DNA.

  19. Platelet Activation and Biofilm Formation by Aerococcus urinae, an Endocarditis-Causing Pathogen▿

    PubMed Central

    Shannon, Oonagh; Mörgelin, Matthias; Rasmussen, Magnus

    2010-01-01

    The Gram-positive bacterium Aerococcus urinae can cause infectious endocarditis (IE) in older persons. Biofilm formation and platelet aggregation are believed to contribute to bacterial virulence in IE. Five A. urinae isolates from human blood were shown to form biofilms in vitro, and biofilm formation was enhanced by the presence of human plasma. Four of the A. urinae isolates caused platelet aggregation in platelet-rich plasma from healthy donors. The Au3 isolate, which induced platelet aggregation in all donors, also activated platelets, as determined by flow cytometry. Platelet aggregation was dependent on bacterial protein structures and on platelet activation since it was sensitive to both trypsin and prostaglandin E1. Plasma proteins at the bacterial surface were needed for platelet aggregation; and roles of the complement system, fibrinogen, and immunoglobulin G were demonstrated. Complement-depleted serum was unable to support platelet aggregation by Au3 and complement blockade using compstatin-inhibited platelet activation. Platelet activation by Au3 was inhibited by blocking of the platelet fibrinogen receptor, and this isolate was also shown to bind to radiolabeled fibrinogen. Removal of IgG from platelet-rich plasma by a specific protease inhibited the platelet aggregation induced by A. urinae, and blockade of the platelet FcRγIIa hindered platelet activation induced by Au3. Convalescent-phase serum from a patient with A. urinae IE transferred the ability of the bacterium to aggregate platelets in an otherwise nonresponsive donor. Our results show that A. urinae exhibits virulence strategies of importance for IE. PMID:20696834

  20. Active layer dynamics in three sites with contrasted topography in the Byers Peninsula (Livingston Island, Antarctica)

    NASA Astrophysics Data System (ADS)

    Oliva, Marc; Ruiz-Fernández, Jesús; Vieira, Gonçalo

    2015-04-01

    Topography exerts a key role on permafrost distribution in areas where mean annual temperatures are slightly negative. This is the case of low-altitude environments in Maritime Antarctica, namely in the South Shetland Islands, where permafrost is marginal to discontinuous until elevations of 20-40 m asl turning to continuous at higher areas. Consequently, the active layer dynamics is also strongly conditioned by the geomorphological setting. In January 2014 we installed three sites for monitoring the active layer dynamics across the Byers Peninsula (Livingston Island, South Shetland Islands) in different geomorphological environments at elevations between 60 and 100 m. The purpose was to examine the role of the topography and microclimatic conditions on the active layer dynamics. At each site a set of loggers was set up to monitor: air temperatures, snow thickness, ground temperatures until 80 cm together with the coupling atmosphere-ground temperatures. During the first year of monitoring the mean annual air temperatures show similar values in the three sites, in all cases slightly below freezing. The snowy conditions during this year in this archipelago have resulted in a late melting of snow, which has also conditioned the duration of frozen conditions in the uppermost soil layers. Topography has a strong influence on snow cover duration, which in turn affects frozen ground conditions. The Domo site is located in a higher position with respect to the central plateau of Byers; here, the wind is stronger and snow cover thinner, which has conditioned a longer thawing season than in the other two sites (Cerro Negro, Escondido). These two sites are located in topographically protected areas favouring snow accumulation. The longer persistence of snow conditions a longer duration of negative temperatures in the active layer of the permafrost. This research was financially supported by the HOLOANTAR project (Portuguese Science Foundation) and the AXA Research Fund.

  1. 78 FR 8190 - Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-02-05

    ... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF THE INTERIOR Bureau of Ocean Energy Management Commercial Wind Leasing and Site Assessment Activities on the Atlantic... Notice of Intent to Prepare an Environmental Assessment (EA) for Commercial Wind Leasing and...

  2. Organized Agents: Canadian Teacher Unions as Alternative Sites for Social Justice Activism

    ERIC Educational Resources Information Center

    Rottmann, Cindy

    2008-01-01

    Historically teachers' federations have been some of the major organizational sites for social justice leadership in K-12 public education. Despite this history of activism, social justice teacher unionism remains a relatively underdeveloped concept. This article merges four philosophical conceptions of social justice in education: liberal…

  3. Penicillin Use in Meningococcal Disease Management: Active Bacterial Core Surveillance Sites, 2009

    PubMed Central

    Blain, Amy E.; Mandal, Sema; Wu, Henry; MacNeil, Jessica R.; Harrison, Lee H.; Farley, Monica M.; Lynfield, Ruth; Miller, Lisa; Nichols, Megin; Petit, Sue; Reingold, Arthur; Schaffner, William; Thomas, Ann; Zansky, Shelley M.; Anderson, Raydel; Harcourt, Brian H.; Mayer, Leonard W.; Clark, Thomas A.; Cohn, Amanda C.

    2016-01-01

    In 2009, in the Active Bacterial Core surveillance sites, penicillin was not commonly used to treat meningococcal disease. This is likely because of inconsistent availability of antimicrobial susceptibility testing and ease of use of third-generation cephalosporins. Consideration of current practices may inform future meningococcal disease management guidelines. PMID:27704009

  4. 77 FR 5830 - Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-02-06

    ... Bureau of Ocean Energy Management Commercial Wind Leasing and Site Assessment Activities on the Atlantic... of involving Federal agencies, states, tribes, local governments, offshore wind energy developers, and the public in the Department of the Interior's (DOI) ``Smart from the Start'' wind...

  5. Penicillin Use in Meningococcal Disease Management: Active Bacterial Core Surveillance Sites, 2009.

    PubMed

    Blain, Amy E; Mandal, Sema; Wu, Henry; MacNeil, Jessica R; Harrison, Lee H; Farley, Monica M; Lynfield, Ruth; Miller, Lisa; Nichols, Megin; Petit, Sue; Reingold, Arthur; Schaffner, William; Thomas, Ann; Zansky, Shelley M; Anderson, Raydel; Harcourt, Brian H; Mayer, Leonard W; Clark, Thomas A; Cohn, Amanda C

    2016-09-01

    In 2009, in the Active Bacterial Core surveillance sites, penicillin was not commonly used to treat meningococcal disease. This is likely because of inconsistent availability of antimicrobial susceptibility testing and ease of use of third-generation cephalosporins. Consideration of current practices may inform future meningococcal disease management guidelines.

  6. The active site of cytochrome P-450 nifedipine oxidase: a model-building study.

    PubMed

    Ferenczy, G G; Morris, G M

    1989-12-01

    A model of the active site of cytochrome P-450 nifedipine oxidase is built on the basis of sequence homology with cytochrome P-450CAM. Substrates are docked into the binding pocket, and molecular mechanical energy minimization is performed to analyze the forces between the substrates and the enzyme.

  7. 77 FR 74218 - Commercial Wind Leasing and Site Assessment Activities on the Atlantic Outer Continental Shelf...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-12-13

    ... identified in the document entitled, Commercial Leasing for Wind Power on the Outer Continental Shelf... Bureau of Ocean Energy Management Commercial Wind Leasing and Site Assessment Activities on the Atlantic... agencies, states, tribes, local governments, offshore wind energy developers, and the public in...

  8. Archaeological Activity Report: Post-Review Discoveries Within 45BN431 at Solid Waste Site 128-F-2

    SciTech Connect

    T. E. Marceau; J. J. Sharpe

    2006-12-21

    During monitoring of remedial activities at Solid Waste Site 128-F-2 on August 19, 2005, a concentration of mussel shell was discovered in the west wall of a trench in the northen section of the waste site.

  9. A facile reflux procedure to increase active surface sites form highly active and durable supported palladium@platinum bimetallic nanodendrites

    NASA Astrophysics Data System (ADS)

    Wang, Qin; Li, Yingjun; Liu, Baocang; Xu, Guangran; Zhang, Geng; Zhao, Qi; Zhang, Jun

    2015-11-01

    A series of well-dispersed bimetallic Pd@Pt nanodendrites uniformly supported on XC-72 carbon black are fabricated by using different capping agents. These capping agents are essential for the branched morphology control. However, the surfactant adsorbed on the nanodendrites surface blocks the access of reactant molecules to the active surface sites, and the catalytic activities of these bimetallic nanodendrites are significantly restricted. Herein, a facile reflux procedure to effectively remove the capping agent molecules without significantly affecting their sizes is reported for activating supported nanocatalysts. More significantly, the structure and morphology of the nanodendrites can also be retained, enhancing the numbers of active surface sites, catalytic activity and stability toward methanol and ethanol electro-oxidation reactions. The as-obtained hot water reflux-treated Pd@Pt/C catalyst manifests superior catalytic activity and stability both in terms of surface and mass specific activities, as compared to the untreated catalysts and the commercial Pt/C and Pd/C catalysts. We anticipate that this effective and facile removal method has more general applicability to highly active nanocatalysts prepared with various surfactants, and should lead to improvements in environmental protection and energy production.

  10. Kinetic and Spectroscopic Studies of Bicupin Oxalate Oxidase and Putative Active Site Mutants

    PubMed Central

    Moomaw, Ellen W.; Hoffer, Eric; Moussatche, Patricia; Salerno, John C.; Grant, Morgan; Immelman, Bridget; Uberto, Richard; Ozarowski, Andrew; Angerhofer, Alexander

    2013-01-01

    Ceriporiopsis subvermispora oxalate oxidase (CsOxOx) is the first bicupin enzyme identified that catalyzes manganese-dependent oxidation of oxalate. In previous work, we have shown that the dominant contribution to catalysis comes from the monoprotonated form of oxalate binding to a form of the enzyme in which an active site carboxylic acid residue must be unprotonated. CsOxOx shares greatest sequence homology with bicupin microbial oxalate decarboxylases (OxDC) and the 241-244DASN region of the N-terminal Mn binding domain of CsOxOx is analogous to the lid region of OxDC that has been shown to determine reaction specificity. We have prepared a series of CsOxOx mutants to probe this region and to identify the carboxylate residue implicated in catalysis. The pH profile of the D241A CsOxOx mutant suggests that the protonation state of aspartic acid 241 is mechanistically significant and that catalysis takes place at the N-terminal Mn binding site. The observation that the D241S CsOxOx mutation eliminates Mn binding to both the N- and C- terminal Mn binding sites suggests that both sites must be intact for Mn incorporation into either site. The introduction of a proton donor into the N-terminal Mn binding site (CsOxOx A242E mutant) does not affect reaction specificity. Mutation of conserved arginine residues further support that catalysis takes place at the N-terminal Mn binding site and that both sites must be intact for Mn incorporation into either site. PMID:23469254

  11. Transfer hydrogenation over sodium-modified ceria: Enrichment of redox sites active for alcohol dehydrogenation

    DOE PAGES

    Nelson, Nicholas C.; Boote, Brett W.; Naik, Pranjali; ...

    2017-01-17

    Ceria (CeO2) and sodium-modified ceria (Ce-Na) were prepared through combustion synthesis. Palladium was deposited onto the supports (Pd/CeO2 and Pd/Ce-Na) and their activity for the aqueous-phase transfer hydrogenation of phenol using 2-propanol under liquid flow conditions was studied. Pd/Ce-Na showed a marked increase (6×) in transfer hydrogenation activity over Pd/CeO2. Material characterization indicated that water-stable sodium species were not doped into the ceria lattice, but rather existed as subsurface carbonates. Modification of ceria by sodium provided more adsorption and redox active sites (i.e. defects) for 2-propanol dehydrogenation. This effect was an intrinsic property of the Ce-Na support and independent ofmore » Pd. The redox sites active for 2-propanol dehydrogenation were thermodynamically equivalent on both supports/catalysts. At high phenol concentrations, the reaction was limited by 2-propanol adsorption. Furthermore, the difference in catalytic activity was attributed to the different numbers of 2-propanol adsorption and redox active sites on each catalyst.« less

  12. A Common Carcinogen Benzo[a]pyrene Causes Neuronal Death in Mouse via Microglial Activation

    PubMed Central

    Nazmi, Arshed; Kumawat, Kanhaiya Lal; Basu, Anirban

    2010-01-01

    Background Benzo[a]pyrene (B[a]P) belongs to a class of polycyclic aromatic hydrocarbons that serve as micropollutants in the environment. B[a]P has been reported as a probable carcinogen in humans. Exposure to B[a]P can take place by ingestion of contaminated (especially grilled, roasted or smoked) food or water, or inhalation of polluted air. There are reports available that also suggests neurotoxicity as a result of B[a]P exposure, but the exact mechanism of action is unknown. Methodology/Principal Findings Using neuroblastoma cell line and primary cortical neuron culture, we demonstrated that B[a]P has no direct neurotoxic effect. We utilized both in vivo and in vitro systems to demonstrate that B[a]P causes microglial activation. Using microglial cell line and primary microglial culture, we showed for the first time that B[a]P administration results in elevation of reactive oxygen species within the microglia thereby causing depression of antioxidant protein levels; enhanced expression of inducible nitric oxide synthase, that results in increased production of NO from the cells. Synthesis and secretion of proinflammatory cytokines were also elevated within the microglia, possibly via the p38MAP kinase pathway. All these factors contributed to bystander death of neurons, in vitro. When administered to animals, B[a]P was found to cause microglial activation and astrogliosis in the brain with subsequent increase in proinflammatory cytokine levels. Conclusions/Significance Contrary to earlier published reports we found that B[a]P has no direct neurotoxic activity. However, it kills neurons in a bystander mechanism by activating the immune cells of the brain viz the microglia. For the first time, we have provided conclusive evidence regarding the mechanism by which the micropollutant B[a]P may actually cause damage to the central nervous system. In today's perspective, where rising pollution levels globally are a matter of grave concern, our study throws light on

  13. Layer-specific optogenetic activation of pyramidal neurons causes beta–gamma entrainment of neonatal networks

    PubMed Central

    Bitzenhofer, Sebastian H; Ahlbeck, Joachim; Wolff, Amy; Wiegert, J. Simon; Gee, Christine E.; Oertner, Thomas G.; Hanganu-Opatz, Ileana L.

    2017-01-01

    Coordinated activity patterns in the developing brain may contribute to the wiring of neuronal circuits underlying future behavioural requirements. However, causal evidence for this hypothesis has been difficult to obtain owing to the absence of tools for selective manipulation of oscillations during early development. We established a protocol that combines optogenetics with electrophysiological recordings from neonatal mice in vivo to elucidate the substrate of early network oscillations in the prefrontal cortex. We show that light-induced activation of layer II/III pyramidal neurons that are transfected by in utero electroporation with a high-efficiency channelrhodopsin drives frequency-specific spiking and boosts network oscillations within beta–gamma frequency range. By contrast, activation of layer V/VI pyramidal neurons causes nonspecific network activation. Thus, entrainment of neonatal prefrontal networks in fast rhythms relies on the activation of layer II/III pyramidal neurons. This approach used here may be useful for further interrogation of developing circuits, and their behavioural readout. PMID:28216627

  14. Site-directed mutagenesis of the Anabaena sp. strain PCC 7120 nitrogenase active site to increase photobiological hydrogen production.

    PubMed

    Masukawa, Hajime; Inoue, Kazuhito; Sakurai, Hidehiro; Wolk, C Peter; Hausinger, Robert P

    2010-10-01

    Cyanobacteria use sunlight and water to produce hydrogen gas (H₂), which is potentially useful as a clean and renewable biofuel. Photobiological H₂ arises primarily as an inevitable by-product of N₂ fixation by nitrogenase, an oxygen-labile enzyme typically containing an iron-molybdenum cofactor (FeMo-co) active site. In Anabaena sp. strain 7120, the enzyme is localized to the microaerobic environment of heterocysts, a highly differentiated subset of the filamentous cells. In an effort to increase H₂ production by this strain, six nitrogenase amino acid residues predicted to reside within 5 Å of the FeMo-co were mutated in an attempt to direct electron flow selectively toward proton reduction in the presence of N₂. Most of the 49 variants examined were deficient in N₂-fixing growth and exhibited decreases in their in vivo rates of acetylene reduction. Of greater interest, several variants examined under an N₂ atmosphere significantly increased their in vivo rates of H₂ production, approximating rates equivalent to those under an Ar atmosphere, and accumulated high levels of H₂ compared to the reference strains. These results demonstrate the feasibility of engineering cyanobacterial strains for enhanced photobiological production of H₂ in an aerobic, nitrogen-containing environment.

  15. A novel biallelic splice site mutation of TECTA causes moderate to severe hearing impairment in an Algerian family.

    PubMed

    Behlouli, Asma; Bonnet, Crystel; Abdi, Samia; Hasbellaoui, Mokhtar; Boudjenah, Farid; Hardelin, Jean-Pierre; Louha, Malek; Makrelouf, Mohamed; Ammar-Khodja, Fatima; Zenati, Akila; Petit, Christine

    2016-08-01

    Congenital deafness is certainly one of the most common monogenic diseases in humans, but it is also one of the most genetically heterogeneous, which makes molecular diagnosis challenging in most cases. Whole-exome sequencing in two out of three Algerian siblings affected by recessively-inherited, moderate to severe sensorineural deafness allowed us to identify a novel splice donor site mutation (c.5272+1G > A) in the gene encoding α-tectorin, a major component of the cochlear tectorial membrane. The mutation was present at the homozygous state in the three affected siblings, and at the heterozygous state in their unaffected, consanguineous parents. To our knowledge, this is the first reported TECTA mutation leading to the DFNB21 form of hearing impairment among Maghrebian individuals suffering from congenital hearing impairment, which further illustrates the diversity of the genes involved in congenital deafness in the Maghreb.

  16. Antifungal activity of Syzygium cumini against Ascochyta rabiei-the cause of chickpea blight.

    PubMed

    Jabeen, Khajista; Javaid, Arshad

    2010-07-01

    Aqueous, ethanol and n-hexane extracts from leaves, fruit, root-bark and stem-bark of Syzygium cumini (L.) Skeels were tested for their antifungal activity against Ascochyta rabiei (Pass.) Lab., the cause of blight disease of the chickpea (Cicer arietinum L.). Different concentrations, namely 1, 2, ..., 5% of both aqueous and the two organic solvent extracts were used in this study. Aqueous extracts of all the four test plant parts, namely leaves, fruit, stem-bark and root-bark, showed significant antifungal activity resulting in 7-30%, 22-59%, 23-39% and 21-64% reduction in fungal growth, respectively. Similarly, n-hexane stem-bark extract, and ethanol root- and stem-bark extracts also significantly suppressed the growth of target fungal species, resulting in 17-39%, 24-30% and 12-32% suppression in fungal growth.

  17. Structure-activity relations between alkyl nucleophilic chemicals causing duodenal ulcer and adrenocortical necrosis

    SciTech Connect

    Szabo, S.; Reynolds, E.S.; Unger, S.H.

    1982-10-01

    Structure-activity relationships were qualitatively and quantitatively examined for 56 chemicals (e.g., derivatives of propionitrile, acrylonitrile and cysteamine) which caused duodenal ulcer and/or adrenocortical necrosis in rats. For the first time the duodenal ulcerogenic property of numerous chemicals has been studied in a rational and predictive manner. Ulcerogenic activity was most intense in the carbonitriles attached to two or three carbon backbones and diminished by shortening, lengthening, branching, unsaturating, halogenating or hydroxylating the carbon chains. Different modes of action are implied. Adrenocorticolytic potency was associated with unsaturation of the carbon chain and substitution of the nitrile by thiol or amine radicals. An action of these chemicals on the central nervous system has been suggested.

  18. Site-specific modification of calmodulin Ca²(+) affinity tunes the skeletal muscle ryanodine receptor activation profile.

    PubMed

    Jiang, Jie; Zhou, Yubin; Zou, Jin; Chen, Yanyi; Patel, Priya; Yang, Jenny J; Balog, Edward M

    2010-11-15

    The skeletal muscle isoform of the ryanodine receptor Ca²(+)-release channel (RyR1) is regulated by Ca²(+) and CaM (calmodulin). CaM shifts the biphasic Ca²(+)-dependence of RyR1 activation leftward, effectively increasing channel opening at low Ca²(+) and decreasing channel opening at high Ca²(+). The conversion of CaM from a RyR1 activator into an inhibitor is due to the binding of Ca²(+) to CaM; however, which of CaM's four Ca²(+)-binding sites serves as the switch for this conversion is unclear. We engineered a series of mutant CaMs designed to individually increase the Ca²(+) affinity of each of CaM's EF-hands by increasing the number of acidic residues in Ca²(+)-chelating positions. Domain-specific Ca²(+) affinities of each CaM variant were determined by equilibrium fluorescence titration. Mutations in sites I (T26D) or II (N60D) in CaM's N-terminal domain had little effect on CaM Ca²(+) affinity and regulation of RyR1. However, the site III mutation N97D increased the Ca²(+)-binding affinity of CaM's C-terminal domain and caused CaM to inhibit RyR1 at a lower Ca²(+) concentration than wild-type CaM. Conversely, the site IV mutation Q135D decreased the Ca²(+)-binding affinity of CaM's C-terminal domain and caused CaM to inhibit RyR1 at higher Ca²(+) concentrations. These results support the hypothesis that Ca²(+) binding to CaM's C-terminal acts as the switch converting CaM from a RyR1 activator into a channel inhibitor. These results indicate further that targeting CaM's Ca²(+) affinity may be a valid strategy to tune the activation profile of CaM-regulated ion channels.

  19. Site-directed mutagenesis of an alkaline phytase: influencing specificity, activity and stability in acidic milieu.

    PubMed

    Tran, Thuy T; Mamo, Gashaw; Búxo, Laura; Le, Nhi N; Gaber, Yasser; Mattiasson, Bo; Hatti-Kaul, Rajni

    2011-07-10

    Site-directed mutagenesis of a thermostable alkaline phytase from Bacillus sp. MD2 was performed with an aim to increase its specific activity and activity and stability in an acidic environment. The mutation sites are distributed on the catalytic surface of the enzyme (P257R, E180N, E229V and S283R) and in the active site (K77R, K179R and E227S). Selection of the residues was based on the idea that acid active phytases are more positively charged around their catalytic surfaces. Thus, a decrease in the content of negatively charged residues or an increase in the positive charges in the catalytic region of an alkaline phytase was assumed to influence the enzyme activity and stability at low pH. Moreover, widening of the substrate-binding pocket is expected to improve the hydrolysis of substrates that are not efficiently hydrolysed by wild type alkaline phytase. Analysis of the phytase variants revealed that E229V and S283R mutants increased the specific activity by about 19% and 13%, respectively. Mutation of the active site residues K77R and K179R led to severe reduction in the specific activity of the enzyme. Analysis of the phytase mutant-phytate complexes revealed increase in hydrogen bonding between the enzyme and the substrate, which might retard the release of the product, resulting in decreased activity. On the other hand, the double mutant (K77R-K179R) phytase showed higher stability at low pH (pH 2.6-3.0). The E227S variant was optimally active at pH 5.5 (in contrast to the wild type enzyme that had an optimum pH of 6) and it exhibited higher stability in acidic condition. This mutant phytase, displayed over 80% of its initial activity after 3h incubation at pH 2.6 while the wild type phytase retained only about 40% of its original activity. Moreover, the relative activity of this mutant phytase on calcium phytate, sodium pyrophosphate and p-nitro phenyl phosphate was higher than that of the wild type phytase.

  20. Short baseline variations in site response and wave-propagation effects and their structural causes: Four examples in and around the santa clara valley, California

    USGS Publications Warehouse

    Hartzell, S.; Ramirez-Guzman, L.; Carver, D.; Liu, P.

    2010-01-01

    Ground motion records of local and regional events from a portable array are used to investigate the structural causes of variations in ground motion over distances of a few hundred meters to a few kilometers in the sedimentary basin environment of the Santa Clara Valley, California, and its margins. Arrays of portable seismic stations are used to target four study areas with different ground motion patterns: (1) an edge of the alluvial basin extending up onto a marginal ridge (Blossom Hill), (2) a Cenozoic basin with a nearly flat bottom (Cupertino Basin), (3) a long, narrow Cenozoic basin with a steep V profile (Evergreen Basin), and (4) a line perpendicular to the trace of the Hayward fault. Average peak velocities on Blossom Hill from local earthquakes are a factor of 2.5 times higher than nearby valley sites. Three-dimensional (3D) modeling is used to conclude that the majority of the amplification is due to lower shear-wave velocities along a local fault zone (Shannon–Berrocal). Site amplification over the Cupertino Basin in the frequency band 0.5–4 Hz is generally low (less than 2.0 relative to a Mesozoic rock site) and spatially uniform. This response is attributed to the shallow, flat-bottomed shape of the basin and the uniform, flat-laying sedimentary fill. In contrast, site amplification in the Evergreen Basin generally exceeds 3.0 and is attributed to the deep, V-shaped geometry of the basin and younger sedimentary fill. 3D waveform modeling shows the elongated shape of the Evergreen Basin causes more efficient trapping of long-period waves for sources along the long axis of the basin. A low-velocity zone is postulated along the Hayward fault with a width between 100 and 200 m, based on elevated site response along the fault trace and 4.5-Hz fault zone guided waves on the horizontal components of stations near the fault.

  1. Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase*

    PubMed Central

    Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W.

    2016-01-01

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites. PMID:26893379

  2. Multiple DNA binding activities of the novel site-specific recombinase, Piv, from Moraxella lacunata.

    PubMed

    Tobiason, D M; Lenich, A G; Glasgow, A C

    1999-04-02

    The recombinase, Piv, is essential for site-specific DNA inversion of the type IV pilin DNA segment in Moraxella lacunata and Moraxella bovis. Piv shows significant homology with the transposases of the IS110/IS492 family of insertion elements, but, surprisingly, Piv contains none of the conserved amino acid motifs of the lambda Int or Hin/Res families of site-specific recombinases. Therefore, Piv may mediate site-specific recombination by a novel mechanism. To begin to determine how Piv may assemble a synaptic nucleoprotein structure for DNA cleavage and strand exchange, we have characterized the interaction of Piv with the DNA inversion region of M. lacunata. Gel shift and nuclease/chemical protection assays, competition and dissociation rate analyses, and cooperativity studies indicate that Piv binds two distinct recognition sequences. One recognition sequence, found at multiple sites within and outside of the invertible segment, is bound by Piv protomers with high affinity. The second recognition sequence is located at the recombination cross-over sites at the ends of the invertible element; Piv interacts with this sequence as an oligomer with apparent low affinity. A model is proposed for the role of the different Piv binding sites of the M. lacunata inversion region in the formation of an active synaptosome.

  3. Fibromodulin Interacts with Collagen Cross-linking Sites and Activates Lysyl Oxidase.

    PubMed

    Kalamajski, Sebastian; Bihan, Dominique; Bonna, Arkadiusz; Rubin, Kristofer; Farndale, Richard W

    2016-04-08

    The hallmark of fibrotic disorders is a highly cross-linked and dense collagen matrix, a property driven by the oxidative action of lysyl oxidase. Other fibrosis-associated proteins also contribute to the final collagen matrix properties, one of which is fibromodulin. Its interactions with collagen affect collagen cross-linking, packing, and fibril diameter. We investigated the possibility that a specific relationship exists between fibromodulin and lysyl oxidase, potentially imparting a specific collagen matrix phenotype. We mapped the fibromodulin-collagen interaction sites using the collagen II and III Toolkit peptide libraries. Fibromodulin interacted with the peptides containing the known collagen cross-linking sites and the MMP-1 cleavage site in collagens I and II. Interestingly, the interaction sites are closely aligned within the quarter-staggered collagen fibril, suggesting a multivalent interaction between fibromodulin and several collagen helices. Furthermore, we detected an interaction between fibromodulin and lysyl oxidase (a major collagen cross-linking enzyme) and mapped the interaction site to 12 N-terminal amino acids on fibromodulin. This interaction also increases the activity of lysyl oxidase. Together, the data suggest a fibromodulin-modulated collagen cross-linking mechanism where fibromodulin binds to a specific part of the collagen domain and also forms a complex with lysyl oxidase, targeting the enzyme toward specific cross-linking sites.

  4. Locomotor activity influences muscle architecture and bone growth but not muscle attachment site morphology

    PubMed Central

    Rabey, Karyne N.; Green, David J.; Taylor, Andrea B.; Begun, David R.; Richmond, Brian G.; McFarlin, Shannon C.

    2014-01-01

    The ability to make behavioural inferences from skeletal remains is critical to understanding the lifestyles and activities of past human populations and extinct animals. Muscle attachment site (enthesis) morphology has long been assumed to reflect muscle strength and activity during life, but little experimental evidence exists to directly link activity patterns with muscle development and the morphology of their attachments to the skeleton. We used a mouse model to experimentally test how the level and type of activity influences forelimb muscle architecture of spinodeltoideus, acromiodeltoideus, and superficial pectoralis, bone growth rate and gross morphology of their insertion sites. Over an 11-week period, we collected data on activity levels in one control group and two experimental activity groups (running, climbing) of female wild-type mice. Our results show that both activity type and level increased bone growth rates influenced muscle architecture, including differences in potential muscular excursion (fibre length) and potential force production (physiological cross-sectional area). However, despite significant influences on muscle architecture and bone development, activity had no observable effect on enthesis morphology. These results suggest that the gross morphology of entheses is less reliable than internal bone structure for making inferences about an individual’s past behaviour. PMID:25467113

  5. Roles of s3 site residues of nattokinase on its activity and substrate specificity.

    PubMed

    Wu, Shuming; Feng, Chi; Zhong, Jin; Huan, Liandong

    2007-09-01

    Nattokinase (Subtilisin NAT, NK) is a bacterial serine protease with high fibrinolytic activity. To probe their roles on protease activity and substrate specificity, three residues of S3 site (Gly(100), Ser(101) and Leu(126)) were mutated by site-directed mutagenesis. Kinetics parameters of 20 mutants were measured using tetrapeptides as substrates, and their fibrinolytic activities were determined by fibrin plate method. Results of mutation analysis showed that Gly(100) and Ser(101) had reverse steric and electrostatic effects. Residues with bulky or positively charged side chains at position 100 decreased the substrate binding and catalytic activity drastically, while residues with the same characters at position 101 could obviously enhance protease and fibrinolytic activity of NK. Mutation of Leu(126) might impair the structure of the active cleft and drastically decreased the activity of NK. Kinetics studies of the mutants showed that S3 residues were crucial to keep protease activity while they moderately affected substrate specificity of NK. The present study provided some original insight into the P3-S3 interaction in NK and other subtilisins, as well as showed successful protein engineering cases to improve NK as a potential therapeutic agent.

  6. Locomotor activity influences muscle architecture and bone growth but not muscle attachment site morphology.

    PubMed

    Rabey, Karyne N; Green, David J; Taylor, Andrea B; Begun, David R; Richmond, Brian G; McFarlin, Shannon C

    2015-01-01

    The ability to make behavioural inferences from skeletal remains is critical to understanding the lifestyles and activities of past human populations and extinct animals. Muscle attachment site (enthesis) morphology has long been assumed to reflect muscle strength and activity during life, but little experimental evidence exists to directly link activity patterns with muscle development and the morphology of their attachments to the skeleton. We used a mouse model to experimentally test how the level and type of activity influences forelimb muscle architecture of spinodeltoideus, acromiodeltoideus, and superficial pectoralis, bone growth rate and gross morphology of their insertion sites. Over an 11-week period, we collected data on activity levels in one control group and two experimental activity groups (running, climbing) of female wild-type mice. Our results show that both activity type and level increased bone growth rates influenced muscle architecture, including differences in potential muscular excursion (fibre length) and potential force production (physiological cross-sectional area). However, despite significant influences on muscle architecture and bone development, activity had no observable effect on enthesis morphology. These results suggest that the gross morphology of entheses is less reliable than internal bone structure for making inferences about an individual's past behaviour.

  7. Structure of Saccharomyces cerevisiae Rtr1 reveals an active site for an atypical phosphatase.

    PubMed

    Irani, Seema; Yogesha, S D; Mayfield, Joshua; Zhang, Mengmeng; Zhang, Yong; Matthews, Wendy L; Nie, Grace; Prescott, Nicholas A; Zhang, Yan Jessie

    2016-03-01

    Changes in the phosphorylation status of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) correlate with the process of eukaryotic transcription. The yeast protein regulator of transcription 1 (Rtr1) and the human homolog RNAPII-associated protein 2 (RPAP2) may function as CTD phosphatases; however, crystal structures of Kluyveromyces lactis Rtr1 lack a consensus active site. We identified a phosphoryl transfer domain in Saccharomyces cerevisiae Rtr1 by obtaining and characterizing a 2.6 Å resolution crystal structure. We identified a putative substrate-binding pocket in a deep groove between the zinc finger domain and a pair of helices that contained a trapped sulfate ion. Because sulfate mimics the chemistry of a phosphate group, this structural data suggested that this groove represents the phosphoryl transfer active site. Mutagenesis of the residues lining this groove disrupted catalytic activity of the enzyme assayed in vitro with a fluorescent chemical substrate, and expression of the mutated Rtr1 failed to rescue growth of yeast lacking Rtr1. Characterization of the phosphatase activity of RPAP2 and a mutant of the conserved putative catalytic site in the same chemical assay indicated a conserved reaction mechanism. Our data indicated that the structure of the phosphoryl transfer domain and reaction mechanism for the phosphoryl transfer activity of Rtr1 is distinct from those of other phosphatase families.

  8. Mutational analysis of the lac regulatory region: second-site changes that activate mutant promoters.

    PubMed Central

    Rothmel, R K; LeClerc, J E

    1989-01-01

    Second-site mutations that restored activity to severe lacP1 down-promoter mutants were isolated. This was accomplished by using a bacteriophage f1 vector containing a fusion of the mutant E. coli lac promoters with the structural gene for chloramphenicol acetyltransferase (CAT), so that a system was provided for selecting phage revertants (or pseudorevertants) that conferred resistance of phage-infected cells to chloramphenicol. Among the second-site changes that relieved defects in mutant lac promoters, the only one that restored lacP1 activity was a T----G substitution at position -14, a weakly conserved site in E. coli promoters. Three other sequence changes, G----A at -2, A----T at +1, and C----A at +10, activated nascent promoters in the lac regulatory region. The nascent promoters conformed to the consensus rule, that activity is gained by sequence changes toward homology with consensus sequences at the -35 and -10 regions of the promoter. However, the relative activities of some promoters cannot be explained solely by consideration of their conserved sequence elements. Images PMID:2660105

  9. Structure of Saccharomyces cerevisiae Rtr1 reveals an active site for an atypical phosphatase

    PubMed Central

    Mayfield, Joshua; Zhang, Mengmeng; Zhang, Yong; Matthews, Wendy L.; Nie, Grace; Prescott, Nicholas A.; Zhang, Yan Jessie

    2016-01-01

    Changes in the phosphorylation status of the carboxyl-terminal domain (CTD) of RNA polymerase II (RNAPII) correlate with the process of eukaryotic transcription. The yeast protein regulator of transcription 1 (Rtr1) and the human homolog RNAPII-associated protein 2 (RPAP2) may function as CTD phosphatases; however, crystal structures of Kluyveromyces lactis Rtr1 lack a consensus active site. We identified a phosphoryl transfer domain in Saccharomyces cerevisiae Rtr1 by obtaining and characterizing a 2.6 Å resolution crystal structure. We identified a putative substrate-binding pocket in a deep groove between the zinc finger domain and a pair of helices that contained a trapped sulfate ion. Because sulfate mimics the chemistry of a phosphate group, this structural data suggested that this groove represents the phosphoryl transfer active site. Mutagenesis of the residues lining this groove disrupted catalytic activity of the enzyme assayed in vitro with a fluorescent chemical substrate, and expression of the mutated Rtr1 failed to rescue growth of yeast lacking Rtr1. Characterization of the phosphatase activity of RPAP2 and a mutant of the conserved putative catalytic site in the same chemical assay indicated a conserved reaction mechanism. Our data indicated that the structure of the phosphoryl transfer domain and reaction mechanism for the phosphoryl transfer activity of Rtr1 is distinct from those of other phosphatase families. PMID:26933063

  10. Sequences flanking the core-binding site modulate glucocorticoid receptor structure and activity

    PubMed Central

    Schöne, Stefanie; Jurk, Marcel; Helabad, Mahdi Bagherpoor; Dror, Iris; Lebars, Isabelle; Kieffer, Bruno; Imhof, Petra; Rohs, Remo; Vingron, Martin; Thomas-Chollier, Morgane; Meijsing, Sebastiaan H.

    2016-01-01

    The glucocorticoid receptor (GR) binds as a homodimer to genomic response elements, which have particular sequence and shape characteristics. Here we show that the nucleotides directly flanking the core-binding site, differ depending on the strength of GR-dependent activation of nearby genes. Our study indicates that these flanking nucleotides change the three-dimensional structure of the DNA-binding site, the DNA-binding domain of GR and the quaternary structure of the dimeric complex. Functional studies in a defined genomic context show that sequence-induced changes in GR activity cannot be explained by differences in GR occupancy. Rather, mutating the dimerization interface mitigates DNA-induced changes in both activity and structure, arguing for a role of DNA-induced structural changes in modulating GR activity. Together, our study shows that DNA sequence identity of genomic binding sites modulates GR activity downstream of binding, which may play a role in achieving regulatory specificity towards individual target genes. PMID:27581526

  11. Zinc oxide nanoparticles cause inhibition of microbial denitrification by affecting transcriptional regulation and enzyme activity.

    PubMed

    Zheng, Xiong; Su, Yinglong; Chen, Yinguang; Wan, Rui; Liu, Kun; Li, Mu; Yin, Daqiang

    2014-12-02

    Over the past few decades, human activities have accelerated the rates and extents of water eutrophication and global warming through increasing delivery of biologically available nitrogen such as nitrate and large emissions of anthropogenic greenhouse gases. In particular, nitrous oxide (N2O) is one of the most important greenhouse gases, because it has a 300-fold higher global warming potential than carbon dioxide. Microbial denitrification is a major pathway responsible for nitrate removal, and also a dominant source of N2O emissions from terrestrial or aquatic environments. However, whether the release of zinc oxide nanoparticles (ZnO NPs) into the environment affects microbial denitrification is largely unknown. Here we show that the presence of ZnO NPs lead to great increases in nitrate delivery (9.8-fold higher) and N2O emissions (350- and 174-fold higher in the gas and liquid phases, respectively). Our data further reveal that ZnO NPs significantly change the transcriptional regulations of glycolysis and polyhydroxybutyrate synthesis, which causes the decrease in reducing powers available for the reduction of nitrate and N2O. Moreover, ZnO NPs substantially inhibit the gene expressions and catalytic activities of key denitrifying enzymes. These negative effects of ZnO NPs on microbial denitrification finally cause lower nitrate removal and higher N2O emissions, which is likely to exacerbate water eutrophication and global warming.

  12. Germline NLRP1 Mutations Cause Skin Inflammatory and Cancer Susceptibility Syndromes via Inflammasome Activation.

    PubMed

    Zhong, Franklin L; Mamaï, Ons; Sborgi, Lorenzo; Boussofara, Lobna; Hopkins, Richard; Robinson, Kim; Szeverényi, Ildikó; Takeichi, Takuya; Balaji, Reshmaa; Lau, Aristotle; Tye, Hazel; Roy, Keya; Bonnard, Carine; Ahl, Patricia J; Jones, Leigh Ann; Baker, Paul; Lacina, Lukas; Otsuka, Atsushi; Fournie, Pierre R; Malecaze, François; Lane, E Birgitte; Akiyama, Masashi; Kabashima, Kenji; Connolly, John E; Masters, Seth L; Soler, Vincent J; Omar, Salma Samir; McGrath, John A; Nedelcu, Roxana; Gribaa, Moez; Denguezli, Mohamed; Saad, Ali; Hiller, Sebastian; Reversade, Bruno

    2016-09-22

    Inflammasome complexes function as key innate immune effectors that trigger inflammation in response to pathogen- and danger-associated signals. Here, we report that germline mutations in the inflammasome sensor NLRP1 cause two overlapping skin disorders: multiple self-healing palmoplantar carcinoma (MSPC) and familial keratosis lichenoides chronica (FKLC). We find that NLRP1 is the most prominent inflammasome sensor in human skin, and all pathogenic NLRP1 mutations are gain-of-function alleles that predispose to inflammasome activation. Mechanistically, NLRP1 mutations lead to increased self-oligomerization by disrupting the PYD and LRR domains, which are essential in maintaining NLRP1 as an inactive monomer. Primary keratinocytes from patients experience spontaneous inflammasome activation and paracrine IL-1 signaling, which is sufficient to cause skin inflammation and epidermal hyperplasia. Our findings establish a group of non-fever inflammasome disorders, uncover an unexpected auto-inhibitory function for the pyrin domain, and provide the first genetic evidence linking NLRP1 to skin inflammatory syndromes and skin cancer predisposition.

  13. Selective targeting of the conserved active site cysteine of Mycobacterium tuberculosis methionine aminopeptidase with electrophilic reagents.

    PubMed

    Reddi, Ravikumar; Arya, Tarun; Kishor, Chandan; Gumpena, Rajesh; Ganji, Roopa J; Bhukya, Supriya; Addlagatta, Anthony

    2014-09-01

    Methionine aminopeptidases (MetAPs) cleave initiator methionine from ~ 70% of the newly synthesized proteins in every living cell, and specific inhibition or knockdown of this function is detrimental. MetAPs are metalloenzymes, and are broadly classified into two subtypes, type I and type II. Bacteria contain only type I MetAPs, and the active site of these enzymes contains a conserved cysteine. By contrast, in type II enzymes the analogous position is occupied by a conserved glycine. Here, we report the reactivity of the active site cysteine in a type I MetAP, MetAP1c, of Mycobacterium tuberculosis (MtMetAP1c) towards highly selective cysteine-specific reagents. The authenticity of selective modification of Cys105 of MtMetAP1c was established by using site-directed mutagenesis and crystal structure determination of covalent and noncovalent complexes. On the basis of these observations, we propose that metal ions in the active site assist in the covalent modification of Cys105 by orienting the reagents appropriately for a successful reaction. These studies establish, for the first time, that the conserved cysteine of type I MetAPs can be targeted for selective inhibition, and we believe that this chemistry can be exploited for further drug discovery efforts regarding microbial MetAPs.

  14. The Ribotoxin Restrictocin Recognizes Its RNA Substrate by Selective Engagement of Active Site Residues

    PubMed Central

    2011-01-01

    Restrictocin and related fungal endoribonucleases from the α-sarcin family site-specifically cleave the sarcin/ricin loop (SRL) on the ribosome to inhibit translation and ultimately trigger cell death. Previous studies showed that the SRL folds into a bulged-G motif and tetraloop, with restrictocin achieving a specificity of ∼1000-fold by recognizing both motifs only after the initial binding step. Here, we identify contacts within the protein−RNA interface and determine the extent to which each one contributes to enzyme specificity by examining the effect of protein mutations on the cleavage of the SRL substrate compared to a variety of other RNA substrates. As with other biomolecular interfaces, only a subset of contacts contributes to specificity. One contact of this subset is critical, with the H49A mutation resulting in quantitative loss of specificity. Maximum catalytic activity occurs when both motifs of the SRL are present, with the major contribution involving the bulged-G motif recognized by three lysine residues located adjacent to the active site: K110, K111, and K113. Our findings support a kinetic proofreading mechanism in which the active site residues H49 and, to a lesser extent, Y47 make greater catalytic contributions to SRL cleavage than to suboptimal substrates. This systematic and quantitative analysis begins to elucidate the principles governing RNA recognition by a site-specific endonuclease and may thus serve as a mechanistic model for investigating other RNA modifying enzymes. PMID:21417210

  15. Computational approaches to find the active binding sites of biological targets against busulfan.

    PubMed

    Karthick, T; Tandon, Poonam

    2016-06-01

    Determination of electrophilic and nucleophilic sites of a molecule is the primary task to find the active sites of the lead molecule. In the present study, the active sites of busulfan have been predicted by molecular electrostatic potential surface and Fukui function analysis with the help of dispersion corrected density functional theory. Similarly, the identification of active binding sites of the proteins against lead compound plays a vital role in the field of drug discovery. Rigid and flexible molecular docking approaches are used for this purpose. For rigid docking, Hex 8.0.0 software employing fast Fourier transform (FFT) algorithm has been used. The partial flexible blind docking simulations have been performed with AutoDock 4.2 software; where a Lamarckian genetic algorithm is employed. The results showed that the most electrophilic atoms of busulfan bind with the targets. It is clear from the docking studies that busulfan has inhibition capability toward the targets 12CA and 1BZM. Graphical Abstract Docking of ligand and protein.

  16. A Frontier Molecular Orbital determination of the active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.

    1992-11-01

    An angular overlap calculation has been used to determine the s, p and d orbital energy levels of the different types of surface sites present on a dispersed metal catalysts. The basis for these calculations is the reported finding that a large number of catalyzed reactions take place on single atom active sites on the metal surface. Thus, these sites can be considered as surface complexes made up of the central active atom surrounded by near-neighbor metal atom ``ligands`` with localized surface orbitals perturbed only by these ``ligands``. These ``complexes`` are based on a twelve coordinate species with the ``ligands`` attached to the t{sub 2g} orbitals and the coordinate axes coincident with the direction of the e{sub g} orbitals on the central atom. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  17. A Frontier Molecular Orbital determination of the active sites on dispersed metal catalysts

    SciTech Connect

    Augustine, R.L.; Lahanas, K.M.

    1992-01-01

    An angular overlap calculation has been used to determine the s, p and d orbital energy levels of the different types of surface sites present on a dispersed metal catalysts. The basis for these calculations is the reported finding that a large number of catalyzed reactions take place on single atom active sites on the metal surface. Thus, these sites can be considered as surface complexes made up of the central active atom surrounded by near-neighbor metal atom ligands'' with localized surface orbitals perturbed only by these ligands''. These complexes'' are based on a twelve coordinate species with the ligands'' attached to the t{sub 2g} orbitals and the coordinate axes coincident with the direction of the e{sub g} orbitals on the central atom. These data can permit a Frontier Molecular Orbital treatment of specific site activities as long as the surface orbital availability for overlap with adsorbed substrates is considered along with its energy value and symmetry.

  18. Rapid maxillary expansion causes neuronal activation in brain structures of rats.

    PubMed

    Joviliano, P; Junqueira, A A; Stabile, A C; Leite-Panissi, C R A; Rocha, M J A

    2008-07-01

    A correlation between pain sensation and neuronal c-fos expression has been analyzed following experimental rapid maxillar expansion (RME). Adult male Wistar rats were anaesthetized and divided into three groups: animals that received an orthodontic apparatus, which was immediately removed after the insertion (control), animals that received an inactivated orthodontic apparatus (without force), and animals that received an orthodontic apparatus previously activated (140 g force). After 6, 24, 48, or 72 h, the animals were re-anaesthetized, and perfused with 4% paraformaldehyde. The brains were removed, fixed, and sections containing brain structures related to nociception were processed for Fos protein immunohistochemistry (IHC). The insertion of the orthodontic apparatus with 140 g was able to cause RME that could be seen by radiography. The IHC results showed that the number of activated neurons in the different nuclei changed according to the duration of appliance insertion and followed a temporal pattern similar to that of sensations described in clinics. The animals that received the orthodontic apparatus without force did not show RME but a smaller c-fos expression in the same brain structures. In conclusion, we demonstrate that orthodontic force used for palate disjunction activates brain structures that are related to nociception, and that this activation is related to the pain sensation described during orthodontic treatment.

  19. Lymphokine-activated killer (LAK) cells can be focused at sites of tumor growth by products of macrophage activation

    SciTech Connect

    Migliori, R.J.; Gruber, S.A.; Sawyer, M.D.; Hoffman, R.; Ochoa, A.; Bach, F.H.; Simmons, R.L.

    1987-08-01

    Successful adoptive cancer immunotherapy presumably depends on the accumulation of tumoricidal leukocytes at the sites of tumor growth. Large numbers of lymphokine-activated killer (LAK) cells can be generated in vitro by growth in high concentrations of interleukin-2 (IL-2), but relatively few arrive at the tumor site after intravenous injection. We hypothesize that the delivery of LAK cells to tumor sites may be augmented by previously demonstrated lymphocyte-recruiting factors, including activated macrophage products such as interleukin-1 (IL-1) and tumor necrosis factor. /sup 111/Indium-labeled LAK cells were injected intravenously into syngeneic mice bearing the macrophage activator endotoxin (LPS) in one hind footpad, and saline solution was injected into the contralateral footpad. Significantly more activity was recovered from the LPS-bearing footpad at all times during a 96-hour period. Recombinant IL-1 also attracted more LAK cells after injection into tumor-free hind footpads. Furthermore, LAK cells preferentially homed to hind footpads that were bearing 3-day established sarcomas after intralesional injections of LPS, IL-1, or tumor necrosis factor when compared with contralateral tumor-bearing footpads injected with saline solution alone. In preliminary experiments, mice with hind-footpad tumors appeared to survive longer after combined systemic IL-2 and LAK therapy if intralesional LPS was administered. These studies demonstrate that macrophage activation factors that have been shown capable of attracting circulating normal lymphocytes can also effectively attract LAK cells from the circulation. By the stimulation of macrophages at the sites of tumor growth, more LAK cells can be attracted. It is hoped that by focusing the migration of LAK cells to tumors, LAK cells and IL-2 would effect tumor regression more efficiently and with less toxicity.

  20. Technical basis for classification of low-activity waste fraction from Hanford site tanks

    SciTech Connect

    Petersen, C.A., Westinghouse Hanford

    1996-07-17

    The overall objective of this report is to provide a technical basis to support a U.S. Nuclear Regulatory Commission determination to classify the low-activity waste from the Hanford Site single-shell and double-shell tanks as `incidental` wastes after removal of additional radionuclides and immobilization.The proposed processing method, in addition to the previous radionuclide removal efforts, will remove the largest practical amount of total site radioactivity, attributable to high-level wastes, for disposal in a deep geologic repository. The remainder of the waste would be considered `incidental` waste and could be disposed onsite.

  1. Technical basis for classification of low-activity waste fraction from Hanford site tanks

    SciTech Connect

    Petersen, C.A.

    1996-09-20

    The overall objective of this report is to provide a technical basis to support a U.S. Nuclear Regulatory Commission determination to classify the low-activity waste from the Hanford Site single-shell and double-shell tanks as `incidental` wastes after removal of additional radionuclides and immobilization.The proposed processing method, in addition to the previous radionuclide removal efforts, will remove the largest practical amount of total site radioactivity, attributable to high-level waste, for disposal is a deep geologic repository. The remainder of the waste would be considered `incidental` waste and could be disposed onsite.

  2. Particle Size Distribution Data From Existing Boreholes at the Immobilized Low-Activity Waste Site

    SciTech Connect

    Valenta, Michelle M.; Martin, Maria B.; Moreno, Jorge R.; Ferri, Rosalie M.; Horton, Duane G.; Reidel, Stephen P.

    2000-09-25

    This report provides particle size distribution data for samples near the Immobilized Low-Activity Waste (ILAW) Site that were archived in the Hanford Geotechnical Sample Library. Seventy-nine sediment samples were analyzed from four boreholes. Samples were collected from every ten feet in the boreholes. Eightly percent of the samples were classified as slightly gravelly sand. Fifteen percent were classified as gravelly sand, gravelly silty sand, or sandy gravels. These data indicate that the particle size of the sediment is consistent across the ILAW site and is dominated by sand in the upper part of the Hanford formation with more gravel rich units in the lower part.

  3. Flow-induced activation of TRPV5 and TRPV6 channels stimulates Ca(2+)-activated K(+) channel causing membrane hyperpolarization.

    PubMed

    Cha, Seung-Kuy; Kim, Ji-Hee; Huang, Chou-Long

    2013-12-01

    TRPV5 and TRPV6 channels are expressed in distal renal tubules and play important roles in the transcellular Ca(2+) reabsorption in kidney. They are regulated by multiple intracellular factors including protein kinases A and C, membrane phospholipid PIP2, protons, and divalent ions Ca(2+) and Mg(2+). Here, we report that fluid flow that generates shear force within the physiological range of distal tubular fluid flow activated TRPV5 and TRPV6 channels expressed in HEK cells. Flow-induced activation of channel activity was reversible and did not desensitize over 2min. Fluid flow stimulated TRPV5 and 6-mediated Ca(2+) entry and increased intracellular Ca(2+) concentration. N-glycosylation-deficient TRPV5 channel was relatively insensitive to fluid flow. In cells coexpressing TRPV5 (or TRPV6) and Slo1-encoded maxi-K channels, fluid flow induced membrane hyperpolarization, which could be prevented by the maxi-K blocker iberiotoxin or TRPV5 and 6 blocker La(3+). In contrast, fluid flow did not cause membrane hyperpolarization in cells coexpressing ROMK1 and TRPV5 or 6 channel. These results reveal a new mechanism for the regulation of TRPV5 and TRPV6 channels. Activation of TRPV5 and TRPV6 by fluid flow may play a role in the regulation of flow-stimulated K(+) secretion via maxi-K channels in distal renal tubules and in the mechanism of pathogenesis of thiazide-induced hypocalciuria.

  4. Flow-Induced Activation of TRPV5 and TRPV6 Channel Stimulates Ca2+-Activated K+ Channel Causing Membrane Hyperpolarization

    PubMed Central

    Cha, Seung-Kuy; Kim, Ji-Hee; Huang, Chou-Long

    2014-01-01

    TRPV5 and TRPV6 channels are expressed in distal renal tubules and play important roles in the transcellular Ca2+ reabsorption in kidney. They are regulated by multiple intracellular factors including protein kinase A and C, membrane phospholipid PIP2, protons, and divalent ions Ca2+ and Mg2+. Here, we report that fluid flow that generates shear force within the physiological range of distal tubular fluid flow activated TRPV5 and TRPV6 channels expressed in HEK cells. Flow-induced activation of channel activity was reversible and did not desensitize over 2 minutes. Fluid flow stimulated TRPV5 and 6-mediated Ca2+ entry and increased intracellular Ca2+ concentration. N-glycosylation-deficient TRPV5 channel was relatively insensitive to fluid flow. In cells coexpressing TRPV5 (or TRPV6) and Slo1-encoded maxi-K channels, fluid flow induced membrane hyperpolarization, which could be prevented by the maxi-K blocker iberiotoxin or TRPV5 and 6 blocker La3+. In contrast, fluid flow did not cause membrane hyperpolarization in cells coexpressing ROMK1 and TRPV5 or 6 channels. These results reveal a new mechanism for regulation of TRPV5 and TRPV6 channels. Activation of TRPV5 and TRPV6 by fluid flow may play a role in the regulation of flow-stimulated K+ secretion via maxi-K channels in distal renal tubules and in the mechanism of pathogenesis of thiazide-induced hypocalciuria. PMID:24001793

  5. Communication: Active space decomposition with multiple sites: Density matrix renormalization group algorithm

    SciTech Connect

    Parker, Shane M.; Shiozaki, Toru

    2014-12-07

    We extend the active space decomposition method, recently developed by us, to more than two active sites using the density matrix renormalization group algorithm. The fragment wave functions are described by complete or restricted active-space wave functions. Numerical results are shown on a benzene pentamer and a perylene diimide trimer. It is found that the truncation errors in our method decrease almost exponentially with respect to the number of renormalization states M, allowing for numerically exact calculations (to a few μE{sub h} or less) with M = 128 in both cases. This rapid convergence is because the renormalization steps are used only for the interfragment electron correlation.

  6. A single-base change at a splice site in a beta 0-thalassemic gene causes abnormal RNA splicing.

    PubMed

    Treisman, R; Proudfoot, N J; Shander, M; Maniatis, T

    1982-07-01

    We have studied the structure and transcription of a cloned human beta-globin gene from a fetus diagnosed for beta 0 thalassemia. The sequence of the beta 0 gene differs from that of a normal beta-globin gene at positions 1 and 74 of the second intervening sequence (IVS2). The position 1 change alters the GT dinucleotide conserved at 5' splice sites, while the position 74 change is a common sequence polymorphism. When the cloned beta 0 gene is introduced into HeLa cells by use of an SV40-derived plasmid vector, two abnormally spliced cytoplasmic beta-globin RNAs are detected. The predominant RNA differs from normal beta-globin mRNA by the insertion of the first 47 nucleotides of IVS2 between exons 2 and 3. The less abundant RNA comprises the normal first exon spliced directly to the third. Analysis of nuclear RNA suggests that the beta 0 transcript is inefficiently spliced and that the removal of the two intervening sequences is coupled.

  7. Albinism-Causing Mutations in Recombinant Human Tyrosinase Alter Intrinsic Enzymatic Activity

    PubMed Central

    Dolinska, Monika B.; Kovaleva, Elena; Backlund, Peter; Wingfield, Paul T.; Brooks, Brian P.; Sergeev, Yuri V.

    2014-01-01

    Background Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (TYR) is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes. Methodology/Principal Findings The intra-melanosomal domain of human tyrosinase (residues 19–469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure. Conclusions/Significance The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure – function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1. PMID:24392141

  8. Developmental Deltamethrin Exposure Causes Persistent Changes in Dopaminergic Gene Expression, Neurochemistry, and Locomotor Activity in Zebrafish

    PubMed Central

    Kung, Tiffany S.; Richardson, Jason R.; Cooper, Keith R.; White, Lori A.

    2015-01-01

    Pyrethroids are commonly used insecticides that are considered to pose little risk to human health. However, there is an increasing concern that children are more susceptible to the adverse effects of pesticides. We used the zebrafish model to test the hypothesis that developmental exposure to low doses of the pyrethroid deltamethrin results in persistent alterations in dopaminergic gene expression, neurochemistry, and locomotor activity. Zebrafish embryos were treated with deltamethrin (0.25–0.50 μg/l), at concentrations below the LOAEL, during the embryonic period [3–72 h postfertilization (hpf)], after which transferred to fresh water until the larval stage (2-weeks postfertilization). Deltamethrin exposure resulted in decreased transcript levels of the D1 dopamine (DA) receptor (drd1) and increased levels of tyrosine hydroxylase at 72 hpf. The reduction in drd1 transcripts persisted to the larval stage and was associated with decreased D2 dopamine receptor transcripts. Larval fish, exposed developmentally to deltamethrin, had increased levels of homovanillic acid, a DA metabolite. Since the DA system is involved in locomotor activity, we measured the swim activity of larval fish following a transition to darkness. Developmental exposure to deltamethrin significantly increased larval swim activity which was attenuated by concomitant knockdown of the DA transporter. Acute exposure to methylphenidate, a DA transporter inhibitor, increased swim activity in control larva, while reducing swim activity in larva developmentally exposed to deltamethrin. Developmental exposure to deltamethrin causes locomotor deficits in larval zebrafish, which is likely mediated by dopaminergic dysfunction. This highlights the need to understand the persistent effects of low-dose neurotoxicant exposure during development. PMID:25912032

  9. Antimicrobial Activity of Seven Essential Oils From Iranian Aromatic Plants Against Common Causes of Oral Infections

    PubMed Central

    Zomorodian, Kamiar; Ghadiri, Pooria; Saharkhiz, Mohammad Jamal; Moein, Mohammad Reza; Mehriar, Peiman; Bahrani, Farideh; Golzar, Tahereh; Pakshir, Keyvan; Fani, Mohammad Mehdi

    2015-01-01

    Background: Over the past two decades, there has been a growing trend in using oral hygienic products originating from natural resources such as essential oils (EOs) and plant extracts. Seven aromatic plants used in this study are among popular traditional Iranian medicinal plants with potential application in modern medicine as anti-oral infectious diseases. Objectives: This study was conducted to determine the chemical composition and antimicrobial activities of essential oils from seven medicinal plants against pathogens causing oral infections. Materials and Methods: The chemical compositions of EOs distilled from seven plants were analyzed by gas chromatography/mass spectrometry (GC/MS). These plants included Satureja khuzestanica, S. bachtiarica, Ocimum sanctum, Artemisia sieberi, Zataria multiflora, Carum copticum and Oliveria decumbens. The antimicrobial activity of the essential oils was evaluated by broth micro-dilution in 96 well plates as recommended by the Clinical and Laboratory Standards Institute (CLSI) methods. Results: The tested EOs inhibited the growth of examined oral pathogens at concentrations of 0.015-16 µL/mL. Among the examined oral pathogens, Enterococcus faecalis had the highest Minimum Inhibitory Concentrations (MICs) and Minimum Microbicidal Concentrations (MMCs). Of the examined EOs, S. khuzestanica, Z. multiflora and S. bachtiarica, showed the highest antimicrobial activities, respectively, while Artemisia sieberi exhibited the lowest antimicrobial activity. Conclusions: The excellent antimicrobial activities of the tested EOs might be due to their major phenolic or alcoholic monoterpenes with known antimicrobial activities. Hence, these EOs can be possibly used as an antimicrobial agent in treatment and control of oral pathogens. PMID:25793100

  10. Controlling activation site density by low-energy far-field stimulation in cardiac tissue.

    PubMed

    Hörning, Marcel; Takagi, Seiji; Yoshikawa, Kenichi

    2012-06-01

    Tachycardia and fibrillation are potentially fatal arrhythmias associated with the formation of rotating spiral waves in the heart. Presently, the termination of these types of arrhythmia is achieved by use of antitachycardia pacing or cardioversion. However, these techniques have serious drawbacks, in that they either have limited application or produce undesirable side effects. Low-energy far-field stimulation has recently been proposed as a superior therapy. This proposed therapeutic method would exploit the phenomenon in which the application of low-energy far-field shocks induces a large number of activation sites ("virtual electrodes") in tissue. It has been found that the formation of such sites can lead to the termination of undesired states in the heart and the restoration of normal beating. In this study we investigate a particular aspect of this method. Here we seek to determine how the activation site density depends on the applied electric field through in vitro experiments carried out on neonatal rat cardiac tissue cultures. The results indicate that the activation site density increases exponentially as a function of the intracellular conductivity and the level of cell isotropy. Additionally, we report numerical results obtained from bidomain simulations of the Beeler-Reuter model that are quantitatively consistent with our experimental results. Also, we derive an intuitive analytical framework that describes the activation site density and provides useful information for determining the ratio of longitudinal to transverse conductivity in a cardiac tissue culture. The results obtained here should be useful in the development of an actual therapeutic method based on low-energy far-field pacing. In addition, they provide a deeper understanding of the intrinsic properties of cardiac cells.

  11. Probing Oxygen Activation Sites in Two Flavoprotein Oxidases Using Chloride as an Oxygen Surrogate

    SciTech Connect

    Kommoju, Phaneeswara-Rao; Chen, Zhi-wei; Bruckner, Robert C.; Mathews, F. Scott; Jorns, Marilyn Schuman

    2011-08-16

    A single basic residue above the si-face of the flavin ring is the site of oxygen activation in glucose oxidase (GOX) (His516) and monomeric sarcosine oxidase (MSOX) (Lys265). Crystal structures of both flavoenzymes exhibit a small pocket at the oxygen activation site that might provide a preorganized binding site for superoxide anion, an obligatory intermediate in the two-electron reduction of oxygen. Chloride binds at these polar oxygen activation sites, as judged by solution and structural studies. First, chloride forms spectrally detectable complexes with GOX and MSOX. The protonated form of His516 is required for tight binding of chloride to oxidized GOX and for rapid reaction of reduced GOX with oxygen. Formation of a binary MSOX-chloride complex requires Lys265 and is not observed with Lys265Met. Binding of chloride to MSOX does not affect the binding of a sarcosine analogue (MTA, methylthioactetate) above the re-face of the flavin ring. Definitive evidence is provided by crystal structures determined for a binary MSOX-chloride complex and a ternary MSOX-chloride-MTA complex. Chloride binds in the small pocket at a position otherwise occupied by a water molecule and forms hydrogen bonds to four ligands that are arranged in approximate tetrahedral geometry: Lys265:NZ, Arg49:NH1, and two water molecules, one of which is hydrogen bonded to FAD:N5. The results show that chloride (i) acts as an oxygen surrogate, (ii) is an effective probe of polar oxygen activation sites, and (iii) provides a valuable complementary tool to the xenon gas method that is used to map nonpolar oxygen-binding cavities.

  12. Controlling activation site density by low-energy far-field stimulation in cardiac tissue

    NASA Astrophysics Data System (ADS)

    Hörning, Marcel; Takagi, Seiji; Yoshikawa, Kenichi

    2012-06-01

    Tachycardia and fibrillation are potentially fatal arrhythmias associated with the formation of rotating spiral waves in the heart. Presently, the termination of these types of arrhythmia is achieved by use of antitachycardia pacing or cardioversion. However, these techniques have serious drawbacks, in that they either have limited application or produce undesirable side effects. Low-energy far-field stimulation has recently been proposed as a superior therapy. This proposed therapeutic method would exploit the phenomenon in which the application of low-energy far-field shocks induces a large number of activation sites (“virtual electrodes”) in tissue. It has been found that the formation of such sites can lead to the termination of undesired states in the heart and the restoration of normal beating. In this study we investigate a particular aspect of this method. Here we seek to determine how the activation site density depends on the applied electric field through in vitro experiments carried out on neonatal rat cardiac tissue cultures. The results indicate that the activation site density increases exponentially as a function of the intracellular conductivity and the level of cell isotropy. Additionally, we report numerical results obtained from bidomain simulations of the Beeler-Reuter model that are quantitatively consistent with our experimental results. Also, we derive an intuitive analytical framework that describes the activation site density and provides useful information for determining the ratio of longitudinal to transverse conductivity in a cardiac tissue culture. The results obtained here should be useful in the development of an actual therapeutic method based on low-energy far-field pacing. In addition, they provide a deeper understanding of the intrinsic properties of cardiac cells.

  13. Threshold occupancy and specific cation binding modes in the hammerhead ribozyme active site are required for active conformation

    PubMed Central

    Lee, Tai-Sung; Giambaşu, George M.; Sosa, Carlos P.; Martick, Monika; Scott, William G.; York, Darrin M.

    2009-01-01

    The relationship between formation of active in-line attack conformations and monovalent (Na+) and divalent (Mg2+) metal ion binding in the hammerhead ribozyme has been explored with molecular dynamics simulations. To stabilize repulsions between negatively charged groups, different requirements of threshold occupancy of metal ions were observed in the reactant and activated precursor states both in the presence or absence of a Mg2+ in the active site. Specific bridging coordination patterns of the ions are correlated with the formation of active in-line attack conformations and can be accommodated in both cases. Furthermore, simulation results suggest that the hammerhead ribozyme folds to form an electronegative recruiting pocket that attracts high local concentrations of positive charge. The present simulations help to reconcile experiments that probe the metal ion sensitivity of hammerhead ribozyme catalysis and support the supposition that Mg2+, in addition to stabilizing active conformations, plays a specific chemical role in catalysis. PMID:19265710

  14. Localization of the active site of an enzyme, bacterial luciferase, using two-quantum affinity modification

    NASA Astrophysics Data System (ADS)

    Benimetskaya, L. Z.; Gitelzon, I. I.; Kozionov, Andrew L.; Novozhilov, S. Y.; Petushkov, V. N.; Rodionova, N. S.; Stockman, Mark I.

    1991-11-01

    For the first time the method of two-quantum affinity modification has been employed to probe the structure of an enzyme, bacterial luciferase. Position of the flavin-binding site of this enzyme, which was previously unknown, has been established. The obtained data indicate that the flavin site is positioned on the (alpha) -subunit. The closest contact of the protein chain of the enzyme with the chromophoric group of the flavin takes place near 80 +/- 10 and 120 +/- 10 amino acid residues; the regions 50 +/- 10 and 215 +/- 10 are also close to the flavin. The established localization does not contradict suggestions on positions of the flavin and phosphate sites of the bacterial luciferase, which had earlier been made from the data on evolutionary stability of various luciferases. The present method can, in principle, be applied to a great number of enzymes, including all flavin-dependent enzymes. Enzymatic catalysis has high speed and specificity. Creation of a method of determination of the elements of the primary structure of a protein, making up the active site (in which substratum conversion occurs), could be a significant advance in clearing up mechanisms of enzymatic catalysis. It was proposed to localize active sites of the enzymes, whose substrata are chromophores, using this method of two-quantum affinity modification. An enzyme- substratum complex is irradiated with laser light of sufficiently long wavelength ((lambda) 300 nm) which is not directly absorbed by the enzyme. Two-quantum quasiresonant excitation of the substratum activates it to the state with energy 5-7 eV, which is then radiativelessly transferred to neighboring protein groups. This energy exceeds the energy of activation of peptide bond breakage. Therefore, the enzyme will be disrupted in the vicinity of its active site. In the present paper the above approach has been implemented for the first time. Information has been obtained about the position of the flavin-binding site of bacterial

  15. Impact of single-site axonal GABAergic synaptic events on cerebellar interneuron activity

    PubMed Central

    Zorrilla de San Martin, Javier; Jalil, Abdelali

    2015-01-01

    Axonal ionotropic receptors are present in a variety of neuronal types, and their function has largely been associated with the modulation of axonal activity and synaptic release. It is usually assumed that activation of axonal GABAARs comes from spillover, but in cerebellar molecular layer interneurons (MLIs) the GABA source is different: in these cells, GABA release activates presynaptic GABAA autoreceptors (autoRs) together with postsynaptic targets, producing an autoR-mediated synaptic event. The frequency of presynaptic, autoR-mediated miniature currents is twice that of their somatodendritic counterparts, suggesting that autoR-mediated responses have an important effect on interneuron activity. Here, we used local Ca2+ photolysis in MLI axons of juvenile rats to evoke GABA release from individual varicosities to study the activation of axonal autoRs in single release sites. Our data show that single-site autoR conductances are similar to postsynaptic dendritic conductances. In conditions of high [Cl−]i, autoR-mediated conductances range from 1 to 5 nS; this corresponds to ∼30–150 GABAA channels per presynaptic varicosity, a value close to the number of channels in postsynaptic densities. Voltage responses produced by the activation of autoRs in single varicosities are amplified by a Nav-dependent mechanism and propagate along the axon with a length constant of 91 µm. Immunolabeling determination of synapse location shows that on average, one third of the synapses produce autoR-mediated signals that are large enough to reach the axon initial segment. Finally, we show that single-site activation of presynaptic GABAA autoRs leads to an increase in MLI excitability and thus conveys a strong feedback signal that contributes to spiking activity. PMID:26621773

  16. Impact of single-site axonal GABAergic synaptic events on cerebellar interneuron activity.

    PubMed

    de San Martin, Javier Zorrilla; Jalil, Abdelali; Trigo, Federico F

    2015-12-01

    Axonal ionotropic receptors are present in a variety of neuronal types, and their function has largely been associated with the modulation of axonal activity and synaptic release. It is usually assumed that activation of axonal GABA(A)Rs comes from spillover, but in cerebellar molecular layer interneurons (MLIs) the GABA source is different: in these cells, GABA release activates presynaptic GABA(A) autoreceptors (autoRs) together with postsynaptic targets, producing an autoR-mediated synaptic event. The frequency of presynaptic, autoR-mediated miniature currents is twice that of their somatodendritic counterparts, suggesting that autoR-mediated responses have an important effect on interneuron activity. Here, we used local Ca(2+) photolysis in MLI axons of juvenile rats to evoke GABA release from individual varicosities to study the activation of axonal autoRs in single release sites. Our data show that single-site autoR conductances are similar to postsynaptic dendritic conductances. In conditions of high [Cl(-)](i), autoR-mediated conductances range from 1 to 5 nS; this corresponds to ∼30-150 GABA(A) channels per presynaptic varicosity, a value close to the number of channels in postsynaptic densities. Voltage responses produced by the activation of autoRs in single varicosities are amplified by a Na(v)-dependent mechanism and propagate along the axon with a length constant of 91 µm. Immunolabeling determination of synapse location shows that on average, one third of the synapses produce autoR-mediated signals that are large enough to reach the axon initial segment. Finally, we show that single-site activation of presynaptic GABA(A) autoRs leads to an increase in MLI excitability and thus conveys a strong feedback signal that contributes to spiking activity.

  17. Abnormal Activation of BMP Signaling Causes Myopathy in Fbn2 Null Mice.

    PubMed

    Sengle, Gerhard; Carlberg, Valerie; Tufa, Sara F; Charbonneau, Noe L; Smaldone, Silvia; Carlson, Eric J; Ramirez, Francesco; Keene, Douglas R; Sakai, Lynn Y

    2015-06-01

    Fibrillins are large extracellular macromolecules that polymerize to form the backbone structure of connective tissue microfibrils. Mutations in the gene for fibrillin-1 cause the Marfan syndrome, while mutations in the gene for fibrillin-2 cause Congenital Contractural Arachnodactyly. Both are autosomal dominant disorders, and both disorders affect musculoskeletal tissues. Here we show that Fbn2 null mice (on a 129/Sv background) are born with reduced muscle mass, abnormal muscle histology, and signs of activated BMP signaling in skeletal muscle. A delay in Myosin Heavy Chain 8, a perinatal myosin, was found in Fbn2 null forelimb muscle tissue, consistent with the notion that muscle defects underlie forelimb contractures in these mice. In addition, white fat accumulated in the forelimbs during the early postnatal period. Adult Fbn2 null mice are already known to demonstrate persistent muscle weakness. Here we measured elevated creatine kinase levels in adult Fbn2 null mice, indicating ongoing cycles of muscle injury. On a C57Bl/6 background, Fbn2 null mice showed severe defects in musculature, leading to neonatal death from respiratory failure. These new findings demonstrate that loss of fibrillin-2 results in phenotypes similar to those found in congenital muscular dystrophies and that FBN2 should be considered as a candidate gene for recessive congenital muscular dystrophy. Both in vivo and in vitro evidence associated muscle abnormalities and accumulation of white fat in Fbn2 null mice with abnormally activated BMP signaling. Genetic rescue of reduced muscle mass and accumulation of white fat in Fbn2 null mice was accomplished by deleting a single allele of Bmp7. In contrast to other reports that activated BMP signaling leads to muscle hypertrophy, our findings demonstrate the exquisite sensitivity of BMP signaling to the fibrillin-2 extracellular environment during early postnatal muscle development. New evidence presented here suggests that fibrillin-2 can

  18. Low-frequency physiological activation of the vestibular utricle causes biphasic modulation of skin sympathetic nerve activity in humans.

    PubMed

    Grewal, Tarandeep; Dawood, Tye; Hammam, Elie; Kwok, Kenny; Macefield, Vaughan G

    2012-07-01

    We have previously shown that sinusoidal galvanic vestibular stimulation, a means of selectively modulating vestibular afferent activity, can cause partial entrainment of sympathetic outflow to muscle and skin in human subjects. However, it influences the firing of afferents from the entire vestibular apparatus, including the semicircular canals. Here, we tested the hypothesis that selective stimulation of one set of otolithic organs-those located in the utricle, which are sensitive to displacement in the horizontal axis-could entrain sympathetic nerve activity. Skin sympathetic nerve activity (SSNA) was recorded via tungsten microelectrodes inserted into cutaneous fascicles of the common peroneal nerve in 10 awake subjects, seated (head vertical, eyes closed) on a motorised platform. Slow sinusoidal accelerations-decelerations (~4 mG) were applied in the X (antero-posterior) or Y (medio-lateral) direction at 0.08 Hz; composite movements in both directions were also applied. Subjects either reported feeling a vague sense of movement (with no sense of direction) or no movement at all. Nevertheless, cross-correlation analysis revealed a marked entrainment of SSNA for all types of movements: vestibular modulation was 97 ± 3 % for movements in the X axis and 91 ± 5 % for displacements in the Y axis. For each sinusoidal cycle, there were two major peaks of modulation-one associated with acceleration as the platform moved forward or to the side, and one associated with acceleration in the opposite direction. We interpret these observations as reflecting inertial displacement of the stereocilia within the utricle during acceleration, which causes a robust vestibulosympathetic reflex.

  19. Crystallographic Analysis of Active Site Contributions to Regiospecificity in the Diiron Enzyme Toluene 4-Monooxygenase

    SciTech Connect

    Bailey, Lucas J.; Acheson, Justin F.; McCoy, Jason G.; Elsen, Nathaniel L.; Phillips, Jr., George N.; Fox, Brian G.

    2014-10-02

    Crystal structures of toluene 4-monooxygenase hydroxylase in complex with reaction products and effector protein reveal active site interactions leading to regiospecificity. Complexes with phenolic products yield an asymmetric {mu}-phenoxo-bridged diiron center and a shift of diiron ligand E231 into a hydrogen bonding position with conserved T201. In contrast, complexes with inhibitors p-NH{sub 2}-benzoate and p-Br-benzoate showed a {mu}-1,1 coordination of carboxylate oxygen between the iron atoms and only a partial shift in the position of E231. Among active site residues, F176 trapped the aromatic ring of products against a surface of the active site cavity formed by G103, E104 and A107, while F196 positioned the aromatic ring against this surface via a {pi}-stacking interaction. The proximity of G103 and F176 to the para substituent of the substrate aromatic ring and the structure of G103L T4moHD suggest how changes in regiospecificity arise from mutations at G103. Although effector protein binding produced significant shifts in the positions of residues along the outer portion of the active site (T201, N202, and Q228) and in some iron ligands (E231 and E197), surprisingly minor shifts (<1 {angstrom}) were produced in F176, F196, and other interior residues of the active site. Likewise, products bound to the diiron center in either the presence or absence of effector protein did not significantly shift the position of the interior residues, suggesting that positioning of the cognate substrates will not be strongly influenced by effector protein binding. Thus, changes in product distributions in the absence of the effector protein are proposed to arise from differences in rates of chemical steps of the reaction relative to motion of substrates within the active site channel of the uncomplexed, less efficient enzyme, while structural changes in diiron ligand geometry associated with cycling between diferrous and diferric states are discussed for their potential

  20. Active-site modifications of adenylation domains lead to hydrolysis of upstream nonribosomal peptidyl thioester intermediates.

    PubMed

    Uguru, Gabriel C; Milne, Claire; Borg, Matthew; Flett, Fiona; Smith, Colin P; Micklefield, Jason

    2004-04-28

    Site-directed mutagenesis of nonribosomal peptide synthetase (NRPS) adenylation (A) domains was investigated as a means to engineer new calcium-dependent antibiotics (CDA) in Streptomyces coelicolor. Single- and double-point mutants of the CDA NRPS module 7, A-domain were generated, which were predicted to alter the specificity of this domain from Asp to Asn. The double-point mutant produced a new peptide CDA2a-7N containing Asn at position 7 as expected. However, in both the single- and the double-point mutants, significant hydrolysis of the CDA-6mer intermediate was evident. One explanation for this is that the mutant module 7 A-domain activates Asn instead of Asp; however, the Asn-thioester intermediate is only weakly recognized by the upstream C-domain acceptor site (a), allowing a water molecule to intercept the hexapeptidyl intermediate in the donor site (d).

  1. Novel active comb-shaped dry electrode for EEG measurement in hairy site.

    PubMed

    Huang, Yan-Jun; Wu, Chung-Yu; Wong, Alice May-Kuen; Lin, Bor-Shyh

    2015-01-01

    Electroencephalography (EEG) is an important biopotential, and has been widely applied in clinical applications. The conventional EEG electrode with conductive gels is usually used for measuring EEG. However, the use of conductive gel also encounters with the issue of drying and hardening. Recently, many dry EEG electrodes based on different conductive materials and techniques were proposed to solve the previous issue. However, measuring EEG in the hairy site is still a difficult challenge. In this study, a novel active comb-shaped dry electrode was proposed to measure EEG in hairy site. Different form other comb-shaped or spike-shaped dry electrodes, it can provide more excellent performance of avoiding the signal attenuation, phase distortion, and the reduction of common mode rejection ratio. Even under walking motion, it can effectively acquire EEG in hairy site. Finally, the experiments for alpha rhythm and steady-state visually evoked potential were also tested to validate the proposed electrode.

  2. A mutation creating an out-of-frame alternative translation initiation site in the GRHPR 5'UTR causing primary hyperoxaluria type II.

    PubMed

    Fu, Y; Rope, R; Fargue, S; Cohen, H T; Holmes, R P; Cohen, D M

    2015-11-01

    Primary hyperoxaluria type II is a recessive genetic disorder caused by mutations in the GRHPR gene. Although several dozen mutations have been described, all affect coding or transcript splicing. A man suspected of having primary hyperoxaluria type II was heterozygous for a novel single-nucleotide deletion (c.694delC) in GRHPR affecting Gln(232) , which introduced a pre-mature termination (p.Gln232Argfs*3). Two 5'untranslated region (UTR) variants of unknown significance were also noted. We show that these two variants occur in cis, on the opposite allele, and introduce - immediately upstream of the canonical translation initiation site - a novel out-of-frame translational start site. In vitro studies using the GRHPR 5'UTR fused to a luciferase reporter show that the variant start site pre-empted initiation at the canonical translational start site, and this was corroborated within the broader context of 1.3 kb of the GRHPR proximal promoter. This latter mechanism may be underappreciated in general; reports of clinically significant functional variation of this type are extremely rare.

  3. Structural role of the active-site metal in the conformation of Trypanosoma brucei phosphoglycerate mutase.

    PubMed

    Mercaldi, Gustavo F; Pereira, Humberto M; Cordeiro, Artur T; Michels, Paul A M; Thiemann, Otavio H

    2012-06-01

    Phosphoglycerate mutases (PGAMs) participate in both the glycolytic and the gluconeogenic pathways in reversible isomerization of 3-phosphoglycerate and 2-phosphoglycerate. PGAMs are members of two distinct protein families: enzymes that are dependent on or independent of the 2,3-bisphosphoglycerate cofactor. We determined the X-ray structure of the monomeric Trypanosoma brucei independent PGAM (TbiPGAM) in its apoenzyme form, and confirmed this observation by small angle X-ray scattering data. Comparing the TbiPGAM structure with the Leishmania mexicana independent PGAM structure, previously reported with a phosphoglycerate molecule bound to the active site, revealed the domain movement resulting from active site occupation. The structure reported here shows the interaction between Asp319 and the metal bound to the active site, and its contribution to the domain movement. Substitution of the metal-binding residue Asp319 by Ala resulted in complete loss of independent PGAM activity, and showed for the first time its involvement in the enzyme's function. As TbiPGAM is an attractive molecular target for drug development, the apoenzyme conformation described here provides opportunities for its use in structure-based drug design approaches. Database Structural data for the Trypanosoma brucei 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) has been deposited with the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank under code 3NVL.

  4. Substrate conformational transitions in the active site of chorismate mutase: their role in the catalytic mechanism.

    PubMed

    Guo, H; Cui, Q; Lipscomb, W N; Karplus, M

    2001-07-31

    Chorismate mutase acts at the first branch-point of aromatic amino acid biosynthesis and catalyzes the conversion of chorismate to prephenate. The results of molecular dynamics simulations of the substrate in solution and in the active site of chorismate mutase are reported. Two nonreactive conformers of chorismate are found to be more stable than the reactive pseudodiaxial chair conformer in solution. It is shown by QM/MM molecular dynamics simulations, which take into account the motions of the enzyme, that when these inactive conformers are bound to the active site, they are rapidly converted to the reactive chair conformer. This result suggests that one contribution of the enzyme is to bind the more prevalent nonreactive conformers and transform them into the active form in a step before the chemical reaction. The motion of the reactive chair conformer in the active site calculated by using the QM/MM potential generates transient structures that are closer to the transition state than is the stable CHAIR conformer.

  5. How Force Might Activate Talin's Vinculin Binding Sites: SMD Reveals a Structural Mechanism

    PubMed Central

    Hytönen, Vesa P; Vogel, Viola

    2008-01-01

    Upon cell adhesion, talin physically couples the cytoskeleton via integrins to the extracellular matrix, and subsequent vinculin recruitment is enhanced by locally applied tensile force. Since the vinculin binding (VB) sites are buried in the talin rod under equilibrium conditions, the structural mechanism of how vinculin binding to talin is force-activated remains unknown. Taken together with experimental data, a biphasic vinculin binding model, as derived from steered molecular dynamics, provides high resolution structural insights how tensile mechanical force applied to the talin rod fragment (residues 486–889 constituting helices H1–H12) might activate the VB sites. Fragmentation of the rod into three helix subbundles is prerequisite to the sequential exposure of VB helices to water. Finally, unfolding of a VB helix into a completely stretched polypeptide might inhibit further binding of vinculin. The first events in fracturing the H1–H12 rods of talin1 and talin2 in subbundles are similar. The proposed force-activated α-helix swapping mechanism by which vinculin binding sites in talin rods are exposed works distinctly different from that of other force-activated bonds, including catch bonds. PMID:18282082

  6. Progress report on decommissioning activities at the Fernald Environmental Management Project (FEMP) site

    SciTech Connect

    1998-07-01

    The Fernald Environmental Management Project (FEMP), is located about 18 miles northwest of Cincinnati, Ohio. Between 1953 and 1989, the facility, then called the Feed Material Production Center or FMPC, produced uranium metal products used in the eventual production of weapons grade material for use by other US Department of Energy (DOE) sites. In 1989, FMPC`s production was suspended by the federal government in order to focus resources on environmental restoration versus defense production. In 1992, Fluor Daniel Fernald assumed responsibility for managing all cleanup activities at the FEMP under contract to the DOE. In 1990, as part of the remediation effort, the site was divided into five operable units based on physical proximity of contaminated areas, similar amounts of types of contamination, or the potential for a similar technology to be used in cleanup activities. This report continues the outline of the decontamination and decommissioning (D and D) activities at the FEMP site Operable Unit 3 (OU3) and provides an update on the status of the decommissioning activities. OU3, the Facilities Closure and Demolition Project, involves the remediation of more than 200 uranium processing facilities. The mission of the project is to remove nuclear materials stored in these buildings, then perform the clean out of the buildings and equipment, and decontaminate and dismantle the facilities.

  7. Structure-based drug design: exploring the proper filling of apolar pockets at enzyme active sites.

    PubMed

    Zürcher, Martina; Diederich, François

    2008-06-20

    The proper filling of apolar pockets at enzyme active sites is central for increasing binding activity and selectivity of hits and leads in medicinal chemistry. In our structure-based design approach toward the generation of potent enzyme inhibitors, we encountered a variety of challenges in gaining suitable binding affinity from the occupation of such pockets. We summarize them here for the first time. A fluorine scan of tricyclic thrombin inhibitors led to the discovery of favorable orthogonal dipolar C-F...CO interactions. Efficient cation-pi interactions were established in the S4 pocket of factor Xa, another serine protease from the blood coagulation cascade. Changing from mono- to bisubstrate inhibitors of catechol O-methyltransferase, a target in the L-Dopa-based treatment of Parkinson's disease, enabled the full exploitation of a previously unexplored hydrophobic pocket. Conformational preorganization of a pocket at an enzyme active site is crucial for harvesting binding affinity. This is demonstrated for two enzymes from the nonmevalonate pathway of isoprenoid biosynthesis, IspE and IspF, which are pursued as antimalarial targets. Disrupting crystallographically defined water networks on the way into a pocket might cost all of the binding free enthalpy gained from its occupation, as revealed in studies with tRNA-guanine transglycosylase, a target against shigellosis. Investigations of the active site of plasmepsin II, another antimalarial target, showed that principles for proper apolar cavity filling, originally developed for synthetic host-guest systems, are also applicable to enzyme environments.

  8. Pose prediction accuracy in docking studies and enrichment of actives in the active site of GSK-3beta.

    PubMed

    Gadakar, Pravin Kumar; Phukan, Samiron; Dattatreya, Prasanna; Balaji, V N

    2007-01-01

    We present molecular docking studies on the inhibitors of GSK-3beta kinase in the enzyme binding sites of the X-ray complexes (1H8F, 1PYX, 1O9U, 1Q4L, 1Q5K, and 1UV5) using the Schrödinger docking tool Glide. Cognate and cross-docking studies using standard precision (SP) and extraprecision (XP) algorithms have been carried out. Cognate docking studies demonstrate that docked poses similar to X-ray poses (root-mean-square deviations of less than 2 A) are found within the top four ranks of the GlideScore and E-model scores. However, cross-docking studies typically produce poses that are significantly deviated from X-ray poses in all but a couple of cases, implying potential for induced fit effects in ligand binding. In this light, we have also carried out induced fit docking studies in the active sites of 1O9U, 1Q4L, and 1Q5K. Specifically, conformational changes have been effected in the active sites of these three protein structures to dock noncognate ligands. Thus, for example, the active site of 1O9U has been induced to fit the ligands of 1Q4L, 1Q5K, and 1UV5. These studies produce ligand docked poses which have significantly lower root-mean-square deviations relative to their X-ray crystallographic poses, when compared to the corresponding values from the cross-docking studies. Furthermore, we have used an ensemble of the induced fit models and X-ray structures to enhance the retrieval of active GSK-3beta inhibitors seeded in a decoy database, normally used in Glide validation studies. Thus, our studies provide valuable insights into computational strategies useful for the identification of potential GSK-3beta inhibitors.

  9. Activation of the succinate receptor GPR91 in macula densa cells causes renin release.

    PubMed

    Vargas, Sarah Laurin; Toma, Ildikó; Kang, Jung Julie; Meer, Elliott James; Peti-Peterdi, János

    2009-05-01

    Macula densa (MD) cells of the juxtaglomerular apparatus (JGA) are salt sensors and generate paracrine signals that control renal blood flow, glomerular filtration, and release of the prohypertensive hormone renin. We hypothesized that the recently identified succinate receptor GPR91 is present in MD cells and regulates renin release. Using immunohistochemistry, we identified GPR91 in the apical plasma membrane of MD cells. Treatment of MD cells with succinate activated mitogen-activated protein kinases (MAPKs; p38 and extracellular signal-regulated kinases 1/2) and cyclooxygenase 2 (COX-2) and induced the synthesis and release of prostaglandin E(2), a potent vasodilator and classic paracrine mediator of renin release. Using microperfused JGA and real-time confocal fluorescence imaging of quinacrine-labeled renin granules, we detected significant renin release in response to tubular succinate (EC(50) 350 microM). Genetic deletion of GPR91 (GPR91(-/-) mice) or pharmacologic inhibition of MAPK or COX-2 blocked succinate-induced renin release. Streptozotocin-induced diabetes caused GPR91-dependent upregulation of renal cortical phospho-p38, extracellular signal-regulated kinases 1/2, COX-2, and renin content. Salt depletion for 1 wk increased plasma renin activity seven-fold in wild-type mice but only 3.4-fold in GPR91(-/-) mice. In summary, MD cells can sense alterations in local tissue metabolism via accumulation of tubular succinate and GPR91 signaling, which involves the activation of MAPKs, COX-2, and the release of prostaglandin E(2). This mechanism may be integral in the regulation of renin release and activation of the renin-angiotensin system in health and disease.

  10. A NMR and MD study of the active site of factor Xa by selective inhibitors

    NASA Astrophysics Data System (ADS)

    Doan, B. T.; Fraternali, F.; Do, Q. T.; Atkinson, R. A.; Palmas, P.; Sklenar, V.; Wildgoose, P.; Strop, P.; Saudek, V.

    1998-02-01

    The structure of two selective inhibitors obtained by the screening of a vast combinatorial library, Ac-Tyr-Ile-Arg-Ile-NH2 and Ac-(4-amino-Phe)-(Cyc.-Gly)-NH2, in the active site of the blood clotting enzyme factor Xa was determined using transferred NOE NMR and simulated annealing (SA) under NMR constraints. The refined structures of the inhibitors were docked in the active site and SA was performed inside the enzyme which has been kept as a rigid charged template. The final structures were optimised by molecular dynamics simulation of the complexes in water. The inhibitors assume a compact, very well defined conformation embedded in the binding site without blocking the catalysis. The model allows to explain the mode of action, affinity and specificity. L'étude structurale d'inhibiteurs du facteur Xa, une enzyme de coagulation, obtenus par chimie combinatoire : Ac-Tyr-Ile-Arg-Ile-NH2, Ac-(4-amino-Phe)-(Cyc.-Gly)-NH2, a été réalisée par RMN NOE de transfert et modélisation moléculaire. Les structures ont été calculées sous contraintes RMN : géométrie de distance, recuit simulé et minimisation, affinées par une recherche conformationnelle et recuit de l'inhibiteur placé dans le site actif et optimisées par simulation de dynamique moléculaire du complexe dans l'eau. L'inhibiteur présente une structure compacte positionnée dans le site d'interaction hors d'accès du site catalytique. Ce modèle permet d'expliquer le mode d'action, l'affinité et la spécificité des peptides.

  11. A unique geometry of the active site of angiotensin-converting enzyme consistent with structure-activity studies

    NASA Astrophysics Data System (ADS)

    Mayer, Dorica; Naylor, Christopher B.; Motoc, Ioan; Marshall, Garland R.

    1987-04-01

    Previous structure-activity studies of captopril and related active angiotensin-converting enzyme (ACE) inhibitors have led to the conclusion that the basic structural requirements for inhibition of ACE involve (a) a terminal carboxyl group; (b) an amido carbonyl group; and (c) different types of effective zinc (Zn) ligand functional groups. Such structural requirements common to a set of compounds acting at the same receptor have been used to define a pharmacophoric pattern of atoms or groups of atoms mutually oriented in space that is necessary for ACE inhibition from a stereochemical point of view. A unique pharmacophore model (within the resolution of approximately 0.15 Å) was observed using a method for systematic search of the conformational hyperspace available to the 28 structurally different molecules under study. The method does not assume a common molecular framework, and, therefore, allows comparison of different compounds that is independent of their absolute orientation. Consequently, by placing the carboxyl binding group, the binding site for amido carbonyl, and the Zn atom site in positions determined by ideal binding geometry with the inhibitors' functional groups, it was possible to clearly specify a geometry for the active site of ACE.

  12. Chronic Inflammation and Neutrophil Activation as Possible Causes of Joint Diseases in Ballet Dancers

    PubMed Central

    Borges, Leandro da Silva; Santos, Vinicius Coneglian; de Moura, Nivaldo Ribeiro; Dermargos, Alexandre; Cury-Boaventura, Maria Fernanda; Gorjão, Renata; Pithon-Curi, Tania Cristina; Hatanaka, Elaine

    2014-01-01

    Herein, we investigated the effects of a ballet class on the kinetic profiles of creatine kinase (CK) and lactate dehydrogenase (LDH) activities, cytokines, complement component 3 (C3), and the concentrations of immunoglobulin (Ig), IgA and IgM, in ballerinas. We also verified neutrophil death and ROS release. Blood samples were taken from 13 dancers before, immediately after, and 18 hours after a ballet class. The ballet class increased the plasma activities of CK-total (2.0-fold) immediately after class, while the activities of CK-cardiac muscle (1.0-fold) and LDH (3.0-fold) were observed to increase 18 hours after the class. Levels of the TNF-α, IL-1β, IgG, and IgA were not affected under the study conditions. The exercise was found to induce neutrophil apoptosis (6.0-fold) 18 hours after the ballet class. Additionally, immediately after the ballet class, the neutrophils from the ballerinas were found to be less responsive to PMA stimulus. Conclusion. Ballet class was found to result in inflammation in dancers. The inflammation caused by the ballet class remained for 18 hours after the exercise. These findings are important in preventing the development of chronic lesions that are commonly observed in dancers, such as those with arthritis and synovitis. PMID:24701035

  13. Somatic activating mutations in Pik3ca cause sporadic venous malformations in mice and humans.

    PubMed

    Castillo, Sandra D; Tzouanacou, Elena; Zaw-Thin, May; Berenjeno, Inma M; Parker, Victoria E R; Chivite, Iñigo; Milà-Guasch, Maria; Pearce, Wayne; Solomon, Isabelle; Angulo-Urarte, Ana; Figueiredo, Ana M; Dewhurst, Robert E; Knox, Rachel G; Clark, Graeme R; Scudamore, Cheryl L; Badar, Adam; Kalber, Tammy L; Foster, Julie; Stuckey, Daniel J; David, Anna L; Phillips, Wayne A; Lythgoe, Mark F; Wilson, Valerie; Semple, Robert K; Sebire, Neil J; Kinsler, Veronica A; Graupera, Mariona; Vanhaesebroeck, Bart

    2016-03-30

    Venous malformations (VMs) are painful and deforming vascular lesions composed of dilated vascular channels, which are present from birth. Mutations in the TEK gene, encoding the tyrosine kinase receptor TIE2, are found in about half of sporadic (nonfamilial) VMs, and the causes of the remaining cases are unknown. Sclerotherapy, widely accepted as first-line treatment, is not fully efficient, and targeted therapy for this disease remains underexplored. We have generated a mouse model that faithfully mirrors human VM through mosaic expression of Pik3ca(H1047R), a constitutively active mutant of the p110α isoform of phosphatidylinositol 3-kinase (PI3K), in the embryonic mesoderm. Endothelial expression of Pik3ca(H1047R)resulted in endothelial cell (EC) hyperproliferation, reduction in pericyte coverage of blood vessels, and decreased expression of arteriovenous specification markers. PI3K pathway inhibition with rapamycin normalized EC hyperproliferation and pericyte coverage in postnatal retinas and stimulated VM regression in vivo. In line with the mouse data, we also report the presence of activating PIK3CA mutations in human VMs, mutually exclusive with TEK mutations. Our data demonstrate a causal relationship between activating Pik3ca mutations and the genesis of VMs, provide a genetic model that faithfully mirrors the normal etiology and development of this human disease, and establish the basis for the use of PI3K-targeted therapies in VMs.

  14. Large zinc cation occupancy of octahedral sites in mechanically activated zinc ferrite powders

    SciTech Connect

    Oliver, S. A.; Harris, V. G.; Hamdeh, H. H.; Ho, J. C.

    2000-05-08

    The cation site occupancy of a mechanically activated nanocrystalline zinc ferrite powder was determined as (Zn{sub 0.55}{sup 2+}Fe{sub 0.18}{sup 3+}){sub tet}[Zr{sub 0.45}{sup 2+}Fe{sub 1.82}{sup 3+}]{sub oct}O{sub 4} through analysis of extended x-ray absorption fine structure measurements, showing a large redistribution of cations between sites compared to normal zinc ferrite samples. The overpopulation of cations in the octahedral sites was attributed to the ascendance in importance of the ionic radii over the crystal energy and bonding coordination in determining which interstitial sites are occupied in this structurally disordered powder. Slight changes are observed in the local atomic environment about the zinc cations, but not the iron cations, with respect to the spinel structure. The presence of Fe{sup 3+} on both sites is consistent with the measured room temperature magnetic properties. (c) 2000 American Institute of Physics.

  15. Foreign Glycoproteins Can Be Actively Recruited to Virus Assembly Sites during Pseudotyping▿

    PubMed Central

    Jorgenson, Rebecca L.; Vogt, Volker M.; Johnson, Marc C.

    2009-01-01

    Retroviruses like human immunodeficiency virus type 1 (HIV-1), as well as many other enveloped viruses, can efficiently produce infectious virus in the absence of their own surface glycoprotein if a suitable glycoprotein from a foreign virus is expressed in the same cell. This process of complementation, known as pseudotyping, often can occur even when the glycoprotein is from an unrelated virus. Although pseudotyping is widely used for engineering chimeric viruses, it has remained unknown whether a virus can actively recruit foreign glycoproteins to budding sites or, alternatively, if a virus obtains the glycoproteins through a passive mechanism. We have studied the specificity of glycoprotein recruitment by immunogold labeling viral glycoproteins and imaging their distribution on the host plasma membrane using scanning electron microscopy. Expressed alone, all tested viral glycoproteins were relatively randomly distributed on the plasma membrane. However, in the presence of budding HIV-1 or Rous sarcoma virus (RSV) particles, some glycoproteins, such as those encoded by murine leukemia virus and vesicular stomatitis virus, were dramatically redistributed to viral budding sites. In contrast, the RSV Env glycoprotein was robustly recruited only to the homologous RSV budding sites. These data demonstrate that viral glycoproteins are not in preformed membrane patches prior to viral assembly but rather that glycoproteins are actively recruited to certain viral assembly sites. PMID:19224995

  16. Barriers to physical activity in an on-site corporate fitness center.

    PubMed

    Schwetschenau, Heather M; O'Brien, William H; Cunningham, Christopher J L; Jex, Steve M

    2008-10-01

    Many corporations provide employees the option of participating in on-site fitness centers, but utilization rates are low. Perceived barriers to physical activity have been established as important correlates of physical activity, and recent research indicates that barriers may vary across settings. Work-site fitness centers may present unique barriers to participation, but there are currently no standardized measures that assess such barriers. Eighty-eight employees of a midwestern corporation completed a survey designed to identify and evaluate the extent to which barriers influence participation in an on-site corporate fitness center. Regression analyses revealed that external environmental barriers (e.g., inadequate exercise facilities) significantly accounted for not joining the fitness center, and for decreased duration of visits to the facility among members. Internal barriers (e.g., feeling embarrassed to exercise around coworkers) significantly accounted for frequency of fitness center visits among members. This corporate specific measure may lead to more effective interventions aimed to increase use of on-site corporate fitness centers.

  17. Den site activity patterns of adult male and female swift foxes, Vulpes velox, in Northwestern Texas

    USGS Publications Warehouse

    Lemons, P.R.; Ballard, W.B.; Sullivan, R.M.; Sovada, M.A.

    2003-01-01

    Activity of Swift Foxes (Vulpes velox) at den sites was studied in northwestern Texas during pup rearing seasons in 2000 and 2001 to determine role of males in parental care. Twenty-four percent of radio-collared females with a potential to breed successfully raised pups to eight weeks of age. We intensively monitored presence and absence of male and female Swift Foxes at two den sites each year. Females were present >2.6 times more at den sites than males during the pup rearing season. Female and male Swift Foxes largely stayed at dens during diurnal hours and were active away from dens during nocturnal and crepuscular hours. Females and males spent 12.4% and 3.0% more time at dens before pups emerged, than after pups emerged, respectively. Following depredation of one male parent, the female spent 29% less time at the den site. Decrease in time spent at the den by the female following loss of her mate suggested that loss of one parent might severely impact recruitment of Swift Foxes. Our observations indicated that intense Coyote (Canis latrans) depredation may severely impact pup-rearing success as well as the parental care within Swift Fox family groups.

  18. The pepsin residue glycine-76 contributes to active-site loop flexibility and participates in catalysis.

    PubMed Central

    Okoniewska, M; Tanaka, T; Yada, R Y

    2000-01-01

    Glycine residues are known to contribute to conformational flexibility of polypeptide chains, and have been found to contribute to flexibility of some loops associated with enzymic catalysis. A comparison of porcine pepsin in zymogen, mature and inhibited forms revealed that a loop (a flap), consisting of residues 71--80, located near the active site changed its position upon substrate binding. The loop residue, glycine-76, has been implicated in the catalytic process and thought to participate in a hydrogen-bond network aligning the substrate. This study investigated the role of glycine-76 using site-directed mutagenesis. Three mutants, G76A, G76V and G76S, were constructed to increase conformational restriction of a polypeptide chain. In addition, the serine mutant introduced a hydrogen-bonding potential at position 76 similar to that observed in human renin. All the mutants, regardless of amino acid size and polarity, had lower catalytic efficiency and activated more slowly than the wild-type enzyme. The slower activation process was associated directly with altered proteolytic activity. Consequently, it was proposed that a proteolytic cleavage represents a limiting step of the activation process. Lower catalytic efficiency of the mutants was explained as a decrease in the flap flexibility and, therefore, a different pattern of hydrogen bonds responsible for substrate alignment and flap conformation. The results demonstrated that flap flexibility is essential for efficient catalytic and activation processes. PMID:10861225

  19. Improving the activity of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation.

    PubMed

    Weng, Meizhi; Deng, Xiongwei; Bao, Wei; Zhu, Li; Wu, Jieyuan; Cai, Yongjun; Jia, Yan; Zheng, Zhongliang; Zou, Guolin

    2015-09-25

    Nattokinase (NK), a bacterial serine protease from Bacillus subtilis var. natto, is a potential cardiovascular drug exhibiting strong fibrinolytic activity. To broaden its commercial and medical applications, we constructed a single-mutant (I31L) and two double-mutants (M222A/I31L and T220S/I31L) by site-directed mutagenesis. Active enzymes were expressed in Escherichia coli with periplasmic secretion and were purified to homogeneity. The kinetic parameters of enzymes were examined by spectroscopy assay and isothermal titration calorimetry (ITC), and their fibrinolytic activities were determined by fibrin plate method. The substitution of Leu(31) for Ile(31) resulted in about 2-fold enhancement of catalytic efficiency (Kcat/KM) compared with wild-type NK. The specific activities of both double-mutants (M222A/I31L and T220S/I31L) were significantly increased when compared with the single-mutants (M222A and T220S) and the oxidative stability of M222A/I31L mutant was enhanced with respect to wild-type NK. This study demonstrates the feasibility of improving activity of NK by site-directed mutagenesis and shows successful protein engineering cases to improve the activity of NK as a potent therapeutic agent.

  20. Linde FUSRAP Site Remediation: Engineering Challenges and Solutions of Remedial Activities on an Active Industrial Facility - 13506

    SciTech Connect

    Beres, Christopher M.; Fort, E. Joseph; Boyle, James D.

    2013-07-01

    The Linde FUSRAP Site (Linde) is located in Tonawanda, New York at a major research and development facility for Praxair, Inc. (Praxair). Successful remediation activities at Linde combines meeting cleanup objectives of radiological contamination while minimizing impacts to Praxair business operations. The unique use of Praxair's property coupled with an array of active and abandoned utilities poses many engineering and operational challenges; each of which has been overcome during the remedial action at Linde. The U.S. Army Corps of Engineers - Buffalo District (USACE) and CABRERA SERVICES, INC. (CABRERA) have successfully faced engineering challenges such as relocation of an aboveground structure, structural protection of an active water line, and installation of active mechanical, electrical, and communication utilities to perform remediation. As remediation nears completion, continued success of engineering challenges is critical as remaining activities exist in the vicinity of infrastructure essential to business operations; an electrical substation and duct bank providing power throughout the Praxair facility. Emphasis on engineering and operations through final remediation and into site restoration will allow for the safe and successful completion of the project. (authors)

  1. PARP promoter-mediated activation of a VSG expression site promoter in insect form Trypanosoma brucei.

    PubMed

    Urményi, T P; Van der Ploeg, L H

    1995-03-25

    In trypanosomes the rRNA, PARP and VSG gene promoters mediate alpha-amanitin-resistant transcription of protein coding genes, presumably by RNA polymerase (pol) I. We compared the activity of PARP and VSG promoters integrated at one of the alleles of the largest subunit of pol II genes in insect form trypanosomes. Even though both promoters are roughly equally active in transient transformation assays in insect form trypanosomes, only the PARP promoter functioned effectively when integrated at the pol II largest subunit or other loci. Promoter activity in transient transformation assays is therefore not necessarily predictive of transcriptional activity once integrated into the trypanosome genome. The integrated fully active PARP promoter could upregulate in cis an otherwise poorly active integrated VSG promoter. The PARP promoter nucleotide sequence elements responsible for VSG promoter activation coincided with most of the important PARP promoter elements mapped previously by linker scanning mutagenesis, indicating that it is not a single unique promoter element that was responsible for VSG promoter activation. The data suggest that PARP promoter-mediated activation of the VSG promoter does not result from complementation of the VSG promoter with a single insect form-specific transcription factor whose binding site is missing from the VSG promoter and present in the PARP promoter. We favor a model in which chromatin structure at the locus is altered by the PARP promoter, allowing VSG promoter activation in insect form trypanosomes. We discuss the significance of these observations for the control of VSG promoters in insect form trypanosomes.

  2. Active-site titration analysis of surface influence on immobilized Candida antarctica Lipase B activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Matrix morphology and surface polarity effects were investigated for Candida antarctica lipase B immobilization. Measurements of the amount of lipase immobilized (bicinchoninic acid method) and the catalyst’s tributyrin hydrolysis activity, coupled with a determination of the lipase’s functional fr...

  3. Mutation of active site residues in synthetic T4-lysozyme gene and their effect on lytic activity.

    PubMed

    Anand, N N; Stephen, E R; Narang, S A

    1988-06-16

    The active site amino acids (Glu11 and Asp20) in T4-lysozyme have been mutated to their isosteric residues Gln or Asn and/or acidic residues such as Glu----Asp or Asp----Glu by the oligonucleotide-replacement method. Out of eight mutants so generated the mutant T4-lysozyme obtained from pTLY.Asp11 retains maximum amount of activity (approximately 16%), pTLY.Asn20 the least (0.9%) whereas pTLY.Gln11 lost completely. A systematic study of the active and inactive mutants thus generated supports the important role of Glu11 and Asp20 in T4-lysozyme activity as predicted in earlier studies.

  4. 78 FR 21352 - Update on Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-04-10

    ... on Reimbursement for Costs of Remedial Action at Active Uranium and Thorium Processing Sites AGENCY... reimbursement for cleanup work performed by licensees at eligible uranium and thorium processing sites in... licensees of eligible uranium and thorium processing sites. If licensees submit claims in FY 2013,...

  5. Leukocyte adhesion deficiency-III is caused by mutations in KINDLIN3 affecting integrin activation

    PubMed Central

    Svensson, Lena; Howarth, Kimberley; McDowall, Alison; Patzak, Irene; Evans, Rachel; Ussar, Siegfried; Moser, Markus; Metin, Ayse; Fried, Mike; Tomlinson, Ian; Hogg, Nancy

    2009-01-01

    Integrins are the major adhesion receptors of leukocytes and platelets. β1 and β2 integrin function on leukocytes is crucial for a successful immune response and the platelet integrin αIIbβ3 initiates the process of blood clotting through binding fibrinogen1-3. Integrins on circulating cells bind poorly to their ligands but become active after ‘inside-out’ signaling through other membrane receptors4,5. Subjects with leukocyte adhesion deficiency-1 (LAD-I) do not express β2 integrins because of mutations in the gene specifying the β2 subunit, and they suffer recurrent bacterial infections6,7. Mutations affecting αIIbβ3 integrin cause the bleeding disorder termed Glanzmann’s thrombasthenia3. Subjects with LAD-III show symptoms of both LAD-I and Glanzmann’s thrombasthenia. Their hematopoietically-derived cells express β1, β2 and β3 integrins, but defective inside-out signaling causes immune deficiency and bleeding problems8. The LAD-III lesion has been attributed to a C→A mutation in the gene encoding calcium and diacylglycerol guanine nucleotide exchange factor (CALDAGGEF1; official symbol RASGRP2) specifying the CALDAG-GEF1 protein9, but we show that this change is not responsible for the LAD-III disorder. Instead, we identify mutations in the KINDLIN3 (official symbol FERMT3) gene specifying the KINDLIN-3 protein as the cause of LAD-III in Maltese and Turkish subjects. Two independent mutations result in decreased KINDLIN3 messenger RNA levels and loss of protein expression. Notably, transfection of the subjects’ lymphocytes with KINDLIN3 complementary DNA but not CALDAGGEF1 cDNA reverses the LAD-III defect, restoring integrin-mediated adhesion and migration. PMID:19234463

  6. Discovery of Active Hydrothermal Sites Along the Mariana Volcanic Arc, Western Pacific Ocean

    NASA Astrophysics Data System (ADS)

    Baker, E. T.; Embley, R. W.; Resing, J. A.; Lupton, J. E.; Massoth, G. J.; de Ronde, C. E.; Nakamura, K.; Walker, S. L.

    2003-12-01

    Some 20,000 km of volcanic arcs, roughly one-third the total length of the global midocean ridge (MOR) system, rim the western Pacific Ocean. But compared to 25 years of hydrothermal investigations along MORs, exploration of similar activity on the estimated 600 submarine arc volcanoes is only beginning. In February 2003, as part of the Submarine Ring of Fire project funded by NOAA's Ocean Exploration Program, we made the first systematic survey of hydrothermal activity along the 1270-km-long Mariana intraoceanic volcanic arc, which lies almost entirely within the US EEZ. Prior fieldwork had documented active (but low-temperature) hydrothermal discharge on only three volcanoes: Kasuga 2, Kasuga 3, and Esmeralda Bank. During the cruise, we conducted 70 CTD operations over more than 50 individual volcanoes from 13° N to 23° N, plus a continuous CTD survey along 75 km of the back-arc spreading center (13° 15'N to 13° 41'N) adjacent to the southern end of the arc. We found evidence for active hydrothermal venting at 11 submarine volcanoes with summit (or caldera floor) depths ranging from 50 to 1550 m. Two additional sites were identified on the back-arc spreading center. Ongoing analyses of collected water samples could increase these totals. Our results confirmed continuing hydrothermal activity at Kasuga 2 (but not Kasuga 3) and Esmeralda Bank, in addition to newly discovered sites on nine other volcanoes. Many of these sites produce intense and widely dispersed plumes indicative of vigorous, high-temperature discharge. The volcanoes with active hydrothermal systems are about equally divided between those with and without summit calderas. The addition of the Marianas data greatly improves our view of hydrothermal sources along arcs. The 20,000 km of Pacific arcs can be divided between 6380 km of intraoceanic (i.e., mostly submarine) arcs and 13,880 km of island (i.e., mostly subaerial) arcs. At present, ˜15% of the total length of Pacific arcs has been surveyed

  7. Synthesis of supported bimetallic nanoparticles with controlled size and composition distributions for active site elucidation

    SciTech Connect

    Hakim, Sikander H.; Sener, Canan; Alba Rubio, Ana C.; Gostanian, Thomas M.; O'neill, Brandon J; Ribeiro, Fabio H.; Miller, Jeffrey T.; Dumesic, James A

    2015-08-01

    Elucidation of active sites in supported bimetallic catalysts is complicated by the high level of dispersity in the nanoparticle size and composition that is inherent in conventional methods of catalyst preparation. We present a synthesis strategy that leads to highly dispersed, bimetallic nanoparticles with uniform particle size and composition by means of controlled surface reactions. We demonstrate the synthesis of three systems, RhMo, PtMo, and RhRe, consisting of a highly reducible metal with an oxophilic promoter. These catalysts are characterized by FTIR, CO chemisorption, STEM/EDS, TPR, and XAS analysis. The catalytic properties of these bimetallic nanoparticles were probed for the selective CO hydrogenolysis of (hydroxymethyl)tetrahydropyran to produce 1,6 hexanediol. Based on the characterization results and reactivity trends, the active sites in the hydrogenolysis reaction are identified to be small ensembles of the more noble metal (Rh, Pt) adjacent to highly reduced moieties of the more oxophilic metal (Mo, Re).

  8. Dynamics and Mechanism of Efficient DNA Repair Reviewed by Active-Site Mutants

    NASA Astrophysics Data System (ADS)

    Tan, Chuang; Liu, Zheyun; Li, Jiang; Guo, Xunmin; Wang, Lijuan; Zhong, Dongping

    2010-06-01

    Photolyases repair the UV-induced pyrimidine dimers in damage DNA via a photoreaction which includes a series of light-driven electron transfers between the two-electron-reduced flavin cofactor FADH^- and the dimer. We report here our systematic studies of the repair dynamics in E. coli photolyase with mutation of several active-site residues. With femtosecond resolution, we observed the significant change in the forward electron transfer from the excited FADH^- to the dimer and the back electron transfer from the repaired thymines by mutation of E274A, R226A, R342A, N378S and N378C. We also found that the mutation of E274A accelerates the bond-breaking of the thymine dimer. The dynamics changes are consistent with the quantum yield study of these mutants. These results suggest that the active-site residues play a significant role, structurally and chemically, in the DNA repair photocycle.

  9. Extreme electric fields power catalysis in the active site of ketosteroid isomerase.

    PubMed

    Fried, Stephen D; Bagchi, Sayan; Boxer, Steven G

    2014-12-19

    Enzymes use protein architecture to impose specific electrostatic fields onto their bound substrates, but the magnitude and catalytic effect of these electric fields have proven difficult to quantify with standard experimental approaches. Using vibrational Stark effect spectroscopy, we found that the active site of the enzyme ketosteroid isomerase (KSI) exerts an extremely large electric field onto the C=O chemical bond that undergoes a charge rearrangement in KSI's rate-determining step. Moreover, we found that the magnitude of the electric field exerted by the active site strongly correlates with the enzyme's catalytic rate enhancement, enabling us to quantify the fraction of the catalytic effect that is electrostatic in origin. The measurements described here may help explain the role of electrostatics in many other enzymes and biomolecular systems.

  10. The active site of RNA polymerase II participates in transcript cleavage within arrested ternary complexes.

    PubMed Central

    Rudd, M D; Izban, M G; Luse, D S

    1994-01-01

    RNA polymerase II may become arrested during transcript elongation, in which case the ternary complex remains intact but further RNA synthesis is blocked. To relieve arrest, the nascent transcript must be cleaved from the 3' end. RNAs of 7-17 nt are liberated and transcription continues from the newly exposed 3' end. Factor SII increases elongation efficiency by strongly stimulating the transcript cleavage reaction. We show here that arrest relief can also occur by the addition of pyrophosphate. This generates the same set of cleavage products as factor SII, but the fragments produced with pyrophosphate have 5'-triphosphate termini. Thus, the active site of RNA polymerase II, in the presence of pyrophosphate, appears to be capable of cleaving phosphodiester linkages as far as 17 nt upstream of the original site of polymerization, leaving the ternary complex intact and transcriptionally active. Images PMID:8058756

  11. Twinning in fcc lattice creates low-coordinated catalytically active sites in porous gold

    NASA Astrophysics Data System (ADS)

    Krajčí, Marian; Kameoka, Satoshi; Tsai, An-Pang

    2016-08-01

    We describe a new mechanism for creation of catalytically active sites in porous gold. Samples of porous gold prepared by de-alloying Al2Au exhibit a clear correlation between the catalytic reactivity towards CO oxidation and structural defects in the fcc lattice of Au. We have found that on the stepped {211} surfaces quite common twin boundary defects in the bulk structure of porous gold can form long close-packed rows of atoms with the coordination number CN = 6. DFT calculations confirm that on these low-coordinated Au sites dioxygen chemisorbs and CO oxidation can proceed via the Langmuir-Hinshelwood mechanism with the activation energy of 37 kJ/mol or via the CO-OO intermediate with the energy barrier of 19 kJ/mol. The existence of the twins in porous gold is stabilized by the surface energy.

  12. Twinning in fcc lattice creates low-coordinated catalytically active sites in porous gold.

    PubMed

    Krajčí, Marian; Kameoka, Satoshi; Tsai, An-Pang

    2016-08-28

    We describe a new mechanism for creation of catalytically active sites in porous gold. Samples of porous gold prepared by de-alloying Al2Au exhibit a clear correlation between the catalytic reactivity towards CO oxidation and structural defects in the fcc lattice of Au. We have found that on the stepped {211} surfaces quite common twin boundary defects in the bulk structure of porous gold can form long close-packed rows of atoms with the coordination number CN = 6. DFT calculations confirm that on these low-coordinated Au sites dioxygen chemisorbs and CO oxidation can proceed via the Langmuir-Hinshelwood mechanism with the activation energy of 37 kJ/mol or via the CO-OO intermediate with the energy barrier of 19 kJ/mol. The existence of the twins in porous gold is stabilized by the surface energy.

  13. 13C-Methyl isocyanide as an NMR probe for cytochrome P450 active sites

    PubMed Central

    McCullough, Christopher R.; Pullela, Phani Kumar; Im, Sang-Choul; Waskell, Lucy

    2012-01-01

    The cytochromes P450 (CYPs) play a central role in many biologically important oxidation reactions, including the metabolism of drugs and other xenobiotic compounds. Because they are often assayed as both drug targets and anti-targets, any tools that provide: (a) confirmation of active site binding and (b) structural data, would be of great utility, especially if data could be obtained in reasonably high throughput. To this end, we have developed an analog of the promiscuous heme ligand, cyanide, with a 13CH3-reporter attached. This 13C-methyl isocyanide ligand binds to bacterial (P450cam) and membrane-bound mammalian (CYP2B4) CYPs. It can be used in a rapid 1D experiment to identify binders, and provides a qualitative measure of structural changes in the active site. PMID:19199046

  14. Computation of Rate Constants for Diffusion of Small Ligands to and from Buried Protein Active Sites.

    PubMed

    Wang, P-H; De Sancho, D; Best, R B; Blumberger, J

    2016-01-01

    The diffusion of ligands to actives sites of proteins is essential to enzyme catalysis and many cellular signaling processes. In this contribution we review our recently developed methodology for calculation of rate constants for diffusion and binding of small molecules to buried protein active sites. The diffusive dynamics of the ligand obtained from molecular dynamics simulation is coarse grained and described by a Markov state model. Diffusion and binding rate constants are then obtained either from the reactive flux formalism or by fitting the time-dependent population of the Markov state model to a phenomenological rate law. The method is illustrated by applications to diffusion of substrate and inhibitors in [NiFe] hydrogenase, CO-dehydrogenase, and myoglobin. We also discuss a recently developed sensitivity analysis that allows one to identify hot spots in proteins, where mutations are expected to have the strongest effects on ligand diffusion rates.

  15. Evaluation of larvicidal activity of biogenic nanoparticles against filariasis causing Culex mosquito vector

    PubMed Central

    Dhanasekaran, Dharumadurai; Thangaraj, Ramasamy

    2013-01-01

    Objective To evaluate the larvicidal activity of biogenic nanoparticles against filariasis causing Culex mosquito vector. Methods The synthesized AgNPs were characterized by UV-vis. spectrum, Fourier transform infrared and X-ray diffraction. Larvae were exposed to varying concentrations of aqueous extract of synthesized AgNPs for 10 min. The different concentrations of 5, 2.5, 1.25, 0.625 and 0.312 mg/L silver nanoparticles were tested against the Culex larvae. Results The mortality rate of Agaricus bisporus biogenic nanoparticles against Culex larvae are 5 mg/L (100%), 2.5 mg/L (81%), 1.25 mg/L (62%), 0.625 mg/L (28%) and 0.312 mg/L (11%). Conclusions These results suggest that the synthesized biogenic AgNPs have the potential to be used as an ideal eco-friendly approach for controlling Culex larvae.

  16. Is autoimmune diabetes caused by aberrant immune activity or defective suppression of physiological self-reactivity?

    PubMed

    Askenasy, Enosh M; Askenasy, Nadir

    2013-03-01

    Two competing hypotheses are proposed to cause autoimmunity: evasion of a sporadic self-reactive clone from immune surveillance and ineffective suppression of autoreactive clones that arise physiologically. We question the relevance of these hypotheses to the study of type 1 diabetes, where autoreactivity may accompany the cycles of physiological adjustment of β-cell mass to body weight and nutrition. Experimental evidence presents variable and conflicting data concerning the activities of both effector and regulatory T cells, arguing in favor and against: quantitative dominance and deficit, aberrant reactivity and expansion, sensitivity to negative regulation and apoptosis. The presence of autoantibodies in umbilical cord blood of healthy subjects and low incidence of the disease following early induction suggest that suppression of self-reactivity is the major determinant factor.

  17. Activation of Helicobacter pylori causes either autoimmune thyroid diseases or carcinogenesis in the digestive tract.

    PubMed

    Astl, J; Šterzl, I

    2015-01-01

    Helicobacter pylori has been implicated in stimulation of immune system, development of autoimmune endocrinopathies as autoimmune thyroiditis (AT) and on other hand induction of immunosupresion activates gastric and extra-gastric diseases such as gastric ulcer or cancer. It causes persistent lifelong infection despite local and systemic immune response. Our results indicate that Helicobacter pylori might cause inhibition of the specific cellular immune response in Helicobacter pylori-infected patients with or without autoimmune diseases such as AT. We cannot also declare the carcinogenic effect in oropharynx. However the association of any infection agents and cancerogenesis exists. The adherence of Helicobacter pylori expression and enlargement of benign lymphatic tissue and the high incidence of the DNA of Helicobacter pylori in laryngopharyngeal and oropharyngeal cancer is reality. LTT appears to be a good tool for detection of immune memory cellular response in patients with Helicobacter pylori infection and AT. All these complications of Helicobacter pylori infection can be abrogated by successful eradication of Helicobacter pylori.

  18. A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior.

    PubMed

    Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H; Muyldermans, Serge; Declerck, Paul J; Huang, Mingdong; Andreasen, Peter A; Ngo, Jacky Chi Ki

    2016-07-15

    A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors.

  19. Myopathic Lamin Mutations Cause Reductive Stress and Activate the Nrf2/Keap-1 Pathway

    PubMed Central

    Dialynas, George; Shrestha, Om K.; Ponce, Jessica M.; Zwerger, Monika; Thiemann, Dylan A.; Young, Grant H.; Moore, Steven A.; Yu, Liping; Lammerding, Jan; Wallrath, Lori L.

    2015-01-01

    Mutations in the human LMNA gene cause muscular dystrophy by mechanisms that are incompletely understood. The LMNA gene encodes A-type lamins, intermediate filaments that form a network underlying the inner nuclear membrane, providing structural support for the nucleus and organizing the genome. To better understand the pathogenesis caused by mutant lamins, we performed a structural and functional analysis on LMNA missense mutations identified in muscular dystrophy patients. These mutations perturb the tertiary structure of the conserved A-type lamin Ig-fold domain. To identify the effects of these structural perturbations on lamin function, we modeled these mutations in Drosophila Lamin C and expressed the mutant lamins in muscle. We found that the structural perturbations had minimal dominant effects on nuclear stiffness, suggesting that the muscle pathology was not accompanied by major structural disruption of the peripheral nuclear lamina. However, subtle alterations in the lamina network and subnuclear reorganization of lamins remain possible. Affected muscles had cytoplasmic aggregation of lamins and additional nuclear envelope proteins. Transcription profiling revealed upregulation of many Nrf2 target genes. Nrf2 is normally sequestered in the cytoplasm by Keap-1. Under oxidative stress Nrf2 dissociates from Keap-1, translocates into the nucleus, and activates gene expression. Unexpectedly, biochemical analyses revealed high levels of reducing agents, indicative of reductive stress. The accumulation of cytoplasmic lamin aggregates correlated with elevated levels of the autophagy adaptor p62/SQSTM1, which also binds Keap-1, abrogating Nrf2 cytoplasmic sequestration, allowing Nrf2 nuclear translocation and target gene activation. Elevated p62/SQSTM1 and nuclear enrichment of Nrf2 were identified in muscle biopsies from the corresponding muscular dystrophy patients, validating the disease relevance of our Drosophila model. Thus, novel connections were made

  20. Cox-dependent fatty acid metabolites cause pain through activation of the irritant receptor TRPA1.

    PubMed

    Materazzi, Serena; Nassini, Romina; Andrè, Eunice; Campi, Barbara; Amadesi, Silvia; Trevisani, Marcello; Bunnett, Nigel W; Patacchini, Riccardo; Geppetti, Pierangelo

    2008-08-19

    Prostaglandins (PG) are known to induce pain perception indirectly by sensitizing nociceptors. Accordingly, the analgesic action of nonsteroidal anti-inflammatory drugs (NSAIDs) results from inhibition of cyclooxygenases and blockade of PG biosynthesis. Cyclopentenone PGs, 15-d-PGJ(2), PGA(2), and PGA(1), formed by dehydration of their respective parent PGs, PGD(2), PGE(2), and PGE(1), possess a highly reactive alpha,beta-unsaturated carbonyl group that has been proposed to gate the irritant transient receptor potential A1 (TRPA1) channel. Here, by using TRPA1 wild-type (TRPA1(+/+)) or deficient (TRPA1(-/-)) mice, we show that cyclopentenone PGs produce pain by direct stimulation of nociceptors via TRPA1 activation. Cyclopentenone PGs caused a robust calcium response in dorsal root ganglion (DRG) neurons of TRPA1(+/+), but not of TRPA1(-/-) mice, and a calcium-dependent release of sensory neuropeptides from the rat dorsal spinal cord. Intraplantar injection of cyclopentenone PGs stimulated c-fos expression in spinal neurons of the dorsal horn and evoked an instantaneous, robust, and transient nociceptive response in TRPA1(+/+) but not in TRPA1(-/-) mice. The classical proalgesic PG, PGE(2), caused a slight calcium response in DRG neurons, increased c-fos expression in spinal neurons, and induced a delayed and sustained nociceptive response in both TRPA1(+/+) and TRPA1(-/-) mice. These results expand the mechanism of NSAID analgesia from blockade of indirect nociceptor sensitization by classical PGs to inhibition of direct TRPA1-dependent nociceptor activation by cyclopentenone PGs. Thus, TRPA1 antagonism may contribute to suppress pain evoked by PG metabolites without the adverse effects of inhibiting cyclooxygenases.

  1. Cannabidiol causes endothelium-dependent vasorelaxation of human mesenteric arteries via CB1 activation

    PubMed Central

    Stanley, Christopher P.; Hind, William H.; Tufarelli, Cristina; O'Sullivan, Saoirse E.

    2015-01-01

    Aims The protective effects of cannabidiol (CBD) have been widely shown in preclinical models and have translated into medicines for the treatment of multiple sclerosis and epilepsy. However, the direct vascular effects of CBD in humans are unknown. Methods and results Using wire myography, the vascular effects of CBD were assessed in human mesenteric arteries, and the mechanisms of action probed pharmacologically. CBD-induced intracellular signalling was characterized using human aortic endothelial cells (HAECs). CBD caused acute, non-recoverable vasorelaxation of human mesenteric arteries with an Rmax of ∼40%. This was inhibited by cannabinoid receptor 1 (CB1) receptor antagonists, desensitization of transient receptor potential channels using capsaicin, removal of the endothelium, and inhibition of potassium efflux. There was no role for cannabinoid receptor-2 (CB2) receptor, peroxisome proliferator activated receptor (PPAR)γ, the novel endothelial cannabinoid receptor (CBe), or cyclooxygenase. CBD-induced vasorelaxation was blunted in males, and in patients with type 2 diabetes or hypercholesterolemia. In HAECs, CBD significantly reduced phosphorylated JNK, NFκB, p70s6 K and STAT5, and significantly increased phosphorylated CREB, ERK1/2, and Akt levels. CBD also increased phosphorylated eNOS (ser1177), which was correlated with increased levels of ERK1/2 and Akt levels. CB1 receptor antagonism prevented the increase in eNOS phosphorylation. Conclusion This study shows, for the first time, that CBD causes vasorelaxation of human mesenteric arteries via activation of CB1 and TRP channels, and is endothelium- and nitric oxide-dependent. PMID:26092099

  2. Interaction of mining activities and aquatic environment: A review from Greek mine sites.

    NASA Astrophysics Data System (ADS)

    Vasileiou, Eleni; Kallioras, Andreas

    2016-04-01

    In Greece a significant amount of mineral and ore deposits have been recorded accompanied by large industrial interest and a long mining history. Today many active and/or abandoned mine sites are scattered within the country; while mining activities take place in different sites for exploiting various deposits (clay, limestone, slate, gypsum, kaolin, mixed sulphide ores (lead, zinc, olivine, pozzolan, quartz lignite, nickel, magnesite, aluminum, bauxite, gold, marbles etc). The most prominent recent ones are: (i) the lignite exploitation that is extended in the area of Ptolemais (Western Macedonia) and Megalopolis (Central Peloponnese); and (ii) the major bauxite deposits located in central Greece within the Parnassos-Ghiona geotectonic zone and on Euboea Island. In the latter area, significant ores of magnesite were exploited and mixed sulphide ores. Centuries of intensive mining exploitation and metallurgical treatment of lead-silver deposits in Greece, have also resulted in significant abandoned sites, such as the one in Lavrion. Mining activities in Lavrio, were initiated in ancient times and continued until the 1980s, resulting in the production of significant waste stockpiles deposited in the area, crucial for the local water resources. Ιn many mining sites, environmental pressures are also recorded after the mine closure to the aquatic environment, as the surface waters flow through waste dump areas and contaminated soils. This paper aims to the geospatial visualization of the mining activities in Greece, in connection to their negative (surface- and/or ground-water pollution; overpumping due to extensive dewatering practices) or positive (enhanced groundwater recharge; pit lakes, improvement of water budget in the catchment scale) impacts on local water resources.

  3. BIOPHYSICS. Comment on "Extreme electric fields power catalysis in the active site of ketosteroid isomerase".

    PubMed

    Natarajan, Aditya; Yabukarski, Filip; Lamba, Vandana; Schwans, Jason P; Sunden, Fanny; Herschlag, Daniel

    2015-08-28

    Fried et al. (Reports, 19 December 2014, p. 1510) demonstrated a strong correlation between reaction rate and the carbonyl stretching frequency of a product analog bound to ketosteroid isomerase oxyanion hole mutants and concluded that the active-site electric field provides 70% of catalysis. Alternative comparisons suggest a smaller contribution, relative to the corresponding solution reaction, and highlight the importance of atomic-level descriptions.

  4. Disposal Activities and the Unique Waste Streams at the Nevada National Security Site (NNSS)

    SciTech Connect

    Arnold, P.

    2012-10-31

    This slide show documents waste disposal at the Nevada National Security Site. Topics covered include: radionuclide requirements for waste disposal; approved performance assessment (PA) for depleted uranium disposal; requirements; program approval; the Waste Acceptance Review Panel (WARP); description of the Radioactive Waste Acceptance Program (RWAP); facility evaluation; recent program accomplishments, nuclear facility safety changes; higher-activity waste stream disposal; and, large volume bulk waste streams.

  5. Analysis of Hydrogen Tunneling in an Enzyme Active Site using von Neumann Measurements

    PubMed Central

    Sumner, Isaiah; Iyengar, Srinivasan S.

    2010-01-01

    We build on our earlier quantum wavepacket study of hydrogen transfer in the biological enzyme, soybean lipoxygenase-1, by using von Neumann quantum measurement theory to gain qualitative insights into the transfer event. We treat the enzyme active site as a measurement device which acts on the tunneling hydrogen nucleus via the potential it exerts at each configuration. A series of changing active site geometries during the tunneling process effects a sequential projection of the initial, reactant state onto the final, product state. We study this process using several different kinds of von Neumann measurements and show how a discrete sequence of such measurements not only progressively increases the projection of the hydrogen nuclear wavepacket onto the product side but also favors proton over deuteron transfer. Several qualitative features of the hydrogen tunneling problem found in wavepacket dynamics studies are also recovered here. These include the shift in the “transition state” towards the reactant as a result of nuclear quantization, greater participation of excited states in the case of deuterium, and presence of critical points along the reaction coordinate that facilitate hydrogen and deuterium transfer and coincide with surface crossings. To further “tailor” the dynamics, we construct a perturbation to the sequence of measurements, that is a perturbation to the dynamical sequence of active site geometry evolution, which leads us to insight on the existence of sensitive regions of the reaction profile where subtle changes to the dynamics of the active site can have an effect on the hydrogen and deuterium transfer process. PMID:22933858

  6. Non-specific binding sites help to explain mixed inhibition in mushroom tyrosinase activities.

    PubMed

    Hassani, Sorour; Haghbeen, Kamahldin; Fazli, Mostafa

    2016-10-21

    Inhibition and activation studies of tyrosinase could prove beneficial to agricultural, food, cosmetic, and pharmaceutical industries. Although non-competitive and mixed-inhibition are frequent modes observed in kinetics studies on mushroom tyrosinase (MT) activities, the phenomena are left unexplained. In this study, dual effects of phthalic acid (PA) and cinnamic acid (CA) on MT during mono-phenolase activity were demonstrated. PA activated and inhibited MT at concentrations lower and higher than 150 μM, respectively. In contrast, CA inhibited and activated MT at concentrations lower and higher than 5 μM. The mode of inhibition for both effectors was mixed-type. Complex kinetics of MT in the presence of a modulator could partly be ascribed to its mixed-cooperativity. However, to explain mixed-inhibition mode, it is necessary to demonstrate how the ternary complex of substrate/enzyme/effector is formed. Therefore, we looked for possible non-specific binding sites using MT tropolone-bound PDB (2Y9X) in the computational studies. When tropolone was in MTPa (active site), PA and CA occupied different pockets (named MTPb and MTPc, respectively). The close Moldock scores of PA binding posed in MTPb and MTPa suggested that MTPb could be a secondary binding site for PA. Similar results were obtained for CA. Ensuing results from 10 ns molecular dynamics simulations for 2Y9X-effector complexes indicated that the structures were gradually stabilized during simulation. Tunnel analysis by using CAVER Analyst and CHEXVIS resulted in identifying two distinct channels that assumingly participate in exchanging the effectors when the direct channel to MTPa is not accessible.

  7. Crystal Structure of Albaflavenone Monooxygenase Containing a Moonlighting Terpene Synthase Active Site*

    PubMed Central

    Zhao, Bin; Lei, Li; Vassylyev, Dmitry G.; Lin, Xin; Cane, David E.; Kelly, Steven L.; Yuan, Hang; Lamb, David C.; Waterman, Michael R.

    2009-01-01

    Albaflavenone synthase (CYP170A1) is a monooxygenase catalyzing the final two steps in the biosynthesis of this antibiotic in the soil bacterium, Streptomyces coelicolor A3(2). Interestingly, CYP170A1 shows no stereo selection forming equal amounts of two albaflavenol epimers, each of which is oxidized in turn to albaflavenone. To explore the structural basis of the reaction mechanism, we have studied the crystal structures of both ligand-free CYP170A1 (2.6 Å) and complex of endogenous substrate (epi-isozizaene) with CYP170A1 (3.3 Å). The structure of the complex suggests that the proximal epi-isozizaene molecules may bind to the heme iron in two orientations. In addition, much to our surprise, we have found that albaflavenone synthase also has a second, completely distinct catalytic activity corresponding to the synthesis of farnesene isomers from farnesyl diphosphate. Within the cytochrome P450 α-helical domain both the primary sequence and x-ray structure indicate the presence of a novel terpene synthase active site that is moonlighting on the P450 structure. This includes signature sequences for divalent cation binding and an α-helical barrel. This barrel is unusual because it consists of only four helices rather than six found in all other terpene synthases. Mutagenesis establishes that this barrel is essential for the terpene synthase activity of CYP170A1 but not for the monooxygenase activity. This is the first bifunctional P450 discovered to have another active site moonlighting on it and the first time a terpene synthase active site is found moonlighting on another protein. PMID:19858213

  8. Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

    SciTech Connect

    Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele; Goedken, Eric R.; Gum, Rebecca J.; Borhani, David W.; Argiriadi, Maria; Groebe, Duncan R.; Jia, Yong; Clampit, Jill E.; Haasch, Deanna L.; Smith, Harriet T.; Wang, Sanyi; Song, Danying; Coen, Michael L.; Cloutier, Timothy E.; Tang, Hua; Cheng, Xueheng; Quinn, Christopher; Liu, Bo; Xin, Zhili; Liu, Gang; Fry, Elizabeth H.; Stoll, Vincent; Ng, Teresa I.; Banach, David; Marcotte, Doug; Burns, David J.; Calderwood, David J.; Hajduk, Philip J.

    2012-03-02

    Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in

  9. Site-specific mutagenesis and functional analysis of active sites of sulfur oxygenase reductase from Gram-positive moderate thermophile Sulfobacillus acidophilus TPY.

    PubMed

    Zhang, Huijun; Guo, Wenbin; Xu, Changan; Zhou, Hongbo; Chen, Xinhua

    2013-12-14

    Sequence alignments revealed that the conserved motifs of SORSa which formed an independent branch between archaea and Gram-negative bacteria SORs according to the phylogenetic relationship were similar with the archaea and Gram-negative bacteria SORs. In order to investigate the active sites of SORSa, cysteines 31, 101 and 104 (C31, C101, C104), histidines 86 and 90 (H86 and H90) and glutamate 114 (E114) of SORSa were chosen as the target amino acid residues for site-specific mutagenesis. The wild type and six mutant SORs were expressed in E. coli BL21, purified and confirmed by SDS-PAGE and Western blotting analysis. Enzyme activity determination revealed that the active sites of SORSa were identical with the archaea and Gram-negative bacteria SORs reported. Replacement of any cysteine residues reduced SOR activity by 53-100%, while the mutants of H86A, H90A and E114A lost their enzyme activities largely, only remaining 20%, 19% and 32% activity of the wild type SOR respectively. This study will enrich our awareness for active sites of SOR in a Gram-positive bacterium.

  10. Differential Assembly of Catalytic Interactions within the Conserved Active Sites of Two Ribozymes

    PubMed Central

    Herschlag, Daniel

    2016-01-01

    Molecular recognition is central to biology and a critical aspect of RNA function. Yet structured RNAs typically lack the preorganization needed for strong binding and precise positioning. A striking example is the group I ribozyme from Tetrahymena, which binds its guanosine substrate (G) orders of magnitude slower than diffusion. Binding of G is also thermodynamically coupled to binding of the oligonucleotide substrate (S) and further work has shown that the transition from E•G to E•S•G accompanies a conformational change that allows G to make the active site interactions required for catalysis. The group I ribozyme from Azoarcus has a similarly slow association rate but lacks the coupled binding observed for the Tetrahymena ribozyme. Here we test, using G analogs and metal ion rescue experiments, whether this absence of coupling arises from a higher degree of preorganization within the Azoarcus active site. Our results suggest that the Azoarcus ribozyme forms cognate catalytic metal ion interactions with G in the E•G complex, interactions that are absent in the Tetrahymena E•G complex. Thus, RNAs that share highly similar active site architectures and catalyze the same reactions can differ in the assembly of transition state interactions. More generally, an ability to readily access distinct local conformational states may have facilitated the evolutionary exploration needed to attain RNA machines that carry out complex, multi-step processes. PMID:27501145

  11. Non-canonical active site architecture of the radical SAM thiamin pyrimidine synthase

    NASA Astrophysics Data System (ADS)

    Fenwick, Michael K.; Mehta, Angad P.; Zhang, Yang; Abdelwahed, Sameh H.; Begley, Tadhg P.; Ealick, Steven E.

    2015-03-01

    Radical S-adenosylmethionine (SAM) enzymes use a [4Fe-4S] cluster to generate a 5‧-deoxyadenosyl radical. Canonical radical SAM enzymes are characterized by a β-barrel-like fold and SAM anchors to the differentiated iron of the cluster, which is located near the amino terminus and within the β-barrel, through its amino and carboxylate groups. Here we show that ThiC, the thiamin pyrimidine synthase in plants and bacteria, contains a tethered cluster-binding domain at its carboxy terminus that moves in and out of the active site during catalysis. In contrast to canonical radical SAM enzymes, we predict that SAM anchors to an additional active site metal through its amino and carboxylate groups. Superimposition of the catalytic domains of ThiC and glutamate mutase shows that these two enzymes share similar active site architectures, thus providing strong evidence for an evolutionary link between the radical SAM and adenosylcobalamin-dependent enzyme superfamilies.

  12. Substrate-binding specificity of chitinase and chitosanase as revealed by active-site architecture analysis.

    PubMed

    Liu, Shijia; Shao, Shangjin; Li, Linlin; Cheng, Zhi; Tian, Li; Gao, Peiji; Wang, Lushan

    2015-12-11

    Chitinases and chitosanases, referred to as chitinolytic enzymes, are two important categories of glycoside hydrolases (GH) that play a key role in degrading chitin and chitosan, two naturally abundant polysaccharides. Here, we investigate the active site architecture of the major chitosanase (GH8, GH46) and chitinase families (GH18, GH19). Both charged (Glu, His, Arg, Asp) and aromatic amino acids (Tyr, Trp, Phe) are observed with higher frequency within chitinolytic active sites as compared to elsewhere in the enzyme structure, indicating significant roles related to enzyme function. Hydrogen bonds between chitinolytic enzymes and the substrate C2 functional groups, i.e. amino groups and N-acetyl groups, drive substrate recognition, while non-specific CH-π interactions between aromatic residues and substrate mainly contribute to tighter binding and enhanced processivity evident in GH8 and GH18 enzymes. For different families of chitinolytic enzymes, the number, type, and position of substrate atoms bound in the active site vary, resulting in different substrate-binding specificities. The data presented here explain the synergistic action of multiple enzyme families at a molecular level and provide a more reasonable method for functional annotation, which can be further applied toward the practical engineering of chitinases and chitosanases.

  13. A Relaxed Active Site After Exon Ligation by the Group I Intron

    SciTech Connect

    Lipchock,S.; Strobel, S.

    2008-01-01

    During RNA maturation, the group I intron promotes two sequential phosphorotransfer reactions resulting in exon ligation and intron release. Here, we report the crystal structure of the intron in complex with spliced exons and two additional structures that examine the role of active-site metal ions during the second step of RNA splicing. These structures reveal a relaxed active site, in which direct metal coordination by the exons is lost after ligation, while other tertiary interactions are retained between the exon and the intron. Consistent with these structural observations, kinetic and thermodynamic measurements show that the scissile phosphate makes direct contact with metals in the ground state before exon ligation and in the transition state, but not after exon ligation. Despite no direct exonic interactions and even in the absence of the scissile phosphate, two metal ions remain bound within the active site. Together, these data suggest that release of the ligated exons from the intron is preceded by a change in substrate-metal coordination before tertiary hydrogen bonding contacts to the exons are broken.

  14. Kinetic Study of the Active Site Structure of β-Amylase from Bacillus cereus var. mycoides.

    PubMed

    Nitta, Y; Shirakawa, M; Takasaki, Y

    1996-01-01

    The subsite affinities of the active site of β-amylase from Bacillus cereus var. mycoides were evaluated based on Hiromi's theory, using (14)C-radiolabeled maltooligosaccharides as substrate. It was estimated that the active site consisted of six subsites, and all subsite affinities could be evaluated. The active site had a common subsite arrangement with those of β -amylases from soybean and wheat bran. The intrinsic breakdown rate constant of α-1,4 glucosidic linkage (kint) was five to seven times as large as those of the other enzymes.From the pH dependence of log[k0/Km], pK values of two functional ionizable groups were pK1 =4.0 and pK2 = 8.4. The pK values were 0.5-0.6 units for pK1 and 0.2-0.3 units for pK2 larger than those of the other enzymes. For the affinity-labeling of this enzyme by 2, 3 epoxypropyl α-D-glucopyranoside (α-EPG), the binding affinity of α-EPG was 1-1.6kcal/mol larger than those of the other β-amylases.

  15. Identification of active site residues of Fenugreek β-amylase: chemical modification and in silico approach.

    PubMed

    Srivastava, Garima; Singh, Vinay K; Kayastha, Arvind M

    2014-10-01

    The amino acid sequence of Fenugreek β-amylase is not available in protein data bank. Therefore, an attempt has been made to identify the catalytic amino acid residues of enzyme by employing studies of pH dependence of enzyme catalysis, chemical modification and bioinformatics. Treatment of purified Fenugreek β-amylase with EDAC in presence of glycine methyl ester and sulfhydryl group specific reagents (IAA, NEM and p-CMB), followed a pseudo first-order kinetics and resulted in effective inactivation of enzyme. The reaction with EDAC in presence of NTEE (3-nitro-l-tyrosine ethylester) resulted into modification of two carboxyl groups per molecule of enzyme and presence of one accessible sulfhydryl group at the active site, per molecule of enzyme was ascertained by titration with DTNB. The above results were supported by the prevention of inactivation of enzyme in presence of substrate. Based on MALDI-TOF analysis of purified Fenugreek β-amylase and MASCOT search, β-amylase of Medicago sativa was found to be the best match. To further confirm the amino acid involved in catalysis, homology modelling of β-amylase of M. sativa was performed. The sequence alignment, superimposition of template and target models, along with study of interactions involved in docking of sucrose and maltose at the active site, led to identification of Glu187, Glu381 and Cys344 as active site residues.

  16. Active site structure and catalytic mechanism of phosphodiesterase for degradation of intracellular second messengers

    NASA Astrophysics Data System (ADS)

    Zhan, Chang-Guo

    2002-03-01

    Phosphodiesterases are clinical targets for a variety of biological disorders, because this superfamily of enzymes regulate intracellular concentration of cyclic nucleotides that serve as the second messengers playing a critical role in a variety of physiological processes. Understanding structure and mechanism of a phosphodiesterase will provide a solid basis for rational design of the more efficient therapeutics. Although a three-dimensional X-ray crystal structure of the catalytic domain of human phosphodiesterase 4B2B was recently reported, it was uncertain whether a critical bridging ligand in the active site is a water molecule or a hydroxide ion. The identity of this bridging ligand has been determined by performing first-principles quantum chemical calculations on models of the active site. All the results obtained indicate that this critical bridging ligand in the active site of the reported X-ray crystal structure is a hydroxide ion, rather than a water molecule, expected to serve as the nucleophile to initialize the catalytic degradation of the intracellular second messengers.

  17. Aerosol measurements at a high-elevation site: composition, size, and cloud condensation nuclei activity

    SciTech Connect

    Friedman, Beth; Zelenyuk, Alla; Beranek, Josef; Kulkarni, Gourihar R.; Pekour, Mikhail S.; Hallar, Anna G.; McCubbin, Ian; Thornton, Joel A.; Cziczo, D. J.

    2013-12-09

    We present measurements of CCN concentrations and associated aerosol composition and size properties at a high-elevation research site in March 2011. CCN closure and aerosol hygroscopicity were assessed using simplified assumptions of bulk aerosol properties as well as a new method utilizing single particle composition and size to assess the importance of particle mixing state in CCN activation. Free troposphere analysis found no significant difference between the CCN activity of free tropospheric aerosol and boundary layer aerosol at this location. Closure results indicate that using only size and number information leads to adequate prediction, in the majority of cases within 50%, of CCN concentrations, while incorporating the hygroscopicity parameters of the individual aerosol components measured by single particle mass spectrometry adds to the agreement, in most cases within 20%, between predicted and measured CCN concentrations. For high-elevation continental sites, with largely aged aerosol and low amounts of local area emissions, a lack of chemical knowledge and hygroscopicity may not hinder models in predicting CCN concentrations. At sites influenced by fresh emissions or more heterogeneous particle types, single particle composition information may be more useful in predicting CCN concentrations and understanding the importance of particle mixing state on CCN activation.

  18. Thermal regime of active layer at two lithologically contrasting sites on James Ross Island, Antarctic Peninsula.

    NASA Astrophysics Data System (ADS)

    Hrbáček, Filip; Nývlt, Daniel; Láska, Kamil

    2016-04-01

    Antarctic Peninsula region (AP) represents one of the most rapidly warming parts of our planet in the last 50 years. Despite increasing research activities along both western and eastern sides of AP in last decades, there is still a lot of gaps in our knowledge relating to permafrost, active layer and its thermal and physical properties. This study brings new results of active layer monitoring on James Ross Island, which is the largest island in northern AP. Its northern part, Ulu Peninsula, is the largest ice-free area (more than 200 km2) in the region. Due its large area, we focused this study on sites located in different lithologies, which would affect local thermal regime of active layer. Study site (1) at Abernethy Flats area (41 m a.s.l.) lies ~7 km from northern coast. Lithologically is formed by disintegrated Cretaceous calcareous sandstones and siltstones of the Santa Marta Formation. Study site (2) is located at the northern slopes of Berry Hill (56 m a.s.l.), about 0.4 km from northern coastline. Lithology is composed of muddy to intermediate diamictites, tuffaceous siltstones to fine grained sandstones of the Mendel Formation. Data of air temperature at 2 meters above ground and the active layer temperatures at 75 cm deep profiles were obtained from both sites in period 1 January 2012 to 31 December 2014. Small differences were found when comparing mean air temperatures and active temperatures at 5 and 75 cm depth in the period 2012-2014. While the mean air temperatures varied between -7.7 °C and -7.0 °C, the mean ground temperatures fluctuated between -6.6 °C and -6.1 °C at 5 cm and -6.9 °C and -6.0 °C at 75 cm at Abernethy Flats and Berry Hill slopes respectively. Even though ground temperature differences along the profiles weren't pronounced during thawing seasons, the maximum active layer thickness was significantly larger at Berry Hill slopes (80 to 82 cm) than at Abernethy Flats (52 to 64 cm). We assume this differences are affected by

  19. Alkynol natural products target ALDH2 in cancer cells by irreversible binding to the active site.

    PubMed

    Heydenreuter, Wolfgang; Kunold, Elena; Sieber, Stephan A

    2015-11-11

    Falcarinol and stipudiol are natural products with potent anti-cancer activity found in several vegetables. Here, we use a chemical proteomic strategy to identify ALDH2 as a molecular target of falcarinol in cancer cells and confirm enzyme inhibition via covalent alkylation of the active site. Furthermore, the synthesis of stipudiol led to the observation that ALDH2 exhibits preference for alkynol-based binders. Inhibition of ALDH2 impairs detoxification of reactive aldehydes and limits oxidative stress response, two crucial pathways for cellular viability.

  20. Altered binding of thioflavin t to the peripheral anionic site of acetylcholinesterase after phosphorylation of the active site by chlorpyrifos oxon or dichlorvos

    SciTech Connect

    Sultatos, L.G. Kaushik, R.

    2008-08-01

    The peripheral anionic site of acetylcholinesterase, when occupied by a ligand, is known to modulate reaction rates at the active site of this important enzyme. The current report utilized the peripheral anionic site specific fluorogenic probe thioflavin t to determine if the organophosphates chlorpyrifos oxon and dichlorvos bind to the peripheral anionic site of human recombinant acetylcholinesterase, since certain organophosphates display concentration-dependent kinetics when inhibiting this enzyme. Incubation of 3 nM acetylcholinesterase active sites with 50 nM or 2000 nM inhibitor altered both the B{sub max} and K{sub d} for thioflavin t binding to the peripheral anionic site. However, these changes resulted from phosphorylation of Ser203 since increasing either inhibitor from 50 nM to 2000 nM did not alter further thioflavin t binding kinetics. Moreover, the organophosphate-induced decrease in B{sub max} did not represent an actual reduction in binding sites, but instead likely resulted from conformational interactions between the acylation and peripheral anionic sites that led to a decrease in the rigidity of bound thioflavin t. A drop in fluorescence quantum yield, leading to an apparent decrease in B{sub max}, would accompany the decreased rigidity of bound thioflavin t molecules. The organophosphate-induced alterations in K{sub d} represented changes in binding affinity of thioflavin t, with diethylphosphorylation of Ser203 increasing K{sub d}, and dimethylphosphorylation of Ser203 decreasing K{sub d}. These results indicate that chlorpyrifos oxon and dichlorvos do not bind directly to the peripheral anionic site of acetylcholinesterase, but can affect binding to that site through phosphorylation of Ser203.

  1. Stochastic analysis of myoelectric temporal signatures for multifunctional single-site activation of prostheses and orthoses.

    PubMed

    Graupe, D; Salahi, J; Zhang, D S

    1985-01-01

    This paper is concerned with a stochastic time-series analysis of the temporal signatures of myoelectric (ME) signals including the determination of model order and sampling rate. The paper considers the use of time-series parameters for the activation of artificial limbs for high-level amputees, of stimulation electrodes or of powered braces for paralysed persons, in several degrees of freedom, from a single or two surface-electrode pairs at locations where considerable ME cross-talk exists. The multifunctional capability from a single site is based on the differences between the time-series (TS) parameters for different muscle activation patterns at the same ME site, these differences being thus used for limb function discrimination via easily trainable muscle activation patterns at the vicinity of the electrode site. Specifically, the analysis is in terms of identifying the AR parameters of a time-domain autoregressive (AR) signature model both for the complete ME spectrum and for parts thereof, and in terms of the autocorrelation of the signal and of the models residual. Determination of sampling rate and of model orders is discussed in detail. It is shown that, using online real-time analysis, differences in the AR time-series parameters can be observed for different trainable patterns of muscle activation, at the same electrode location, even at the same ME power levels, as long as considerable cross-talk exists at the electrode site. These parameter differences can be accentuated if one considers the AR parameters for lower-frequency spectral windows. A case is made in this paper for employing TS analysis to squeeze out information in a distinct but low-level ripple of the low frequency spectrum of the signal. This information tends to be ignored in frequency domain, but is all that the AR parameters care for in TS analysis, since they are not concerned, with a flat-average low-frequency spectrum, i.e., its white-noise-like part, which is the residual term

  2. Flexibility Matters: Cooperative Active Sites in Covalent Organic Framework and Threaded Ionic Polymer.

    PubMed

    Sun, Qi; Aguila, Briana; Perman, Jason; Nguyen, Nicholas; Ma, Shengqian

    2016-12-07

    The combination of two or more reactive centers working in concert on a substrate to facilitate the reaction is now considered state of the art in catalysis, yet there still remains a tremendous challenge. Few heterogeneous systems of this sort have been exploited, as the active sites spatially separated within the rigid framework are usually difficult to cooperate. It is now shown that this roadblock can be surpassed. The underlying principle of the strategy presented here is the integration of catalytic components with excellent flexibility and porous heterogeneous catalysts, as demonstrated by the placement of linear ionic polymers in close proximity to surface Lewis acid active sites anchored on the walls of a covalent organic framework (COF). Using the cycloaddition of the epoxides and CO2 as a model reaction, dramatic activity improvements have been achieved for the composite catalysts in relation to the individual catalytic component. Furthermore, they also clearly outperform the benchmark catalytic systems formed by the combination of the molecular organocatalysts and heterogeneous Lewis acid catalysts, while affording additional recyclability. The extraordinary flexibility and enriched concentration of the catalytically active moieties on linear polymers facilitate the concerted catalysis, thus leading to superior catalytic performance. This work therefore uncovers an entirely new strategy for designing bifunctional catalysts with double-activation behavior and opens a new avenue in the design of multicapable systems that mimic biocatalysis.

  3. Litter type affects the activity of aerobic decomposers in a boreal peatland more than site nutrient and water table regimes

    NASA Astrophysics Data System (ADS)

    Straková, P.; Niemi, R. M.; Freeman, C.; Peltoniemi, K.; Toberman, H.; Heiskanen, I.; Fritze, H.; Laiho, R.

    2011-09-01

    Peatlands are carbon (C) storage ecosystems sustained by a high water table (WT). High WT creates anoxic conditions that suppress the activity of aerobic decomposers and provide conditions for peat accumulation. Peatland function can be dramatically affected by WT drawdown caused by climate and/or land-use change. Aerobic decomposers are directly affected by WT drawdown through environmental factors such as increased oxygenation and nutrient availability. Additionally, they are indirectly affected via changes in plant community composition and litter quality. We studied the relative importance of direct and indirect effects of WT drawdown on aerobic decomposer activity in plant litter at two stages of decomposition (incubated in the field for 1 or 2 years). We did this by profiling 11 extracellular enzymes involved in the mineralization of organic C, nitrogen (N), phosphorus (P) and sulphur. Our study sites represented a three-stage chronosequence from pristine to short-term (years) and long-term (decades) WT drawdown conditions under two nutrient regimes (bog and fen). The litter types included reflected the prevalent vegetation: Sphagnum mosses, graminoids, shrubs and trees. Litter type was the main factor shaping microbial activity patterns and explained about 30 % of the variation in enzyme activities and activity allocation. Overall, enzyme activities were higher in vascular plant litters compared to Sphagnum litters, and the allocation of enzyme activities towards C or nutrient acquisition was related to the initial litter quality (chemical composition). Direct effects of WT regime, site nutrient regime and litter decomposition stage (length of incubation period) summed to only about 40 % of the litter type effect. WT regime alone explained about 5 % of the variation in enzyme activities and activity allocation. Generally, enzyme activity increased following the long-term WT drawdown and the activity allocation turned from P and N acquisition towards C

  4. Orthogonal electrode catheter array for mapping of endocardial focal site of ventricular activation

    SciTech Connect

    Desai, J.M.; Nyo, H.; Vera, Z.; Seibert, J.A.; Vogelsang, P.J. )

    1991-04-01

    Precise location of the endocardial site of origin of ventricular tachycardia may facilitate surgical and catheter ablation of this arrhythmia. The endocardial catheter mapping technique can locate the site of ventricular tachycardia within 4-8 cm2 of the earliest site recorded by the catheter. This report describes an orthogonal electrode catheter array (OECA) for mapping and radiofrequency ablation (RFA) of endocardial focal site of origin of a plunge electrode paced model of ventricular activation in dogs. The OECA is an 8 F five pole catheter with four peripheral electrodes and one central electrode (total surface area 0.8 cm{sup 2}). In eight mongrel dogs, mapping was performed by arbitrarily dividing the left ventricle (LV) into four segments. Each segment was mapped with OECA to find the earliest segment. Bipolar and unipolar electrograms were obtained. The plunge electrode (not visible on fluoroscopy) site was identified by the earliest wave front arrival times of -30 msec or earlier at two or more electrodes (unipolar electrograms) with reference to the earliest recorded surface ECG (I, AVF, and V1). Validation of the proximity of the five electrodes of the OECA to the plunge electrode was performed by digital radiography and RFA. Pathological examination was performed to document the proximity of the OECA to the plunge electrode and also for the width, depth, and microscopic changes of the ablation. To find the segment with the earliest LV activation a total of 10 {plus minus} 3 (mean {plus minus} SD) positions were mapped. Mean arrival times at the two earlier electrodes were -39 {plus minus} 4 msec and -35 {plus minus} 3 msec. Digital radiography showed the plunge electrode to be within the area covered by all five electrodes in all eight dogs. The plunge electrode was within 1 cm2 area of the region of RFA in all eight dogs.

  5. Spatial patterns of microbial diversity and activity in an aged creosote-contaminated site

    PubMed Central

    Mukherjee, Shinjini; Juottonen, Heli; Siivonen, Pauli; Lloret Quesada, Cosme; Tuomi, Pirjo; Pulkkinen, Pertti; Yrjälä, Kim

    2014-01-01

    Restoration of polluted sites via in situ bioremediation relies heavily on the indigenous microbes and their activities. Spatial heterogeneity of microbial populations, contaminants and soil chemical parameters on such sites is a major hurdle in optimizing and implementing an appropriate bioremediation regime. We performed a grid-based sampling of an aged creosote-contaminated site followed by geostatistical modelling to illustrate the spatial patterns of microbial diversity and activity and to relate these patterns to the distribution of pollutants. Spatial distribution of bacterial groups unveiled patterns of niche differentiation regulated by patchy distribution of pollutants and an east-to-west pH gradient at the studied site. Proteobacteria clearly dominated in the hot spots of creosote pollution, whereas the abundance of Actinobacteria, TM7 and Planctomycetes was considerably reduced from the hot spots. The pH preferences of proteobacterial groups dominating in pollution could be recognized by examining the order and family-level responses. Acidobacterial classes came across as generalists in hydrocarbon pollution whose spatial distribution seemed to be regulated solely by the pH gradient. Although the community evenness decreased in the heavily polluted zones, basal respiration and fluorescein diacetate hydrolysis rates were higher, indicating the adaptation of specific indigenous microbial populations to hydrocarbon pollution. Combining the information from the kriged maps of microbial and soil chemistry data provided a comprehensive understanding of the long-term impacts of creosote pollution on the subsurface microbial communities. This study also highlighted the prospect of interpreting taxa-specific spatial patterns and applying them as indicators or proxies for monitoring polluted sites. PMID:25105905

  6. Three-dimensional representations of salt-dome margins at four active strategic petroleum reserve sites.

    SciTech Connect

    Rautman, Christopher Arthur; Stein, Joshua S.

    2003-01-01

    Existing paper-based site characterization models of salt domes at the four active U.S. Strategic Petroleum Reserve sites have been converted to digital format and visualized using modern computer software. The four sites are the Bayou Choctaw dome in Iberville Parish, Louisiana; the Big Hill dome in Jefferson County, Texas; the Bryan Mound dome in Brazoria County, Texas; and the West Hackberry dome in Cameron Parish, Louisiana. A new modeling algorithm has been developed to overcome limitations of many standard geological modeling software packages in order to deal with structurally overhanging salt margins that are typical of many salt domes. This algorithm, and the implementing computer program, make use of the existing interpretive modeling conducted manually using professional geological judgement and presented in two dimensions in the original site characterization reports as structure contour maps on the top of salt. The algorithm makes use of concepts of finite-element meshes of general engineering usage. Although the specific implementation of the algorithm described in this report and the resulting output files are tailored to the modeling and visualization software used to construct the figures contained herein, the algorithm itself is generic and other implementations and output formats are possible. The graphical visualizations of the salt domes at the four Strategic Petroleum Reserve sites are believed to be major improvements over the previously available two-dimensional representations of the domes via conventional geologic drawings (cross sections and contour maps). Additionally, the numerical mesh files produced by this modeling activity are available for import into and display by other software routines. The mesh data are not explicitly tabulated in this report; however an electronic version in simple ASCII format is included on a PC-based compact disk.

  7. Mushroom toxicosis in dogs in general practice causing gastroenteritis, ptyalism and elevated serum lipase activity.

    PubMed

    Hall, J; Barton, L

    2013-05-01

    Mushroom toxicosis is rarely diagnosed in dogs and is poorly reported in the veterinary literature. This report suggests that mushroom toxicosis is a potentially under-diagnosed condition in first opinion practice in the UK. Nine dogs with clinical signs consistent with mushroom toxicosis were identified from the records of an out-of-hours emergency service between August 2010 and January 2011. Four dogs were later excluded because of clinical inconsistencies. Clinical signs included acute profuse ptyalism (5/5), diarrhoea (5/5), vomiting (4/5), hypovolaemia (4/5), stuporous (3/5) or obtunded mentation (1/5), miosis (2/5) and hypothermia (2/5). Serum lipase activity was elevated in 4/4 dogs; canine-specific pancreatic lipase was elevated in the remaining dog. Four dogs recovered with aggressive intravenous fluid therapy, analgesia and supportive care; the remaining dog was euthanased due to severe clinical signs and financial constraints. Mushroom toxicosis is an important differential diagnosis for acute gastroenteritis and one possible cause of some cases of "Seasonal Canine Illness". Affected dogs may demonstrate elevated pancreatic enzymes and mushroom toxicosis should be considered in cases of elevated lipase or abnormal semi-quantitative canine-specific pancreatic lipase activities.

  8. Antibacterial activity of leaves extracts of Trifolium alexandrinum Linn. against pathogenic bacteria causing tropical diseases

    PubMed Central

    Khan, Abdul Viqar; Ahmed, Qamar Uddin; Shukla, Indu; Khan, Athar Ali

    2012-01-01

    Objective To investigate antibacterial potential of Trifolium alexandrinum (T. alexandrinum) Linn. against seven gram positive and eleven gram negative hospital isolated human pathogenic bacterial strains responsible for many tropical diseases. Methods Non-polar and polar extracts of the leaves of T. alexandrinum i.e., hexane, dichloromethane (DCM), ethyl acetate (EtOAc), methanol (MeOH) and aqueous (AQ) extracts at five different concentrations (1, 2, 5, 10 and 15 mg/mL) were prepared to evaluate their antibacterial value. NCCL standards were strictly followed to perform antimicrobial disc susceptibility test using disc diffusion method. Results Polar extracts demonstrated significant antibacterial activity against tested pathogens. EtOAc and MeOH extracts showed maximum antibacterial activity with higher inhibition zone and were found effective against seventeen of the tested pathogens. While AQ plant extract inhibited the growth of sixteen of the test strains. EtOAc and MeOH plant extracts inhibited the growth of all seven gram positive and ten of the gram negative bacterial strains. Conclusions The present study strongly confirms the effectiveness of crude leaves extracts against tested human pathogenic bacterial strains causing several tropical diseases. Since Egyptian clover is used as a fodder plant, it could be helpful in controlling various infectious diseases associated with cattle as well. PMID:23569896

  9. Aging causes decreased resistance to multiple stresses and a failure to activate specific stress response pathways

    PubMed Central

    Bergsma, Alexis L.; Senchuk, Megan M.; Van Raamsdonk, Jeremy M.

    2016-01-01

    In this work, we examine the relationship between stress resistance and aging. We find that resistance to multiple types of stress peaks during early adulthood and then declines with age. To dissect the underlying mechanisms, we use C. elegans transcriptional reporter strains that measure the activation of different stress responses including: the heat shock response, mitochondrial unfolded protein response, endoplasmic reticulum unfolded protein response, hypoxia response, SKN-1-mediated oxidative stress response, and the DAF-16-mediated stress response. We find that the decline in stress resistance with age is at least partially due to a decreased ability to activate protective mechanisms in response to stress. In contrast, we find that any baseline increase in stress caused by the advancing age is too mild to detectably upregulate any of the stress response pathways. Further exploration of how worms respond to stress with increasing age revealed that the ability to mount a hormetic response to heat stress is also lost with increasing age. Overall, this work demonstrates that resistance to all types of stress declines with age. Based on our data, we speculate that the decrease in stress resistance with advancing age results from a genetically-programmed inactivation of stress response pathways, not accumulation of damage. PMID:27053445

  10. Could a change in magnetic field geometry cause the break in the wind-activity relation?

    NASA Astrophysics Data System (ADS)

    Vidotto, A. A.; Donati, J.-F.; Jardine, M.; See, V.; Petit, P.; Boisse, I.; Boro Saikia, S.; Hébrard, E.; Jeffers, S. V.; Marsden, S. C.; Morin, J.

    2016-01-01

    Wood et al. suggested that mass-loss rate is a function of X-ray flux (dot{M}∝ F_x^{1.34}) for dwarf stars with Fx ≲ Fx,6 ≡ 106 erg cm-2 s-1. However, more active stars do not obey this relation. These authors suggested that the break at Fx,6 could be caused by significant changes in magnetic field topology that would inhibit stellar wind generation. Here, we investigate this hypothesis by analysing the stars in Wood et al. sample that had their surface magnetic fields reconstructed through Zeeman-Doppler Imaging (ZDI). Although the solar-like outliers in the dot{M} - Fx relation have higher fractional toroidal magnetic energy, we do not find evidence of a sharp transition in magnetic topology at Fx,6. To confirm this, further wind measurements and ZDI observations at both sides of the break are required. As active stars can jump between states with highly toroidal to highly poloidal fields, we expect significant scatter in magnetic field topology to exist for stars with Fx ≳ Fx,6. This strengthens the importance of multi-epoch ZDI observations. Finally, we show that there is a correlation between Fx and magnetic energy, which implies that dot{M} - magnetic energy relation has the same qualitative behaviour as the original dot{M} - Fx relation. No break is seen in any of the Fx - magnetic energy relations.

  11. Aging causes decreased resistance to multiple stresses and a failure to activate specific stress response pathways.

    PubMed

    Dues, Dylan J; Andrews, Emily K; Schaar, Claire E; Bergsma, Alexis L; Senchuk, Megan M; Van Raamsdonk, Jeremy M

    2016-04-01

    In this work, we examine the relationship between stress resistance and aging. We find that resistance to multiple types of stress peaks during early adulthood and then declines with age. To dissect the underlying mechanisms, we use C. elegans transcriptional reporter strains that measure the activation of different stress responses including: the heat shock response, mitochondrial unfolded protein response, endoplasmic reticulum unfolded protein response, hypoxia response, SKN-1-mediated oxidative stress response, and the DAF-16-mediated stress response. We find that the decline in stress resistance with age is at least partially due to a decreased ability to activate protective mechanisms in response to stress. In contrast, we find that any baseline increase in stress caused by the advancing age is too mild to detectably upregulate any of the stress response pathways. Further exploration of how worms respond to stress with increasing age revealed that the ability to mount a hormetic response to heat stress is also lost with increasing age. Overall, this work demonstrates that resistance to all types of stress declines with age. Based on our data, we speculate that the decrease in stress resistance with advancing age results from a genetically-programmed inactivation of stress response pathways, not accumulation of damage.

  12. Control of the active site structure of giant bilayer hemoglobin from the Annelid Eisenia foetida using hierarchic assemblies

    SciTech Connect

    Girasole, Marco; Arcovito, Alessandro; Marconi, Augusta; Davoli, Camilla; Congiu-Castellano, Agostina; Bellelli, Andrea; Amiconi, Gino

    2005-12-05

    The active site structure of the oxygenated derivative of the main subassemblies (whole protein, dodecamers, and trimers) of the giant haemoglobin from Eisenia foetida has been characterized by x-ray absorption near edge structure spectroscopy. The data revealed a remarkable effect of the hierarchic assemblies on the active site of the subunit. Specifically, the whole protein has the same site structure of the dodecamer, while a sharp conformational transition occurs when the dodecamer is disassembled into trimers (and monomers) revealing that constraints due to the protein matrix determine the active site geometry and, consequently, the protein function in these large complexes.

  13. Extracellular matrix-specific focal adhesions in vascular smooth muscle produce mechanically active adhesion sites

    PubMed Central

    Sun, Zhe; Martinez-Lemus, Luis A.; Hill, Michael A.; Meininger, Gerald A.

    2008-01-01

    Integrin-mediated mechanotransduction in vascular smooth muscle cells (VSMCs) plays an important role in the physiological control of tissue blood flow and vascular resistance. To test whether force applied to specific extracellular matrix (ECM)-integrin interactions could induce myogenic-like mechanical activity at focal adhesion sites, we used atomic force microscopy (AFM) to apply controlled forces to specific ECM adhesion sites on arteriolar VSMCs. The tip of AFM probes were fused with a borosilicate bead (2∼5 μm) coated with fibronectin (FN), collagen type I (CNI), laminin (LN), or vitronectin (VN). ECM-coated beads induced clustering of α5- and β3-integrins and actin filaments at sites of bead-cell contact indicative of focal adhesion formation. Step increases of an upward (z-axis) pulling force (800∼1,600 pN) applied to the bead-cell contact site for FN-specific focal adhesions induced a myogenic-like, force-generating response from the VSMC, resulting in a counteracting downward pull by the cell. This micromechanical event was blocked by cytochalasin D but was enhanced by jasplakinolide. Function-blocking antibodies to α5β1- and αvβ3-integrins also blocked the micromechanical cell event in a concentration-dependent manner. Similar pulling experiments with CNI, VN, or LN failed to induce myogenic-like micromechanical events. Collectively, these results demonstrate that mechanical force applied to integrin-FN adhesion sites induces an actin-dependent, myogenic-like, micromechanical event. Focal adhesions formed by different ECM proteins exhibit different mechanical characteristics, and FN appears of particular relevance in its ability to strongly attach to VSMCs and to induce myogenic-like, force-generating reactions from sites of focal adhesion in response to externally applied forces. PMID:18495809

  14. Potential landslide activity affecting the archaeological site of Orongo (Easter Island-Chile): preliminary analysis

    NASA Astrophysics Data System (ADS)

    Margottini, C.; Delmonaco, G.; Spizzichino, D.; Pandolfi, O.; Crisostomo, R.; Nohe, S.

    2009-04-01

    Easter Island forms part of the Easter Line, a continuous latitudinal chain of volcanic seamounts and islands in the Pacific Sea. The island's roughly triangular shape is determined by the merging of lava flows produced by its three main volcanoes (Rano Kau, Terevaka, Poike) which form its main mass. The Rano Kau volcano, sited in the SW vertex of the island, is made up of numerous basaltic lava flows and has been reduced in size by faulting and marine erosion. Its crater (1.4 km wide) is a small caldera that collapsed after a late, large explosive phase, as attested by the presence of breccia deposits around the eastern rim of the crater. The archaeological stone village of Orongo is located above the inner wall of the crater at an altitude of ca. 300m a.s.l. Prominent historical remains are the numerous petroglyphs that represent the ancient ceremonial of the birdman cult (tangata manu). Rano Kau is mainly composed of sequences of basaltic and intermediate lavas and pyroclastics. Most of the of the original caldera area, especially in the southern flank, has been disrupted by marine erosion. This has caused a dramatic change of the original morphology, resulting in a sub-vertical cliff and steep slopes, especially in the middle-low portions. In the upper part of the slopes weathered soils and regolith are outcropping. Topographical and geomorphological analysis of the area conducted by a direct field surveys in January and July 2008 have provided clear evidences of slope instability along the southern external flank of the caldera. Different landslide areas have been detected. The most active area is located at east of the village in correspondence of the crest zone of Rano Kau where a debris slide/fall has recently occurred. The analysis of photos taken in Nov. 2007 in the same area evidences that the landslide crown area was originated at an elevation of ca. 200m a.s.l. along a probable contact between basaltic layers on the top and weathered lava. Other minor

  15. Spontaneous activation of [FeFe]-hydrogenases by an inorganic [2Fe] active site mimic

    PubMed Central

    Esselborn, Julian; Berggren, Gustav; Noth, Jens; Siebel, Judith; Hemschemeier, Anja; Artero, Vincent; Reijerse, Edward; Fontecave, Marc; Lubitz, Wolfgang; Happe, Thomas

    2013-01-01

    Hydrogenases catalyze the formation of hydrogen. The cofactor (H-cluster) of [FeFe]-hydrogenases consists of a [4Fe-4S]-cluster bridged to a unique [2Fe]-subcluster whose biosynthesis in vivo requires hydrogenase-specific maturases. Here we show that a chemical mimic of the [2Fe]-subcluster can reconstitute apo-hydrogenase to full activity, independent of helper proteins. The assembled H-cluster is virtually indistinguishable from the native cofactor. This procedure will be a powerful tool for developing novel artificial H2-producing catalysts. PMID:23934246

  16. Different cleavage sites are aligned differently in the active site of M1 RNA, the catalytic subunit of Escherichia coli RNase P.

    PubMed Central

    Kufel, J; Kirsebom, L A

    1996-01-01

    We have studied RNase P RNA (M1 RNA) cleavage of model tRNA precursors that are cleaved at two independent positions. Here we present data demonstrating that cleavage at both sites depends on the 2'-OH immediately 5' of the respective cleavage site. However, we show that the 2-amino group of a guanosine at the cleavage site plays a significant role in cleavage at one of these sites but not at the other. These data suggest that these two cleavage sites are handled differently by the ribozyme. This theory is supported by our finding that the cross-linking pattern between Ml RNA and tRNA precursors carrying 4-thioU showed distinct differences, depending on the location of the 4-thioU relative to the respective cleavage site. These findings lead us to suggest that different cleavage sites are aligned differently in the active site, possibly as a result of different binding modes of a substrate to M1 RNA. We discuss a model in which the interaction between the 3'-terminal "RCCA" motif (first three residues interact) of a tRNA precursor and M1 RNA plays a significant role in this process. Images Fig. 2 Fig. 3 Fig. 4 PMID:8650223

  17. Substrate specificity engineering of beta-mannosidase and beta-glucosidase from Pyrococcus by exchange of unique active site residues.

    PubMed

    Kaper, Thijs; van Heusden, Hester H; van Loo, Bert; Vasella, Andrea; van der Oost, John; de Vos, Willem M

    2002-03-26

    A beta-mannosidase gene (PH0501) was identified in the Pyrococcus horikoshii genome and cloned and expressed in E. coli. The purified enzyme (BglB) was most specific for the hydrolysis of p-nitrophenyl-beta-D-mannopyranoside (pNP-Man) (Km: 0.44 mM) with a low turnover rate (kcat: 4.3 s(-1)). The beta-mannosidase has been classified as a member of family 1 of glycoside hydrolases. Sequence alignments and homology modeling showed an apparent conservation of its active site region with, remarkably, two unique active site residues, Gln77 and Asp206. These residues are an arginine and asparagine residue in all other known family 1 enzymes, which interact with the catalytic nucleophile and equatorial C2-hydroxyl group of substrates, respectively. The unique residues of P. horikoshii BglB were introduced in the highly active beta-glucosidase CelB of Pyrococcus furiosus and vice versa, yielding two single and one double mutant for each enzyme. In CelB, both substitutions R77Q and N206D increased the specificity for mannosides and reduced hydrolysis rates 10-fold. In contrast, BglB D206N showed 10-fold increased hydrolysis rates and 35-fold increased affinity for the hydrolysis of glucosides. In combination with inhibitor studies, it was concluded that the substituted residues participate in the ground-state binding of substrates with an equatorial C2-hydroxyl group, but contribute most to transition-state stabilization. The unique activity profile of BglB seems to be caused by an altered interaction between the enzyme and C2-hydroxyl of the substrate and a specifically increased affinity for mannose that results from Asp206.

  18. Altered neuronal mitochondrial coenzyme A synthesis in neurodegeneration with brain iron accumulation caused by abnormal processing, stability, and catalytic activity of mutant pantothenate kinase 2.

    PubMed

    Kotzbauer, Paul T; Truax, Adam C; Trojanowski, John Q; Lee, Virginia M-Y

    2005-01-19

    Mutations in the pantothenate kinase 2 (PANK2) gene have been identified in patients with neurodegeneration with brain iron accumulation (NBIA; formerly Hallervorden-Spatz disease). However, the mechanisms by which these mutations cause neurodegeneration are unclear, especially given the existence of multiple pantothenate kinase genes in humans and multiple PanK2 transcripts with potentially different subcellular localizations. We demonstrate that PanK2 protein is localized to mitochondria of neurons in human brain, distinguishing it from other pantothenate kinases that do not possess mitochondrial-targeting sequences. PanK2 protein translated from the most 5' start site is sequentially cleaved at two sites by the mitochondrial processing peptidase, generating a long-lived 48 kDa mature protein identical to that found in human brain extracts. The mature protein catalyzes the initial step in coenzyme A (CoA) synthesis but displays feedback inhibition in response to species of acyl CoA rather than CoA itself. Some, but not all disease-associated point mutations result in significantly reduced catalytic activity. The most common mutation, G521R, results in marked instability of the intermediate PanK2 isoform and reduced production of the mature isoform. These results suggest that NBIA is caused by altered neuronal mitochondrial lipid metabolism caused by mutations disrupting PanK2 protein levels and catalytic activity.

  19. Active-Site Structure of Class IV Adenylyl Cyclase and Transphyletic Mechanism

    SciTech Connect

    Gallagher, D.T.; Robinson, H.; Kim, S.-K.; Reddy, P. T.

    2011-01-21

    Adenylyl cyclases (ACs) belonging to three nonhomologous classes (II, III, and IV) have been structurally characterized, enabling a comparison of the mechanisms of cyclic adenosine 3',5'-monophosphate biosynthesis. We report the crystal structures of three active-site complexes for Yersinia pestis class IV AC (AC-IV)-two with substrate analogs and one with product. Mn{sup 2+} binds to all three phosphates, and to Glu12 and Glu136. Electropositive residues Lys14, Arg63, Lys76, Lys111, and Arg113 also form hydrogen bonds to phosphates. The conformation of the analogs is suitable for in-line nucleophilic attack by the ribose O3' on {alpha}-phosphate (distance {approx} 4 {angstrom}). In the product complex, a second Mn ion is observed to be coordinated to both ribose 2' oxygen and ribose 3' oxygen. Observation of both metal sites, together with kinetic measurements, provides strong support for a two-cation mechanism. Eleven active-site mutants were also made and kinetically characterized. These findings and comparisons with class II and class III enzymes enable a detailed transphyletic analysis of the AC mechanism. Consistent with its lack of coordination to purine, Y. pestis AC-IV cyclizes both ATP and GTP. As in other classes of AC, the ribose is loosely bound, and as in class III, no base appears