The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate

DOE PAGES

Poudel, Suresh; Giannone, Richard J.; Basen, Mirko; ...

2018-03-23

Background: Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates.Results:Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs),more » ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. In conclusion, this study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii’s utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.« less

The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate

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

Poudel, Suresh; Giannone, Richard J.; Basen, Mirko

Background: Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates.Results:Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs),more » ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. In conclusion, this study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii’s utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.« less

The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate.

PubMed

Poudel, Suresh; Giannone, Richard J; Basen, Mirko; Nookaew, Intawat; Poole, Farris L; Kelly, Robert M; Adams, Michael W W; Hettich, Robert L

2018-01-01

Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates. Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs), ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. This study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii 's utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.

Identification of amino acid residues involved in substrate specificity of plant acyl-ACP thioesterases using a bioinformatics-guided approach

PubMed Central

Mayer, Kimberly M; Shanklin, John

2007-01-01

Background The large amount of available sequence information for the plant acyl-ACP thioesterases (TEs) made it possible to use a bioinformatics-guided approach to identify amino acid residues involved in substrate specificity. The Conserved Property Difference Locator (CPDL) program allowed the identification of putative specificity-determining residues that differ between the FatA and FatB TE classes. Six of the FatA residue differences identified by CPDL were incorporated into the FatB-like parent via site-directed mutagenesis and the effect of each on TE activity was determined. Variants were expressed in E. coli strain K27 that allows determination of enzyme activity by GCMS analysis of fatty acids released into the medium. Results Substitutions at four of the positions (74, 86, 141, and 174) changed substrate specificity to varying degrees while changes at the remaining two positions, 110 and 221, essentially inactivated the thioesterase. The effects of substitutions at positions 74, 141, and 174 (3-MUT) or 74, 86, 141, 174 (4-MUT) were not additive with respect to specificity. Conclusion Four of six putative specificity determining positions in plant TEs, identified with the use of CPDL, were validated experimentally; a novel colorimetric screen that discriminates between active and inactive TEs is also presented. PMID:17201914

An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis*

PubMed Central

Kayode, Olumide; Wang, Ruiying; Pendlebury, Devon F.; Cohen, Itay; Henin, Rachel D.; Hockla, Alexandra; Soares, Alexei S.; Papo, Niv; Caulfield, Thomas R.; Radisky, Evette S.

2016-01-01

The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. Although considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here, we examine the importance of substrate dynamics in the cleavage of Kunitz-bovine pancreatic trypsin inhibitor protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4-Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals a dramatic conformational change in the substrate upon proteolysis. By using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning 3 orders of magnitude, we identify global and local dynamic features of substrates on the nanosecond-microsecond time scale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substrate-like and product-like states, linking substrate dynamics on the nanosecond-microsecond time scale with large collective substrate motions on the much slower time scale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis. PMID:27810896

Neural Substrates of Processing Path and Manner Information of a Moving Event

ERIC Educational Resources Information Center

Wu, Denise H.; Morganti, Anne; Chatterjee, Anjan

2008-01-01

Languages consistently distinguish the path and the manner of a moving event in different constituents, even if the specific constituents themselves vary across languages. Children also learn to categorize moving events according to their path and manner at different ages. Motivated by these linguistic and developmental observations, we employed…

An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis

DOE PAGES

Kayode, Olumide; Wang, Ruiying; Pendlebury, Devon F.; ...

2016-11-03

The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. While considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here in this paper, we examine the importance of substrate dynamics in the cleavage of Kunitz-BPTI protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4 Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals amore » dramatic conformational change in the substrate upon proteolysis. Using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning three orders of magnitude, we identify global and local dynamic features of substrates on the ns-μs timescale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substratelike and product-like states, linking substrate dynamics on the ns-μs timescale with large collective substrate motions on the much slower timescale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis.« less

An enzyme-mediated protein-fragment complementation assay for substrate screening of sortase A.

PubMed

Li, Ning; Yu, Zheng; Ji, Qun; Sun, Jingying; Liu, Xiao; Du, Mingjuan; Zhang, Wei

2017-04-29

Enzyme-mediated protein conjugation has gained great attention recently due to the remarkable site-selectivity and mild reaction condition affected by the nature of enzyme. Among all sorts of enzymes reported, sortase A from Staphylococcus aureus (SaSrtA) is the most popular enzyme due to its selectivity and well-demonstrated applications. Position scanning has been widely applied to understand enzyme substrate specificity, but the low throughput of chemical synthesis of peptide substrates and analytical methods (HPLC, LC-ESI-MS) have been the major hurdle to fully decode enzyme substrate profile. We have developed a simple high-throughput substrate profiling method to reveal novel substrates of SaSrtA 7M, a widely used hyperactive peptide ligase, by modified protein-fragment complementation assay (PCA). A small library targeting the LPATG motif recognized by SaSrtA 7M was generated and screened against proteins carrying N-terminal glycine. Using this method, we have confirmed all currently known substrates of the enzyme, and moreover identified some previously unknown substrates with varying activities. The method provides an easy, fast and highly-sensitive way to determine substrate profile of a peptide ligase in a high-throughput manner. Copyright © 2017 Elsevier Inc. All rights reserved.

Sequence specificity of the human mRNA N6-adenosine methylase in vitro.

PubMed Central

Harper, J E; Miceli, S M; Roberts, R J; Manley, J L

1990-01-01

N6-adenosine methylation is a frequent modification of mRNAs and their precursors, but little is known about the mechanism of the reaction or the function of the modification. To explore these questions, we developed conditions to examine N6-adenosine methylase activity in HeLa cell nuclear extracts. Transfer of the methyl group from S-[3H methyl]-adenosylmethionine to unlabeled random copolymer RNA substrates of varying ribonucleotide composition revealed a substrate specificity consistent with a previously deduced consensus sequence, Pu[G greater than A]AC[A/C/U]. 32-P labeled RNA substrates of defined sequence were used to examine the minimum sequence requirements for methylation. Each RNA was 20 nucleotides long, and contained either the core consensus sequence GGACU, or some variation of this sequence. RNAs containing GGACU, either in single or multiple copies, were good substrates for methylation, whereas RNAs containing single base substitutions within the GGACU sequence gave dramatically reduced methylation. These results demonstrate that the N6-adenosine methylase has a strict sequence specificity, and that there is no requirement for extended sequences or secondary structures for methylation. Recognition of this sequence does not require an RNA component, as micrococcal nuclease pretreatment of nuclear extracts actually increased methylation efficiency. Images PMID:2216767

Batch growth kinetic studies of locally isolated cyanide-degrading Serratia marcescens strain AQ07.

PubMed

Karamba, Kabiru Ibrahim; Ahmad, Siti Aqlima; Zulkharnain, Azham; Yasid, Nur Adeela; Ibrahim, Salihu; Shukor, Mohd Yunus

2018-01-01

The evaluation of degradation and growth kinetics of Serratia marcescens strain AQ07 was carried out using three half-order models at all the initial concentrations of cyanide with the values of regression exceeding 0.97. The presence of varying cyanide concentrations reveals that the growth and degradation of bacteria were affected by the increase in cyanide concentration with a total halt at 700 ppm KCN after 72 h incubation. In this study, specific growth and degradation rates were found to trail the substrate inhibition kinetics. These two rates fitted well to the kinetic models of Teissier, Luong, Aiba and Heldane, while the performance of Monod model was found to be unsatisfactory. These models were used to clarify the substrate inhibition on the bacteria growth. The analyses of these models have shown that Luong model has fitted the experimental data with the highest coefficient of determination ( R 2 ) value of 0.9794 and 0.9582 with the lowest root mean square error (RMSE) value of 0.000204 and 0.001, respectively, for the specific rate of degradation and growth. It is the only model that illustrates the maximum substrate concentration ( S m ) of 713.4 and empirical constant ( n ) of 1.516. Tessier and Aiba fitted the experimental data with a R 2 value of 0.8002 and 0.7661 with low RMSE of 0.0006, respectively, for specific biodegradation rate, while having a R 2 value of 0.9 and RMSE of 0.001, respectively, for specific growth rate. Haldane has the lowest R 2 value of 0.67 and 0.78 for specific biodegradation and growth rate with RMSE of 0.0006 and 0.002, respectively. This indicates the level of the bacteria stability in varying concentrations of cyanide and the maximum cyanide concentration it can tolerate within a specific time period. The biokinetic constant predicted from this model demonstrates a good ability of the locally isolated bacteria in cyanide remediation in industrial effluents.

Molecular Evolution of the Substrate Specificity of Chloroplastic Aldolases/Rubisco Lysine Methyltransferases in Plants.

PubMed

Ma, Sheng; Martin-Laffon, Jacqueline; Mininno, Morgane; Gigarel, Océane; Brugière, Sabine; Bastien, Olivier; Tardif, Marianne; Ravanel, Stéphane; Alban, Claude

2016-04-04

Rubisco and fructose-1,6-bisphosphate aldolases (FBAs) are involved in CO2 fixation in chloroplasts. Both enzymes are trimethylated at a specific lysine residue by the chloroplastic protein methyltransferase LSMT. Genes coding LSMT are present in all plant genomes but the methylation status of the substrates varies in a species-specific manner. For example, chloroplastic FBAs are naturally trimethylated in both Pisum sativum and Arabidopsis thaliana, whereas the Rubisco large subunit is trimethylated only in the former species. The in vivo methylation status of aldolases and Rubisco matches the catalytic properties of AtLSMT and PsLSMT, which are able to trimethylate FBAs or FBAs and Rubisco, respectively. Here, we created chimera and site-directed mutants of monofunctional AtLSMT and bifunctional PsLSMT to identify the molecular determinants responsible for substrate specificity. Our results indicate that the His-Ala/Pro-Trp triad located in the central part of LSMT enzymes is the key motif to confer the capacity to trimethylate Rubisco. Two of the critical residues are located on a surface loop outside the methyltransferase catalytic site. We observed a strict correlation between the presence of the triad motif and the in vivo methylation status of Rubisco. The distribution of the motif into a phylogenetic tree further suggests that the ancestral function of LSMT was FBA trimethylation. In a recent event during higher plant evolution, this function evolved in ancestors of Fabaceae, Cucurbitaceae, and Rosaceae to include Rubisco as an additional substrate to the archetypal enzyme. Our study provides insight into mechanisms by which SET-domain protein methyltransferases evolve new substrate specificity. Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.

Bio-inert interfaces via biomimetic anchoring of a zwitterionic copolymer on versatile substrates.

PubMed

Dizon, Gian Vincent; Chou, Ying-Nien; Yeh, Lu-Chen; Venault, Antoine; Huang, James; Chang, Yung

2018-05-22

Bio-inert biomaterial design is vital for fields like biosensors, medical implants, and drug delivery systems. Bio-inert materials are generally hydrophilic and electrical neutral. One limitation faced in the design of bio-inert materials is that most of the modifiers used are specific to their substrate. In this work, we synthesized a novel zwitterionic copolymer containing a catechol group, a non-substrate dependent biomimetic anchoring segment, that can form a stable coating on various materials. No previous study was conducted using a grafting-to approach and determined the critical amount of catechol groups needed to effectively modify a material. The synthesized copolymers of sulfobetaine acrylamide (SBAA) and dopamine methacrylamide (DMA) in this work contains varying numbers of catechol groups, in which the critical number of catechol groups that had effectively modified substrates to have the bio-inert property was determined. The bio-inert property and capability to do coating on versatile substrates were evaluated in contact with human blood by coating different material groups such as ceramic, metallic, and polymeric groups. The novel structure and the simple grafting-to approach provides bio-inert property on various materials, giving them non-specific adsorption and attachment of biomolecules such as plasma proteins, erythrocytes, thrombocytes, bacteria, and tissue cells (85-95% reduction). Copyright © 2018 Elsevier Inc. All rights reserved.

An Acrobatic Substrate Metamorphosis Reveals a Requirement for Substrate Conformational Dynamics in Trypsin Proteolysis.

PubMed

Kayode, Olumide; Wang, Ruiying; Pendlebury, Devon F; Cohen, Itay; Henin, Rachel D; Hockla, Alexandra; Soares, Alexei S; Papo, Niv; Caulfield, Thomas R; Radisky, Evette S

2016-12-16

The molecular basis of enzyme catalytic power and specificity derives from dynamic interactions between enzyme and substrate during catalysis. Although considerable effort has been devoted to understanding how conformational dynamics within enzymes affect catalysis, the role of conformational dynamics within protein substrates has not been addressed. Here, we examine the importance of substrate dynamics in the cleavage of Kunitz-bovine pancreatic trypsin inhibitor protease inhibitors by mesotrypsin, finding that the varied conformational dynamics of structurally similar substrates can profoundly impact the rate of catalysis. A 1.4-Å crystal structure of a mesotrypsin-product complex formed with a rapidly cleaved substrate reveals a dramatic conformational change in the substrate upon proteolysis. By using long all-atom molecular dynamics simulations of acyl-enzyme intermediates with proteolysis rates spanning 3 orders of magnitude, we identify global and local dynamic features of substrates on the nanosecond-microsecond time scale that correlate with enzymatic rates and explain differential susceptibility to proteolysis. By integrating multiple enhanced sampling methods for molecular dynamics, we model a viable conformational pathway between substrate-like and product-like states, linking substrate dynamics on the nanosecond-microsecond time scale with large collective substrate motions on the much slower time scale of catalysis. Our findings implicate substrate flexibility as a critical determinant of catalysis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Evaluation of preparation methods for suspended nano-objects on substrates for dimensional measurements by atomic force microscopy

PubMed Central

Göhler, Daniel; Wessely, Benno; Stintz, Michael; Lazzerini, Giovanni Mattia; Yacoot, Andrew

2017-01-01

Dimensional measurements on nano-objects by atomic force microscopy (AFM) require samples of safely fixed and well individualized particles with a suitable surface-specific particle number on flat and clean substrates. Several known and proven particle preparation methods, i.e., membrane filtration, drying, rinsing, dip coating as well as electrostatic and thermal precipitation, were performed by means of scanning electron microscopy to examine their suitability for preparing samples for dimensional AFM measurements. Different suspensions of nano-objects (with varying material, size and shape) stabilized in aqueous solutions were prepared therefore on different flat substrates. The drop-drying method was found to be the most suitable one for the analysed suspensions, because it does not require expensive dedicated equipment and led to a uniform local distribution of individualized nano-objects. Traceable AFM measurements based on Si and SiO2 coated substrates confirmed the suitability of this technique. PMID:28904839

Evaluation of preparation methods for suspended nano-objects on substrates for dimensional measurements by atomic force microscopy.

PubMed

Fiala, Petra; Göhler, Daniel; Wessely, Benno; Stintz, Michael; Lazzerini, Giovanni Mattia; Yacoot, Andrew

2017-01-01

Dimensional measurements on nano-objects by atomic force microscopy (AFM) require samples of safely fixed and well individualized particles with a suitable surface-specific particle number on flat and clean substrates. Several known and proven particle preparation methods, i.e., membrane filtration, drying, rinsing, dip coating as well as electrostatic and thermal precipitation, were performed by means of scanning electron microscopy to examine their suitability for preparing samples for dimensional AFM measurements. Different suspensions of nano-objects (with varying material, size and shape) stabilized in aqueous solutions were prepared therefore on different flat substrates. The drop-drying method was found to be the most suitable one for the analysed suspensions, because it does not require expensive dedicated equipment and led to a uniform local distribution of individualized nano-objects. Traceable AFM measurements based on Si and SiO 2 coated substrates confirmed the suitability of this technique.

miCLIP-MaPseq, a Substrate Identification Approach for Radical SAM RNA Methylating Enzymes.

PubMed

Stojković, Vanja; Chu, Tongyue; Therizols, Gabriel; Weinberg, David E; Fujimori, Danica Galonić

2018-06-13

Although present across bacteria, the large family of radical SAM RNA methylating enzymes is largely uncharacterized. Escherichia coli RlmN, the founding member of the family, methylates an adenosine in 23S rRNA and several tRNAs to yield 2-methyladenosine (m 2 A). However, varied RNA substrate specificity among RlmN enzymes, combined with the ability of certain family members to generate 8-methyladenosine (m 8 A), makes functional predictions across this family challenging. Here, we present a method for unbiased substrate identification that exploits highly efficient, mechanism-based cross-linking between the enzyme and its RNA substrates. Additionally, by determining that the thermostable group II intron reverse transcriptase introduces mismatches at the site of the cross-link, we have identified the precise positions of RNA modification using mismatch profiling. These results illustrate the capability of our method to define enzyme-substrate pairs and determine modification sites of the largely uncharacterized radical SAM RNA methylating enzyme family.

Elucidating the substrate specificities of acyl-lipid thioesterases from diverse plant taxa.

PubMed

Kalinger, Rebecca S; Pulsifer, Ian P; Rowland, Owen

2018-06-01

Acyl-ACP thioesterase enzymes, which cleave fatty acyl thioester bonds to release free fatty acids, contribute to much of the fatty acid diversity in plants. In Arabidopsis thaliana, a family of four single hot-dog fold domain, plastid-localized acyl-lipid thioesterases (AtALT1-4) generate medium-chain (C6-C14) fatty and β-keto fatty acids as secondary metabolites. These volatile products may serve to attract insect pollinators or deter predatory insects. Homologs of AtALT1-4 are present in all plant taxa, but are nearly all uncharacterized. Despite high sequence identity, AtALT1-4 generate different lipid products, suggesting that ALT homologs in other plants also have highly varied activities. We investigated the catalytic diversity of ALT-like thioesterases by screening the substrate specificities of 15 ALT homologs from monocots, eudicots, a lycophyte, a green microalga, and the ancient gymnosperm Gingko biloba, via expression in Escherichia coli. Overall, these enzymes had highly varied substrate preferences compared to one another and to AtALT1-4, and could be classified into four catalytic groups comprising members from diverse taxa. Group 1 ALTs primarily generated 14:1 β-keto fatty acids, Group 2 ALTs produced 6-10 carbon fatty/β-keto fatty acids, Group 3 ALTs predominantly produced 12-14 carbon fatty acids, and Group 4 ALTs mainly generated 16 carbon fatty acids. Enzymes in each group differed significantly in the quantities of lipids and types of minor products they generated in E. coli. Medium-chain fatty acids are used to manufacture insecticides, pharmaceuticals, and biofuels, and ALT-like proteins are ideal candidates for metabolic engineering to produce specific fatty acids in significant quantities. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Molecular cloning, characterization and comparison of bile salt hydrolases from Lactobacillus johnsonii PF01.

PubMed

Chae, J P; Valeriano, V D; Kim, G-B; Kang, D-K

2013-01-01

To clone, characterize and compare the bile salt hydrolase (BSH) genes of Lactobacillus johnsonii PF01. The BSH genes were amplified by polymerase chain reaction (PCR) using specific oligonucleotide primers, and the products were inserted into the pET21b expression vector. Escherichia coli BLR (DE3) cells were transformed with pET21b vectors containing the BSH genes and induced using 0·1 mmol l(-1) isopropylthiolgalactopyranoside. The overexpressed BSH enzymes were purified using a nickel-nitrilotriacetic acid (Ni(2+) -NTA) agarose column and their activities characterized. BSH A hydrolysed tauro-conjugated bile salts optimally at pH 5·0 and 55°C, whereas BSH C hydrolysed glyco-conjugated bile salts optimally at pH 5·0 and 70°C. The enzymes had no preferential activities towards a specific cholyl moiety. BSH enzymes vary in their substrate specificities and characteristics to broaden its activity. Despite the lack of conservation in their putative substrate-binding sites, these remain functional through motif conservation. This is to our knowledge the first report of isolation of BSH enzymes from a single strain, showing hydrolase activity towards either glyco-conjugated or tauro-conjugated bile salts. Future structural homology studies and site-directed mutagenesis of sites associated with substrate specificity may elucidate specificities of BSH enzymes. © 2012 The Society for Applied Microbiology.

The Aldo-Keto Reductase Superfamily and its Role in Drug Metabolism and Detoxification

PubMed Central

Barski, Oleg A.; Tipparaju, Srinivas M.; Bhatnagar, Aruni

2008-01-01

The Aldo-Keto Reductase (AKR) superfamily comprises of several enzymes that catalyze redox transformations involved in biosynthesis, intermediary metabolism and detoxification. Substrates of the family include glucose, steroids, glycosylation end products, lipid peroxidation products, and environmental pollutants. These proteins adopt a (β/α)8 barrel structural motif interrupted by a number of extraneous loops and helixes that vary between proteins and bring structural identity to individual families. The human AKR family differs from the rodent families. Due to their broad substrate specificity, AKRs play an important role in the Phase II detoxification of a large number of pharmaceuticals, drugs, and xenobiotics. PMID:18949601

Understanding Which Residues of the Active Site and Loop Structure of a Tyrosine Aminomutase Define Its Mutase and Lyase Activities.

PubMed

Attanayake, Gayanthi; Walter, Tyler; Walker, Kevin D

2018-05-30

Site-directed mutations and substrate analogues were used to gain insights into the branch-point reaction of the 3,5-dihydro-5-methylidene-4 H-imidazol-4-one (MIO)-tyrosine aminomutase from Oryza sativa ( OsTAM). Exchanging the active residues of OsTAM (Y125C/N446K) for those in a phenylalanine aminomutase TcPAM altered its substrate specificity from tyrosine to phenylalanine. The aminomutase mechanism of OsTAM surprisingly changed almost exclusively to that of an ammonia lyase making cinnamic acid (>95%) over β-phenylalanine [Walter, T., et al. (2016) Biochemistry 55, 3497-3503]. We hypothesized that the missing electronics or sterics on the aryl ring of the phenylalanine substrate, compared with the sizable electron-donating hydroxyl of the natural tyrosine substrate, influenced the unexpected lyase reactivity of the OsTAM mutant. The double mutant was incubated with 16 α-phenylalanine substituent analogues of varying electronic strengths and sterics. The mutant converted each analogue principally to its acrylate with ∼50% conversion of the p-Br substrate, making only a small amount of the β-amino acid. The inner loop structure over the entrance to the active site was also mutated to assess how the lyase and mutase activities are affected. An OsTAM loop mutant, matching the loop residues of TcPAM, still chiefly made >95% of the acrylate from each substrate. A combined active site:loop mutant was most reactive but remained a lyase, making 10-fold more acrylates than other mutants did. While mutations within the active site changed the substrate specificity of OsTAM, continued exploration is needed to fully understand the interplay among the inner loop, the substrate, and the active site in defining the mutase and lyase activities.

On-chip ultra-thin layer chromatography and surface enhanced Raman spectroscopy.

PubMed

Chen, Jing; Abell, Justin; Huang, Yao-wen; Zhao, Yiping

2012-09-07

We demonstrate that silver nanorod (AgNR) array substrates can be used for on-chip separation and detection of chemical mixtures by combining ultra-thin layer chromatography (UTLC) and surface enhanced Raman spectroscopy (SERS). The UTLC-SERS plate consists of an AgNR array fabricated by oblique angle deposition. The capability of the AgNR substrates to separate the different compounds in a mixture was explored using a mixture of four dyes and a mixture of melamine and Rhodamine 6G at varied concentrations with different mobile phase solvents. After UTLC separation, spatially-resolved SERS spectra were collected along the mobile phase development direction and the intensities of specific SERS peaks from each component were used to generate chromatograms. The AgNR substrates demonstrate the potential for separating the test dyes with plate heights as low as 9.6 μm. The limits of detection are between 10(-5)-10(-6) M. Furthermore, we show that the coupling of UTLC with SERS improves the SERS detection specificity, as small amounts of target analytes can be separated from the interfering background components.

Deposition of Nanostructured CdS Thin Films by Thermal Evaporation Method: Effect of Substrate Temperature

PubMed Central

Memarian, Nafiseh; Rozati, Seyeed Mohammad; Concina, Isabella

2017-01-01

Nanocrystalline CdS thin films were grown on glass substrates by a thermal evaporation method in a vacuum of about 2 × 10−5 Torr at substrate temperatures ranging between 25 °C and 250 °C. The physical properties of the layers were analyzed by transmittance spectra, XRD, SEM, and four-point probe measurements, and exhibited strong dependence on substrate temperature. The XRD patterns of the films indicated the presence of single-phase hexagonal CdS with (002) orientation. The structural parameters of CdS thin films (namely crystallite size, number of grains per unit area, dislocation density and the strain of the deposited films) were also calculated. The resistivity of the as-deposited films were found to vary in the range 3.11–2.2 × 104 Ω·cm, depending on the substrate temperature. The low resistivity with reasonable transmittance suggest that this is a reliable way to fine-tune the functional properties of CdS films according to the specific application. PMID:28773133

Molecular and thermodynamic mechanisms of the chloride-dependent human angiotensin-I-converting enzyme (ACE).

PubMed

Yates, Christopher J; Masuyer, Geoffrey; Schwager, Sylva L U; Akif, Mohd; Sturrock, Edward D; Acharya, K Ravi

2014-01-17

Somatic angiotensin-converting enzyme (sACE), a key regulator of blood pressure and electrolyte fluid homeostasis, cleaves the vasoactive angiotensin-I, bradykinin, and a number of other physiologically relevant peptides. sACE consists of two homologous and catalytically active N- and C-domains, which display marked differences in substrate specificities and chloride activation. A series of single substitution mutants were generated and evaluated under varying chloride concentrations using isothermal titration calorimetry. The x-ray crystal structures of the mutants provided details on the chloride-dependent interactions with ACE. Chloride binding in the chloride 1 pocket of C-domain ACE was found to affect positioning of residues from the active site. Analysis of the chloride 2 pocket R522Q and R522K mutations revealed the key interactions with the catalytic site that are stabilized via chloride coordination of Arg(522). Substrate interactions in the S2 subsite were shown to affect chloride affinity in the chloride 2 pocket. The Glu(403)-Lys(118) salt bridge in C-domain ACE was shown to stabilize the hinge-bending region and reduce chloride affinity by constraining the chloride 2 pocket. This work demonstrated that substrate composition to the C-terminal side of the scissile bond as well as interactions of larger substrates in the S2 subsite moderate chloride affinity in the chloride 2 pocket of the ACE C-domain, providing a rationale for the substrate-selective nature of chloride dependence in ACE and how this varies between the N- and C-domains.

Molecular and Thermodynamic Mechanisms of the Chloride-dependent Human Angiotensin-I-converting Enzyme (ACE)*

PubMed Central

Yates, Christopher J.; Masuyer, Geoffrey; Schwager, Sylva L. U.; Akif, Mohd; Sturrock, Edward D.; Acharya, K. Ravi

2014-01-01

Somatic angiotensin-converting enzyme (sACE), a key regulator of blood pressure and electrolyte fluid homeostasis, cleaves the vasoactive angiotensin-I, bradykinin, and a number of other physiologically relevant peptides. sACE consists of two homologous and catalytically active N- and C-domains, which display marked differences in substrate specificities and chloride activation. A series of single substitution mutants were generated and evaluated under varying chloride concentrations using isothermal titration calorimetry. The x-ray crystal structures of the mutants provided details on the chloride-dependent interactions with ACE. Chloride binding in the chloride 1 pocket of C-domain ACE was found to affect positioning of residues from the active site. Analysis of the chloride 2 pocket R522Q and R522K mutations revealed the key interactions with the catalytic site that are stabilized via chloride coordination of Arg522. Substrate interactions in the S2 subsite were shown to affect chloride affinity in the chloride 2 pocket. The Glu403-Lys118 salt bridge in C-domain ACE was shown to stabilize the hinge-bending region and reduce chloride affinity by constraining the chloride 2 pocket. This work demonstrated that substrate composition to the C-terminal side of the scissile bond as well as interactions of larger substrates in the S2 subsite moderate chloride affinity in the chloride 2 pocket of the ACE C-domain, providing a rationale for the substrate-selective nature of chloride dependence in ACE and how this varies between the N- and C-domains. PMID:24297181

Guide-substrate base-pairing requirement for box H/ACA RNA-guided RNA pseudouridylation.

PubMed

De Zoysa, Meemanage D; Wu, Guowei; Katz, Raviv; Yu, Yi-Tao

2018-06-05

Box H/ACA RNAs are a group of small RNAs found in abundance in eukaryotes (as well as in archaea). Although their sequences differ, eukaryotic box H/ACA RNAs all share the same unique hairpin-hinge-hairpin-tail structure. Almost all of them function as guides that primarily direct pseudouridylation of rRNAs and spliceosomal snRNAs at specific sites. Although box H/ACA RNA-guided pseudouridylation has been extensively studied, the detailed rules governing this reaction, especially those concerning the guide RNA-substrate RNA base-pairing interactions that determine the specificity and efficiency of pseudouridylation, are still not exactly clear. This is particularly relevant given that the lengths of the guide sequences involved in base-pairing vary from one box H/ACA RNA to another. Here, we carry out a detailed investigation into guide-substrate base-pairing interactions, and identify the minimum number of base-pairs (8), required for RNA-guided pseudouridylation. In addition, we find that the pseudouridylation pocket, present in each hairpin of box H/ACA RNA, exhibits flexibility in fitting slightly different substrate sequences. Our results are consistent across three independent pseudouridylation pockets tested, suggesting that our findings are generally applicable to box H/ACA RNA-guided RNA pseudouridylation. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Microbial Breakdown of Organic Carbon in the Diverse Sediments of Guaymas Basin

NASA Astrophysics Data System (ADS)

Hoarfrost, A.; Snider, R.; Arnosti, C.

2015-12-01

Guaymas Basin is characterized by sediments under conditions ranging from hemipelagic to hydrothermal. This wide range in geochemical contexts results in diverse microbial communities that may have varying abilities to access organic matter. We can address these functional differences by comparing enzyme activities initializing the breakdown of organic matter across these sediment types; however, previous direct measurements of the extracellular hydrolysis of complex organic carbon in sediments are sparse. We measured this first step of heterotrophic processing of organic matter in sediments at 5-10cm and 55-60cm depth from a wide range of environmental settings in Guaymas Basin. Sediment sources included sulfidic seeps on the Sonora Margin, hemipelagic ridge flank sediments, and hydrothermically altered Sonora Margin sediments bordering a methane seep site. Hydrolysis of organic substrates varied by depth and by sediment source, but despite high energy potential and organic carbon load in sulfidic sediments, activity was not highest where hydrothermal influence was highest. These results suggest that heterotrophic breakdown of organic carbon in Guaymas Basin sediments may be sensitive to factors including varying composition of organic carbon available in different sediment types, or differences in microbial community capacities to access specific organic substrates.

Single-Molecule Probing the Energy Landscape of Enzymatic Reaction and Non-Covalent Interactions

NASA Astrophysics Data System (ADS)

Lu, H. Peter; Hu, Dehong; Chen, Yu; Vorpagel, Erich R.

2002-03-01

We have applied single-molecule spectroscopy under physiological conditions to study the mechanisms and dynamics of T4 lysozyme enzymatic reactions, characterizing mode-specific protein conformational dynamics. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time. The overall reaction rates were found to vary widely from molecule-to-molecule, and the initial non-specific binding of the enzyme to the substrate was seen to dominate this inhomogeneity. The reaction steps subsequent to the initial binding were found to have homogeneous rates. Molecular dynamics simulation has been applied to elucidate the mechanism and intermediate states of the single-molecule enzymatic reaction. Combining the analysis of single-molecule experimental trajectories, MD simulation trajectories, and statistical modeling, we have revealed the nature of multiple intermediate states involved in the active enzyme-substrate complex formation and the associated conformational change mechanism and dynamics.

Fabricating amorphous silicon solar cells by varying the temperature _of the substrate during deposition of the amorphous silicon layer

DOEpatents

Carlson, David E.

1982-01-01

An improved process for fabricating amorphous silicon solar cells in which the temperature of the substrate is varied during the deposition of the amorphous silicon layer is described. Solar cells manufactured in accordance with this process are shown to have increased efficiencies and fill factors when compared to solar cells manufactured with a constant substrate temperature during deposition of the amorphous silicon layer.

The novel Legionella pneumophila type II secretion substrate NttC contributes to infection of amoebae Hartmannella vermiformis and Willaertia magna.

PubMed

Tyson, Jessica Y; Vargas, Paloma; Cianciotto, Nicholas P

2014-12-01

The type II protein secretion (T2S) system of Legionella pneumophila secretes over 25 proteins, including novel proteins that have no similarity to proteins of known function. T2S is also critical for the ability of L. pneumophila to grow within its natural amoebal hosts, including Acanthamoeba castellanii, Hartmannella vermiformis and Naegleria lovaniensis. Thus, T2S has an important role in the natural history of legionnaires' disease. Our previous work demonstrated that the novel T2S substrate NttA promotes intracellular infection of A. castellanii, whereas the secreted RNase SrnA, acyltransferase PlaC, and metalloprotease ProA all promote infection of H. vermiformis and N. lovaniensis. In this study, we determined that another novel T2S substrate that is specific to Legionella, designated NttC, is unique in being required for intracellular infection of H. vermiformis but not for infection of N. lovaniensis or A. castellanii. Expanding our repertoire of amoebal hosts, we determined that Willaertia magna is susceptible to infection by L. pneumophila strains 130b, Philadelphia-1 and Paris. Furthermore, T2S and, more specifically, NttA, NttC and PlaC were required for infection of W. magna. Taken together, these data demonstrate that the T2S system of L. pneumophila is critical for infection of at least four types of aquatic amoebae and that the importance of the individual T2S substrates varies in a host cell-specific fashion. Finally, it is now clear that novel T2S-dependent proteins that are specific to the genus Legionella are particularly important for L. pneumophila infection of key, environmental hosts. © 2014 The Authors.

Electric field induced microstructures in thin films on physicochemically heterogeneous and patterned substrates.

PubMed

Srivastava, Samanvaya; Reddy, P Dinesh Sankar; Wang, Cindy; Bandyopadhyay, Dipankar; Sharma, Ashutosh

2010-05-07

We study by nonlinear simulations the electric field induced pattern formation in a thin viscous film resting on a topographically or chemically patterned substrate. The thin film microstructures can be aligned to the substrate patterns within a window of parameters where the spinodal length scale of the field induced instability is close to the substrate periodicity. We investigate systematically the change in the film morphology and order when (i) the substrate pattern periodicity is varied at a constant film thickness and (ii) the film thickness is varied at a constant substrate periodicity. Simulations show two distinct pathway of evolution when the substrate-topography changes from protrusions to cavities. The isolated substrate defects generate locally ordered ripplelike structures distinct from the structures on a periodically patterned substrate. In the latter case, film morphology is governed by a competition between the pattern periodicity and the length scale of instability. Relating the thin film morphologies to the underlying substrate pattern has implications for field induced patterning and robustness of inter-interface pattern transfer, e.g., coding-decoding of information printed on a substrate.

Fabrication of Semiconductor ZnO Nanostructures for Versatile SERS Application

PubMed Central

Yang, Lili; Yang, Yong; Ma, Yunfeng; Li, Shuai; Wei, Yuquan; Huang, Zhengren; Long, Nguyen Viet

2017-01-01

Since the initial discovery of surface-enhanced Raman scattering (SERS) in the 1970s, it has exhibited a huge potential application in many fields due to its outstanding advantages. Since the ultra-sensitive noble metallic nanostructures have increasingly exposed themselves as having some problems during application, semiconductors have been gradually exploited as one of the critical SERS substrate materials due to their distinctive advantages when compared with noble metals. ZnO is one of the most representative metallic oxide semiconductors with an abundant reserve, various and cost-effective fabrication techniques, as well as special physical and chemical properties. Thanks to the varied morphologies, size-dependent exciton, good chemical stability, a tunable band gap, carrier concentration, and stoichiometry, ZnO nanostructures have the potential to be exploited as SERS substrates. Moreover, other distinctive properties possessed by ZnO such as biocompatibility, photocatcalysis and self-cleaning, and gas- and chemo-sensitivity can be synergistically integrated and exerted with SERS activity to realize the multifunctional potential of ZnO substrates. In this review, we discuss the inevitable development trend of exploiting the potential semiconductor ZnO as a SERS substrate. After clarifying the root cause of the great disparity between the enhancement factor (EF) of noble metals and that of ZnO nanostructures, two specific methods are put forward to improve the SERS activity of ZnO, namely: elemental doping and combination of ZnO with noble metals. Then, we introduce a distinctive advantage of ZnO as SERS substrate and illustrate the necessity of reporting a meaningful average EF. We also summarize some fabrication methods for ZnO nanostructures with varied dimensions (0–3 dimensions). Finally, we present an overview of ZnO nanostructures for the versatile SERS application. PMID:29156600

Method and system using power modulation for maskless vapor deposition of spatially graded thin film and multilayer coatings with atomic-level precision and accuracy

DOEpatents

Montcalm, Claude [Livermore, CA; Folta, James Allen [Livermore, CA; Tan, Swie-In [San Jose, CA; Reiss, Ira [New City, NY

2002-07-30

A method and system for producing a film (preferably a thin film with highly uniform or highly accurate custom graded thickness) on a flat or graded substrate (such as concave or convex optics), by sweeping the substrate across a vapor deposition source operated with time-varying flux distribution. In preferred embodiments, the source is operated with time-varying power applied thereto during each sweep of the substrate to achieve the time-varying flux distribution as a function of time. A user selects a source flux modulation recipe for achieving a predetermined desired thickness profile of the deposited film. The method relies on precise modulation of the deposition flux to which a substrate is exposed to provide a desired coating thickness distribution.

Characterisation and treatment of VOCs in process water from upgrading facilities for compressed biogas (CBG).

PubMed

Nilsson Påledal, S; Arrhenius, K; Moestedt, J; Engelbrektsson, J; Stensen, K

2016-02-01

Compression and upgrading of biogas to vehicle fuel generates process water, which to varying degrees contains volatile organic compounds (VOCs) originating from the biogas. The compostion of this process water has not yet been studied and scientifically published and there is currently an uncertainty regarding content of VOCs and how the process water should be managed to minimise the impact on health and the environment. The aim of the study was to give an overview about general levels of VOCs in the process water. Characterisation of process water from amine and water scrubbers at plants digesting waste, sewage sludge or agricultural residues showed that both the average concentration and composition of particular VOCs varied depending on the substrate used at the biogas plant, but the divergence was high and the differences for total concentrations from the different substrate groups were only significant for samples from plants using waste compared to residues from agriculture. The characterisation also showed that the content of VOCs varied greatly between different sampling points for same main substrate and between sampling occasions at the same sampling point, indicating that site-specific conditions are important for the results which also indicates that a number of analyses at different times are required in order to make an more exact characterisation with low uncertainty. Inhibition of VOCs in the anaerobic digestion (AD) process was studied in biomethane potential tests, but no inhibition was observed during addition of synthetic process water at concentrations of 11.6 mg and 238 mg VOC/L. Copyright © 2015 Elsevier Ltd. All rights reserved.

Electromagnetic energy coupling mechanism with matrix architecture control

NASA Technical Reports Server (NTRS)

Hughes, Eli (Inventor); Knowles, Gareth (Inventor)

2006-01-01

The present invention relates generally to reconfigurable, solid-state matrix arrays comprising multiple rows and columns of reconfigurable secondary mechanisms that are independently tuned. Specifically, the invention relates to reconfigurable devices comprising multiple, solid-state mechanisms characterized by at least one voltage-varied parameter disposed within a flexible, multi-laminate film, which are suitable for use as magnetic conductors, ground surfaces, antennas, varactors, ferrotunable substrates, or other active or passive electronic mechanisms.

17Beta-hydroxysteroid dehydrogenase (17beta-HSD) in scleractinian corals and zooxanthellae.

PubMed

Blomquist, Charles H; Lima, P H; Tarrant, A M; Atkinson, M J; Atkinson, S

2006-04-01

Steroid metabolism studies have yielded evidence of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity in corals. This project was undertaken to clarify whether there are multiple isoforms of 17beta-HSD, whether activity levels vary seasonally, and if zooxanthellae contribute to activity. 17Beta-HSD activity was characterized in zooxanthellate and azooxanthellate coral fragments collected in summer and winter and in zooxanthellae cultured from Montipora capitata. More specifically, 17beta-HSD activity was characterized with regard to steroid substrate and inhibitor specificity, coenzyme specificity, and Michaelis constants for estradiol (E2) and NADP+. Six samples each of M. capitata and Tubastrea coccinea (three summers, three winters) were assayed with E2 and NADP+. Specific activity levels (pmol/mg protein) varied 10-fold among M. capitata samples and 6-fold among T. coccinea samples. There was overlap of activity levels between summer and winter samples. NADP+/NAD+ activity ratios varied from 1.6 to 22.2 for M. capatita, 2.3 to 3.8 for T. coccinea and 0.7 to 1.1 for zooxanthellae. Coumestrol was the most inhibitory of the steroids and phytoestrogens tested. Our data confirm that corals and zooxanthellae contain 17beta-HSD and are consistent with the presence of more than one isoform of the enzyme.

Intersexual and temporal variation in foraging ecology of prothonotary warblers during the breeding season

USGS Publications Warehouse

Petit, L.J.; Petit, D.R.; Petit, K.E.; Fleming, W.J.

1990-01-01

We studied foraging ecology of Prothonotary Warblers (Protonotaria citrea) over four breeding seasons to determine if this species exhibited sex-specific or temporal variation in foraging behavior. Significant differences between sexes during the prenestling period were found for foraging height and substrate height (foraging method, plant species/substrate, perch diameter, horizontal location from trunk, and prey location were not significantly different). During the nestling period, this divergence between sexes was evident for foraging height, substrate height, substrate / tree species, and prey location. Additionally, male warblers significantly altered their behavior for all seven foraging variables between the two periods, whereas females exhibited changes similar to those of males for five of the foraging variables. This parallel shift suggests a strong behavioral response by both sexes to proximate factors (such as vegetation structure, and prey abundance and distribution) that varied throughout the breeding season. Sex-specific foraging behavior during the prenestling period was best explained by differences in reproductive responsibilities rather than by the theory of intersexual competition for limited resources. During the nestling period, neither hypothesis by itself explained foraging divergences adequately. However, when integrated with the temporal responses of the warblers to changes in prey availability, reproductive responsibilities seemed to be of primary importance in explaining intersexual niche partitioning during the nestling period. We emphasize the importance of considering both intersexual and intraseasonal variation when quantifying a species' foraging ecology.

The Effects of Noncellulosic Compounds on the Nanoscale Interaction Forces Measured between Carbohydrate-Binding Module and Lignocellulosic Biomass.

PubMed

Arslan, Baran; Colpan, Mert; Ju, Xiaohui; Zhang, Xiao; Kostyukova, Alla; Abu-Lail, Nehal I

2016-05-09

The lack of fundamental understanding of the types of forces that govern how cellulose-degrading enzymes interact with cellulosic and noncellulosic components of lignocellulosic surfaces limits the design of new strategies for efficient conversion of biomass to bioethanol. In a step to improve our fundamental understanding of such interactions, nanoscale forces acting between a model cellulase-a carbohydrate-binding module (CBM) of cellobiohydrolase I (CBH I)-and a set of lignocellulosic substrates with controlled composition were measured using atomic force microscopy (AFM). The three model substrates investigated were kraft (KP), sulfite (SP), and organosolv (OPP) pulped substrates. These substrates varied in their surface lignin coverage, lignin type, and xylan and acetone extractives' content. Our results indicated that the overall adhesion forces of biomass to CBM increased linearly with surface lignin coverage with kraft lignin showing the highest forces among lignin types investigated. When the overall adhesion forces were decoupled into specific and nonspecific component forces via the Poisson statistical model, hydrophobic and Lifshitz-van der Waals (LW) forces dominated the binding forces of CBM to kraft lignin, whereas permanent dipole-dipole interactions and electrostatic forces facilitated the interactions of lignosulfonates to CBM. Xylan and acetone extractives' content increased the attractive forces between CBM and lignin-free substrates, most likely through hydrogen bonding forces. When the substrates treated differently were compared, it was found that both the differences in specific and nonspecific forces between lignin-containing and lignin-free substrates were the least for OPP. Therefore, cellulase enzymes represented by CBM would weakly bind to organosolv lignin. This will facilitate an easy enzyme recovery compared to other substrates treated with kraft or sulfite pulping. Our results also suggest that altering the surface hydrophobicity and the surface energy of lignin that facilitates the LW forces should be a priori to avoid nonproductive binding of cellulase to kraft lignin.

Mapping the Complex Morphology of Cell Interactions with Nanowire Substrates Using FIB-SEM

PubMed Central

Jensen, Mikkel R. B.; Łopacińska, Joanna; Schmidt, Michael S.; Skolimowski, Maciej; Abeille, Fabien; Qvortrup, Klaus; Mølhave, Kristian

2013-01-01

Using high resolution focused ion beam scanning electron microscopy (FIB-SEM) we study the details of cell-nanostructure interactions using serial block face imaging. 3T3 Fibroblast cellular monolayers are cultured on flat glass as a control surface and on two types of nanostructured scaffold substrates made from silicon black (Nanograss) with low- and high nanowire density. After culturing for 72 hours the cells were fixed, heavy metal stained, embedded in resin, and processed with FIB-SEM block face imaging without removing the substrate. The sample preparation procedure, image acquisition and image post-processing were specifically optimised for cellular monolayers cultured on nanostructured substrates. Cells display a wide range of interactions with the nanostructures depending on the surface morphology, but also greatly varying from one cell to another on the same substrate, illustrating a wide phenotypic variability. Depending on the substrate and cell, we observe that cells could for instance: break the nanowires and engulf them, flatten the nanowires or simply reside on top of them. Given the complexity of interactions, we have categorised our observations and created an overview map. The results demonstrate that detailed nanoscale resolution images are required to begin understanding the wide variety of individual cells’ interactions with a structured substrate. The map will provide a framework for light microscopy studies of such interactions indicating what modes of interactions must be considered. PMID:23326412

Quantitative framework for ordered degradation of APC/C substrates.

PubMed

Lu, Dan; Girard, Juliet R; Li, Weihan; Mizrak, Arda; Morgan, David O

2015-11-16

During cell-cycle progression, substrates of a single master regulatory enzyme can be modified in a specific order. Here, we used experimental and computational approaches to dissect the quantitative mechanisms underlying the ordered degradation of the substrates of the ubiquitin ligase APC/C(Cdc20), a key regulator of chromosome segregation in mitosis. We show experimentally that the rate of catalysis varies with different substrates of APC/C(Cdc20). Using a computational model based on multi-step ubiquitination, we then show how changes in the interaction between a single substrate and APC/C(Cdc20) can alter the timing of degradation onset relative to APC/C(Cdc20) activation, while ensuring a fast degradation rate. Degradation timing and dynamics depend on substrate affinity for the enzyme as well as the catalytic rate at which the substrate is modified. When two substrates share the same pool of APC/C(Cdc20), their relative enzyme affinities and rates of catalysis influence the partitioning of APC/C(Cdc20) among substrates, resulting in substrate competition. Depending on how APC/C(Cdc20) is partitioned among its substrates, competition can have minor or major effects on the degradation of certain substrates. We show experimentally that increased expression of the early APC/C(Cdc20) substrate Clb5 does not delay the degradation of the later substrate securin, arguing against a role for competition with Clb5 in establishing securin degradation timing. The degradation timing of APC/C(Cdc20) substrates depends on the multi-step nature of ubiquitination, differences in substrate-APC/C(Cdc20) interactions, and competition among substrates. Our studies provide a conceptual framework for understanding how ordered modification can be established among substrates of the same regulatory enzyme, and facilitate our understanding of how precise temporal control is achieved by a small number of master regulators to ensure a successful cell division cycle.

Transport of steroid 3-sulfates and steroid 17-sulfates by the sodium-dependent organic anion transporter SOAT (SLC10A6).

PubMed

Grosser, Gary; Bennien, Josefine; Sánchez-Guijo, Alberto; Bakhaus, Katharina; Döring, Barbara; Hartmann, Michaela; Wudy, Stefan A; Geyer, Joachim

2018-05-01

The sodium-dependent organic anion transporter SOAT/Soat shows highly specific transport activity for sulfated steroids. SOAT substrates identified so far include dehydroepiandrosterone sulfate, 16α-hydroxydehydroepiandrosterone sulfate, estrone-3-sulfate, pregnenolone sulfate, 17β-estradiol-3-sulfate, and androstenediol sulfate. Apart from these compounds, many other sulfated steroids occur in mammals. Therefore, we aimed to expand the substrate spectrum of SOAT and analyzed the SOAT-mediated transport of eight different sulfated steroids by combining in vitro transport experiments in SOAT-transfected HEK293 cells with LC-MS/MS analytics of cell lysates. In addition, we aimed to better understand the structural requirements for SOAT substrates and so selected structural pairs varying only at specific positions: 3α/3β-sulfate, 17α/17β-sulfate, mono-sulfate/di-sulfate, and 17α-hydroxylation. We found significant and sodium-dependent SOAT-mediated transport of 17α-hydroxypregnenolone sulfate, 17β-estradiol-17-sulfate, androsterone sulfate, epiandrosterone sulfate, testosterone sulfate, epitestosterone sulfate, and 5α-dihydrotestosterone sulfate. However, 17β-estradiol-3,17-disulfate was not transported by SOAT. SOAT substrates from the group of sulfated steroids are characterized by a planar and lipophilic steroid backbone in trans-trans-trans conformation of the rings and a negatively charged mono-sulfate group at positions 3' or 17' with flexibility for α- or β- orientation. Furthermore, 5α-reduction, 16α-hydroxylation, and 17α-hydroxylation are acceptable for SOAT substrate recognition, whereas addition of a second negatively charged sulfate group seems to abolish substrate binding to SOAT, and so 17β-estradiol-3,17-disulfate is not transported by SOAT. Copyright © 2017 Elsevier Ltd. All rights reserved.

Aldehyde Dehydrogenases in Arabidopsis thaliana: Biochemical Requirements, Metabolic Pathways, and Functional Analysis.

PubMed

Stiti, Naim; Missihoun, Tagnon D; Kotchoni, Simeon O; Kirch, Hans-Hubert; Bartels, Dorothea

2011-01-01

Aldehyde dehydrogenases (ALDHs) are a family of enzymes which catalyze the oxidation of reactive aldehydes to their corresponding carboxylic acids. Here we summarize molecular genetic and biochemical analyses of selected ArabidopsisALDH genes. Aldehyde molecules are very reactive and are involved in many metabolic processes but when they accumulate in excess they become toxic. Thus activity of aldehyde dehydrogenases is important in regulating the homeostasis of aldehydes. Overexpression of some ALDH genes demonstrated an improved abiotic stress tolerance. Despite the fact that several reports are available describing a role for specific ALDHs, their precise physiological roles are often still unclear. Therefore a number of genetic and biochemical tools have been generated to address the function with an emphasis on stress-related ALDHs. ALDHs exert their functions in different cellular compartments and often in a developmental and tissue specific manner. To investigate substrate specificity, catalytic efficiencies have been determined using a range of substrates varying in carbon chain length and degree of carbon oxidation. Mutational approaches identified amino acid residues critical for coenzyme usage and enzyme activities.

COMT Genetic Reduction Produces Sexually Divergent Effects on Cortical Anatomy and Working Memory in Mice and Humans.

PubMed

Sannino, Sara; Gozzi, Alessandro; Cerasa, Antonio; Piras, Fabrizio; Scheggia, Diego; Managò, Francesca; Damiano, Mario; Galbusera, Alberto; Erickson, Lucy C; De Pietri Tonelli, Davide; Bifone, Angelo; Tsaftaris, Sotirios A; Caltagirone, Carlo; Weinberger, Daniel R; Spalletta, Gianfranco; Papaleo, Francesco

2015-09-01

Genetic variations in catechol-O-methyltransferase (COMT) that modulate cortical dopamine have been associated with pleiotropic behavioral effects in humans and mice. Recent data suggest that some of these effects may vary among sexes. However, the specific brain substrates underlying COMT sexual dimorphisms remain unknown. Here, we report that genetically driven reduction in COMT enzyme activity increased cortical thickness in the prefrontal cortex (PFC) and postero-parieto-temporal cortex of male, but not female adult mice and humans. Dichotomous changes in PFC cytoarchitecture were also observed: reduced COMT increased a measure of neuronal density in males, while reducing it in female mice. Consistent with the neuroanatomical findings, COMT-dependent sex-specific morphological brain changes were paralleled by divergent effects on PFC-dependent working memory in both mice and humans. These findings emphasize a specific sex-gene interaction that can modulate brain morphological substrates with influence on behavioral outcomes in healthy subjects and, potentially, in neuropsychiatric populations. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Aldehyde Dehydrogenases in Arabidopsis thaliana: Biochemical Requirements, Metabolic Pathways, and Functional Analysis

PubMed Central

Stiti, Naim; Missihoun, Tagnon D.; Kotchoni, Simeon O.; Kirch, Hans-Hubert; Bartels, Dorothea

2011-01-01

Aldehyde dehydrogenases (ALDHs) are a family of enzymes which catalyze the oxidation of reactive aldehydes to their corresponding carboxylic acids. Here we summarize molecular genetic and biochemical analyses of selected Arabidopsis ALDH genes. Aldehyde molecules are very reactive and are involved in many metabolic processes but when they accumulate in excess they become toxic. Thus activity of aldehyde dehydrogenases is important in regulating the homeostasis of aldehydes. Overexpression of some ALDH genes demonstrated an improved abiotic stress tolerance. Despite the fact that several reports are available describing a role for specific ALDHs, their precise physiological roles are often still unclear. Therefore a number of genetic and biochemical tools have been generated to address the function with an emphasis on stress-related ALDHs. ALDHs exert their functions in different cellular compartments and often in a developmental and tissue specific manner. To investigate substrate specificity, catalytic efficiencies have been determined using a range of substrates varying in carbon chain length and degree of carbon oxidation. Mutational approaches identified amino acid residues critical for coenzyme usage and enzyme activities. PMID:22639603

Water Use and Growth of Two Woody Taxa Produced in Varying Indigenous Douglas-Fir Based Soilless Substrates

USDA-ARS?s Scientific Manuscript database

In the Pacific Northwest (PNW) container crops are grown in soilless substrates that contain different percentages of Douglas-fir bark (DFB), sphagnum peat moss and pumice. Previous research conducted by Gabriel et al. found varying combinations and ratios of these components result in differing phy...

Signal processing for the detection of explosive residues on varying substrates using laser-induced breakdown spectroscopy

NASA Astrophysics Data System (ADS)

Morton, Kenneth D., Jr.; Torrione, Peter A.; Collins, Leslie

2011-05-01

Laser induced breakdown spectroscopy (LIBS) can provide rapid, minimally destructive, chemical analysis of substances with the benefit of little to no sample preparation. Therefore, LIBS is a viable technology for the detection of substances of interest in near real-time fielded remote sensing scenarios. Of particular interest to military and security operations is the detection of explosive residues on various surfaces. It has been demonstrated that LIBS is capable of detecting such residues, however, the surface or substrate on which the residue is present can alter the observed spectra. Standard chemometric techniques such as principal components analysis and partial least squares discriminant analysis have previously been applied to explosive residue detection, however, the classification techniques developed on such data perform best against residue/substrate pairs that were included in model training but do not perform well when the residue/substrate pairs are not in the training set. Specifically residues in the training set may not be correctly detected if they are presented on a previously unseen substrate. In this work, we explicitly model LIBS spectra resulting from the residue and substrate to attempt to separate the response from each of the two components. This separation process is performed jointly with classifier design to ensure that the classifier that is developed is able to detect residues of interest without being confused by variations in the substrates. We demonstrate that the proposed classification algorithm provides improved robustness to variations in substrate compared to standard chemometric techniques for residue detection.

Crystal structure and substrate specificity of the [beta]-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus

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

Qiu, Xiayang; Choudhry, Anthony E.; Janson, Cheryl A.

{beta}-Ketoacyl-ACP synthase III (FabH), an essential enzyme for bacterial viability, catalyzes the initiation of fatty acid elongation by condensing malonyl-ACP with acetyl-CoA. We have determined the crystal structure of FabH from Staphylococcus aureus, a Gram-positive human pathogen, to 2 {angstrom} resolution. Although the overall structure of S. aureus FabH is similar to that of Escherichia coli FabH, the primer binding pocket in S. aureus FabH is significantly larger than that present in E. coli FabH. The structural differences, which agree with kinetic parameters, provide explanation for the observed varying substrate specificity for E. coli and S. aureus FabH. The rankmore » order of activity of S. aureus FabH with various acyl-CoA primers was as follows: isobutyryl- > hexanoyl- > butyryl- > isovaleryl- >> acetyl-CoA. The availability of crystal structure may aid in designing potent, selective inhibitors of S. aureus FabH.« less

Uniform modeling of bacterial colony patterns with varying nutrient and substrate

NASA Astrophysics Data System (ADS)

Schwarcz, Deborah; Levine, Herbert; Ben-Jacob, Eshel; Ariel, Gil

2016-04-01

Bacteria develop complex patterns depending on growth condition. For example, Bacillus subtilis exhibit five different patterns depending on substrate hardness and nutrient concentration. We present a unified integro-differential model that reproduces the entire experimentally observed morphology diagram at varying nutrient concentrations and substrate hardness. The model allows a comprehensive and quantitative comparison between experimental and numerical variables and parameters, such as colony growth rate, nutrient concentration and diffusion constants. As a result, the role of the different physical mechanisms underlying and regulating the growth of the colony can be evaluated.

Structural basis of RND-type multidrug exporters

PubMed Central

Yamaguchi, Akihito; Nakashima, Ryosuke; Sakurai, Keisuke

2015-01-01

Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient ejection of drugs from the cell. Substrates are transported through dual multidrug-binding pockets via the peristaltic motion of the substrate translocation channel. Although there are no clinically available inhibitors of bacterial multidrug exporters, efforts to develop inhibitors based on structural information are underway. PMID:25941524

Structural basis of RND-type multidrug exporters.

PubMed

Yamaguchi, Akihito; Nakashima, Ryosuke; Sakurai, Keisuke

2015-01-01

Bacterial multidrug exporters are intrinsic membrane transporters that act as cellular self-defense mechanism. The most notable characteristics of multidrug exporters is that they export a wide range of drugs and toxic compounds. The overexpression of these exporters causes multidrug resistance. Multidrug-resistant pathogens have become a serious problem in modern chemotherapy. Over the past decade, investigations into the structure of bacterial multidrug exporters have revealed the multidrug recognition and export mechanisms. In this review, we primarily discuss RND-type multidrug exporters particularly AcrAB-TolC, major drug exporter in Gram-negative bacteria. RND-type drug exporters are tripartite complexes comprising a cell membrane transporter, an outer membrane channel and an adaptor protein. Cell membrane transporters and outer membrane channels are homo-trimers; however, there is no consensus on the number of adaptor proteins in these tripartite complexes. The three monomers of a cell membrane transporter have varying conformations (access, binding, and extrusion) during transport. Drugs are exported following an ordered conformational change in these three monomers, through a functional rotation mechanism coupled with the proton relay cycle in ion pairs, which is driven by proton translocation. Multidrug recognition is based on a multisite drug-binding mechanism, in which two voluminous multidrug-binding pockets in cell membrane exporters recognize a wide range of substrates as a result of permutations at numerous binding sites that are specific for the partial structures of substrate molecules. The voluminous multidrug-binding pocket may have numerous binding sites even for a single substrate, suggesting that substrates may move between binding sites during transport, an idea named as multisite-drug-oscillation hypothesis. This hypothesis is consistent with the apparently broad substrate specificity of cell membrane exporters and their highly efficient ejection of drugs from the cell. Substrates are transported through dual multidrug-binding pockets via the peristaltic motion of the substrate translocation channel. Although there are no clinically available inhibitors of bacterial multidrug exporters, efforts to develop inhibitors based on structural information are underway.

Cell-Bound Lipase and Esterase of Brevibacterium linens

PubMed Central

Sørhaug, Terje; Ordal, Z. John

1974-01-01

The activities of glycerol ester hydrolase, lipase (EC 3.1.1.3) and carboxylesterase, and esterase (EC 3.1.1.1) were determined for whole cell preparations of Brevibacterium linens by using the pH-stat assay. The culture growth liquors were inactive against the three substrates, tributyrin emulsion, triacetin, and methyl butyrate. Cells washed in water had less activity than cells washed in 5% NaCl; the ratio of activities was close to 1:2 for all strains using tributyrin emulsion as the substrate. For the esterase substrates, this relationship varied widely and was strain dependent. The ability to hydrolyze the two esterase substrates varied independently of the level of lipase activity. PMID:4824883

Quantification of Transporter and Receptor Proteins in Dog Brain Capillaries and Choroid Plexus: Relevance for the Distribution in Brain and CSF of Selected BCRP and P-gp Substrates.

PubMed

Braun, Clemens; Sakamoto, Atsushi; Fuchs, Holger; Ishiguro, Naoki; Suzuki, Shinobu; Cui, Yunhai; Klinder, Klaus; Watanabe, Michitoshi; Terasaki, Tetsuya; Sauer, Achim

2017-10-02

Transporters at the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) play a pivotal role as gatekeepers for efflux or uptake of endogenous and exogenous molecules. The protein expression of a number of them has already been determined in the brains of rodents, nonhuman primates, and humans using quantitative targeted absolute proteomics (QTAP). The dog is an important animal model for drug discovery and development, especially for safety evaluations. The purpose of the present study was to clarify the relevance of the transporter protein expression for drug distribution in the dog brain and CSF. We used QTAP to examine the protein expression of 17 selected transporters and receptors at the dog BBB and BCSFB. For the first time, we directly linked the expression of two efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), to regional brain and CSF distribution using specific substrates. Two cocktails, each containing one P-gp substrate (quinidine or apafant) and one BCRP substrate (dantrolene or daidzein) were infused intravenously prior to collection of the brain. Transporter expression varied only slightly between the capillaries of different brain regions and did not result in region-specific distribution of the investigated substrates. There were, however, distinct differences between brain capillaries and choroid plexus. Largest differences were observed for BCRP and P-gp: both were highly expressed in brain capillaries, but no BCRP and only low amounts of P-gp were detected in the choroid plexus. K p,uu,brain and K p,uu,CSF of both P-gp substrates were indicative of drug efflux. Also, K p,uu,brain for the BCRP substrates was low. In contrast, K p,uu,CSF for both BCRP substrates was close to unity, resulting in K p,uu,CSF /K p,uu,brain ratios of 7 and 8, respectively. We conclude that the drug transporter expression profiles differ between the BBB and BCSFB in dogs, that there are species differences in the expression profiles, and that CSF is not a suitable surrogate for unbound brain concentrations of BCRP substrates in dogs.

Effect of pretreatment severity in continuous steam explosion on enzymatic conversion of wheat straw: Evidence from kinetic analysis of hydrolysis time courses.

PubMed

Monschein, Mareike; Nidetzky, Bernd

2016-01-01

Focusing on continuous steam explosion, the influence of pretreatment severity due to varied acid loading on hydrolysis of wheat straw by Trichoderma reesei cellulases was investigated based on kinetic evaluation of the saccharification of each pretreated substrate. Using semi-empirical descriptors of the hydrolysis time course, key characteristics of saccharification efficiency were captured in a quantifiable fashion. Not only hydrolysis rates per se, but also the transition point of their bi-phasic decline was crucial for high saccharification degree. After 48h the highest saccharification was achieved for substrate pretreated at relatively low severity (1.2% acid). Higher severity increased enzyme binding to wheat straw, but reduced the specific hydrolysis rates. Higher affinity of the lignocellulosic material for cellulases does not necessarily result in increased saccharification, probably because of lignin modifications occurring at high pretreatment severities. At comparable severity, continuous pretreatment produced a substrate more susceptible to enzymatic hydrolysis than the batch process. Copyright © 2015 Elsevier Ltd. All rights reserved.

Quantitative regulation of bone-mimetic, oriented collagen/apatite matrix structure depends on the degree of osteoblast alignment on oriented collagen substrates.

PubMed

Matsugaki, Aira; Isobe, Yoshihiro; Saku, Taro; Nakano, Takayoshi

2015-02-01

Bone tissue has a specific anisotropic morphology derived from collagen fiber alignment and the related apatite crystal orientation as a bone quality index. However, the precise mechanism of cellular regulation of the crystallographic orientation of apatite has not been clarified. In this study, anisotropic construction of cell-produced mineralized matrix in vitro was established by initiating organized cellular alignment and subsequent oriented bone-like matrix (collagen/apatite) production. The oriented collagen substrates with three anisotropic levels were prepared by a hydrodynamic method. Primary osteoblasts were cultured on the fabricated substrates until mineralized matrix formation is confirmed. Osteoblast alignment was successfully regulated by the level of substrate collagen orientation, with preferential alignment along the direction of the collagen fibers. Notably, both fibrous orientation of newly synthesized collagen matrix and c-axis of produced apatite crystals showed preferential orientation along the cell direction. Because the degree of anisotropy of the deposited apatite crystals showed dependency on the directional distribution of osteoblasts cultured on the oriented collagen substrates, the cell orientation determines the crystallographic anisotropy of produced apatite crystals. To the best of our knowledge, this is the first report demonstrating that bone tissue anisotropy, even the alignment of apatite crystals, is controllable by varying the degree of osteoblast alignment via regulating the level of substrate orientation. © 2014 Wiley Periodicals, Inc.

Strategies to characterize fungal lipases for applications in medicine and dairy industry.

PubMed

Gopinath, Subash C B; Anbu, Periasamy; Lakshmipriya, Thangavel; Hilda, Azariah

2013-01-01

Lipases are water-soluble enzymes that act on insoluble substrates and catalyze the hydrolysis of long-chain triglycerides. Lipases play a vital role in the food, detergent, chemical, and pharmaceutical industries. In the past, fungal lipases gained significant attention in the industries due to their substrate specificity and stability under varied chemical and physical conditions. Fungal enzymes are extracellular in nature, and they can be extracted easily, which significantly reduces the cost and makes this source preferable over bacteria. Soil contaminated with spillage from the products of oil and dairy harbors fungal species, which have the potential to secrete lipases to degrade fats and oils. Herein, the strategies involved in the characterization of fungal lipases, capable of degrading fatty substances, are narrated with a focus on further applications.

Strategies to Characterize Fungal Lipases for Applications in Medicine and Dairy Industry

PubMed Central

Gopinath, Subash C. B.; Anbu, Periasamy; Lakshmipriya, Thangavel; Hilda, Azariah

2013-01-01

Lipases are water-soluble enzymes that act on insoluble substrates and catalyze the hydrolysis of long-chain triglycerides. Lipases play a vital role in the food, detergent, chemical, and pharmaceutical industries. In the past, fungal lipases gained significant attention in the industries due to their substrate specificity and stability under varied chemical and physical conditions. Fungal enzymes are extracellular in nature, and they can be extracted easily, which significantly reduces the cost and makes this source preferable over bacteria. Soil contaminated with spillage from the products of oil and dairy harbors fungal species, which have the potential to secrete lipases to degrade fats and oils. Herein, the strategies involved in the characterization of fungal lipases, capable of degrading fatty substances, are narrated with a focus on further applications. PMID:23865040

Assessing Carbon-Based Anodes for Lithium-Ion Batteries: A Universal Description of Charge-Transfer Binding

DOE PAGES

Liu, Yuanyue; Wang, Y. Morris; Yakobson, Boris I.; ...

2014-07-11

Many key performance characteristics of carbon-based lithium-ion battery anodes are largely determined by the strength of binding between lithium (Li) and sp 2 carbon (C), which can vary significantly with subtle changes in substrate structure, chemistry, and morphology. We use density functional theory calculations to investigate the interactions of Li with a wide variety of sp 2 C substrates, including pristine, defective, and strained graphene, planar C clusters, nanotubes, C edges, and multilayer stacks. In almost all cases, we find a universal linear relation between the Li-C binding energy and the work required to fill previously unoccupied electronic states withinmore » the substrate. This suggests that Li capacity is predominantly determined by two key factors—namely, intrinsic quantum capacitance limitations and the absolute placement of the Fermi level. This simple descriptor allows for straightforward prediction of the Li-C binding energy and related battery characteristics in candidate C materials based solely on the substrate electronic structure. It further suggests specific guidelines for designing more effective C-based anodes. Furthermore, this method should be broadly applicable to charge-transfer adsorption on planar substrates, and provides a phenomenological connection to established principles in supercapacitor and catalyst design.« less

Variable substrate preference among phospholipase D toxins from sicariid spiders

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

Lajoie, Daniel M.; Roberts, Sue A.; Zobel-Thropp, Pamela A.

Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used 31P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, andmore » all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. Lastly, the evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey.« less

Variable substrate preference among phospholipase D toxins from sicariid spiders

DOE PAGES

Lajoie, Daniel M.; Roberts, Sue A.; Zobel-Thropp, Pamela A.; ...

2015-03-09

Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used 31P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, andmore » all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. Lastly, the evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey.« less

Variable Substrate Preference among Phospholipase D Toxins from Sicariid Spiders*

PubMed Central

Lajoie, Daniel M.; Roberts, Sue A.; Zobel-Thropp, Pamela A.; Delahaye, Jared L.; Bandarian, Vahe; Binford, Greta J.; Cordes, Matthew H. J.

2015-01-01

Venoms of the sicariid spiders contain phospholipase D enzyme toxins that can cause severe dermonecrosis and even death in humans. These enzymes convert sphingolipid and lysolipid substrates to cyclic phosphates by activating a hydroxyl nucleophile present in both classes of lipid. The most medically relevant substrates are thought to be sphingomyelin and/or lysophosphatidylcholine. To better understand the substrate preference of these toxins, we used 31P NMR to compare the activity of three related but phylogenetically diverse sicariid toxins against a diverse panel of sphingolipid and lysolipid substrates. Two of the three showed significantly faster turnover of sphingolipids over lysolipids, and all three showed a strong preference for positively charged (choline and/or ethanolamine) over neutral (glycerol and serine) headgroups. Strikingly, however, the enzymes vary widely in their preference for choline, the headgroup of both sphingomyelin and lysophosphatidylcholine, versus ethanolamine. An enzyme from Sicarius terrosus showed a strong preference for ethanolamine over choline, whereas two paralogous enzymes from Loxosceles arizonica either preferred choline or showed no significant preference. Intrigued by the novel substrate preference of the Sicarius enzyme, we solved its crystal structure at 2.1 Å resolution. The evolution of variable substrate specificity may help explain the reduced dermonecrotic potential of some natural toxin variants, because mammalian sphingolipids use primarily choline as a positively charged headgroup; it may also be relevant for sicariid predatory behavior, because ethanolamine-containing sphingolipids are common in insect prey. PMID:25752604

Targeting Inhibition of Fibroblast Activation Protein-α and Prolyl Oligopeptidase Activities on Cells Common to Metastatic Tumor Microenvironments1

PubMed Central

Christiansen, Victoria J; Jackson, Kenneth W; Lee, Kyung N; Downs, Tamyra D; McKee, Patrick A

2013-01-01

Fibroblast activation protein (FAP), a membrane prolyl-specific proteinase with both dipeptidase and endopeptidase activities, is overexpressed by reactive stromal fibroblasts during epithelial-derived cancer growth. FAP digests extracellular matrix as tissue is remodeled during cancer expansion and may also promote an immunotolerant tumor microenvironment. Recent studies suggest that nonspecific FAP inhibitors suppress human cancer xenografts in mouse models. Prolyl oligopeptidase (POP), another prolyl-specific serine proteinase, is also elevated in many cancers and may have a regulatory role in angiogenesis promotion. FAP and POP cell-associated activities may be targets for diagnosis and treatment of various cancers, but their accessibilities to highly effective specific inhibitors have not been shown for cells important to cancer growth. Despite their frequent simultaneous expression in many cancers and their overlapping activities toward commonly used substrates, precise, separate measurement of FAP or POP activity has largely been ignored. To distinguish each of the two activities, we synthesized highly specific substrates and inhibitors for FAP or POP based on amino acid sequences surrounding the scissile bonds of their respective putative substrates. We found varying amounts of FAP and POP protein and activities on activated fibroblasts, mesenchymal cells, normal breast cells, and one breast cancer cell line, with some cells exhibiting more POP than FAP activity. Replicating endothelial cells (ECs) expressed POP but not FAP until tubulogenesis began. Targeting FAP-positive cells, especially mesenchymal stem cells and cancer-associated fibroblasts for inactivation or destruction, and inhibiting POP-producing EC may abrogate stromal invasion and angiogenesis simultaneously and thereby diminish cancer growth. PMID:23555181

[Characterization of polygalacturonase covalently bonded to Sepharose].

PubMed

Bock, W; Krause, M; Anger, H; Flemming, C

1981-01-01

For the soluble endo-polygalacturonase (EC 3.2.1.15.) from Aspergillus spec., investigated in the present work, the defined substrate turnover U at 50% loss of viscosity if 0.2% and is independent on the reaction temperature. In the case of the covalently-bonded enzyme, the following linear equation applies to U, depending on the specific activity A and in the limits from A = O [U] and Amax: U = [U] + S square root of A. U is influenced by the kind of linkage, the conditions of immobilization and the properties of the carrier: it is a measure of the postulated conformational change of the polygalacturonase. The characteristic limiting value for the substrate turnover at A = O [U] is also temperature-independent and proves to be a true increment of binding, whereas Amax depends essentially on the porosity of the carrier. Polygalacturonase-sepharose complexes with a real substrate turnover U of 3--20% were prepared by varying systematically the kind of linkage and the specific activity A. It was found that with increasing U these complexes were, as a rule, inhibited to a lesser extent by a non-competitive pectinase inhibitor than the soluble polygalacturonase. Furthermore, their ability to liberate or enrich oligomeric galacturonic acids with a degree of polymerization greater than 3 was markedly reduced.

Annexins - scaffolds modulating PKC localization and signaling.

PubMed

Hoque, Monira; Rentero, Carles; Cairns, Rose; Tebar, Francesc; Enrich, Carlos; Grewal, Thomas

2014-06-01

Spatial and temporal organization of signal transduction is critical to link different extracellular stimuli with distinct cellular responses. A classical example of hormones and growth factors creating functional diversity is illustrated by the multiple signaling pathways activated by the protein kinase C (PKC) family of serine/threonine protein kinases. The molecular requirements for diacylglycerol (DAG) and calcium (Ca(2+)) to promote PKC membrane translocation, the hallmark of PKC activation, have been clarified. However, the underlying mechanisms that establish selectivity of individual PKC family members to facilitate differential substrate phosphorylation and varied signal output are still not fully understood. It is now well believed that the coordinated control and functional diversity of PKC signaling involves the formation of PKC isozyme-specific protein complexes in certain subcellular sites. In particular, interaction of PKC isozymes with compartment and signal-organizing scaffolds, including receptors for activated C-kinase (RACKs), A-kinase-anchoring proteins (AKAPs), 14-3-3, heat shock proteins (HSP), and importins target PKC isozymes to specific cellular locations, thereby delivering PKC isozymes into close proximity of their substrates. In addition, several annexins (Anx), including AnxA1, A2, A5 and A6, display specific and distinct abilities to interact and promote membrane targeting of different PKC isozymes. Together with the ability of annexins to create specific membrane microenvironments, this is likely to enable PKCs to phosphorylate certain substrates and regulate their downstream effector pathways in specific cellular sites. This review aims to summarize the capacity of annexins to modulate the localization and activity of PKC family members and participate in the spatiotemporal regulation of PKC signaling in health and disease. Copyright © 2014 Elsevier Inc. All rights reserved.

Refurbishment of SRB aluminum components by walnut hull blast removal of protective coatings

NASA Technical Reports Server (NTRS)

Colberg, W. R.; Gordon, G. H.; Jackson, C. H.

1982-01-01

A test program was conducted to develop, optimize, and scale up an abrasive blasting procedure was developed for refurbishment of specific SRB components: aft skirt, forward skirt, frustrum, and painted piece parts. Test specimens utilizing 2219 T87 aluminum substrate of varying thicknesses were prepared and blasted at progressively increasing pressures with selected abrasives. Specimens were analyzed for material response. The optimum blasting parameters were determined on panel specimens and verified on a large cylindrical integrated test bed.

Microbes on a Bottle: Substrate, Season and Geography Influence Community Composition of Microbes Colonizing Marine Plastic Debris.

PubMed

Oberbeckmann, Sonja; Osborn, A Mark; Duhaime, Melissa B

2016-01-01

Plastic debris pervades in our oceans and freshwater systems and the potential ecosystem-level impacts of this anthropogenic litter require urgent evaluation. Microbes readily colonize aquatic plastic debris and members of these biofilm communities are speculated to include pathogenic, toxic, invasive or plastic degrading-species. The influence of plastic-colonizing microorganisms on the fate of plastic debris is largely unknown, as is the role of plastic in selecting for unique microbial communities. This work aimed to characterize microbial biofilm communities colonizing single-use poly(ethylene terephthalate) (PET) drinking bottles, determine their plastic-specificity in contrast with seawater and glass-colonizing communities, and identify seasonal and geographical influences on the communities. A substrate recruitment experiment was established in which PET bottles were deployed for 5-6 weeks at three stations in the North Sea in three different seasons. The structure and composition of the PET-colonizing bacterial/archaeal and eukaryotic communities varied with season and station. Abundant PET-colonizing taxa belonged to the phylum Bacteroidetes (e.g. Flavobacteriaceae, Cryomorphaceae, Saprospiraceae-all known to degrade complex carbon substrates) and diatoms (e.g. Coscinodiscophytina, Bacillariophytina). The PET-colonizing microbial communities differed significantly from free-living communities, but from particle-associated (>3 μm) communities or those inhabiting glass substrates. These data suggest that microbial community assembly on plastics is driven by conventional marine biofilm processes, with the plastic surface serving as raft for attachment, rather than selecting for recruitment of plastic-specific microbial colonizers. A small proportion of taxa, notably, members of the Cryomorphaceae and Alcanivoraceae, were significantly discriminant of PET but not glass surfaces, conjuring the possibility that these groups may directly interact with the PET substrate. Future research is required to investigate microscale functional interactions at the plastic surface.

Microbes on a Bottle: Substrate, Season and Geography Influence Community Composition of Microbes Colonizing Marine Plastic Debris

PubMed Central

Osborn, A. Mark

2016-01-01

Plastic debris pervades in our oceans and freshwater systems and the potential ecosystem-level impacts of this anthropogenic litter require urgent evaluation. Microbes readily colonize aquatic plastic debris and members of these biofilm communities are speculated to include pathogenic, toxic, invasive or plastic degrading-species. The influence of plastic-colonizing microorganisms on the fate of plastic debris is largely unknown, as is the role of plastic in selecting for unique microbial communities. This work aimed to characterize microbial biofilm communities colonizing single-use poly(ethylene terephthalate) (PET) drinking bottles, determine their plastic-specificity in contrast with seawater and glass-colonizing communities, and identify seasonal and geographical influences on the communities. A substrate recruitment experiment was established in which PET bottles were deployed for 5–6 weeks at three stations in the North Sea in three different seasons. The structure and composition of the PET-colonizing bacterial/archaeal and eukaryotic communities varied with season and station. Abundant PET-colonizing taxa belonged to the phylum Bacteroidetes (e.g. Flavobacteriaceae, Cryomorphaceae, Saprospiraceae—all known to degrade complex carbon substrates) and diatoms (e.g. Coscinodiscophytina, Bacillariophytina). The PET-colonizing microbial communities differed significantly from free-living communities, but from particle-associated (>3 μm) communities or those inhabiting glass substrates. These data suggest that microbial community assembly on plastics is driven by conventional marine biofilm processes, with the plastic surface serving as raft for attachment, rather than selecting for recruitment of plastic-specific microbial colonizers. A small proportion of taxa, notably, members of the Cryomorphaceae and Alcanivoraceae, were significantly discriminant of PET but not glass surfaces, conjuring the possibility that these groups may directly interact with the PET substrate. Future research is required to investigate microscale functional interactions at the plastic surface. PMID:27487037

Numerical investigations on the rebound phenomena and the bonding mechanisms in cold spray processes

NASA Astrophysics Data System (ADS)

Viscusi, A.

2018-05-01

Cold spray technology is a relatively new additive process allowing to create high quality metallic coatings, on both metallic and non-metallic substrates, without extensive heating of the powders sprayed. Upon impact with a target surface, conversion of kinetic energy to plastic deformation occurs, the solid particles deform and bond together. The actual bonding mechanism for cold spray particles is still not well understood, a high number of works has been carried out during the past two decades, several theories have been proposed to explain the adhesion/rebound mechanisms making the system ineffective for industrial applications. Therefore, the aim of this research activity is to better explain the complex adhesion/rebound phenomena into cold spray impact processes through numerical simulations; for this purpose, on the base of simplified hypothesis and results found in literature, an original 3D Finite Element Method (FEM) model of an aluminium particle impacting on an aluminium substrate was proposed. A cohesive behaviour algorithm was implemented in the particle-substrate contact regions aiming to simulate the bonding between the impacting particle and the substrate under specific working conditions. A rebound coefficient was also defined representing the particle residual energy. Different simulations were performed using a range of impact velocities and varying the interfacial cohesive strength. It was shown that at low impact velocities the rebound phenomenon is governed by the elastic energy stored in the system, meanwhile at high impact velocities, the rebound phenomenon is mainly due to the strain rate effects making the system mechanically stronger; therefore, a specific range of bonding velocities depending on substrate-particle contact area were found.

Identification of functional domains within the alpha and beta subunits of beta-hexosaminidase A through the expression of alpha-beta fusion proteins.

PubMed

Tse, R; Wu, Y J; Vavougios, G; Hou, Y; Hinek, A; Mahuran, D J

1996-08-20

There are three human beta-hexosaminidase isozymes which are composed of all possible dimeric combinations of an alpha and/or a beta subunit; A (alpha beta), and B (beta beta), and S (alpha alpha). The amino acid sequences of the two subunits are 60% identical. The homology between the two chains varies with the middle > the carboxy-terminal > > the amino-terminal portions. Although dimerization is required for activity, each subunit contains its own active site and differs in its substrate specificity and thermal stability. The presence of the beta subunit in hexosaminidase A also influences the substrate specificity of the alpha subunit; e.g., in vivo only the A heterodimer can hydrolyze GM2 ganglioside. In this report, we localize functional regions in the two subunits by cellular expression of alpha/beta fusion proteins joined at adjacently aligned residues. First, a chimeric alpha/beta chain was made by replacing the least well-conserved amino-terminal section of the beta chain with the corresponding alpha section. The biochemical characteristics of this protein were nearly identical to hexosaminidase B. Therefore, the most dissimilar regions in the subunits are not responsible for their dissimilar biochemical properties. A second fusion protein was made that also included the more homologous middle section of the alpha chain. This protein expressed the substrate specificity unique to isozymes containing an alpha subunit (A and S). We conclude that the region responsible for the ability of the alpha subunit to bind negatively charged substrates is located within residues alpha 132-283. Interestingly, the remaining carboxy-terminal section from the beta chain, beta 316-556, was sufficient to allow this chimera to hydrolyze GM2 ganglioside with 10% the specific activity of heterodimeric hexosaminidase A. Thus, the carboxy-terminal section of each subunit is likely involved in subunit-subunit interactions.

Regulation of ATP production: dependence on calcium concentration and respiratory state.

PubMed

Fink, Brian D; Bai, Fan; Yu, Liping; Sivitz, William I

2017-08-01

Nanomolar free calcium enhances oxidative phosphorylation. However, the effects over a broad concentration range, at different respiratory states, or on specific energy substrates are less clear. We examined the action of varying [Ca 2+ ] over respiratory states ranging 4 to 3 on skeletal muscle mitochondrial respiration, potential, ATP production, and H 2 O 2 production using ADP recycling to clamp external [ADP]. Calcium at 450 nM enhanced respiration in mitochondria energized by the complex I substrates, glutamate/malate (but not succinate), at [ADP] of 4-256 µM, but more substantially at intermediate respiratory states and not at all at state 4. Using varied [Ca 2+ ], we found that the stimulatory effects on respiration and ATP production were most prominent at nanomolar concentrations, but inhibitory at 10 µM or higher. ATP production decreased more than respiration at 10 µM calcium. However, potential continued to increase up to 10 µM; suggesting a calcium-induced inability to utilize potential for phosphorylation independent of opening of the mitochondrial permeability transition pore (MTP). This effect of 10 µM calcium was confirmed by direct determination of ATP production over a range of potential created by differing substrate concentrations. Consistent with past reports, nanomolar [Ca 2+ ] had a stimulatory effect on utilization of potential for phosphorylation. Increasing [Ca 2+ ] was positively and continuously associated with H 2 O 2 production. In summary, the stimulatory effect of calcium on mitochondrial function is substrate dependent and most prominent over intermediate respiratory states. Calcium stimulates or inhibits utilization of potential for phosphorylation dependent on concentration with inhibition at higher concentration independent of MTP opening.

Methodological constraints in interpreting serum paraoxonase-1 activity measurements: an example from a study in HIV-infected patients.

PubMed

Parra, Sandra; Marsillach, Judit; Aragonès, Gerard; Rull, Anna; Beltrán-Debón, Raúl; Alonso-Villaverde, Carlos; Joven, Jorge; Camps, Jordi

2010-03-25

Paraoxonase-1 (PON1) is an antioxidant enzyme that attenuates the production of the monocyte chemoattractant protein-1 (MCP-1) in vitro. Although oxidation and inflammation are closely related processes, the association between PON1 and MCP-1 has not been completely characterised due, probably, to that the current use of synthetic substrates for PON1 measurement limits the interpretation of the data. In the present study, we explored the relationships between the circulating levels of PON1 and MCP-1 in human immunodeficiency virus-infected patients in relation to the multifunctional capabilities of PON1. We measured selected variables in 227 patients and in a control group of 409 participants. Serum PON1 esterase and lactonase activities were measured as the rates of hydrolysis of paraoxon and of 5-(thiobutyl)-butyrolactone, respectively. Oxidised LDL and MCP-1 concentrations were determined by enzyme-linked immunosorbent assay. High-density lipoproteins cholesterol, apolipoprotein A-I, and C-reactive protein concentrations were measured by standard automated methods. There were significant relationships between PON1 activity and several indices of oxidation and inflammation in control subjects and in infected patients. However, these relationships varied not only with disease status but also on the type of substrate used for PON1 measurement. The present study is a cautionary tale highlighting that results of clinical studies on PON1 may vary depending on the methods used as well as the disease studied. Until more specific methods using physiologically-akin substrates are developed for PON1 measurement, we suggest the simultaneous employment of at least two different substrates in order to improve the reliability of the results obtained.

Ran-dependent nuclear export mediators: a structural perspective

PubMed Central

Güttler, Thomas; Görlich, Dirk

2011-01-01

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

Electrowetting of liquid polymer on petal-mimetic microbowl-array surfaces for formation of microlens array with varying focus on a single substrate

NASA Astrophysics Data System (ADS)

Li, Xiangmeng; Shao, Jinyou; Li, Xiangming; Tian, Hongmiao

2015-03-01

In this paper, microlens array with varying focal lengths were fabricated on a single microbowl-array textured substrate. The solid microbowl-arrayed NOA61 (kind of polyurethane-based polymer with UV curablity) surface was resulted from nanoimprinting by polydimethylsiloxane (PDMS) mold. The PDMS mold was replicated from an SU-8 master which was generated by electron beam lithography. Such microbowl-arrayed surfaces demonstrate petal-mimetic highly adhesive hydrophobic wetting properties, which can promote an irreversible electrowetting (EW) effect and a dereased contact angle of water droplets as well as other liquid droplets by applying direct current (DC) voltage. To fabricate a microlens array with varying focal-lengths, liquid NOA61 was supplied from a syringe on the solid NOA61 microtextured film and DC voltage was applied succesively. After removing the DC voltage, these liquid NOA61 microdrops deposited on the solid microtextured NOA61 surface on tin-indium-oxide coated substrate could be solidified via UV irradiation, thus leading to microlens array with uneven numerical apertures on a single substrate. Numerical simulation was also done to verify the EW effect. Finally, optical imaging characterization was performed to confirm the varied focus of the NOA61 microdrops.

Active Site Metal Identity Alters Histone Deacetylase 8 Substrate Selectivity: A Potential Novel Regulatory Mechanism.

PubMed

Castaneda, Carol Ann; Lopez, Jeffrey E; Joseph, Caleb G; Scholle, Michael D; Mrksich, Milan; Fierke, Carol A

2017-10-24

Histone deacetylase 8 (HDAC8) is a well-characterized member of the class I acetyl-lysine deacetylase (HDAC) family. Previous work has shown that the efficiency of HDAC8-catalyzed deacetylation of a methylcoumarin peptide varies depending on the identity of the divalent metal ion in the HDAC8 active site. Here we demonstrate that both HDAC8 activity and substrate selectivity for a diverse range of peptide substrates depend on the identity of the active site metal ion. Varied deacetylase activities of Fe(II)- and Zn(II)-HDAC8 toward an array of peptide substrates were identified using self-assembled monolayers for matrix-assisted laser desorption ionization (SAMDI) mass spectrometry. Subsequently, the metal dependence of deacetylation of peptides of biological interest was measured using an in vitro peptide assay. While Fe(II)-HDAC8 is generally more active than Zn(II)-HDAC8, the Fe(II)/Zn(II) HDAC8 activity ratio varies widely (from 2 to 150) among the peptides tested. These data provide support for the hypothesis that HDAC8 may undergo metal switching in vivo that, in turn, may regulate its activity. However, future studies are needed to explore the identity of the metal ion bound to HDAC8 in cells under varied conditions.

Neural changes in control implementation of a continuous task.

PubMed

Lungu, Ovidiu V; Binenstock, Meagan M; Pline, Megan A; Yeaton, Jennifer R; Carey, James R

2007-03-14

It is commonly agreed that control implementation, being a resource-consuming endeavor, is not exerted continuously or in simple tasks. However, most research in the field was done using tasks that varied the need for control on a trial-by-trial basis (e.g., Stroop, flanker) in a discrete manner. In this case, the anterior cingulate cortex (ACC) was found to monitor the need for control, whereas regions in the prefrontal cortex (PFC) were found to be involved in control implementation. Whether or not the same control mechanism would be used in continuous tasks was an open question. In our study, we found that in a continuous task, the same neural substrate subserves control monitoring (ACC) but that the neural substrate of control implementation changes over time. Early in the task, regions in the PFC were involved in control implementation, whereas later the control was taken over by subcortical structures, specifically the caudate. Our results suggest that humans possess a flexible control mechanism, with a specific structure dedicated to monitoring the need for control and with multiple structures involved in control implementation.

Measurements of weak interactions between truncated substrates and a hammerhead ribozyme by competitive kinetic analyses: implications for the design of new and efficient ribozymes with high sequence specificity

PubMed Central

Kasai, Yasuhiro; Shizuku, Hideki; Takagi, Yasuomi; Warashina, Masaki; Taira, Kazunari

2002-01-01

Exploitation of ribozymes in a practical setting requires high catalytic activity and strong specificity. The hammerhead ribozyme R32 has considerable potential in this regard since it has very high catalytic activity. In this study, we have examined how R32 recognizes and cleaves a specific substrate, focusing on the mechanism behind the specificity. Comparing rates of cleavage of a substrate in a mixture that included the correct substrate and various substrates with point mutations, we found that R32 cleaved the correct substrate specifically and at a high rate. To clarify the source of this strong specificity, we quantified the weak interactions between R32 and various truncated substrates, using truncated substrates as competitive inhibitors since they were not readily cleaved during kinetic measurements of cleavage of the correct substrate, S11. We found that the strong specificity of the cleavage reaction was due to a closed form of R32 with a hairpin structure. The self-complementary structure within R32 enabled the ribozyme to discriminate between the correct substrate and a mismatched substrate. Since this hairpin motif did not increase the Km (it did not inhibit the binding interaction) or decrease the kcat (it did not decrease the cleavage rate), this kind of hairpin structure might be useful for the design of new ribozymes with strong specificity and high activity. PMID:12034825

"Phylogenetic and evolutionary analysis of functional divergence among Gamma glutamyl transpeptidase (GGT) subfamilies".

PubMed

Verma, Ved Vrat; Gupta, Rani; Goel, Manisha

2015-09-14

γ-glutamyltranspeptidase (GGT) is a bi-substrate enzyme conserved in all three domains of life. It catalyzes the cleavage and transfer of γ-glutamyl moiety of glutathione to either water (hydrolysis) or substrates like peptides (transpeptidation). GGTs exhibit great variability in their enzyme kinetics although the mechanism of catalysis is conserved. Recently, GGT has been shown to be a virulence factor in microbes like Helicobacter pylori and Bacillus anthracis. In mammalian cells also, GGT inhibition prior to chemotherapy has been shown to sensitize tumors to the therapy. Therefore, lately both bacterial and eukaryotic GGTs have emerged as potential drug targets, but the efforts directed towards finding suitable inhibitors have not yielded any significant results yet. We propose that delineating the residues responsible for the functional diversity associated with these proteins could help in design of species/clade specific inhibitors. In the present study, we have carried out phylogenetic analysis on a set of 47 GGT-like proteins to address the functional diversity. These proteins segregate into various subfamilies, forming separate clades on the tree. Sequence conservation and motif prediction studies show that even though most of the highly conserved residues have been characterized biochemically in previous studies, a significant number of novel putative sites and motifs are discovered that vary in a clade specific manner. Many of the putative sites predicted during the functional divergence type I and type II analysis, lie close to the known catalytic residues and line the walls of the substrate binding cavity, reinforcing their role in modulating the substrate specificity, catalytic rates and stability of this protein. The study offers interesting insights into the evolution of GGT-like proteins in pathogenic vs. non-pathogenic bacteria, archaea and eukaryotes. Our analysis delineates residues that are highly specific to each GGT subfamily. We propose that these sites not only explain the differences in stability and catalytic variability of various GGTs but can also aid in design of specific inhibitors against particular GGTs. Thus, apart from the commonly used in-silico inhibitor screening approaches, evolutionary analysis identifying the functional divergence hotspots in GGT proteins could augment the structure based drug design approaches.

Neural substrates of socioemotional self-awareness in neurodegenerative disease

PubMed Central

Sollberger, Marc; Rosen, Howard J; Shany-Ur, Tal; Ullah, Jerin; Stanley, Christine M; Laluz, Victor; Weiner, Michael W; Wilson, Stephen M; Miller, Bruce L; Rankin, Katherine P

2014-01-01

Background Neuroimaging studies examining neural substrates of impaired self-awareness in patients with neurodegenerative diseases have shown divergent results depending on the modality (cognitive, emotional, behavioral) of awareness. Evidence is accumulating to suggest that self-awareness arises from a combination of modality-specific and large-scale supramodal neural networks. Methods We investigated the structural substrates of patients' tendency to overestimate or underestimate their own capacity to demonstrate empathic concern for others. Subjects' level of empathic concern was measured using the Interpersonal Reactivity Index, and subject-informant discrepancy scores were used to predict regional atrophy pattern, using voxel-based morphometry analysis. Of the 102 subjects, 83 were patients with neurodegenerative diseases such as behavioral variant frontotemporal dementia (bvFTD) or semantic variant primary progressive aphasia (svPPA); the other 19 were healthy older adults. Results bvFTD and svPPA patients typically overestimated their level of empathic concern compared to controls, and overestimating one's empathic concern predicted damage to predominantly right-hemispheric anterior infero-lateral temporal regions, whereas underestimating one's empathic concern showed no neuroanatomical basis. Conclusions These findings suggest that overestimation and underestimation of one's capacity for empathic concern cannot be interpreted as varying degrees of the same phenomenon, but may arise from different pathophysiological processes. Damage to anterior infero-lateral temporal regions has been associated with semantic self-knowledge, emotion processing, and social perspective taking; neuropsychological functions partly associated with empathic concern itself. These findings support the hypothesis that—at least in the socioemotional domain—neural substrates of self-awareness are partly modality-specific. PMID:24683513

[Reading and writing Japanese: Kanji versus Kana].

PubMed

Kawamura, Mitsuru

2006-11-01

In my talk, I reviewed studies on the neural substrates of Kanji vs. Kana, two types of Japanese characters, written since the 1980s. More Specifically, I reviewed the development of the studies on (1) Kanji and Kana in pure alexia/agraphia, (2) alexia with agraphia of Kanji and (3) 'musical letters' vs. 'literary letters', and reported new findings from those studies. In the 1980s, we frequently studied patients with partial callosal lesions and those with pure alexia, and many of the studies were on the neural substrates of Kanji vs. Kana. Later, we discovered cases of alexia with agraphia of Kanji caused by lesions in the posterior part of the left inferior temporal gyrus, leading us to understand the neural substrates of Kanji and Kana in more detail. In addition to the reading and writing of 'literary letters', we studied the neural mechanisms of the reading and writing of 'musical letters', i.e. musical scores. Our study showed that the neural mechanisms of reading and writing musical scores were similar to those of reading and writing 'literary letters' in professional musicians, although those neural mechanisms varied slightly.

Effect of Reduced Graphene Oxide Reinforcement on the Wear Characteristics of Electroless Ni-P Coatings

NASA Astrophysics Data System (ADS)

Tamilarasan, T. R.; Sanjith, U.; Rajendran, R.; Rajagopal, G.; Sudagar, J.

2018-03-01

Electroless composite coatings with various concentrations of reduced graphene oxide (rGO) particles were deposited onto mild steel substrate. The effects of adding rGO particles by varying their concentration from 0 to 100 mg/L on morphology, composition, microhardness, adhesion, wear and friction of the electroless composite coatings were investigated. Among the various parameters that influence the tribological behavior, sliding velocity was varied within a specific range for definite concentrations of rGO to obtain enhanced wear resistance in this study. The micrographs of the worn surfaces and indented spots were examined for the nature of wear mechanism and interfacial adhesion. The wear rate increased with increasing sliding velocity but was relatively stable for coatings with lower concentrations of rGO.

Modulation of substrate specificities of D-sialic acid aldolase through single mutations of Val-251.

PubMed

Chou, Chien-Yu; Ko, Tzu-Ping; Wu, Kuan-Jung; Huang, Kai-Fa; Lin, Chun-Hung; Wong, Chi-Huey; Wang, Andrew H-J

2011-04-22

In a recent directed-evolution study, Escherichia coli D-sialic acid aldolase was converted by introducing eight point mutations into a new enzyme with relaxed specificity, denoted RS-aldolase (also known formerly as L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase), which showed a preferred selectivity toward L-KDO. To investigate the underlying molecular basis, we determined the crystal structures of D-sialic acid aldolase and RS-aldolase. All mutations are away from the catalytic center, except for V251I, which is near the opening of the (α/β)(8)-barrel and proximal to the Schiff base-forming Lys-165. The change of specificity from D-sialic acid to RS-aldolase can be attributed mainly to the V251I substitution, which creates a narrower sugar-binding pocket, but without altering the chirality in the reaction center. The crystal structures of D-sialic acid aldolase·l-arabinose and RS-aldolase·hydroxypyruvate complexes and five mutants (V251I, V251L, V251R, V251W, and V251I/V265I) of the D-sialic acid aldolase were also determined, revealing the location of substrate molecules and how the contour of the active site pocket was shaped. Interestingly, by mutating Val251 alone, the enzyme can accept substrates of varying size in the aldolase reactions and still retain stereoselectivity. The engineered D-sialic acid aldolase may find applications in synthesizing unnatural sugars of C(6) to C(10) for the design of antagonists and inhibitors of glycoenzymes.

High Carbon Use Efficiency is Not Explained by Production of Storage Compounds

NASA Astrophysics Data System (ADS)

Dijkstra, Paul; van Groenigen, Kees-Jan

2015-04-01

The efficiency with which microbes use substrate to make new microbial biomass (Carbon Use Efficiency or CUE; mol C / mol C) is an important variable in soil and ecosystem C cycling models. Estimates of CUE in soil microbial communities vary widely. It has been hypothesized that high values of CUE are associated with production of storage compounds following a sudden increases in substrate availability during CUE measurements. In that case, these high CUE values would not be representative for balanced microbial growth (i.e. the production of all compounds needed to make new microbial cells). To test this hypothesis, we added position-specific 13C-labeled glucose isotopomers in parallel incubations of a ponderosa pine and piñon-juniper soil. We compared the measured pattern of CO2 release for the six glucose C atoms with patterns of CO2 production expected for balanced growth with a low, medium, or high CUE, and with CO2 production patterns associated with production of storage compounds (glycogen, lipids, or polyhydroxybutyrate). The measured position-specific CO2 production did not match that for production of glycogen, lipids, or polyhydroxybutyrate, but agreed closely with that expected for balanced growth at high CUE and high pentose phosphate pathway activity. We conclude that soil microbial communities utilize glucose substrate for biomass growth with high CUE, and that addition of small amounts of 13C-labeled glucose tracers do not affect CUE or induce storage compounds production. We submit that the measurement of position-specific CO2 production offers a quick and easy way to test biochemically explicit hypotheses concerning microbial growth metabolism.

Organic photochemical storage of solar energy. Progress report, July 1, 1977--Feburary 28, 1978

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

Jones, G. II

1978-03-01

The prospects for driving endoergic reactions of simple, relatively abundant organic chemicals by photochemical means have been examined. Strategies for utilization of light of varying wavelength involve sensitization mechanisms which depend on the redox properties of energy storing substrates and photosensitizers. Of principal interest is valence isomerization which can be induced by electron donor-acceptor interaction between substrate and sensitizer in an excited complex or exciplex. Photophysical studies show that potentially isomerizable substrates efficiently intercept redox photosensitizers. The quenching of emission of electron acceptor sensitizers by non conjugated hydrocarbon dienes is indeed a function of the reduction potential of the acceptorsmore » (a series of aromatics with varying absorption characteristics) and the oxidation potentials of the substrates. Electron deficient dienes have been shown alternatively to be efficient quenchers of excited donor sensitizers. That exciplexes are formed between isomerizable substrates and donor or acceptor sensitizers has been confirmed by emission spectroscopy. The rearrangement of hexamethyldewarbenzene, a model exciplex isomerization has been examined in some detail.« less

Bul Proteins, a Nonredundant, Antagonistic Family of Ubiquitin Ligase Regulatory Proteins

PubMed Central

Novoselova, Tatiana V.; Zahira, Kiran; Rose, Ruth-Sarah

2012-01-01

Like other Nedd4 ligases, Saccharomyces cerevisiae E3 Rsp5p utilizes adaptor proteins to interact with some substrates. Previous studies have indentified Bul1p and Bul2p as adaptor proteins that facilitate the ligase-substrate interaction. Here, we show the identification of a third member of the Bul family, Bul3p, the product of two adjacent open reading frames separated by a stop codon that undergoes readthrough translation. Combinatorial analysis of BUL gene deletions reveals that they regulate some, but not all, of the cellular pathways known to involve Rsp5p. Surprisingly, we find that Bul proteins can act antagonistically to regulate the same ubiquitin-dependent process, and the nature of this antagonistic activity varies between different substrates. We further show, using in vitro ubiquitination assays, that the Bul proteins have different specificities for WW domains and that the two forms of Bul3p interact differently with Rsp5p, potentially leading to alternate functional outcomes. These data introduce a new level of complexity into the regulatory interactions that take place between Rsp5p and its adaptors and substrates and suggest a more critical role for the Bul family of proteins in controlling adaptor-mediated ubiquitination. PMID:22307975

Diversification in substrate usage by glutathione synthetases from soya bean (Glycine max), wheat (Triticum aestivum) and maize (Zea mays)

PubMed Central

2005-01-01

Unlike animals which accumulate glutathione (γ-glutamyl-L-cysteinyl-glycine) alone as their major thiol antioxidant, several crops synthesize alternative forms of glutathione by varying the carboxy residue. The molecular basis of this variation is not well understood, but the substrate specificity of the respective GSs (glutathione synthetases) has been implicated. To investigate their substrate tolerance, five GS-like cDNAs have been cloned from plants that can accumulate alternative forms of glutathione, notably soya bean [hGSH (homoglutathione or γ-glutamyl-L-cysteinyl-β-alanine)], wheat (hydroxymethylglutathione or γ-glutamyl-L-cysteinyl-serine) and maize (γ-Glu-Cys-Glu). The respective recombinant GSs were then assayed for the incorporation of differing C-termini into γ-Glu-Cys. The soya bean enzyme primarily incorporated β-alanine to form hGSH, whereas the GS enzymes from cereals preferentially catalysed the formation of glutathione. However, when assayed with other substrates, several GSs and one wheat enzyme in particular were able to synthesize a diverse range of glutathione variants by incorporating unusual C-terminal moieties including D-serine, non-natural amino acids and α-amino alcohols. Our results suggest that plant GSs are capable of producing a diverse range of glutathione homologues depending on the availability of the acyl acceptor. PMID:16008521

The Neural Basis of Event Simulation: An fMRI Study

PubMed Central

Yomogida, Yukihito; Sugiura, Motoaki; Akimoto, Yoritaka; Miyauchi, Carlos Makoto; Kawashima, Ryuta

2014-01-01

Event simulation (ES) is the situational inference process in which perceived event features such as objects, agents, and actions are associated in the brain to represent the whole situation. ES provides a common basis for various cognitive processes, such as perceptual prediction, situational understanding/prediction, and social cognition (such as mentalizing/trait inference). Here, functional magnetic resonance imaging was used to elucidate the neural substrates underlying important subdivisions within ES. First, the study investigated whether ES depends on different neural substrates when it is conducted explicitly and implicitly. Second, the existence of neural substrates specific to the future-prediction component of ES was assessed. Subjects were shown contextually related object pictures implying a situation and performed several picture–word-matching tasks. By varying task goals, subjects were made to infer the implied situation implicitly/explicitly or predict the future consequence of that situation. The results indicate that, whereas implicit ES activated the lateral prefrontal cortex and medial/lateral parietal cortex, explicit ES activated the medial prefrontal cortex, posterior cingulate cortex, and medial/lateral temporal cortex. Additionally, the left temporoparietal junction plays an important role in the future-prediction component of ES. These findings enrich our understanding of the neural substrates of the implicit/explicit/predictive aspects of ES-related cognitive processes. PMID:24789353

Removal of clay by stingless bees: load size and moisture selection.

PubMed

Costa-Pereira, Raul

2014-09-01

Some organisms disperse energy, associated with the transportation of resource, which is not necessarily food. Stingless bees of Central Amazonia (Melipona flavolineata and M. lateralis) collect clay in banks along streams for nest building. The moisture of the clay varies along the bank, and bees collect clay from specific location, indicating that there is some sort of preference regarding their selection. This study aims at identifying: if larger bees carry more clay; if there is a preference for moisture of substrates; and if bees are less efficient accumulating and transporting clay when it is wet. In order to do so, I measured the size of the bees and of the pellets of clay found in the corbicula. I set up a field experiment to test substrate preferences. The amount of clay transported, increased exponentially in accordance to the size of the bee, and the preferred substrate was the driest clay. The amount and the efficiency of removal of clay were not affected by the moisture of the substrate. Despite the wet clay being denser, it does not reduce the efficiency of exploitation of the resource, but suggests that bees spend more energy to carry the same quantity of wet clay, which may be the underlying mechanism explaining their preference for removing drier clay.

Characterization of 1-Aminobenzotriazole and Ketoconazole as Novel Inhibitors of Monoamine Oxidase (MAO): An In Vitro Investigation.

PubMed

Shaik, Abdul Naveed; LeDuc, Barbara W; Khan, Ansar A

2017-10-01

1-Aminobenzotriazole, a known time-dependent inhibitor of cytochrome P450 (CYP) enzymes, and ketoconazole, a strong inhibitor of the human CYP3A4 isozyme, are used as standard probe inhibitors to characterize the CYP and/or non-CYP-mediated metabolism of xenobiotics. In the present investigation, 1-Aminobenzotriazole and ketoconazole are characterized as potent monoamine oxidase (MAO) inhibitors in vitro using mouse, rat and human liver microsomes and S9 fractions. Inhibition potential of 1-aminobenzotriazole and ketoconazole was studied in mice, rat and human liver microsomes, S9 fractions, MAO-A and MAO-B expressed enzymes by monitoring the formation of 4-hydroxyquinoline (4-HQ) from kynuramine, a specific substrate of MAO by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Mechanism of MAO inhibition was studied by incubating varying concentration of kynuramine with mouse, rat and human S9 fractions at varying concentration of 1-aminobenzatriazole and ketoconazole and monitoring the formation of 4-HQ. 1-aminobenzotriazole and ketoconazole inhibited both MAO isozymes (MAO-A and MAO-B) with more specificity towards MAO-B. Kynuramine substrate kinetics in mouse, rat and human S9 fractions with varying 1-aminobenzotriazole and ketoconazole concentrations showed decreased maximum rate (V max ) for 4-HQ formation without affecting the Michaelis-Menten constant (K m ). A non-competitive inhibition model was constructed and inhibition constants (K i ) for 1-aminobenzotriazole (7.87 ± 0.61, 8.61 ± 0.92, 65.2 ± 1.61 µM for mice, rat and humans, respectively) and ketoconazole (0.12 ± 0.01, 2.04 ± 0.08, 5.52 ± 0.47 µM for mice, rat and humans, respectively) were determined. 1-Aminobenzotriazole and ketoconazole are characterized as non-competitive inhibitors of mice, rat and human MAO in vitro and the extent of their MAO inhibition potential is species specific. 1-Aminobenzotriazole or ketoconazole can be used as a probe inhibitor in vitro for screening the involvement of MAO-dependent metabolism of new chemical entities (NCE) in early drug discovery.

Gray scale x-ray mask

DOEpatents

Morales, Alfredo M [Livermore, CA; Gonzales, Marcela [Seattle, WA

2006-03-07

The present invention describes a method for fabricating an embossing tool or an x-ray mask tool, providing microstructures that smoothly vary in height from point-to-point in etched substrates, i.e., structure which can vary in all three dimensions. The process uses a lithographic technique to transfer an image pattern in the surface of a silicon wafer by exposing and developing the resist and then etching the silicon substrate. Importantly, the photoresist is variably exposed so that when developed some of the resist layer remains. The remaining undeveloped resist acts as an etchant barrier to the reactive plasma used to etch the silicon substrate and therefore provides the ability etch structures of variable depths.

Modeling Thermal Noise From Crystalline Coatings For Gravitational-Wave Detectors

NASA Astrophysics Data System (ADS)

Demos, Nicholas; Lovelace, Geoffrey; LSC Collaboration

2017-01-01

In 2015, Advanced LIGO made the first direct detection of gravitational waves. The sensitivity of current and future ground-based gravitational-wave detectors is limited by thermal noise in each detector's test mass substrate and coating. This noise can be modeled using the fluctuation-dissipation theorem, which relates thermal noise to an auxiliary elastic problem. I will present results from a new code that numerically models thermal noise for different crystalline mirror coatings. The thermal noise in crystalline mirror coatings could be significantly lower but is challenging to model analytically. The code uses a finite element method with adaptive mesh refinement to model the auxiliary elastic problem which is then related to thermal noise. Specifically, I will show results for a crystal coating on an amorphous substrate of varying sizes and elastic properties. This and future work will help develop the next generation of ground-based gravitational-wave detectors.

Quantitative and qualitative effects of phosphorus on extracts and exudates of sudangrass roots in relation to vesicular-arbuscular mycorrhiza formation.

PubMed

Schwab, S M; Menge, J A; Leonard, R T

1983-11-01

A comparison was made of water-soluble root exudates and extracts of Sorghum vulgare Pers. grown under two levels of P nutrition. An increase in P nutrition significantly decreased the concentration of carbohydrates, carboxylic acids, and amino acids in exudates, and decreased the concentration of carboxylic acids in extracts. Higher P did not affect the relative proportions of specific carboxylic acids and had little effect on proportions of specific amino acids in both extracts and exudates. Phosphorus amendment resulted in an increase in the relative proportion of arabinose and a decrease in the proportion of fructose in exudates, but did not have a large effect on the proportion of individual sugars in extracts. The proportions of specific carbohydrates, carboxylic acids, and amino acids varied between exudates and extracts. Therefore, the quantity and composition of root extracts may not be a reliable predictor of the availability of substrate for symbiotic vesicular-arbuscular mycorrhizal fungi. Comparisons of the rate of leakage of compounds from roots with the growth rate of vesicular-arbuscular mycorrhizal fungi suggest that the fungus must either be capable of using a variety of organic substrates for growth, or be capable of inducing a much higher rate of movement of specific organic compounds across root cell membranes than occurs through passive exudation as measured in this study.

A possible mechanism of metabolic regulation in Gibberella fujikuroi using a mixed carbon source of glucose and corn oil inferred from analysis of the kinetics data obtained in a stirrer tank bioreactor.

PubMed

Rios-Iribe, Erika Y; Hernández-Calderón, Oscar M; Reyes-Moreno, C; Contreras-Andrade, I; Flores-Cotera, Luis B; Escamilla-Silva, Eleazar M

2013-01-01

A nonstructured model was used to study the dynamics of gibberellic acid production in a stirred tank bioreactor. Experimental data were obtained from submerged batch cultures of Gibberella fujikuroi (CDBB H-984) grown in varying ratios of glucose-corn oil as the carbon source. The nitrogen depletion effect was included in mathematical model by considering the specific kinetic constants as a linear function of the normalized nitrogen consumption rate. The kinetics of biomass growth and consumption of phosphate and nitrogen were based on the logistic model. The traditional first-order kinetic model was used to describe the specific consumption of glucose and corn oil. The nitrogen effect was solely included in the phosphate and corn oil consumption and biomass growth. The model fit was satisfactory, revealing the dependence of the kinetics with respect to the nitrogen assimilation rate. Through simulations, it was possible to make diagrams of specific growth rate and specific rate of substrate consumptions, which was a powerful tool for understanding the metabolic interactions that occurred during the various stages of fermentation process. This kinetic analysis provided the proposal of a possible mechanism of regulation on growth, substrate consumptions, and production of gibberellic acid (GA3 ) in G. fujikuroi. © 2013 American Institute of Chemical Engineers.

Both Intrinsic Substrate Preference and Network Context Contribute to Substrate Selection of Classical Tyrosine Phosphatases*

PubMed Central

Tinti, Michele; Paoluzi, Serena; Santonico, Elena; Masch, Antonia; Schutkowski, Mike

2017-01-01

Reversible tyrosine phosphorylation is a widespread post-translational modification mechanism underlying cell physiology. Thus, understanding the mechanisms responsible for substrate selection by kinases and phosphatases is central to our ability to model signal transduction at a system level. Classical protein-tyrosine phosphatases can exhibit substrate specificity in vivo by combining intrinsic enzymatic specificity with the network of protein-protein interactions, which positions the enzymes in close proximity to their substrates. Here we use a high throughput approach, based on high density phosphopeptide chips, to determine the in vitro substrate preference of 16 members of the protein-tyrosine phosphatase family. This approach helped identify one residue in the substrate binding pocket of the phosphatase domain that confers specificity for phosphopeptides in a specific sequence context. We also present a Bayesian model that combines intrinsic enzymatic specificity and interaction information in the context of the human protein interaction network to infer new phosphatase substrates at the proteome level. PMID:28159843

Determinants of fish assemblage structure in Northwestern Great Plains streams

USGS Publications Warehouse

Mullen, J.A.; Bramblett, R.G.; Guy, C.S.; Zale, A.V.; Roberts, D.W.

2011-01-01

Prairie streams are known for their harsh and stochastic physical conditions, and the fish assemblages therein have been shown to be temporally variable. We assessed the spatial and temporal variation in fish assemblage structure in five intermittent, adventitious northwestern Great Plains streams representing a gradient of watershed areas. Fish assemblages and abiotic conditions varied more spatially than temporally. The most important variables explaining fish assemblage structure were longitudinal position and the proportion of fine substrates. The proportion of fine substrates increased proceeding upstream, approaching 100% in all five streams, and species richness declined upstream with increasing fine substrates. High levels of fine substrate in the upper reaches appeared to limit the distribution of obligate lithophilic fish species to reaches further downstream. Species richness and substrates were similar among all five streams at the lowermost and uppermost sites. However, in the middle reaches, species richness increased, the amount of fine substrate decreased, and connectivity increased as watershed area increased. Season and some dimensions of habitat (including thalweg depth, absolute distance to the main-stem river, and watershed size) were not essential in explaining the variation in fish assemblages. Fish species richness varied more temporally than overall fish assemblage structure did because common species were consistently abundant across seasons, whereas rare species were sometimes absent or perhaps not detected by sampling. The similarity in our results among five streams varying in watershed size and those from other studies supports the generalization that spatial variation exceeds temporal variation in the fish assemblages of prairie and warmwater streams. Furthermore, given longitudinal position, substrate, and stream size, general predictions regarding fish assemblage structure and function in prairie streams are possible. ?? American Fisheries Society 2011.

Human variation in CYP-specific chlorpyrifos metabolism.

PubMed

Croom, Edward L; Wallace, Andrew D; Hodgson, Ernest

2010-10-29

Chlorpyrifos, an organophophorothioate insecticide, is bioactivated to the neurotoxic metabolite, chlorpyrifos-oxon (CPO) by cytochromes P450 (CYPs). To determine the variability in chlorpyrifos bioactivation, CPO production by human liver microsomes from 17 individual donors was compared relative to phenotype and genotype. CPO production varied over 14-fold between individuals in incubations utilizing 20 μM chlorpyrifos as substrate, while CPO production varied 57-fold in incubations with 100 μM chlorpyrifos. For all but two samples, the formation of the less toxic metabolite, 3,5,6-trichloro-2-pyridinol (TCP), was greater than CPO production. TCP production varied 9-fold in incubations utilizing 20 μM chlorpyrifos as substrate and 19-fold using 100 μM chlorpyrifos. Chlorpyrifos metabolism by individual human liver microsomes was significantly correlated with CYP2B6, CYP2C19 and CYP3A4 related activity. CPO formation was best correlated with CYP2B6 related activity at low (20 μM) chlorpyrifos concentrations while CYP3A4 related activity was best correlated with CPO formation at high concentrations (100 μM) of chlorpyrifos. TCP production was best correlated with CYP3A4 activity at all substrate concentrations of chlorpyrifos. The production of both CPO and TCP was significantly lower at a concentration of 20 μM chlorpyrifos as compared to 100 μM chlorpyrifos. Calculations of percent total normalized rates (% TNR) and the chemical inhibitors ketoconazole and ticlopidine were used to confirm the importance of CYP2B6, CYP2C19, and CYP3A4 for the metabolism of chlorpyrifos. The combination of ketoconazole and ticlopidine inhibited the majority of TCP and CPO formation. CPO formation did not differ by CYP2B6 genotype. Individual variations in CPO production may need to be considered in determining the risk of chlorpyrifos poisoning. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Controlling carbon-nanotube-phospholipid solubility by curvature-dependent self-assembly.

PubMed

Määttä, Jukka; Vierros, Sampsa; Sammalkorpi, Maria

2015-03-12

Control of aqueous dispersion is central in the processing and usage of nanoscale hydrophobic objects. However, selecting dispersive agents based on the size and form of the hydrophobic object and the role of coating morphology in dispersion efficiency remain important open questions. Here, the effect of the substrate and the dispersing molecule curvature, as well as, the influence of dispersant concentration on the adsorption morphology are examined by molecular simulations of graphene and carbon nanotube (CNT) substrates with phospholipids of varying curvature as the dispersing agents. Lipid spontaneous curvature is increased from close to zero (effectively cylindrical lipid) to highly positive (effectively conical lipid) by studying double tailed dipalmitoylphosphadidylcholine (DPPC) and single tailed lysophosphadidylcholine (LPC) which differ in the number of acyl chains but have identical headgroup. We find that lipids are good dispersion agents for both planar and curved nanoparticles and induce a dispersive barrier nonsize selectively. Differences in dispersion efficiency arise from lipid headgroup density and their extension from the hydrophobic substrate in the adsorption morphology. We map the packing morphology contributing factors and report that the aggregate morphologies depend on the competition of interactions rising from (1) hydrophobicity driven maximization of lipid-substrate contacts and lipid self-adhesion, (2) tail bending energy cost, (3) preferential alignment along the graphitic substrate principal axes, and (4) lipid headgroup preferential packing. Curved substrates adjust the morphology by changing the balance between the interaction strengths. Jointly, the findings show substrate curvature and dimensions are a way to tune lipid adsorption to desired, self-assembling patterns. Besides engineering dispersion efficiency, the findings could bear significance in designing materials with defined molecular scale, molecular coatings for orientation specific CNT assembly or lipid-based molecular masks and patterning on graphene.

Printing quality control automation

NASA Astrophysics Data System (ADS)

Trapeznikova, O. V.

2018-04-01

One of the most important problems in the concept of standardizing the process of offset printing is the control the quality rating of printing and its automation. To solve the problem, a software has been developed taking into account the specifics of printing system components and the behavior in printing process. In order to characterize the distribution of ink layer on the printed substrate the so-called deviation of the ink layer thickness on the sheet from nominal surface is suggested. The geometric data construction the surface projections of the color gamut bodies allows to visualize the color reproduction gamut of printing systems in brightness ranges and specific color sectors, that provides a qualitative comparison of the system by the reproduction of individual colors in a varying ranges of brightness.

Fiber Optic Couplers.

DTIC Science & Technology

1979-11-01

over a 1 x 4 inch glass plate. A further problem has been that the surface over the ion diffused region is submerged 2 pm below that of the substrate...of varying mask openings (35, 45, 55, 65, 78, 85 pm ). The ion exchange processing time was varied using 20, 25, 35 and 40 minutes. We found that the...pattern is then overcoated with a thick layer of SiO 2 (lO0 pm thick). This thick layer and the SiO 2 substrate thus com- pletely surround the dopant

Enzyme specificity under dynamic control

NASA Astrophysics Data System (ADS)

Ota, Nobuyuki; Agard, David A.

2002-03-01

The contributions of conformational dynamics to substrate specificity have been examined by the application of principal component analysis to molecular dynamics trajectories of alpha-lytic protease. The wild-type alpha-lytic protease is highly specific for substrates with small hydrophobic side chains at the specificity pocket, while the Met190Ala binding pocket mutant has a much broader specificity, actively hydrolyzing substrates ranging from Ala to Phe. We performed a principal component analysis using 1-nanosecond molecular dynamics simulations using solvent boundary condition. We found that the walls of the wild-type substrate binding pocket move in tandem with one another, causing the pocket size to remain fixed so that only small substrates are recognized. In contrast, the M190A mutant shows uncoupled movement of the binding pocket walls, allowing the pocket to sample both smaller and larger sizes, which appears to be the cause of the observed broad specificity. The results suggest that the protein dynamics of alpha-lytic protease may play a significant role in defining the patterns of substrate specificity.

Carbon nanotubes on a substrate

DOEpatents

Gao, Yufei [Kennewick, WA; Liu, Jun [West Richland, WA

2002-03-26

The present invention includes carbon nanotubes whose hollow cores are 100% filled with conductive filler. The carbon nanotubes are in uniform arrays on a conductive substrate and are well-aligned and can be densely packed. The uniformity of the carbon nanotube arrays is indicated by the uniform length and diameter of the carbon nanotubes, both which vary from nanotube to nanotube on a given array by no more than about 5%. The alignment of the carbon nanotubes is indicated by the perpendicular growth of the nanotubes from the substrates which is achieved in part by the simultaneous growth of the conductive filler within the hollow core of the nanotube and the densely packed growth of the nanotubes. The present invention provides a densely packed carbon nanotube growth where each nanotube is in contact with at least one nearest-neighbor nanotube. The substrate is a conductive substrate coated with a growth catalyst, and the conductive filler can be single crystals of carbide formed by a solid state reaction between the substrate material and the growth catalyst. The present invention further provides a method for making the filled carbon nanotubes on the conductive substrates. The method includes the steps of depositing a growth catalyst onto the conductive substrate as a prepared substrate, creating a vacuum within a vessel which contains the prepared substrate, flowing H2/inert (e.g. Ar) gas within the vessel to increase and maintain the pressure within the vessel, increasing the temperature of the prepared substrate, and changing the H2/Ar gas to ethylene gas such that the ethylene gas flows within the vessel. Additionally, varying the density and separation of the catalyst particles on the conductive substrate can be used to control the diameter of the nanotubes.

Method of making carbon nanotubes on a substrate

DOEpatents

Gao, Yufei; Liu, Jun

2006-03-14

The present invention includes carbon nanotubes whose hollow cores are 100% filled with conductive filler. The carbon nanotubes are in uniform arrays on a conductive substrate and are well-aligned and can be densely packed. The uniformity of the carbon nanotube arrays is indicated by the uniform length and diameter of the carbon nanotubes, both which vary from nanotube to nanotube on a given array by no more than about 5%. The alignment of the carbon nanotubes is indicated by the perpendicular growth of the nanotubes from the substrates which is achieved in part by the simultaneous growth of the conductive filler within the hollow core of the nanotube and the densely packed growth of the nanotubes. The present invention provides a densely packed carbon nanotube growth where each nanotube is in contact with at least one nearest-neighbor nanotube. The substrate is a conductive substrate coated with a growth catalyst, and the conductive filler can be single crystals of carbide formed by a solid state reaction between the substrate material and the growth catalyst. The present invention further provides a method for making the filled carbon nanotubes on the conductive substrates. The method includes the steps of depositing a growth catalyst onto the conductive substrate as a prepared substrate, creating a vacuum within a vessel which contains the prepared substrate, flowing H2/inert (e.g. Ar) gas within the vessel to increase and maintain the pressure within the vessel, increasing the temperature of the prepared substrate, and changing the H2/Ar gas to ethylene gas such that the ethylene gas flows within the vessel. Additionally, varying the density and separation of the catalyst particles on the conductive substrate can be used to control the diameter of the nanotubes.

Rare earth elements concentration in mushroom cultivation substrates affects the production process and fruit-bodies content of Pleurotus ostreatus and Cyclocybe cylindracea.

PubMed

Koutrotsios, Georgios; Danezis, Georgios P; Georgiou, Constantinos A; Zervakis, Georgios I

2018-04-20

Concentrations of 16 rare earth elements (REEs) and two actinides were determined for the first time both in cultivated mushrooms and in their production substrates by inductively coupled plasma mass spectroscopy. Moreover, the effect of REEs on cultivation parameters and composition of the final product was assessed, together with their potential use for authentication purposes. The concentrations of REEs varied greatly among seven cultivation substrates and correlated with measurements in Cyclocybe cylindracea mushrooms; no such correlation was established in Pleurotus ostreatus. Reduction of hemicellulose, cellulose, and lignin in substrates during P. ostreatus cultivation was positively correlated with REE concentrations, which also affected the production performance depending on the species examined. In all cases, a negative correlation was established between bioconcentration factors (BCF) in mushrooms and REE content in substrates, while the effect of substrate composition on BCF values varied according to the element studied. The estimated daily intake values of REEs through mushroom consumption was at much lower levels than those reported as potentially harmful for human health. The content of REEs in cultivation substrates and in mushrooms revealed that the bioaccumulation of elements differed in each fungus. The nature/origin of substrates seemed to affect the concentration of REEs in mushrooms to a considerable extent. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry.

Parametric studies of metabolic cooperativity in Escherichia coli colonies: Strain and geometric confinement effects

PubMed Central

Cole, John A.; Luthey-Schulten, Zaida

2017-01-01

Characterizing the complex spatial and temporal interactions among cells in a biological system (i.e. bacterial colony, microbiome, tissue, etc.) remains a challenge. Metabolic cooperativity in these systems can arise due to the subtle interplay between microenvironmental conditions and the cells’ regulatory machinery, often involving cascades of intra- and extracellular signalling molecules. In the simplest of cases, as demonstrated in a recent study of the model organism Escherichia coli, metabolic cross-feeding can arise in monoclonal colonies of bacteria driven merely by spatial heterogeneity in the availability of growth substrates; namely, acetate, glucose and oxygen. Another recent study demonstrated that even closely related E. coli strains evolved different glucose utilization and acetate production capabilities, hinting at the possibility of subtle differences in metabolic cooperativity and the resulting growth behavior of these organisms. Taking a first step towards understanding the complex spatio-temporal interactions within microbial populations, we performed a parametric study of E. coli growth on an agar substrate and probed the dependence of colony behavior on: 1) strain-specific metabolic characteristics, and 2) the geometry of the underlying substrate. To do so, we employed a recently developed multiscale technique named 3D dynamic flux balance analysis which couples reaction-diffusion simulations with iterative steady-state metabolic modeling. Key measures examined include colony growth rate and shape (height vs. width), metabolite production/consumption and concentration profiles, and the emergence of metabolic cooperativity and the fractions of cell phenotypes. Five closely related strains of E. coli, which exhibit large variation in glucose consumption and organic acid production potential, were studied. The onset of metabolic cooperativity was found to vary substantially between these five strains by up to 10 hours and the relative fraction of acetate utilizing cells within the colonies varied by a factor of two. Additionally, growth with six different geometries designed to mimic those that might be found in a laboratory, a microfluidic device, and inside a living organism were considered. Geometries were found to have complex, often nonlinear effects on colony growth and cross-feeding with “hard” features resulting in larger effect than “soft” features. These results demonstrate that strain-specific features and spatial constraints imposed by the growth substrate can have significant effects even for microbial populations as simple as isogenic E. coli colonies. PMID:28820904

Parametric studies of metabolic cooperativity in Escherichia coli colonies: Strain and geometric confinement effects.

PubMed

Peterson, Joseph R; Cole, John A; Luthey-Schulten, Zaida

2017-01-01

Characterizing the complex spatial and temporal interactions among cells in a biological system (i.e. bacterial colony, microbiome, tissue, etc.) remains a challenge. Metabolic cooperativity in these systems can arise due to the subtle interplay between microenvironmental conditions and the cells' regulatory machinery, often involving cascades of intra- and extracellular signalling molecules. In the simplest of cases, as demonstrated in a recent study of the model organism Escherichia coli, metabolic cross-feeding can arise in monoclonal colonies of bacteria driven merely by spatial heterogeneity in the availability of growth substrates; namely, acetate, glucose and oxygen. Another recent study demonstrated that even closely related E. coli strains evolved different glucose utilization and acetate production capabilities, hinting at the possibility of subtle differences in metabolic cooperativity and the resulting growth behavior of these organisms. Taking a first step towards understanding the complex spatio-temporal interactions within microbial populations, we performed a parametric study of E. coli growth on an agar substrate and probed the dependence of colony behavior on: 1) strain-specific metabolic characteristics, and 2) the geometry of the underlying substrate. To do so, we employed a recently developed multiscale technique named 3D dynamic flux balance analysis which couples reaction-diffusion simulations with iterative steady-state metabolic modeling. Key measures examined include colony growth rate and shape (height vs. width), metabolite production/consumption and concentration profiles, and the emergence of metabolic cooperativity and the fractions of cell phenotypes. Five closely related strains of E. coli, which exhibit large variation in glucose consumption and organic acid production potential, were studied. The onset of metabolic cooperativity was found to vary substantially between these five strains by up to 10 hours and the relative fraction of acetate utilizing cells within the colonies varied by a factor of two. Additionally, growth with six different geometries designed to mimic those that might be found in a laboratory, a microfluidic device, and inside a living organism were considered. Geometries were found to have complex, often nonlinear effects on colony growth and cross-feeding with "hard" features resulting in larger effect than "soft" features. These results demonstrate that strain-specific features and spatial constraints imposed by the growth substrate can have significant effects even for microbial populations as simple as isogenic E. coli colonies.

Effect of substrate temperature and oxygen partial pressure on RF sputtered NiO thin films

NASA Astrophysics Data System (ADS)

Cheemadan, Saheer; Santhosh Kumar, M. C.

2018-04-01

Nickel oxide (NiO) thin films were deposited by RF sputtering process and the physical properties were investigated for varying substrate temperatures and oxygen partial pressure. The variation of the crystallographic orientation and microstructure of the NiO thin films with an increase in substrate temperature were studied. It was observed that NiO thin films deposited at 350 °C shows relatively good crystalline characteristics with a preferential orientation along (111) plane. With the optimum substrate temperature of 350 °C, the NiO thin films were deposited under various oxygen partial pressures at the same experimental conditions. The structural, optical and electrical properties of NiO thin films under varying oxygen partial pressure of 10%–50% were investigated. From XRD it is clear that the films prepared in the pure argon atmosphere were amorphous while the films in oxygen partial pressure exhibited polycrystalline NiO phase. SEM and AFM investigations unveil that the higher substrate temperature improves the microstructure of the thin films. It is revealed that the NiO thin films deposited at oxygen partial pressure of 40% and a substrate temperature of 350 °C, showed higher electrical conductivity with p-type characteristics.

Colloidal spray method for low cost thin coating deposition

DOEpatents

Pham, Ai-Quoc; Glass, Robert S.; Lee, Tae H.

2005-01-25

A dense or porous coating of material is deposited onto a substrate by forcing a colloidal suspension through an ultrasonic nebulizer and spraying a fine mist of particles in a carrier medium onto a sufficiently heated substrate. The spraying rate is essentially matched to the evaporation rate of the carrier liquid from the substrate to produce a coating that is uniformly distributed over the surface of the substrate. Following deposition to a sufficient coating thickness, a single sintering step may be used to produce a dense ceramic coating. Using this method, coatings ranging in thickness from about one to several hundred microns can be obtained. By using a plurality of compounds in the colloidal suspension, coatings of mixed composition can be obtained. By using a plurality of solutions and separate pumps and a single or multiple ultrasonic nebulizer(s), and varying the individual pumping rates and/or the concentrations of the solutions, a coating of mixed and discontinuously graded (e.g., stepped) or continuously graded layers may be obtained. This method is particularly useful for depositing ceramic coatings. Dense ceramic coating materials on porous substrates are useful in providing improved electrode performance in devices such as high power density solid oxide fuel cells. Dense ceramic coatings obtained by the invention are also useful for gas turbine blade coatings, sensors, steam electrolyzers, etc. The invention has general use in preparation of systems requiring durable and chemically resistant coatings, or coatings having other specific chemical or physical properties.

Colloidal spray method for low cost thin coating deposition

DOEpatents

Pham, Ai-Quoc; Glass, Robert S.; Lee, Tae H.

2002-01-01

A dense or porous coating of material is deposited onto a substrate by forcing a colloidal suspension through an ultrasonic nebulizer and spraying a fine mist of particles in a carrier medium onto a sufficiently heated substrate. The spraying rate is essentially matched to the evaporation rate of the carrier liquid from the substrate to produce a coating that is uniformly distributed over the surface of the substrate. Following deposition to a sufficient coating thickness, a single sintering step may be used to produce a dense ceramic coating. Using this method, coatings ranging in thickness from about one to several hundred microns can be obtained. By using a plurality of compounds in the colloidal suspension, coatings of mixed composition can be obtained. By using a plurality of solutions and separate pumps and a single or multiple ultrasonic nebulizer(s), and varying the individual pumping rates and/or the concentrations of the solutions, a coating of mixed and discontinuously graded (e.g., stepped) or continuously graded layers may be obtained. This method is particularly useful for depositing ceramic coatings. Dense ceramic coating materials on porous substrates are useful in providing improved electrode performance in devices such as high power density solid oxide fuel cells. Dense ceramic coatings obtained by the invention are also useful for gas turbine blade coatings, sensors, steam electrolyzers, etc. The invention has general use in preparation of systems requiring durable and chemically resistant coatings, or coatings having other specific chemical or physical properties.

Control of Growth Rate by Initial Substrate Concentration at Values Below Maximum Rate

PubMed Central

Gaudy, Anthony F.; Obayashi, Alan; Gaudy, Elizabeth T.

1971-01-01

The hyperbolic relationship between specific growth rate, μ, and substrate concentration, proposed by Monod and used since as the basis for the theory of steady-state growth in continuous-flow systems, was tested experimentally in batch cultures. Use of a Flavobacterium sp. exhibiting a high saturation constant for growth in glucose minimal medium allowed direct measurement of growth rate and substrate concentration throughout the growth cycle in medium containing a rate-limiting initial concentration of glucose. Specific growth rates were also measured for a wide range of initial glucose concentrations. A plot of specific growth rate versus initial substrate concentration was found to fit the hyperbolic equation. However, the instantaneous relationship between specific growth rate and substrate concentration during growth, which is stated by the equation, was not observed. Well defined exponential growth phases were developed at initial substrate concentrations below that required for support of the maximum exponential growth rate and a constant doubling time was maintained until 50% of the substrate had been used. It is suggested that the external substrate concentration initially present “sets” the specific growth rate by establishing a steady-state internal concentration of substrate, possibly through control of the number of permeation sites. PMID:5137579

Method for providing an arbitrary three-dimensional microstructure in silicon using an anisotropic deep etch

DOEpatents

Morales, Alfredo M.; Gonzales, Marcela

2004-06-15

The present invention describes a method for fabricating an embossing tool or an x-ray mask tool, providing microstructures that smoothly vary in height from point-to-point in etched substrates, i.e., structure which can vary in all three dimensions. The process uses a lithographic technique to transfer an image pattern in the surface of a silicon wafer by exposing and developing the resist and then etching the silicon substrate. Importantly, the photoresist is variably exposed so that when developed some of the resist layer remains. The remaining undeveloped resist acts as an etchant barrier to the reactive plasma used to etch the silicon substrate and therefore provides the ability etch structures of variable depths.

UO(2) 2+ speciation determines uranium toxicity and bioaccumulation in an environmental Pseudomonas sp. isolate.

PubMed

Vanengelen, Michael R; Field, Erin K; Gerlach, Robin; Lee, Brady D; Apel, William A; Peyton, Brent M

2010-04-01

In the present study, experiments were performed to investigate how representative cellulosic breakdown products, when serving as growth substrates under aerobic conditions, affect hexavalent uranyl cation (UO(2) (2+)) toxicity and bioaccumulation within a Pseudomonas sp. isolate (designated isolate A). Isolate A taken from the Cold Test Pit South (CTPS) region of the Idaho National Laboratory (INL), Idaho Falls, ID, USA. The INL houses low-level uranium-contaminated cellulosic material and understanding how this material, and specifically its breakdown products, affect U-bacterial interactions is important for understanding UO(2) (2+) fate and mobility. Toxicity was modeled using a generalized Monod expression. Butyrate, dextrose, ethanol, and lactate served as growth substrates. The potential contribution of bicarbonate species present in high concentrations was also investigated and compared with toxicity and bioaccumulation patterns seen in low-bicarbonate conditions. Isolate A was significantly more sensitive to UO(2) (2+) and accumulated significantly more UO(2) (2+) in low-bicarbonate concentrations. In addition, UO(2) (2+) growth inhibition and bioaccumulation varied depending on the growth substrate. In the presence of high bicarbonate concentrations, sensitivity to UO(2) (2+) inhibition was greatly mitigated, and did not vary between the four substrates tested. The extent of UO(2) (2+) accumulation was also diminished. The observed patterns were related to UO(2) (2+) aqueous complexation, as predicted by MINTEQ (ver. 2.52) (Easton, PA, USA). In the low- bicarbonate medium, the presence of positively charged and unstable UO(2) (2+)-hydroxide complexes explained both the greater sensitivity of isolate A to UO(2) (2+), and the ability of isolate A to accumulate significant amounts of UO(2) (2+). The exclusive presence of negatively charged and stable UO(2) (2+)-carbonate complexes in the high bi-carbonate medium explained the diminished sensitivity of isolate A to UO(2) (2+) toxicity, and limited ability of isolate A to accumulate UO(2) (2+). (c) 2010 SETAC.

Continuous anaerobic co-digestion of Ulva biomass and cheese whey at varying substrate mixing ratios: Different responses in two reactors with different operating regimes.

PubMed

Jung, Heejung; Kim, Jaai; Lee, Changsoo

2016-12-01

The feasibility of co-digestion of Ulva with whey was investigated at varying substrate mixing ratios in two continuous reactors run with increasing and decreasing proportions of Ulva, respectively. Co-digestion with whey proved beneficial to the biomethanation of Ulva, with the methane yield being greater by up to 1.6-fold in co-digestion phases than in the Ulva mono-digestion phases. The experimental reactors responded differently, in terms of process performance and community structure, to the changes in the substrate mixing ratio. This can be attributed to the different operating regimes between two reactors, which may have caused the microbial communities to develop in different ways to acclimate. Methanosaeta-related populations were the predominant methanogens responsible for the production of methane regardless of different substrate mixing ratios in both reactors. Considering the methane recovery and the Ulva treatment capacity, the optimal fraction of Ulva in the substrate mixture is suggested to be 50-75%. Copyright © 2016 Elsevier Ltd. All rights reserved.

Why the impact of mechanical stimuli on stem cells remains a challenge.

PubMed

Goetzke, Roman; Sechi, Antonio; De Laporte, Laura; Neuss, Sabine; Wagner, Wolfgang

2018-05-04

Mechanical stimulation affects growth and differentiation of stem cells. This may be used to guide lineage-specific cell fate decisions and therefore opens fascinating opportunities for stem cell biology and regenerative medicine. Several studies demonstrated functional and molecular effects of mechanical stimulation but on first sight these results often appear to be inconsistent. Comparison of such studies is hampered by a multitude of relevant parameters that act in concert. There are notorious differences between species, cell types, and culture conditions. Furthermore, the utilized culture substrates have complex features, such as surface chemistry, elasticity, and topography. Cell culture substrates can vary from simple, flat materials to complex 3D scaffolds. Last but not least, mechanical forces can be applied with different frequency, amplitude, and strength. It is therefore a prerequisite to take all these parameters into consideration when ascribing their specific functional relevance-and to only modulate one parameter at the time if the relevance of this parameter is addressed. Such research questions can only be investigated by interdisciplinary cooperation. In this review, we focus particularly on mesenchymal stem cells and pluripotent stem cells to discuss relevant parameters that contribute to the kaleidoscope of mechanical stimulation of stem cells.

Electrodeposition and Capacitive Behavior of Films for Electrodes of Electrochemical Supercapacitors

NASA Astrophysics Data System (ADS)

Shi, C.; Zhitomirsky, I.

2010-03-01

Polypyrrole films were deposited by anodic electropolymerization on stainless steel substrates from aqueous pyrrole solutions containing sodium salicylate and tiron additives. The deposition yield was studied under galvanostatic conditions. The amount of the deposited material was varied by the variation of deposition time at a constant current density. SEM studies showed the formation of porous films with thicknesses in the range of 0-3 μm. Cyclic voltammetry data for the films tested in 0.5 M Na2SO4 solutions showed capacitive behavior and high specific capacitance (SC) in a voltage window of 0.9 V. The films prepared from pyrrole solutions containing tiron showed better capacitive behavior compared to the films prepared from the solutions containing sodium salicylate. A highest SC of 254 F g-1 was observed for the sample with a specific mass of 89 μg cm-2 at a scan rate of 2 mV s-1. The SC decreased with an increasing film thickness and scan rate. The results indicated that the polypyrrole films deposited on the stainless steel substrates by anodic electropolymerization can be used as electrodes for electrochemical supercapacitors (ES).

Electrodeposition and capacitive behavior of films for electrodes of electrochemical supercapacitors.

PubMed

Shi, C; Zhitomirsky, I

2010-01-08

Polypyrrole films were deposited by anodic electropolymerization on stainless steel substrates from aqueous pyrrole solutions containing sodium salicylate and tiron additives. The deposition yield was studied under galvanostatic conditions. The amount of the deposited material was varied by the variation of deposition time at a constant current density. SEM studies showed the formation of porous films with thicknesses in the range of 0-3 μm. Cyclic voltammetry data for the films tested in 0.5 M Na2SO4 solutions showed capacitive behavior and high specific capacitance (SC) in a voltage window of 0.9 V. The films prepared from pyrrole solutions containing tiron showed better capacitive behavior compared to the films prepared from the solutions containing sodium salicylate. A highest SC of 254 F g-1 was observed for the sample with a specific mass of 89 μg cm-2 at a scan rate of 2 mV s-1. The SC decreased with an increasing film thickness and scan rate. The results indicated that the polypyrrole films deposited on the stainless steel substrates by anodic electropolymerization can be used as electrodes for electrochemical supercapacitors (ES).

Electrodeposition and Capacitive Behavior of Films for Electrodes of Electrochemical Supercapacitors

PubMed Central

2010-01-01

Polypyrrole films were deposited by anodic electropolymerization on stainless steel substrates from aqueous pyrrole solutions containing sodium salicylate and tiron additives. The deposition yield was studied under galvanostatic conditions. The amount of the deposited material was varied by the variation of deposition time at a constant current density. SEM studies showed the formation of porous films with thicknesses in the range of 0–3 μm. Cyclic voltammetry data for the films tested in 0.5 M Na2SO4 solutions showed capacitive behavior and high specific capacitance (SC) in a voltage window of 0.9 V. The films prepared from pyrrole solutions containing tiron showed better capacitive behavior compared to the films prepared from the solutions containing sodium salicylate. A highest SC of 254 F g−1 was observed for the sample with a specific mass of 89 μg cm−2 at a scan rate of 2 mV s−1. The SC decreased with an increasing film thickness and scan rate. The results indicated that the polypyrrole films deposited on the stainless steel substrates by anodic electropolymerization can be used as electrodes for electrochemical supercapacitors (ES). PMID:20672082

Classification of Lactococcus lactis cell envelope proteinase based on gene sequencing, peptides formed after hydrolysis of milk, and computer modeling.

PubMed

Børsting, M W; Qvist, K B; Brockmann, E; Vindeløv, J; Pedersen, T L; Vogensen, F K; Ardö, Y

2015-01-01

Lactococcus lactis strains depend on a proteolytic system for growth in milk to release essential AA from casein. The cleavage specificities of the cell envelope proteinase (CEP) can vary between strains and environments and whether the enzyme is released or bound to the cell wall. Thirty-eight Lc. lactis strains were grouped according to their CEP AA sequences and according to identified peptides after hydrolysis of milk. Finally, AA positions in the substrate binding region were suggested by the use of a new CEP template based on Streptococcus C5a CEP. Aligning the CEP AA sequences of 38 strains of Lc. lactis showed that 21 strains, which were previously classified as group d, could be subdivided into 3 groups. Independently, similar subgroupings were found based on comparison of the Lc. lactis CEP AA sequences and based on normalized quantity of identified peptides released from αS1-casein and β-casein. A model structure of Lc. lactis CEP based on the crystal structure of Streptococcus C5a CEP was used to investigate the AA positions in the substrate-binding region. New AA positions were suggested, which could be relevant for the cleavage specificity of CEP; however, these could only explain 2 out of 3 found subgroups. The third subgroup could be explained by 1 to 5 AA positions located opposite the substrate binding region. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

NADP(+)-dependent D-xylose dehydrogenase from pig liver. Purification and properties.

PubMed

Zepeda, S; Monasterio, O; Ureta, T

1990-03-15

An NADP(+)-dependent D-xylose dehydrogenase from pig liver cytosol was purified about 2000-fold to apparent homogeneity with a yield of 15% and specific activity of 6 units/mg of protein. An Mr value of 62,000 was obtained by gel filtration. PAGE in the presence of SDS gave an Mr value of 32,000, suggesting that the native enzyme is a dimer of similar or identical subunits. D-Xylose, D-ribose, L-arabinose, 2-deoxy-D-glucose, D-glucose and D-mannose were substrates in the presence of NADP+ but the specificity constant (ratio kcat./Km(app.)) is, by far, much higher for D-xylose than for the other sugars. The enzyme is specific for NADP+; NAD+ is not reduced in the presence of D-xylose or other sugars. Initial-velocity studies for the forward direction with xylose or NADP+ concentrations varied at fixed concentrations of the nucleotide or the sugar respectively revealed a pattern of parallel lines in double-reciprocal plots. Km values for D-xylose and NADP+ were 8.8 mM and 0.99 mM respectively. Dead-end inhibition studies to confirm a ping-pong mechanism showed that NAD+ acted as an uncompetitive inhibitor versus NADP+ (Ki 5.8 mM) and as a competitive inhibitor versus xylose. D-Lyxose was a competitive inhibitor versus xylose and uncompetitive versus NADP+. These results fit better to a sequential compulsory ordered mechanism with NADP+ as the first substrate, but a ping-pong mechanism with xylose as the first substrate has not been ruled out. The presence of D-xylose dehydrogenase suggests that in mammalian liver D-xylose is utilized by a pathway other than the pentose phosphate pathway.

The core microprocessor component DiGeorge syndrome critical region 8 (DGCR8) is a nonspecific RNA-binding protein.

PubMed

Roth, Braden M; Ishimaru, Daniella; Hennig, Mirko

2013-09-13

MicroRNA (miRNA) biogenesis follows a conserved succession of processing steps, beginning with the recognition and liberation of an miRNA-containing precursor miRNA hairpin from a large primary miRNA transcript (pri-miRNA) by the Microprocessor, which consists of the nuclear RNase III Drosha and the double-stranded RNA-binding domain protein DGCR8 (DiGeorge syndrome critical region protein 8). Current models suggest that specific recognition is driven by DGCR8 detection of single-stranded elements of the pri-miRNA stem-loop followed by Drosha recruitment and pri-miRNA cleavage. Because countless RNA transcripts feature single-stranded-dsRNA junctions and DGCR8 can bind hundreds of mRNAs, we explored correlations between RNA binding properties of DGCR8 and specific pri-miRNA substrate processing. We found that DGCR8 bound single-stranded, double-stranded, and random hairpin transcripts with similar affinity. Further investigation of DGCR8/pri-mir-16 interactions by NMR detected intermediate exchange regimes over a wide range of stoichiometric ratios. Diffusion analysis of DGCR8/pri-mir-16 interactions by pulsed field gradient NMR lent further support to dynamic complex formation involving free components in exchange with complexes of varying stoichiometry, although in vitro processing assays showed exclusive cleavage of pri-mir-16 variants bearing single-stranded flanking regions. Our results indicate that DGCR8 binds RNA nonspecifically. Therefore, a sequential model of DGCR8 recognition followed by Drosha recruitment is unlikely. Known RNA substrate requirements are broad and include 70-nucleotide hairpins with unpaired flanking regions. Thus, specific RNA processing is likely facilitated by preformed DGCR8-Drosha heterodimers that can discriminate between authentic substrates and other hairpins.

The Core Microprocessor Component DiGeorge Syndrome Critical Region 8 (DGCR8) Is a Nonspecific RNA-binding Protein*

PubMed Central

Roth, Braden M.; Ishimaru, Daniella; Hennig, Mirko

2013-01-01

MicroRNA (miRNA) biogenesis follows a conserved succession of processing steps, beginning with the recognition and liberation of an miRNA-containing precursor miRNA hairpin from a large primary miRNA transcript (pri-miRNA) by the Microprocessor, which consists of the nuclear RNase III Drosha and the double-stranded RNA-binding domain protein DGCR8 (DiGeorge syndrome critical region protein 8). Current models suggest that specific recognition is driven by DGCR8 detection of single-stranded elements of the pri-miRNA stem-loop followed by Drosha recruitment and pri-miRNA cleavage. Because countless RNA transcripts feature single-stranded-dsRNA junctions and DGCR8 can bind hundreds of mRNAs, we explored correlations between RNA binding properties of DGCR8 and specific pri-miRNA substrate processing. We found that DGCR8 bound single-stranded, double-stranded, and random hairpin transcripts with similar affinity. Further investigation of DGCR8/pri-mir-16 interactions by NMR detected intermediate exchange regimes over a wide range of stoichiometric ratios. Diffusion analysis of DGCR8/pri-mir-16 interactions by pulsed field gradient NMR lent further support to dynamic complex formation involving free components in exchange with complexes of varying stoichiometry, although in vitro processing assays showed exclusive cleavage of pri-mir-16 variants bearing single-stranded flanking regions. Our results indicate that DGCR8 binds RNA nonspecifically. Therefore, a sequential model of DGCR8 recognition followed by Drosha recruitment is unlikely. Known RNA substrate requirements are broad and include 70-nucleotide hairpins with unpaired flanking regions. Thus, specific RNA processing is likely facilitated by preformed DGCR8-Drosha heterodimers that can discriminate between authentic substrates and other hairpins. PMID:23893406

Functioning of inorganic/organic battery separators in silver-zinc cells

NASA Technical Reports Server (NTRS)

Philipp, W. H.; May, C. E.

1976-01-01

The results of three experimental studies related to the inorganic/organic battery separator operating mechanism are described: saponification of the plasticizer, resistivity of the simulated separators, and zincate diffusion through the separators. The inorganic/organic separator appears to be a particular example of a general class of ionic conducting films composed of inorganic fillers and/or substrates bonded together by an organic polymer containing an incompatible plasticizer that may be leached by the electrolyte. The I/O separator functions as a microporous film of varying tortuosity with essentially no specific chemical inhibition to zincate diffusion.

Cushing's syndrome mutant PKA L205R exhibits altered substrate specificity

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

Lubner, Joshua M.; Dodge-Kafka, Kimberly L.; Carlson, Cathrine R.

The PKA L205R hotspot mutation has been implicated in Cushing's syndrome through hyperactive gain-of-function PKA signaling; however, its influence on substrate specificity has not been investigated. Here, we employ the Proteomic Peptide Library (ProPeL) approach to create high-resolution models for PKA WT and PKA L205R substrate specificity. We reveal that the L205R mutation reduces canonical hydrophobic preference at the substrate P + 1 position, and increases acidic preference in downstream positions. Using these models, we designed peptide substrates that exhibit altered selectivity for specific PKA variants, and demonstrate the feasibility of selective PKA L205R loss-of-function signaling. Through these results, wemore » suggest that substrate rewiring may contribute to Cushing's syndrome disease etiology, and introduce a powerful new paradigm for investigating mutation-induced kinase substrate rewiring in human disease.« less

Cushing's syndrome mutant PKA L205R exhibits altered substrate specificity

DOE PAGES

Lubner, Joshua M.; Dodge-Kafka, Kimberly L.; Carlson, Cathrine R.; ...

2017-02-01

The PKA L205R hotspot mutation has been implicated in Cushing's syndrome through hyperactive gain-of-function PKA signaling; however, its influence on substrate specificity has not been investigated. Here, we employ the Proteomic Peptide Library (ProPeL) approach to create high-resolution models for PKA WT and PKA L205R substrate specificity. We reveal that the L205R mutation reduces canonical hydrophobic preference at the substrate P + 1 position, and increases acidic preference in downstream positions. Using these models, we designed peptide substrates that exhibit altered selectivity for specific PKA variants, and demonstrate the feasibility of selective PKA L205R loss-of-function signaling. Through these results, wemore » suggest that substrate rewiring may contribute to Cushing's syndrome disease etiology, and introduce a powerful new paradigm for investigating mutation-induced kinase substrate rewiring in human disease.« less

Structural basis for the substrate specificity of PepA from Streptococcus pneumoniae, a dodecameric tetrahedral protease.

PubMed

Kim, Doyoun; San, Boi Hoa; Moh, Sang Hyun; Park, Hyejin; Kim, Dong Young; Lee, Sangho; Kim, Kyeong Kyu

2010-01-01

Regulated cytosolic proteolysis is one of the key cellular processes ensuring proper functioning of a cell. M42 family proteases show a broad spectrum of substrate specificities, but the structural basis for such diversity of the substrate specificities is lagging behind biochemical data. Here we report the crystal structure of PepA from Streptococcus pneumoniae, a glutamyl aminopeptidase belonging to M42 family (SpPepA). We found that Arg-257 in the substrate binding pocket is strategically positioned so that Arg-257 can make electrostatic interactions with the acidic residue of a substrate at its N-terminus. Structural comparison of the substrate binding pocket of the M42 family proteases, along with the structure-based multiple sequence alignment, argues that the appropriate electrostatic interactions contribute to the selective substrate specificity of SpPepA. Copyright 2009 Elsevier Inc. All rights reserved.

Chaperone activity of human small heat shock protein-GST fusion proteins.

PubMed

Arbach, Hannah; Butler, Caley; McMenimen, Kathryn A

2017-07-01

Small heat shock proteins (sHsps) are a ubiquitous part of the machinery that maintains cellular protein homeostasis by acting as molecular chaperones. sHsps bind to and prevent the aggregation of partially folded substrate proteins in an ATP-independent manner. sHsps are dynamic, forming an ensemble of structures from dimers to large oligomers through concentration-dependent equilibrium dissociation. Based on structural studies and mutagenesis experiments, it is proposed that the dimer is the smallest active chaperone unit, while larger oligomers may act as storage depots for sHsps or play additional roles in chaperone function. The complexity and dynamic nature of their structural organization has made elucidation of their chaperone function challenging. HspB1 and HspB5 are two canonical human sHsps that vary in sequence and are expressed in a wide variety of tissues. In order to determine the role of the dimer in chaperone activity, glutathione-S-transferase (GST) was genetically linked as a fusion protein to the N-terminus regions of both HspB1 and HspB5 (also known as Hsp27 and αB-crystallin, respectively) proteins in order to constrain oligomer formation of HspB1 and HspB5, by using GST, since it readily forms a dimeric structure. We monitored the chaperone activity of these fusion proteins, which suggest they primarily form dimers and monomers and function as active molecular chaperones. Furthermore, the two different fusion proteins exhibit different chaperone activity for two model substrate proteins, citrate synthase (CS) and malate dehydrogenase (MDH). GST-HspB1 prevents more aggregation of MDH compared to GST-HspB5 and wild type HspB1. However, when CS is the substrate, both GST-HspB1 and GST-HspB5 are equally effective chaperones. Furthermore, wild type proteins do not display equal activity toward the substrates, suggesting that each sHsp exhibits different substrate specificity. Thus, substrate specificity, as described here for full-length GST fusion proteins with MDH and CS, is modulated by both sHsp oligomeric conformation and by variations of sHsp sequences.

Understanding the Specificity and Random Collision of Enzyme-Substrate Interaction

ERIC Educational Resources Information Center

Kin, Ng Hong; Ling, Tan Aik

2016-01-01

The concept of specificity of enzyme action can potentially be abstract for some students as they fail to appreciate how the three-dimensional configuration of enzymes and the active sites confer perfect fit for specific substrates. In science text books, the specificity of enzyme-substrate binding is typically likened to the action of a lock and…

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 constants in the initial intermediate formed during acylation (EAP, where EA is the acyl enzyme and P is the alcohol leaving group cleaved from the ester substrate) may be constrained such that the leaving group P must dissociate before hydrolytic deacylation can occur.

Durable anti-fogging effect and adhesion improvement on polymer surfaces

NASA Astrophysics Data System (ADS)

Moser, E. M.; Gilliéron, D.; Henrion, G.

2010-01-01

The hydrophobic properties of polymeric surfaces may cause fogging in transparent packaging and poor adhesion to printing colours and coatings. Novel plasma processes for durable functionalization of polypropylene and polyethylene terephthalate substrates were developed and analysed using optical emission spectroscopy. A worm-like nano pattern was created on the polypropylene surface prior to the deposition of thin polar plasma polymerised layers. For both substrates, highly polar surfaces exhibiting a surface tension of up to 69 mN/m and a water contact angle of about 10° were produced - providing the anti-fogging effect. The deposition of thin plasma polymerised layers protects the increased surface areas and enables to tailoring the surface energy of the substrate in a wide range. Wetting characteristics were determined by dynamic contact angle measurements. Investigations of the chemical composition of several layers using X-ray photoelectron spectroscopy and FT-infrared spectroscopy were correlated with functional testing. The surface topography was investigated using atomic force microscopy. The weldability and peeling-off characteristics of the plasma treated polymer films could be adjusted by varying the process parameters. Global and specific migration analyses were undertaken in order to ensure the manufacturing of plasma treated polymer surfaces for direct food contact purposes.

Comparison of expression and enzymatic properties of Aspergillus oryzae lysine aminopeptidases ApsA and ApsB.

PubMed

Marui, Junichiro; Matsushita-Morita, Mayumi; Tada, Sawaki; Hattori, Ryota; Suzuki, Satoshi; Amano, Hitoshi; Ishida, Hiroki; Yamagata, Youhei; Takeuchi, Michio; Kusumoto, Ken-Ichi

2012-08-01

The apsA and apsB genes encoding family M1 aminopeptidases were identified in the industrial fungus Aspergillus oryzae. The apsB was transcriptionally up-regulated up to 2.5-fold in response to the deprivation of nitrogen or carbon sources in growth media, while up-regulation of apsA was less significant. The encoded proteins were bacterially expressed and purified to characterize their enzymatic properties. ApsA and ApsB were optimally active at pH 7.0 and 35 °C and stable at pH ranges of 6-10 and 4-10, respectively, up to 40 °C. The enzymes were inhibited by bestatin and EDTA, as has been reported for family M1 aminopeptidases that characteristically contain a zinc-binding catalytic motif. Both enzymes preferentially liberated N-terminal lysine, which is an essential amino acid and an important additive to animal feed. Enzymes that efficiently release N-terminal lysine from peptides could be useful for food and forage industries. Examination of the reactivity toward peptide substrate of varying length revealed that ApsB exhibited broader substrate specificity than ApsA although the reactivity of ApsB decreased as the length of peptide substrate decreased.

Coated semiconductor devices for neutron detection

DOEpatents

Klann, Raymond T.; McGregor, Douglas S.

2002-01-01

A device for detecting neutrons includes a semi-insulated bulk semiconductor substrate having opposed polished surfaces. A blocking Schottky contact comprised of a series of metals such as Ti, Pt, Au, Ge, Pd, and Ni is formed on a first polished surface of the semiconductor substrate, while a low resistivity ("ohmic") contact comprised of metals such as Au, Ge, and Ni is formed on a second, opposed polished surface of the substrate. In one embodiment, n-type low resistivity pinout contacts comprised of an Au/Ge based eutectic alloy or multi-layered Pd/Ge/Ti/Au are also formed on the opposed polished surfaces and in contact with the Schottky and ohmic contacts. Disposed on the Schottky contact is a neutron reactive film, or coating, for detecting neutrons. The coating is comprised of a hydrogen rich polymer, such as a polyolefin or paraffin; lithium or lithium fluoride; or a heavy metal fissionable material. By varying the coating thickness and electrical settings, neutrons at specific energies can be detected. The coated neutron detector is capable of performing real-time neutron radiography in high gamma fields, digital fast neutron radiography, fissile material identification, and basic neutron detection particularly in high radiation fields.

Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease.

PubMed

Alvizo, Oscar; Mittal, Seema; Mayo, Stephen L; Schiffer, Celia A

2012-07-01

HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease. Copyright © 2012 The Protein Society.

Lava-substrate heat transfer: Laboratory experiments and thermodynamic modeling

NASA Astrophysics Data System (ADS)

Rumpf, M.; Fagents, S. A.; Hamilton, C. W.; Wright, R.; Crawford, I.

2012-12-01

We have performed laboratory experiments and numerical modeling to investigate the heat transfer from a lava flow into various substrate materials, focusing on the effects of the differing thermophysical properties of substrate materials. Initial motivation for this project developed from the desire to understand the loss of solar wind volatiles embedded in lunar regolith deposits that were subsequently covered by a lava flow. The Moon lacks a significant atmosphere and magnetosphere, leaving the surface regolith exposed to bombardment by solar flare and solar wind particles, and by the cosmogenic products of galactic cosmic rays. Preservation of particle-rich regolith deposits may have occurred by the emplacement of an active lava flow on top of the regolith layer, provided the embedded particles survive heating by the lava. During future expeditions to the lunar surface, ancient regolith deposits could be sampled through surface drilling to extract the extra-lunar particles, revealing a history of the solar activity and galactic events not available on the Earth. This project also has important implications for terrestrial lava flows, particularly in the prediction of lava flow hazards. Lava erupted on Earth may be emplaced on various substrates, including solid lava rock, volcanic tephra, sands, soils, etc. The composition, grain size, consolidation, moisture content, etc. of these materials will vary greatly and have different effects on the cooling of the flow. Accounting for specific properties of the substrate could be an important improvement in lava flow models We have performed laboratory experiments in collaboration with the Department of Art and Art History at the University of Hawaii at Manoa in which ~5-6 kg of basalt, collected at Kilauea Volcano, Hawaii, is melted to ~1200 °C. The lava is poured into a device constructed of calcium silicate sheeting that has been filled with a solid or particulate substrate material and embedded with thermocouples. Internal temperatures are monitored by the thermocouple array, while external temperatures are monitored by a Forward Looking Infrared Radiometer (FLIR) video camera. The experimental data thus describe the cooling rates of the system, and reveal the release of latent heat of crystallization within the cooling lava. These experiments have been conducted in conjunction with numerical simulations of the heat transfer from a lava flow into various substrates, to quantify the depth reached by the heat pulse as it penetrates the substrate. Models include material-specific, temperature-dependent thermophysical properties, including thermal conductivity, specific heat capacity, and latent heat of crystallization. We find that particulate materials, such as lunar regolith, sand, and soils will be heated to depths shallower than solid materials. In addition, the particulate materials will act as insulators, shielding the lava flow from basal cooling and maintaining high temperatures in the flow core. These results suggest that lava flows emplaced on a dry particulate terrain will remain above solidus for a longer duration, allowing the lava to flow further than when emplaced on a solid substrate.

Specificity of a protein-protein interface: local dynamics direct substrate recognition of effector caspases.

PubMed

Fuchs, Julian E; von Grafenstein, Susanne; Huber, Roland G; Wallnoefer, Hannes G; Liedl, Klaus R

2014-04-01

Proteases are prototypes of multispecific protein-protein interfaces. Proteases recognize and cleave protein and peptide substrates at a well-defined position in a substrate binding groove and a plethora of experimental techniques provide insights into their substrate recognition. We investigate the caspase family of cysteine proteases playing a key role in programmed cell death and inflammation, turning caspases into interesting drug targets. Specific ligand binding to one particular caspase is difficult to achieve, as substrate specificities of caspase isoforms are highly similar. In an effort to rationalize substrate specificity of two closely related caspases, we investigate the substrate promiscuity of the effector Caspases 3 and 7 by data mining (cleavage entropy) and by molecular dynamics simulations. We find a strong correlation between binding site rigidity and substrate readout for individual caspase subpockets explaining more stringent substrate readout of Caspase 7 via its narrower conformational space. Caspase 3 subpockets S3 and S4 show elevated local flexibility explaining the more unspecific substrate readout of that isoform in comparison to Caspase 7. We show by in silico exchange mutations in the S3 pocket of the proteases that a proline residue in Caspase 7 contributes to the narrowed conformational space of the binding site. These findings explain the substrate specificities of caspases via a mechanism of conformational selection and highlight the crucial importance of binding site local dynamics in substrate recognition of proteases. Proteins 2014; 82:546-555. © 2013 Wiley Periodicals, Inc. Copyright © 2013 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

Probing the molecular determinants of aniline dioxygenase substrate specificity by saturation mutagenesis.

PubMed

Ang, Ee L; Obbard, Jeffrey P; Zhao, Huimin

2007-02-01

Aniline dioxygenase is a multicomponent Rieske nonheme-iron dioxygenase enzyme isolated from Acinetobacter sp. strain YAA. Saturation mutagenesis of the substrate-binding pocket residues, which were identified using a homology model of the alpha subunit of the terminal dioxygenase (AtdA3), was used to probe the molecular determinants of AtdA substrate specificity. The V205A mutation widened the substrate specificity of aniline dioxygenase to include 2-isopropylaniline, for which the wild-type enzyme has no activity. The V205A mutation also made 2-isopropylaniline a better substrate for the enzyme than 2,4-dimethylaniline, a native substrate of the wild-type enzyme. The I248L mutation improved the activity of aniline dioxygenase against aniline and 2,4-dimethylaniline approximately 1.7-fold and 2.1-fold, respectively. Thus, it is shown that the alpha subunit of the terminal dioxygenase indeed plays a part in the substrate specificity as well as the activity of aniline dioxygenase. Interestingly, the equivalent residues of V205 and I248 have not been previously reported to influence the substrate specificity of other Rieske dioxygenases. These results should facilitate future engineering of the enzyme for bioremediation and industrial applications.

Effects of near-bed turbulence and micro-topography on macroinvertebrate movements across contrasting gravel-bed surfaces (Invited)

NASA Astrophysics Data System (ADS)

Buffin-Belanger, T. K.; Rice, S. P.; Reid, I.; Lancaster, J.

2009-12-01

Fluvial habitats can be described from a series of physical variables but to adequately address the habitat quality it becomes necessary to develop an understanding that combines the physical variables with the behaviour of the inhabitating organisms. The hypothesis of flow refugia provide a rational that can explain the persistence of macroinvertebrate communities in gravel-bed rivers when spates occur. The movement behaviour of macroinvertebrates is a key element to the flow refugia hypothesis, but little is known about how local near-bed turbulence and bed microtopography may affect macroinvertebrate movements. We reproduced natural gravel-bed substrates with contrasting gravel bed textures in a large flume where we were able to document the movement behaviour of the cased caddisfly Potamophylax latipennis for a specific discharge. The crawling paths and drift events of animals were analysed from video recordings. Characteristics of movements differ from one substrate to another. The crawling speed is higher for the small grain-size substrates but the mean travel distance remains approximately the same between substrates. For each substrate, the animals tended to follow consistent paths across the surface. The number of drift events and mean distance drifted is higher for the small grain-size substrate. ADV measurements close to the boundary allow detailed characterisation of near-bed hydraulic variables, including : skewness coefficients, TKE, UV correlation coefficients and integral time scales from autocorrelation analysis. For these variables, the vertical patterns of turbulence parameters are similar between the substrates but the amplitude of the average values and standard errors vary significantly. The spatial distribution of this variability is considered in relation to the crawling paths. It appears that the animals tend to crawl within areas of the substrate where low flow velocities and low turbulent kinetic energies are found, while sites that insects avoided were characterised by higher elevations, velocities and turbulence.

Investigation of plasma dynamics and spatially varying O and OH concentrations in atmospheric pressure plasma jets impinging on glass, water and metal substrates

NASA Astrophysics Data System (ADS)

Yue, Yuanfu; Pei, Xuekai; Gidon, Dogan; Wu, Fan; Wu, Shuqun; Lu, Xinpei

2018-06-01

Atmospheric pressure plasma jets (APPJs) have attracted considerable attention over the last decade, specifically for use in surface engineering. A comparative study of an APPJ, driven by pulsed DC voltage, is conducted in order to examine the plasma impingement onto different surfaces. In this paper, the effect of gas flow rate and composition is investigated using three kinds of substrates: dielectric glass, distilled water and metal plate using fast imaging. Alongside discharges associated with rising and falling voltage, a so-called third discharge is observed during the pulse for water and metal surfaces which corresponds to a restrike breakdown from surfaces to nozzle. The differences in plasma dynamics observed are mainly attributed to the differences in substrate conductivity. In addition, spatial and temporal distributions of OH and O density are investigated by means of laser induced fluorescence (LIF). The OH/O LIF intensity is found to be much higher for metal and water substrates compared to the glass plate. We attribute this effect to the differences in power dissipation associated with the presence and intensity of the third discharge. Effects of gas flow rate and seed gas (H2O and O2) mixing on the LIF enhancement are also studied. The related results provide additional insights for optimizing the generation of reactive species.

Initial steps of the peroxidase-catalyzed polymerization of coniferyl alcohol and/or sinapyl aldehyde: capillary zone electrophoresis study of pH effect.

PubMed

Fournand, David; Cathala, Bernard; Lapierre, Catherine

2003-01-01

Capillary zone electrophoresis has been used to monitor the first steps of the dehydrogenative polymerization of coniferyl alcohol, sinapyl aldehyde, or a mixture of both, catalyzed by the horseradish peroxidase (HRP)-H(2)O(2) system. When coniferyl alcohol was the unique HRP substrate, three major dimers were observed (beta-5, beta-beta, and beta-O-4 interunit linkages) and their initial formation velocity as well as their relative abundance varied with pH. The beta-O-4 interunit linkage was thus slightly favored at lower pH values. In contrast, sinapyl aldehyde turned out to be a very poor substrate for HRP except in basic conditions (pH 8). The major dimer observed was the beta,beta'-di-sinapyl aldehyde, a red-brown exhibiting compound which might partly participate in the red coloration usually observed in cinnamyl alcohol dehydrogenase-deficient angiosperms. Finally, when a mixture of coniferyl alcohol and sinapyl aldehyde was used, it looked as if sinapyl aldehyde became a very good substrate for HRP. Indeed, coniferyl alcohol turned out to serve as a redox mediator (i.e. "shuttle oxidant") for the sinapyl aldehyde incorporation in the lignin-like polymer. This means that in particular conditions the specificity of oxidative enzymes might not hinder the incorporation of poor substrates into the growing lignin polymer.

Extensive peptide and natural protein substrate screens reveal that mouse caspase-11 has much narrower substrate specificity than caspase-1

PubMed Central

Ramirez, Monica L. Gonzalez; Poreba, Marcin; Snipas, Scott J.; Groborz, Katarzyna; Drag, Marcin; Salvesen, Guy S.

2018-01-01

Inflammatory cell death, or pyroptosis, is triggered by pathogenic infections or events. It is executed by caspase-1 (in the canonical pyroptosis pathway) or caspase-11 (noncanonical pathway), each via production of a cell-lytic domain from the pyroptosis effector protein gasdermin D through specific and limited proteolysis. Pyroptosis is accompanied by the release of inflammatory mediators, including the proteolytically processed forms of interleukin-1β (IL-1β) and IL-18. Given the similar inflammatory outcomes of the canonical and noncanonical pyroptosis pathways, we hypothesized that caspase-1 and -11 should have very similar activities and substrate specificities. To test this hypothesis, we purified recombinant murine caspases and analyzed their primary specificities by massive hybrid combinatorial substrate library (HyCoSuL) screens. We correlated the substrate preferences of each caspase with their activities on the recombinant natural substrates IL-1β, IL-18, and gasdermin D. Although we identified highly selective and robust peptidyl substrates for caspase-1, we were unable to do so for caspase-11, because caspase-1 cleaved even the best caspase-11 substrates equally well. Caspase-1 rapidly processed pro-IL-1β and -18, but caspase-11 processed these two pro-ILs extremely poorly. However, both caspase-1 and -11 efficiently produced the cell-lytic domain from the gasdermin D precursor. We hypothesize that caspase-11 may have evolved a specific exosite to selectively engage pyroptosis without directly activating pro-IL-1β or -18. In summary, comparing the activities of caspase-1 and -11 in HyCoSuL screens and with three endogenous protein substrates, we conclude that caspase-11 has highly restricted substrate specificity, preferring gasdermin D over all other substrates examined. PMID:29414788

Lignocellulolytic enzyme activity, substrate utilization, and mushroom yield by Pleurotus ostreatus cultivated on substrate containing anaerobic digester solids.

PubMed

Isikhuemhen, Omoanghe S; Mikiashvilli, Nona A

2009-11-01

Solid waste from anaerobic digestion of litter from the commercial production of broiler chickens has limited use as fertilizer. Its disposal is a major problem for digester operators who are seeking alternative use for anaerobic digester solids, also referred to as solid waste (SW). The use of SW as substrates for the cultivation of Pleurotus ostreatus strain MBFBL400 was investigated. Lignocellulolytic enzymes activity, substrate utilization, and mushroom yield were evaluated in ten different substrate combinations (SCs) containing varying amounts of solid waste, wheat straw, and millet. Nutritional content of mushrooms produced on the different substrates was also determined. Substrates containing 70-80% wheat straw, 10-20% SW, and 10-20% millet were found to produce the highest mushroom yield (874.8-958.3 g/kg). Loss of organic matter in all SCs tested varied from 45.8% to 56.2%, which had positive correlation with the biological efficiency. Laccase, peroxidase, and carboxymethylcellulase (CMCase) activities were higher before fruiting, whereas xylanase showed higher activities after mushroom fruiting. SW increased the nutritional content in mushrooms harvested, and the combination of wheat straw and SW with millet significantly improved mushroom yield. Our findings demonstrated the possibility of utilizing anaerobic digester solids in mushroom cultivation. The application of SW as such could improve the financial gains in the overall economy of anaerobic digester plants.

Substrate specificity of the ubiquitin and Ubl proteases

PubMed Central

Ronau, Judith A; Beckmann, John F; Hochstrasser, Mark

2016-01-01

Conjugation and deconjugation of ubiquitin and ubiquitin-like proteins (Ubls) to cellular proteins are highly regulated processes integral to cellular homeostasis. Most often, the C-termini of these small polypeptides are attached to lysine side chains of target proteins by an amide (isopeptide) linkage. Deubiquitinating enzymes (DUBs) and Ubl-specific proteases (ULPs) comprise a diverse group of proteases that recognize and remove ubiquitin and Ubls from their substrates. How DUBs and ULPs distinguish among different modifiers, or different polymeric forms of these modifiers, remains poorly understood. The specificity of ubiquitin/Ubl-deconjugating enzymes for particular substrates depends on multiple factors, ranging from the topography of specific substrate features, as in different polyubiquitin chain types, to structural elements unique to each enzyme. Here we summarize recent structural and biochemical studies that provide insights into mechanisms of substrate specificity among various DUBs and ULPs. We also discuss the unexpected specificities of non-eukaryotic proteases in these families. PMID:27012468

An investigation of Au/Ti multilayer thin-films: surface morphology, structure and interfacial/surface migration of constituents under applied thermal stress

NASA Astrophysics Data System (ADS)

Senevirathne, Indrajith; Kemble, Eric; Lavoie, John

2014-03-01

Multilayer thin films are ubiquitous in industry. Au/Ti/substrate is unique due to possible biological applications in proof of concept devices. Material used for substrates include borosilicate glass, and quartz. Typical Ti depositions on substrates give rise to Stanski-Krastonov (SK) like growth while Frank-van der Merwe (FM) like growth is preferred. Ti films with thickness of ~ 100nm were deposited onto varying substrates using a thermal evaporator. The additional Au layer is then deposited via magnetron sputter deposition at 100mtorr at low deposition rates (~ 1ML/min) onto the Ti thin film. These systems were annealed at varying temperatures and at different durations. Systems were investigated via AFM (Atomic Force Microscopy) probes to examine the surface morphology, and structure. Further, the ambient contamination and elemental distribution/diffusion at annealing was investigated via Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray spectroscopy (EDX). PASSHE FPDC Annual Grant (LOU # 2010-LHU-03)

Tunable high-q superconducting notch filter

DOEpatents

Pang, C.S.; Falco, C.M.; Kampwirth, R.T.; Schuller, I.K.

1979-11-29

A superconducting notch filter is made of three substrates disposed in a cryogenic environment. A superconducting material is disposed on one substrate in a pattern of a circle and an annular ring connected together. The second substrate has a corresponding pattern to form a parallel plate capacitor and the second substrate has the circle and annular ring connected by a superconducting spiral that forms an inductor. The third substrate has a superconducting spiral that is placed parallel to the first superconducting spiral to form a transformer. Relative motion of the first substrate with respect to the second is effected from outside the cryogenic environment to vary the capacitance and hence the frequency of the resonant circuit formed by the superconducting devices.

Rapid misfit dislocation characterization in heteroepitaxial III-V/Si thin films by electron channeling contrast imaging

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

Carnevale, Santino D.; Deitz, Julia I.; Carlin, John A.

Electron channeling contrast imaging (ECCI) is used to characterize misfit dislocations in heteroepitaxial layers of GaP grown on Si(100) substrates. Electron channeling patterns serve as a guide to tilt and rotate sample orientation so that imaging can occur under specific diffraction conditions. This leads to the selective contrast of misfit dislocations depending on imaging conditions, confirmed by dynamical simulations, similar to using standard invisibility criteria in transmission electron microscopy (TEM). The onset and evolution of misfit dislocations in GaP films with varying thicknesses (30 to 250 nm) are studied. This application simultaneously reveals interesting information about misfit dislocations in GaP/Si layersmore » and demonstrates a specific measurement for which ECCI is preferable versus traditional plan-view TEM.« less

Germination and seedling establishment in orchids: a complex of requirements

PubMed Central

Rasmussen, Hanne N.; Dixon, Kingsley W.; Jersáková, Jana; Těšitelová, Tamara

2015-01-01

Background Seedling recruitment is essential to the sustainability of any plant population. Due to the minute nature of seeds and early-stage seedlings, orchid germination in situ was for a long time practically impossible to observe, creating an obstacle towards understanding seedling site requirements and fluctuations in orchid populations. The introduction of seed packet techniques for sowing and retrieval in natural sites has brought with it important insights, but many aspects of orchid seed and germination biology remain largely unexplored. Key Considerations The germination niche for orchids is extremely complex, because it is defined by requirements not only for seed lodging and germination, but also for presence of a fungal host and its substrate. A mycobiont that the seedling can parasitize is considered an essential element, and a great diversity of Basidiomycota and Ascomycota have now been identified for their role in orchid seed germination, with fungi identifiable as imperfect Rhizoctonia species predominating. Specificity patterns vary from orchid species employing a single fungal lineage to species associating individually with a limited selection of distantly related fungi. A suitable organic carbon source for the mycobiont constitutes another key requirement. Orchid germination also relies on factors that generally influence the success of plant seeds, both abiotic, such as light/shade, moisture, substrate chemistry and texture, and biotic, such as competitors and antagonists. Complexity is furthermore increased when these factors influence seeds/seedling, fungi and fungal substrate differentially. Conclusions A better understanding of germination and seedling establishment is needed for conservation of orchid populations. Due to the obligate association with a mycobiont, the germination niches in orchid species are extremely complex and varied. Microsites suitable for germination can be small and transient, and direct observation is difficult. An experimental approach using several levels of environmental manipulation/control is recommended. PMID:26271118

Zebrafish Oatp-mediated transport of microcystin congeners.

PubMed

Steiner, Konstanze; Zimmermann, Lisa; Hagenbuch, Bruno; Dietrich, Daniel

2016-05-01

Microcystins (MC), representing >100 congeners being produced by cyanobacteria, are a hazard for aquatic species. As MC congeners vary in their toxicity, the congener composition of a bloom primarily dictates the severity of adverse effects and appears primarily to be governed by toxicokinetics, i.e., whether transport of MCs occurs via organic anion-transporting polypeptides (Oatps). Differences in observed MC toxicity in various fish species suggest differential expression of Oatp subtypes leading to varying tissue distribution of the very same MC congener within different species. The objectives of this study were the functional characterization and analysis of the tissue distribution of Oatp subtypes in zebrafish (Danio rerio) as a surrogate model for cyprinid fish. Zebrafish Oatps (zfOatps) were cloned, and the organ distribution was determined at the mRNA level. zfOatps were transiently expressed in HEK293 cells for functional characterization using the Oatp substrates estrone-3-sulfate, taurocholate and methotrexate and specific MC congeners (MC-LR, MC-RR, MC-LF and MC-LW). Novel zfOatp isoforms were isolated. Among these isoforms, the organ-specific expression of zfOatp1d1 and of members of the zfOatp1f subfamily was identified. At the functional level, zfOatp1d1, zfOatp1f2, zfOatp1f3 and zfOatp1f4 transported at least one of the Oatp substrates, and zfOatp1d1, zfOatp1f2 and zfOatp1f4 were shown to transport MC congeners. MC-LF and MC-LW were generally transported faster than MC-LR and MC-RR. The subtype-specific expression of zfOatp1d1 and of members of the zfOatp1f subfamily as well as differences in the transport of MC congeners could explain the MC congener-dependent differences in toxicity in cyprinids.

RF plasma MOCVD of Y2O3 thin films: Effect of RF self-bias on the substrates during deposition

NASA Astrophysics Data System (ADS)

Chopade, S. S.; Barve, S. A.; Thulasi Raman, K. H.; Chand, N.; Deo, M. N.; Biswas, A.; Rai, Sanjay; Lodha, G. S.; Rao, G. M.; Patil, D. S.

2013-11-01

Yttrium oxide (Y2O3) thin films have been deposited by radio frequency plasma assisted metal organic chemical vapor deposition (MOCVD) process using (2,2,6,6-tetramethyl-3,5-heptanedionate) yttrium (commonly known as Y(thd)3) precursor in a plasma of argon and oxygen gases at a substrate temperature of 350 °C. The films have been deposited under influence of varying RF self-bias (-50 V to -175 V) on silicon, quartz, stainless steel and tantalum substrates. The deposited coatings are characterized by glancing angle X-ray diffraction (GIXRD), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), spectroscopic ellipsometry and scanning electron microscopy (SEM). GIXRD and FTIR results indicate deposition of Y2O3 (BCC structure) in all cases. However, XPS results indicate nonstoichiometric cubic phase deposition on the surface of deposited films. The degree of nonstoichiometry varies with bias during deposition. Ellipsometry results indicate that the refractive index for the deposited films is varying from 1.70 to 1.83 that is typical for Y2O3. All films are transparent in the investigated wavelength range 300-1200 nm. SEM results indicate that the microstructure of the films is changing with applied bias. Results indicate that it is possible to deposit single phase cubic Y2O3 thin films at low substrate temperature by RF plasma MOCVD process. RF self-bias that decides about the energy of impinging ions on the substrates plays an important role in controlling the texture of deposited Y2O3 films on the substrates. Results indicate that to control the structure of films and its texture, it is important to control the bias on the substrate during deposition. The films deposited at high bias level show degradation in the crystallinity and reduction of thickness.

Altered Substrate Specificity of Drug-Resistant Human Immunodeficiency Virus Type 1 Protease

PubMed Central

Dauber, Deborah S.; Ziermann, Rainer; Parkin, Neil; Maly, Dustin J.; Mahrus, Sami; Harris, Jennifer L.; Ellman, Jon A.; Petropoulos, Christos; Craik, Charles S.

2002-01-01

Resistance to human immunodeficiency virus type 1 protease (HIV PR) inhibitors results primarily from the selection of multiple mutations in the protease region. Because many of these mutations are selected for the ability to decrease inhibitor binding in the active site, they also affect substrate binding and potentially substrate specificity. This work investigates the substrate specificity of a panel of clinically derived protease inhibitor-resistant HIV PR variants. To compare protease specificity, we have used positional-scanning, synthetic combinatorial peptide libraries as well as a select number of individual substrates. The subsite preferences of wild-type HIV PR determined by using the substrate libraries are consistent with prior reports, validating the use of these libraries to compare specificity among a panel of HIV PR variants. Five out of seven protease variants demonstrated subtle differences in specificity that may have significant impacts on their abilities to function in viral maturation. Of these, four variants demonstrated up to fourfold changes in the preference for valine relative to alanine at position P2 when tested on individual peptide substrates. This change correlated with a common mutation in the viral NC/p1 cleavage site. These mutations may represent a mechanism by which severely compromised, drug-resistant viral strains can increase fitness levels. Understanding the altered substrate specificity of drug-resistant HIV PR should be valuable in the design of future generations of protease inhibitors as well as in elucidating the molecular basis of regulation of proteolysis in HIV. PMID:11773410

The "handwriting brain": a meta-analysis of neuroimaging studies of motor versus orthographic processes.

PubMed

Planton, Samuel; Jucla, Mélanie; Roux, Franck-Emmanuel; Démonet, Jean-François

2013-01-01

Handwriting is a modality of language production whose cerebral substrates remain poorly known although the existence of specific regions is postulated. The description of brain damaged patients with agraphia and, more recently, several neuroimaging studies suggest the involvement of different brain regions. However, results vary with the methodological choices made and may not always discriminate between "writing-specific" and motor or linguistic processes shared with other abilities. We used the "Activation Likelihood Estimate" (ALE) meta-analytical method to identify the cerebral network of areas commonly activated during handwriting in 18 neuroimaging studies published in the literature. Included contrasts were also classified according to the control tasks used, whether non-specific motor/output-control or linguistic/input-control. These data were included in two secondary meta-analyses in order to reveal the functional role of the different areas of this network. An extensive, mainly left-hemisphere network of 12 cortical and sub-cortical areas was obtained; three of which were considered as primarily writing-specific (left superior frontal sulcus/middle frontal gyrus area, left intraparietal sulcus/superior parietal area, right cerebellum) while others related rather to non-specific motor (primary motor and sensorimotor cortex, supplementary motor area, thalamus and putamen) or linguistic processes (ventral premotor cortex, posterior/inferior temporal cortex). This meta-analysis provides a description of the cerebral network of handwriting as revealed by various types of neuroimaging experiments and confirms the crucial involvement of the left frontal and superior parietal regions. These findings provide new insights into cognitive processes involved in handwriting and their cerebral substrates. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cleavage Entropy as Quantitative Measure of Protease Specificity

PubMed Central

Fuchs, Julian E.; von Grafenstein, Susanne; Huber, Roland G.; Margreiter, Michael A.; Spitzer, Gudrun M.; Wallnoefer, Hannes G.; Liedl, Klaus R.

2013-01-01

A purely information theory-guided approach to quantitatively characterize protease specificity is established. We calculate an entropy value for each protease subpocket based on sequences of cleaved substrates extracted from the MEROPS database. We compare our results with known subpocket specificity profiles for individual proteases and protease groups (e.g. serine proteases, metallo proteases) and reflect them quantitatively. Summation of subpocket-wise cleavage entropy contributions yields a measure for overall protease substrate specificity. This total cleavage entropy allows ranking of different proteases with respect to their specificity, separating unspecific digestive enzymes showing high total cleavage entropy from specific proteases involved in signaling cascades. The development of a quantitative cleavage entropy score allows an unbiased comparison of subpocket-wise and overall protease specificity. Thus, it enables assessment of relative importance of physicochemical and structural descriptors in protease recognition. We present an exemplary application of cleavage entropy in tracing substrate specificity in protease evolution. This highlights the wide range of substrate promiscuity within homologue proteases and hence the heavy impact of a limited number of mutations on individual substrate specificity. PMID:23637583

Dose-dependent fate of GFP-E. coli in the alimentary canal of adult house flies

USDA-ARS?s Scientific Manuscript database

Adult house flies (Diptera: Muscidae; Musca domestica L.) disseminate bacteria from microbe-rich substrates to areas where humans and domesticated animals reside. Because bacterial abundance fluctuates widely across substrates, flies encounter and ingest varying amounts of bacteria. We investigated ...

Quantitative Comparison of Protein Adsorption and Conformational Changes on Dielectric-Coated Nanoplasmonic Sensing Arrays.

PubMed

Ferhan, Abdul Rahim; Jackman, Joshua A; Sut, Tun Naw; Cho, Nam-Joon

2018-04-22

Nanoplasmonic sensors are a popular, surface-sensitive measurement tool to investigate biomacromolecular interactions at solid-liquid interfaces, opening the door to a wide range of applications. In addition to high surface sensitivity, nanoplasmonic sensors have versatile surface chemistry options as plasmonic metal nanoparticles can be coated with thin dielectric layers. Within this scope, nanoplasmonic sensors have demonstrated promise for tracking protein adsorption and substrate-induced conformational changes on oxide film-coated arrays, although existing studies have been limited to single substrates. Herein, we investigated human serum albumin (HSA) adsorption onto silica- and titania-coated arrays of plasmonic gold nanodisks by localized surface plasmon resonance (LSPR) measurements and established an analytical framework to compare responses across multiple substrates with different sensitivities. While similar responses were recorded on the two substrates for HSA adsorption under physiologically-relevant ionic strength conditions, distinct substrate-specific behavior was observed at lower ionic strength conditions. With decreasing ionic strength, larger measurement responses occurred for HSA adsorption onto silica surfaces, whereas HSA adsorption onto titania surfaces occurred independently of ionic strength condition. Complementary quartz crystal microbalance-dissipation (QCM-D) measurements were also performed, and the trend in adsorption behavior was similar. Of note, the magnitudes of the ionic strength-dependent LSPR and QCM-D measurement responses varied, and are discussed with respect to the measurement principle and surface sensitivity of each technique. Taken together, our findings demonstrate how the high surface sensitivity of nanoplasmonic sensors can be applied to quantitatively characterize protein adsorption across multiple surfaces, and outline broadly-applicable measurement strategies for biointerfacial science applications.

Biofunctionalization on alkylated silicon substrate surfaces via "click" chemistry.

PubMed

Qin, Guoting; Santos, Catherine; Zhang, Wen; Li, Yan; Kumar, Amit; Erasquin, Uriel J; Liu, Kai; Muradov, Pavel; Trautner, Barbara Wells; Cai, Chengzhi

2010-11-24

Biofunctionalization of silicon substrates is important to the development of silicon-based biosensors and devices. Compared to conventional organosiloxane films on silicon oxide intermediate layers, organic monolayers directly bound to the nonoxidized silicon substrates via Si-C bonds enhance the sensitivity of detection and the stability against hydrolytic cleavage. Such monolayers presenting a high density of terminal alkynyl groups for bioconjugation via copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC, a "click" reaction) were reported. However, yields of the CuAAC reactions on these monolayer platforms were low. Also, the nonspecific adsorption of proteins on the resultant surfaces remained a major obstacle for many potential biological applications. Herein, we report a new type of "clickable" monolayers grown by selective, photoactivated surface hydrosilylation of α,ω-alkenynes, where the alkynyl terminal is protected with a trimethylgermanyl (TMG) group, on hydrogen-terminated silicon substrates. The TMG groups on the film are readily removed in aqueous solutions in the presence of Cu(I). Significantly, the degermanylation and the subsequent CuAAC reaction with various azides could be combined into a single step in good yields. Thus, oligo(ethylene glycol) (OEG) with an azido tag was attached to the TMG-alkyne surfaces, leading to OEG-terminated surfaces that reduced the nonspecific adsorption of protein (fibrinogen) by >98%. The CuAAC reaction could be performed in microarray format to generate arrays of mannose and biotin with varied densities on the protein-resistant OEG background. We also demonstrated that the monolayer platform could be functionalized with mannose for highly specific capturing of living targets (Escherichia coli expressing fimbriae) onto the silicon substrates.

SERS substrate based on silver nanoparticles and graphene: Dependence on the layer number of graphene

NASA Astrophysics Data System (ADS)

Garg, Preeti; Soni, R. K.; Raman, R.

2018-05-01

In this report, we describe a low-cost fabrication process for highly sensitive SERS substrate by using thermal evaporation technique. The SERS substrate structure consists of silver nanoparticles deposited on monolayer, bilayer and few layer graphene. The fabricated SERS substrates are investigated by field emission scanning electron microscope (FE-SEM), atomic force microscope (AFM), and confocal Raman spectroscope. From the surface morphology we have verified that the fabricated SERS substrate consist of high-density of silver nanoparticles with their size distribution varies from 10 to 150 nm. The surface-enhanced Raman scattering activities of these nanostructures is highest for monolayer graphene.

Kinetics of reactions of the Actinomadura R39 DD-peptidase with specific substrates.

PubMed

Adediran, S A; Kumar, Ish; Nagarajan, Rajesh; Sauvage, Eric; Pratt, R F

2011-01-25

The Actinomadura R39 DD-peptidase catalyzes the hydrolysis and aminolysis of a number of small peptides and depsipeptides. Details of its substrate specificity and the nature of its in vivo substrate are not, however, well understood. This paper describes the interactions of the R39 enzyme with two peptidoglycan-mimetic substrates 3-(D-cysteinyl)propanoyl-D-alanyl-D-alanine and 3-(D-cysteinyl)propanoyl-D-alanyl-D-thiolactate. A detailed study of the reactions of the former substrate, catalyzed by the enzyme, showed DD-carboxypeptidase, DD-transpeptidase, and DD-endopeptidase activities. These results confirm the specificity of the enzyme for a free D-amino acid at the N-terminus of good substrates and indicated a preference for extended D-amino acid leaving groups. The latter was supported by determination of the structural specificity of amine nucleophiles for the acyl-enzyme generated by reaction of the enzyme with the thiolactate substrate. It was concluded that a specific substrate for this enzyme, and possibly the in vivo substrate, may consist of a partly cross-linked peptidoglycan polymer where a free side chain N-terminal un-cross-linked amino acid serves as the specific acyl group in an endopeptidase reaction. The enzyme is most likely a DD-endopeptidase in vivo. pH-rate profiles for reactions of the enzyme with peptides, the thiolactate named above, and β-lactams indicated the presence of complex proton dissociation pathways with sticky substrates and/or protons. The local structure of the active site may differ significantly for reactions of peptides and β-lactams. Solvent kinetic deuterium isotope effects indicate the presence of classical general acid/base catalysis in both acylation and deacylation; there is no evidence of the low fractionation factor active site hydrogen found previously in class A and C β-lactamases.

E3Net: a system for exploring E3-mediated regulatory networks of cellular functions.

PubMed

Han, Youngwoong; Lee, Hodong; Park, Jong C; Yi, Gwan-Su

2012-04-01

Ubiquitin-protein ligase (E3) is a key enzyme targeting specific substrates in diverse cellular processes for ubiquitination and degradation. The existing findings of substrate specificity of E3 are, however, scattered over a number of resources, making it difficult to study them together with an integrative view. Here we present E3Net, a web-based system that provides a comprehensive collection of available E3-substrate specificities and a systematic framework for the analysis of E3-mediated regulatory networks of diverse cellular functions. Currently, E3Net contains 2201 E3s and 4896 substrates in 427 organisms and 1671 E3-substrate specific relations between 493 E3s and 1277 substrates in 42 organisms, extracted mainly from MEDLINE abstracts and UniProt comments with an automatic text mining method and additional manual inspection and partly from high throughput experiment data and public ubiquitination databases. The significant functions and pathways of the extracted E3-specific substrate groups were identified from a functional enrichment analysis with 12 functional category resources for molecular functions, protein families, protein complexes, pathways, cellular processes, cellular localization, and diseases. E3Net includes interactive analysis and navigation tools that make it possible to build an integrative view of E3-substrate networks and their correlated functions with graphical illustrations and summarized descriptions. As a result, E3Net provides a comprehensive resource of E3s, substrates, and their functional implications summarized from the regulatory network structures of E3-specific substrate groups and their correlated functions. This resource will facilitate further in-depth investigation of ubiquitination-dependent regulatory mechanisms. E3Net is freely available online at http://pnet.kaist.ac.kr/e3net.

Glycan microarray screening assay for glycosyltransferase specificities.

PubMed

Peng, Wenjie; Nycholat, Corwin M; Razi, Nahid

2013-01-01

Glycan microarrays represent a high-throughput approach to determining the specificity of glycan-binding proteins against a large set of glycans in a single format. This chapter describes the use of a glycan microarray platform for evaluating the activity and substrate specificity of glycosyltransferases (GTs). The methodology allows simultaneous screening of hundreds of immobilized glycan acceptor substrates by in situ incubation of a GT and its appropriate donor substrate on the microarray surface. Using biotin-conjugated donor substrate enables direct detection of the incorporated sugar residues on acceptor substrates on the array. In addition, the feasibility of the method has been validated using label-free donor substrate combined with lectin-based detection of product to assess enzyme activity. Here, we describe the application of both procedures to assess the specificity of a recombinant human α2-6 sialyltransferase. This technique is readily adaptable to studying other glycosyltransferases.

Carbon Nanotube Patterning on a Metal Substrate

NASA Technical Reports Server (NTRS)

Nguyen, Cattien V. (Inventor)

2016-01-01

A CNT electron source, a method of manufacturing a CNT electron source, and a solar cell utilizing a CNT patterned sculptured substrate are disclosed. Embodiments utilize a metal substrate which enables CNTs to be grown directly from the substrate. An inhibitor may be applied to the metal substrate to inhibit growth of CNTs from the metal substrate. The inhibitor may be precisely applied to the metal substrate in any pattern, thereby enabling the positioning of the CNT groupings to be more precisely controlled. The surface roughness of the metal substrate may be varied to control the density of the CNTs within each CNT grouping. Further, an absorber layer and an acceptor layer may be applied to the CNT electron source to form a solar cell, where a voltage potential may be generated between the acceptor layer and the metal substrate in response to sunlight exposure.

Modeling micromechanical measurements of depth-varying properties with scanning acoustic microscopy

NASA Astrophysics Data System (ADS)

Marangos, Orestes; Misra, Anil

2018-02-01

Scanning acoustic microscopy (SAM) has been applied to measure the near-surface elastic properties of materials. For many substrates, the near-surface property is not constant but varies with depth. In this paper, we aim to interpret the SAM data from such substrates by modeling the interaction of the focused ultrasonic field with a substrate having a near-surface graded layer. The focused ultrasonic field solutions were represented as spherical harmonic expansions while the substrate solutions were represented as plane wave expansions. The bridging of the two solutions was achieved through the decomposition of the ultrasonic pressure fields in their angular spectra. Parametric studies were performed, which showed that near-surface graded layers exhibit distinctive frequency dependence of their reflectance functions. This behavior is characteristic to the material property gradation profile as well as the extent of the property gradation. The developed model was used to explain the frequency-dependent reflection coefficients measured from an acid-etched dentin substrate. Based on the model calculations, the elastic property variations of the acid-etched dentin near-surface indicate that the topmost part of the etched layer is very soft (3-6 GPa) and transitions to the native dentin through a depth of 27 and 36 microns.

Characterizing Protease Specificity: How Many Substrates Do We Need?

PubMed Central

Schauperl, Michael; Fuchs, Julian E.; Waldner, Birgit J.; Huber, Roland G.; Kramer, Christian; Liedl, Klaus R.

2015-01-01

Calculation of cleavage entropies allows to quantify, map and compare protease substrate specificity by an information entropy based approach. The metric intrinsically depends on the number of experimentally determined substrates (data points). Thus a statistical analysis of its numerical stability is crucial to estimate the systematic error made by estimating specificity based on a limited number of substrates. In this contribution, we show the mathematical basis for estimating the uncertainty in cleavage entropies. Sets of cleavage entropies are calculated using experimental cleavage data and modeled extreme cases. By analyzing the underlying mathematics and applying statistical tools, a linear dependence of the metric in respect to 1/n was found. This allows us to extrapolate the values to an infinite number of samples and to estimate the errors. Analyzing the errors, a minimum number of 30 substrates was found to be necessary to characterize substrate specificity, in terms of amino acid variability, for a protease (S4-S4’) with an uncertainty of 5 percent. Therefore, we encourage experimental researchers in the protease field to record specificity profiles of novel proteases aiming to identify at least 30 peptide substrates of maximum sequence diversity. We expect a full characterization of protease specificity helpful to rationalize biological functions of proteases and to assist rational drug design. PMID:26559682

The characterization of photographic materials as substrates for surface enhanced Raman spectroscopy

NASA Astrophysics Data System (ADS)

Vaughan, J.; Hortin, N.; Christie, S.; Kvasnik, F.; Scully, P. J.

2005-06-01

In this study, five types of photographic materials were obtained from commercial sources and characterized for use as substrates for surface enhanced Raman spectroscopy. The substrates are photographic emulsions coated on glass or paper support. The emulsions were developed to maximize the amount of metallic silver aggregated into clusters. The test analyte, Cresyl Violet, was deposited directly onto the substrate surface. The permeable nature of the supporting gelatin matrix enables the interaction between the target analyte and the solid silver clusters. The surface enhanced Raman spectra of a 2.75 × 10-7 M concentration of Cresyl Violet in ethanol were obtained using these photographic substrates. The Raman and resonant Raman enhancement of Cresyl Violet varies from substrate to substrate, as does the ratio of Raman to resonant Raman peak heights.

Quantitative investigation of hydraulic mixing energy input during batch mode anaerobic digestion and its impact on performance.

PubMed

McLeod, James; Othman, Maazuza Z; Parthasarathy, Rajarathinam

2018-05-26

The relationship between mixing energy input and biogas production was investigated by anaerobically digesting sewage sludge in lab scale, hydraulically mixed, batch mode digesters at six different specific energy inputs. The goal was to identify how mixing energy influenced digestion performance at quantitative levels to help explain the varying results in other published works. The results showed that digester homogeneity was largely uninfluenced by energy input, whereas cumulative biogas production and solids destruction were. With similar solids distributions between conditions, the observed differences were attributed to shear forces disrupting substrate-microbe flocs rather than the formation of temperature and/or concentration gradients. Disruption of the substrate-microbe flocs produced less favourable conditions for hydrolytic bacteria, resulting in less production of biomass and more biogas. Overall, this hypothesis explains the current body of research including the inhibitory conditions reported at extreme mixing power inputs. However, further work is required to definitively prove it. Copyright © 2018 Elsevier Ltd. All rights reserved.

Golgi Glycosylation

PubMed Central

Stanley, Pamela

2011-01-01

Glycosylation is a very common modification of protein and lipid, and most glycosylation reactions occur in the Golgi. Although the transfer of initial sugar(s) to glycoproteins or glycolipids occurs in the ER or on the ER membrane, the subsequent addition of the many different sugars that make up a mature glycan is accomplished in the Golgi. Golgi membranes are studded with glycosyltransferases, glycosidases, and nucleotide sugar transporters arrayed in a generally ordered manner from the cis-Golgi to the trans-Golgi network (TGN), such that each activity is able to act on specific substrate(s) generated earlier in the pathway. The spectrum of glycosyltransferases and other activities that effect glycosylation may vary with cell type, and thus the final complement of glycans on glycoconjugates is variable. In addition, glycan synthesis is affected by Golgi pH, the integrity of Golgi peripheral membrane proteins, growth factor signaling, Golgi membrane dynamics, and cellular stress. Knowledge of Golgi glycosylation has fostered the development of assays to identify mechanisms of intracellular vesicular trafficking and facilitated glycosylation engineering of recombinant glycoproteins. PMID:21441588

Kinome signaling through regulated protein-protein interactions in normal and cancer cells.

PubMed

Pawson, Tony; Kofler, Michael

2009-04-01

The flow of molecular information through normal and oncogenic signaling pathways frequently depends on protein phosphorylation, mediated by specific kinases, and the selective binding of the resulting phosphorylation sites to interaction domains present on downstream targets. This physical and functional interplay of catalytic and interaction domains can be clearly seen in cytoplasmic tyrosine kinases such as Src, Abl, Fes, and ZAP-70. Although the kinase and SH2 domains of these proteins possess similar intrinsic properties of phosphorylating tyrosine residues or binding phosphotyrosine sites, they also undergo intramolecular interactions when linked together, in a fashion that varies from protein to protein. These cooperative interactions can have diverse effects on substrate recognition and kinase activity, and provide a variety of mechanisms to link the stimulation of catalytic activity to substrate recognition. Taken together, these data have suggested how protein kinases, and the signaling pathways in which they are embedded, can evolve complex properties through the stepwise linkage of domains within single polypeptides or multi-protein assemblies.

Specificity of lecithin:cholesterol acyltransferase and atherogenic risk: comparative studies on the plasma composition and in vitro synthesis of cholesteryl esters in 14 vertebrate species.

PubMed

Liu, M; Bagdade, J D; Subbaiah, P V

1995-08-01

To determine whether the specificity of lecithin: cholesterol acyltransferase (LCAT) influences the susceptibility to atherosclerosis, we compared the composition and in vitro synthesis of cholesteryl ester (CE) in the plasmas of 14 vertebrate species with varying predisposition to atherosclerosis. The susceptible species (Group I) had significantly higher ratios of 16:0 CE/20:4 CE in their plasma than the resistant species (Group II). The in vitro formation of labeled CE species in native plasma from labeled cholesterol correlated highly with the mass composition, showing that the LCAT reaction is the predominant source of plasma CE in all the animal species examined. Isolated LCATs from Group I species also synthesized CE with higher ratios of 16:0/20:4 than LCATs from Group II when egg phosphatidylcholine (PC) was used as the acyl donor. In addition, the Group I LCATs exhibited lower specificity towards sn-2-20:4 and sn-2-22:6 PCs, and higher specificity towards sn-2-18:2 PC species than Group II LCATs. With 16:0-20:4 PC as the substrate, all Group I LCATs synthesized more 16:0 CE than 20:4 CE, whereas all Group II LCATs, with the exception of dog enzyme, synthesized predominantly 20:4 CE, showing that the two types of LCAT have different positional specificities towards this PC. These results suggest that there are two classes of LCAT in nature that differ from each other in their substrate and positional specificities, possibly because of differences in their active-site architectures. We propose that the presence of one type of LCAT, which cannot efficiently transfer certain long chain polyunsaturated acyl groups and which consequently synthesizes more saturated CE, may increase the risk of atherosclerosis.

Method and system using power modulation and velocity modulation producing sputtered thin films with sub-angstrom thickness uniformity or custom thickness gradients

DOEpatents

Montcalm, Claude [Livermore, CA; Folta, James Allen [Livermore, CA; Walton, Christopher Charles [Berkeley, CA

2003-12-23

A method and system for determining a source flux modulation recipe for achieving a selected thickness profile of a film to be deposited (e.g., with highly uniform or highly accurate custom graded thickness) over a flat or curved substrate (such as concave or convex optics) by exposing the substrate to a vapor deposition source operated with time-varying flux distribution as a function of time. Preferably, the source is operated with time-varying power applied thereto during each sweep of the substrate to achieve the time-varying flux distribution as a function of time. Preferably, the method includes the steps of measuring the source flux distribution (using a test piece held stationary while exposed to the source with the source operated at each of a number of different applied power levels), calculating a set of predicted film thickness profiles, each film thickness profile assuming the measured flux distribution and a different one of a set of source flux modulation recipes, and determining from the predicted film thickness profiles a source flux modulation recipe which is adequate to achieve a predetermined thickness profile. Aspects of the invention include a computer-implemented method employing a graphical user interface to facilitate convenient selection of an optimal or nearly optimal source flux modulation recipe to achieve a desired thickness profile on a substrate. The method enables precise modulation of the deposition flux to which a substrate is exposed to provide a desired coating thickness distribution.

YAP-dependent Mechanotransduction is Required for Proliferation and Migration on Native-like Substrate Topography

PubMed Central

Mascharak, Shamik; Benitez, Patrick L.; Proctor, Amy C.; Madl, Christopher M.; Hu, Kenneth H.; Dewi, Ruby E.; Butte, Manish J.; Heilshorn, Sarah C.

2017-01-01

Native vascular extracellular matrices (vECM) consist of elastic fibers that impart varied topographical properties, yet most in vitro models designed to study the effects of topography on cell behavior are not representative of native architecture. Here, we engineer an electrospun elastin-like protein (ELP) system with independently tunable, vECM-mimetic topography and demonstrate that increasing topographical variation causes loss of endothelial cell-cell junction organization. This loss of VE-cadherin signaling and increased cytoskeletal contractility on more topographically varied ELP substrates in turn promote YAP activation and nuclear translocation, resulting in significantly increased endothelial cell migration and proliferation. Our findings identify YAP as a required signaling factor through which fibrous substrate topography influences cell behavior and highlights topography as a key design parameter for engineered biomaterials. PMID:27889666

Extensive peptide and natural protein substrate screens reveal that mouse caspase-11 has much narrower substrate specificity than caspase-1.

PubMed

Ramirez, Monica L Gonzalez; Poreba, Marcin; Snipas, Scott J; Groborz, Katarzyna; Drag, Marcin; Salvesen, Guy S

2018-05-04

Inflammatory cell death, or pyroptosis, is triggered by pathogenic infections or events. It is executed by caspase-1 (in the canonical pyroptosis pathway) or caspase-11 (noncanonical pathway), each via production of a cell-lytic domain from the pyroptosis effector protein gasdermin D through specific and limited proteolysis. Pyroptosis is accompanied by the release of inflammatory mediators, including the proteolytically processed forms of interleukin-1β (IL-1β) and IL-18. Given the similar inflammatory outcomes of the canonical and noncanonical pyroptosis pathways, we hypothesized that caspase-1 and -11 should have very similar activities and substrate specificities. To test this hypothesis, we purified recombinant murine caspases and analyzed their primary specificities by massive hybrid combinatorial substrate library (HyCoSuL) screens. We correlated the substrate preferences of each caspase with their activities on the recombinant natural substrates IL-1β, IL-18, and gasdermin D. Although we identified highly selective and robust peptidyl substrates for caspase-1, we were unable to do so for caspase-11, because caspase-1 cleaved even the best caspase-11 substrates equally well. Caspase-1 rapidly processed pro-IL-1β and -18, but caspase-11 processed these two pro-ILs extremely poorly. However, both caspase-1 and -11 efficiently produced the cell-lytic domain from the gasdermin D precursor. We hypothesize that caspase-11 may have evolved a specific exosite to selectively engage pyroptosis without directly activating pro-IL-1β or -18. In summary, comparing the activities of caspase-1 and -11 in HyCoSuL screens and with three endogenous protein substrates, we conclude that caspase-11 has highly restricted substrate specificity, preferring gasdermin D over all other substrates examined. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanistic Studies of the Yeast Polyamine Oxidase Fms1: Kinetic Mechanism, Substrate Specificity, and pH Dependence†

PubMed Central

Adachi, Mariya S.; Torres, Jason M.; Fitzpatrick, Paul F.

2010-01-01

The flavoprotein oxidase Fms1 from Saccharomyces cerevisiae catalyzes the oxidation of spermine and N1-acetylspermine to yield spermidine and 3-aminopropanal or N-acetyl-3-aminopropanal. The kinetic mechanism of the enzyme has been determined with both substrates. The initial velocity patterns are ping-pong, consistent with reduction being kinetically irreversible. Reduction of Fms1 by either substrate is biphasic. The rate constant for the rapid phase varies with the substrate concentration, with limiting rates for reduction of the enzyme of 126 and 1410 s−1 and apparent Kd values of 24.3 and 484 μM for spermine and N1-acetylspermine, respectively. The rapid phase is followed by a concentration-independent phase that is slower than turnover. The reaction of the reduced enzyme with oxygen is monophasic, with a rate constant of 402 mM−1 s−1 with spermine at 25 °C, and 204 mM−1 s−1 with N1-acetylspermine at 4 °C, pH 9.0. This step is followed by rate-limiting product dissociation. The kcat/Kamine-pH profiles are bell-shaped, with an average pKa value of 9.3 with spermine and pKa values of 8.3 and 9.6 with N1-acetylspermine. Both profiles are consistent with the active forms of substrates having two charged nitrogens. The pH profiles for the rate constant for flavin reduction show pKa values of 8.3 and 7.2 for spermine and N1-acetylspermine, respectively, for groups that must be unprotonated; these pKa values are assigned to the substrate N4. The kcat/KO2-pH profiles show pKa values of 7.5 for spermine and 6.8 for N1-acetylspermine. With both substrates, the kcat value decreases when a single residue is protonated. PMID:21067138

A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases.

PubMed

Ewing, Tom A; van Noord, Aster; Paul, Caroline E; van Berkel, Willem J H

2018-01-14

Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para -substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para -phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para -phenol oxidases, facilitating the enzyme engineering of known para- phenol oxidases and the evaluation of the substrate specificity of novel para -phenol oxidases.

Production of β-Lactamase by Non-Streptomyces Actinomycetales

PubMed Central

Schwartz, Jeffrey L.; Schwartz, Surya P.

1979-01-01

Supernatants and whole cells from fermentation broths of Micromonospora, Nocardia, Oerskovia, and other genera of the Actinomycetales were examined for the presence of β-lactamase activity by using the chromogenic cephalosporin 87/312. Nearly 60% of the 250 isolates examined produced detectable levels of β-lactamase. All enzyme preparations were active over a range of pH values from 6.5 to 8.2, with maximum activity occurring between 7.0 and 7.8. The preparations varied in their stability at 60°C. An examination of selected enzyme preparations revealed a similarity between substrate specificities of the non-Streptomyces Actinomycetales and gram-negative-bacterial β-lactamases. PMID:311614

Model of depositing layer on cylindrical surface produced by induction-assisted laser cladding process

NASA Astrophysics Data System (ADS)

Kotlan, Václav; Hamar, Roman; Pánek, David; Doležel, Ivo

2017-12-01

A model of hybrid cladding on a cylindrical surface is built and numerically solved. Heating of both substrate and the powder material to be deposited on its surface is realized by laser beam and preheating inductor. The task represents a hard-coupled electromagnetic-thermal problem with time-varying geometry. Two specific algorithms are developed to incorporate this effect into the model, driven by local distribution of temperature and its gradients. The algorithms are implemented into the COMSOL Multiphysics 5.2 code that is used for numerical computations of the task. The methodology is illustrated with a typical example whose results are discussed.

Characterisation of phenol oxidase and peroxidase from maize silk.

PubMed

Sukalović, V Hadzi-Tasković; Veljović-Jovanović, S; Maksimović, J Dragisić; Maksimović, V; Pajić, Z

2010-05-01

Silk of some maize genotypes contains a high level of phenolics that undergo enzymatic oxidation to form quinones, which condense among themselves or with proteins to form brown pigments. Two phenolic oxidizing enzymes, peroxidase (POD; EC 1.11.1.7) and polyphenol oxidase (PPO; EC 1.10.3.1), from maize (Zea mays L.) silk were characterised with respect to their preferred substrate, different isoforms and specific effectors. One browning silk sample with high, and two non-browning samples with low phenolic content were investigated. Although POD oxidizes a wide range of phenolic substrates in vitro, its activity rate was independent of silk phenolic content. PPO activity, detected with o-diphenolic substrates, was abundant only in browning silk, and low or absent in non-browning silk. Pollination increased POD but not PPO activity. Isoelectric-focusing (IEF) and specific staining for POD and PPO showed a high degree of polymorphism that varied with silk origin. The IEF pattern of POD revealed a number of anionic and several cationic isoenzymes, with the most pronounced having neutral pI 7 and a basic isoform with pI 10. Detected isoforms of PPO were anionic, except for one neutral form found only in browning silk, and occupied positions different from those of POD. Different inhibitory effects of NaN(3), EDTA, KCN, and L-cysteine, as well as different impacts of a variety of cations on the oxidation of chlorogenic acid, mediated by PPO or POD, were detected. The findings are discussed in terms of a possible roles of these enzymes in defence and pollination.

Highly sensitive and adaptable fluorescence-quenched pair discloses the substrate specificity profiles in diverse protease families

PubMed Central

Poreba, Marcin; Szalek, Aleksandra; Rut, Wioletta; Kasperkiewicz, Paulina; Rutkowska-Wlodarczyk, Izabela; Snipas, Scott J.; Itoh, Yoshifumi; Turk, Dusan; Turk, Boris; Overall, Christopher M.; Kaczmarek, Leszek; Salvesen, Guy S.; Drag, Marcin

2017-01-01

Internally quenched fluorescent (IQF) peptide substrates originating from FRET (Förster Resonance Energy Transfer) are powerful tool for examining the activity and specificity of proteases, and a variety of donor/acceptor pairs are extensively used to design individual substrates and combinatorial libraries. We developed a highly sensitive and adaptable donor/acceptor pair that can be used to investigate the substrate specificity of cysteine proteases, serine proteases and metalloproteinases. This novel pair comprises 7-amino-4-carbamoylmethylcoumarin (ACC) as the fluorophore and 2,4-dinitrophenyl-lysine (Lys(DNP)) as the quencher. Using caspase-3, caspase-7, caspase-8, neutrophil elastase, legumain, and two matrix metalloproteinases (MMP2 and MMP9), we demonstrated that substrates containing ACC/Lys(DNP) exhibit 7 to 10 times higher sensitivity than conventional 7-methoxy-coumarin-4-yl acetic acid (MCA)/Lys(DNP) substrates; thus, substantially lower amounts of substrate and enzyme can be used for each assay. We therefore propose that the ACC/Lys(DNP) pair can be considered a novel and sensitive scaffold for designing substrates for any group of endopeptidases. We further demonstrate that IQF substrates containing unnatural amino acids can be used to investigate protease activities/specificities for peptides containing post-translationally modified amino acids. Finally, we used IQF substrates to re-investigate the P1-Asp characteristic of caspases, thus demonstrating that some human caspases can also hydrolyze substrates after glutamic acid. PMID:28230157

Chemically Functionalized Carbon Nanotubes as Substrates for Neuronal Growth

PubMed Central

Hu, Hui; Ni, Yingchun; Montana, Vedrana; Haddon, Robert C.; Parpura, Vladimir

2009-01-01

We report the use of chemically modified carbon nanotubes as a substrate for cultured neurons. The morphological features of neurons that directly reflect their potential capability in synaptic transmission are characterized. The chemical properties of carbon nanotubes are systematically varied by attaching different functional groups that confer known characteristics to the substrate. By manipulating the charge carried by functionalized carbon nanotubes we are able to control the outgrowth and branching pattern of neuronal processes. PMID:21394241

Micro and nanocrystalline diamond formation on reticulated vitreous carbon substrate

NASA Astrophysics Data System (ADS)

Diniz, A. V.; Trava-Airoldi, V. J.; Corat, E. J.; Ferreira, N. G.

2005-10-01

High diamond nucleation and a three-dimensional growth on reticulated vitreous carbon substrate are obtained by chemical vapor deposition. Scanning electron microscopy images show continuous films covering the whole substrate including the center of 3.5 mm thick porous samples. It is evident the nanocrystalline diamond (NCD) film formation on deeper substrate regions. The grain size can vary from nano to micro scale for deposition time of 20 h. Raman spectra of sample regions closer to filaments exhibit well-defined diamond line. For central regions of sample (depth between 1.0 and 2.0 mm) Raman spectra also confirm NCD film.

Mueller matrix characterization of flexible plastic substrates

NASA Astrophysics Data System (ADS)

Hong, Nina; Synowicki, Ron A.; Hilfiker, James N.

2017-11-01

This work reports on Mueller matrix spectroscopic ellipsometry characterization of various flexible plastic substrates that are optically anisotropic with varying degrees of birefringence. The samples are divided into three groups according to the suggested characterization strategy: low birefringence, high birefringence, and twisted birefringence. The first group includes poly(methyl methacrylate) and cyclic olefin copolymer substrates. These are modeled with biaxial anisotropy for the real part of the refractive index while the imaginary part is approximated as isotropic due to small light absorption. The second group includes polyethylene terephthalate and polyethylene naphthalate substrates, which are modeled with biaxial anisotropy for both real and imaginary refractive indices. Lastly, a polyimide substrate is described as two birefringent layers with twisted in-plane orientation.

Tree species diversity affects decomposition through modified micro-environmental conditions across European forests.

PubMed

Joly, François-Xavier; Milcu, Alexandru; Scherer-Lorenzen, Michael; Jean, Loreline-Katia; Bussotti, Filippo; Dawud, Seid Muhie; Müller, Sandra; Pollastrini, Martina; Raulund-Rasmussen, Karsten; Vesterdal, Lars; Hättenschwiler, Stephan

2017-05-01

Different tree species influence litter decomposition directly through species-specific litter traits, and indirectly through distinct modifications of the local decomposition environment. Whether these indirect effects on decomposition are influenced by tree species diversity is presently not clear. We addressed this question by studying the decomposition of two common substrates, cellulose paper and wood sticks, in a total of 209 forest stands of varying tree species diversity across six major forest types at the scale of Europe. Tree species richness showed a weak but positive correlation with the decomposition of cellulose but not with that of wood. Surprisingly, macroclimate had only a minor effect on cellulose decomposition and no effect on wood decomposition despite the wide range in climatic conditions among sites from Mediterranean to boreal forests. Instead, forest canopy density and stand-specific litter traits affected the decomposition of both substrates, with a particularly clear negative effect of the proportion of evergreen tree litter. Our study suggests that species richness and composition of tree canopies modify decomposition indirectly through changes in microenvironmental conditions. These canopy-induced differences in the local decomposition environment control decomposition to a greater extent than continental-scale differences in macroclimatic conditions. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

A new buckwheat dihydroflavonol 4-reductase (DFR), with a unique substrate binding structure, has altered substrate specificity.

PubMed

Katsu, Kenjiro; Suzuki, Rintaro; Tsuchiya, Wataru; Inagaki, Noritoshi; Yamazaki, Toshimasa; Hisano, Tomomi; Yasui, Yasuo; Komori, Toshiyuki; Koshio, Motoyuki; Kubota, Seiji; Walker, Amanda R; Furukawa, Kiyoshi; Matsui, Katsuhiro

2017-12-11

Dihydroflavonol 4-reductase (DFR) is the key enzyme committed to anthocyanin and proanthocyanidin biosynthesis in the flavonoid biosynthetic pathway. DFR proteins can catalyse mainly the three substrates (dihydrokaempferol, dihydroquercetin, and dihydromyricetin), and show different substrate preferences. Although relationships between the substrate preference and amino acids in the region responsible for substrate specificity have been investigated in several plant species, the molecular basis of the substrate preference of DFR is not yet fully understood. By using degenerate primers in a PCR, we isolated two cDNA clones that encoded DFR in buckwheat (Fagopyrum esculentum). Based on sequence similarity, one cDNA clone (FeDFR1a) was identical to the FeDFR in DNA databases (DDBJ/Gen Bank/EMBL). The other cDNA clone, FeDFR2, had a similar sequence to FeDFR1a, but a different exon-intron structure. Linkage analysis in an F 2 segregating population showed that the two loci were linked. Unlike common DFR proteins in other plant species, FeDFR2 contained a valine instead of the typical asparagine at the third position and an extra glycine between sites 6 and 7 in the region that determines substrate specificity, and showed less activity against dihydrokaempferol than did FeDFR1a with an asparagine at the third position. Our 3D model suggested that the third residue and its neighbouring residues contribute to substrate specificity. FeDFR1a was expressed in all organs that we investigated, whereas FeDFR2 was preferentially expressed in roots and seeds. We isolated two buckwheat cDNA clones of DFR genes. FeDFR2 has unique structural and functional features that differ from those of previously reported DFRs in other plants. The 3D model suggested that not only the amino acid at the third position but also its neighbouring residues that are involved in the formation of the substrate-binding pocket play important roles in determining substrate preferences. The unique characteristics of FeDFR2 would provide a useful tool for future studies on the substrate specificity and organ-specific expression of DFRs.

Visualizing the complex functions and mechanisms of the anaphase promoting complex/cyclosome (APC/C)

PubMed Central

Alfieri, Claudio; Zhang, Suyang

2017-01-01

The anaphase promoting complex or cyclosome (APC/C) is a large multi-subunit E3 ubiquitin ligase that orchestrates cell cycle progression by mediating the degradation of important cell cycle regulators. During the two decades since its discovery, much has been learnt concerning its role in recognizing and ubiquitinating specific proteins in a cell-cycle-dependent manner, the mechanisms governing substrate specificity, the catalytic process of assembling polyubiquitin chains on its target proteins, and its regulation by phosphorylation and the spindle assembly checkpoint. The past few years have witnessed significant progress in understanding the quantitative mechanisms underlying these varied APC/C functions. This review integrates the overall functions and properties of the APC/C with mechanistic insights gained from recent cryo-electron microscopy (cryo-EM) studies of reconstituted human APC/C complexes. PMID:29167309

Reprogramming caspase-7 specificity by regio-specific mutations and selection provides alternate solutions for substrate recognition

DOE PAGES

Hill, Maureen E.; MacPherson, Derek J.; Wu, Peng; ...

2016-03-31

The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. In this paper, we report a new approach based on a caged green fluorescent protein (CA-GFP) reporter that allows for flow-cytometry-based selection in bacteria or other cell types enabling selection of intracellular protease specificity, regardless of the compositional complexity of the protease. Here, we apply this approach to introduce the specificity of caspase-6 into caspase-7, an intracellular cysteine protease important in cellular remodeling and cell death. We found that substitution of substrate-contacting residues from caspase-6 into caspase-7more » was ineffective, yielding an inactive enzyme, whereas saturation mutagenesis at these positions and selection by directed evolution produced active caspases. The process produced a number of nonobvious mutations that enabled conversion of the caspase-7 specificity to match caspase-6. The structures of the evolved-specificity caspase-7 (esCasp-7) revealed alternate binding modes for the substrate, including reorganization of an active site loop. Profiling the entire human proteome of esCasp-7 by N-terminomics demonstrated that the global specificity toward natural protein substrates is remarkably similar to that of caspase-6. Because the esCasp-7 maintained the core of caspase-7, we were able to identify a caspase-6 substrate, lamin C, that we predict relies on an exosite for substrate recognition. These reprogrammed proteases may be the first tool built with the express intent of distinguishing exosite dependent or independent substrates. Finally, this approach to specificity reprogramming should also be generalizable across a wide range of proteases.« less

Reprogramming caspase-7 specificity by regio-specific mutations and selection provides alternate solutions for substrate recognition

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

Hill, Maureen E.; MacPherson, Derek J.; Wu, Peng

The ability to routinely engineer protease specificity can allow us to better understand and modulate their biology for expanded therapeutic and industrial applications. In this paper, we report a new approach based on a caged green fluorescent protein (CA-GFP) reporter that allows for flow-cytometry-based selection in bacteria or other cell types enabling selection of intracellular protease specificity, regardless of the compositional complexity of the protease. Here, we apply this approach to introduce the specificity of caspase-6 into caspase-7, an intracellular cysteine protease important in cellular remodeling and cell death. We found that substitution of substrate-contacting residues from caspase-6 into caspase-7more » was ineffective, yielding an inactive enzyme, whereas saturation mutagenesis at these positions and selection by directed evolution produced active caspases. The process produced a number of nonobvious mutations that enabled conversion of the caspase-7 specificity to match caspase-6. The structures of the evolved-specificity caspase-7 (esCasp-7) revealed alternate binding modes for the substrate, including reorganization of an active site loop. Profiling the entire human proteome of esCasp-7 by N-terminomics demonstrated that the global specificity toward natural protein substrates is remarkably similar to that of caspase-6. Because the esCasp-7 maintained the core of caspase-7, we were able to identify a caspase-6 substrate, lamin C, that we predict relies on an exosite for substrate recognition. These reprogrammed proteases may be the first tool built with the express intent of distinguishing exosite dependent or independent substrates. Finally, this approach to specificity reprogramming should also be generalizable across a wide range of proteases.« less

Substrate- and isoform-specific proteome stability in normal and stressed cardiac mitochondria.

PubMed

Lau, Edward; Wang, Ding; Zhang, Jun; Yu, Hongxiu; Lam, Maggie P Y; Liang, Xiangbo; Zong, Nobel; Kim, Tae-Young; Ping, Peipei

2012-04-27

Mitochondrial protein homeostasis is an essential component of the functions and oxidative stress responses of the heart. To determine the specificity and efficiency of proteome turnover of the cardiac mitochondria by endogenous and exogenous proteolytic mechanisms. Proteolytic degradation of the murine cardiac mitochondria was assessed by 2-dimensional differential gel electrophoresis and liquid chromatography-tandem mass spectrometry. Mitochondrial proteases demonstrated a substrate preference for basic protein variants, which indicates a possible recognition mechanism based on protein modifications. Endogenous mitochondrial proteases and the cytosolic 20S proteasome exhibited different substrate specificities. The cardiac mitochondrial proteome contains low amounts of proteases and is remarkably stable in isolation. Oxidative damage lowers the proteolytic capacity of cardiac mitochondria and reduces substrate availability for mitochondrial proteases. The 20S proteasome preferentially degrades specific substrates in the mitochondria and may contribute to cardiac mitochondrial proteostasis.

Production of ochratoxin A by Aspergillus ochraceus NRRL-3174 before and after exposures to 60Co irradiation.

PubMed Central

Applegate, K L; Chipley, J R

1976-01-01

Spores from the toxigenic organism Aspergillus ochraceus NRRL-3174 were exposed to specific levels of gamma irradiation and then allowed to germinate on selected media. Increases in ochratoxin A production by irradiated, compared to non-irradiated, spores were observed after inoculation of spores onto a cracked red wheat or into a synthetic liquid medium. Variations in daily ochratoxin production were also observed for control and irradiated spore-derived cultures developing on both media, with maximum toxin production varying from 7 to 11 days of incubation. The most notable increases in ochratoxin A production occurred from cultures developing from spores having been irradiated with 10, 25, or 50 krad. Exposures to 400 or 600 krad resulted in complete inhibition of spore germination and, consequently, no ochratoxin production. Of the two substrates used, wheat and synthetic, the quantities of ochratoxin A produced were significantly lower in the synthetic media than on the natural substrate. Higher and more rapid toxin production occurred from spores having been irradiated with 10, 25, 50, and 100 krad than occurred from the non-irradiated control spores when grown on synthetic media. Cultures derived from spores having been exposed to 10, 25, 50, and 100 krad produced significantly higher levels of ochratoxin A after 8 days of incubation on natural substrate than did the controls. Analysis of variance revealed that substrate, length of incubation, as well as irradiation levels all affected the time required to produce maximum levels of ochratoxin A. PMID:938031

Vertical visual features have a strong influence on cuttlefish camouflage.

PubMed

Ulmer, K M; Buresch, K C; Kossodo, M M; Mäthger, L M; Siemann, L A; Hanlon, R T

2013-04-01

Cuttlefish and other cephalopods use visual cues from their surroundings to adaptively change their body pattern for camouflage. Numerous previous experiments have demonstrated the influence of two-dimensional (2D) substrates (e.g., sand and gravel habitats) on camouflage, yet many marine habitats have varied three-dimensional (3D) structures among which cuttlefish camouflage from predators, including benthic predators that view cuttlefish horizontally against such 3D backgrounds. We conducted laboratory experiments, using Sepia officinalis, to test the relative influence of horizontal versus vertical visual cues on cuttlefish camouflage: 2D patterns on benthic substrates were tested versus 2D wall patterns and 3D objects with patterns. Specifically, we investigated the influence of (i) quantity and (ii) placement of high-contrast elements on a 3D object or a 2D wall, as well as (iii) the diameter and (iv) number of 3D objects with high-contrast elements on cuttlefish body pattern expression. Additionally, we tested the influence of high-contrast visual stimuli covering the entire 2D benthic substrate versus the entire 2D wall. In all experiments, visual cues presented in the vertical plane evoked the strongest body pattern response in cuttlefish. These experiments support field observations that, in some marine habitats, cuttlefish will respond to vertically oriented background features even when the preponderance of visual information in their field of view seems to be from the 2D surrounding substrate. Such choices highlight the selective decision-making that occurs in cephalopods with their adaptive camouflage capability.

Quantification of microscopic surface features of single point diamond turned optics with subsequent chemical polishing

NASA Astrophysics Data System (ADS)

Cardenas, Nelson; Kyrish, Matthew; Taylor, Daniel; Fraelich, Margaret; Lechuga, Oscar; Claytor, Richard; Claytor, Nelson

2015-03-01

Electro-Chemical Polishing is routinely used in the anodizing industry to achieve specular surface finishes of various metals products prior to anodizing. Electro-Chemical polishing functions by leveling the microscopic peaks and valleys of the substrate, thereby increasing specularity and reducing light scattering. The rate of attack is dependent of the physical characteristics (height, depth, and width) of the microscopic structures that constitute the surface finish. To prepare the sample, mechanical polishing such as buffing or grinding is typically required before etching. This type of mechanical polishing produces random microscopic structures at varying depths and widths, thus the electropolishing parameters are determined in an ad hoc basis. Alternatively, single point diamond turning offers excellent repeatability and highly specific control of substrate polishing parameters. While polishing, the diamond tool leaves behind an associated tool mark, which is related to the diamond tool geometry and machining parameters. Machine parameters such as tool cutting depth, speed and step over can be changed in situ, thus providing control of the spatial frequency of the microscopic structures characteristic of the surface topography of the substrate. By combining single point diamond turning with subsequent electro-chemical etching, ultra smooth polishing of both rotationally symmetric and free form mirrors and molds is possible. Additionally, machining parameters can be set to optimize post polishing for increased surface quality and reduced processing times. In this work, we present a study of substrate surface finish based on diamond turning tool mark spatial frequency with subsequent electro-chemical polishing.

Extending pine bark supplies with wholetree and clean chip residual substrates

USDA-ARS?s Scientific Manuscript database

Limited supplies of pine bark (PB) due to a number of reasons over the past several years has caused concern among many nursery producers. This study was developed to evaluate varying ratios of pine bark (PB) with clean chip residual (CCR) or WholeTree substrate (WT), in order to assist growers with...

Rate-determining Step of Flap Endonuclease 1 (FEN1) Reflects a Kinetic Bias against Long Flaps and Trinucleotide Repeat Sequences.

PubMed

Tarantino, Mary E; Bilotti, Katharina; Huang, Ji; Delaney, Sarah

2015-08-21

Flap endonuclease 1 (FEN1) is a structure-specific nuclease responsible for removing 5'-flaps formed during Okazaki fragment maturation and long patch base excision repair. In this work, we use rapid quench flow techniques to examine the rates of 5'-flap removal on DNA substrates of varying length and sequence. Of particular interest are flaps containing trinucleotide repeats (TNR), which have been proposed to affect FEN1 activity and cause genetic instability. We report that FEN1 processes substrates containing flaps of 30 nucleotides or fewer at comparable single-turnover rates. However, for flaps longer than 30 nucleotides, FEN1 kinetically discriminates substrates based on flap length and flap sequence. In particular, FEN1 removes flaps containing TNR sequences at a rate slower than mixed sequence flaps of the same length. Furthermore, multiple-turnover kinetic analysis reveals that the rate-determining step of FEN1 switches as a function of flap length from product release to chemistry (or a step prior to chemistry). These results provide a kinetic perspective on the role of FEN1 in DNA replication and repair and contribute to our understanding of FEN1 in mediating genetic instability of TNR sequences. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Thermally dried ink-jet process for 6,13-bis(triisopropylsilylethynyl)-pentacene for high mobility and high uniformity on a large area substrate

USGS Publications Warehouse

Ryu, Gi Seong; Lee, Myung Won; Jeong, Seung Hyeon; Song, Chung Kun

2012-01-01

In this study we developed a simple ink-jet process for 6,13-bis(triisopropylsilylethynyl)-pentacene (TIPS-pentacene), which is known as a high-mobility soluble organic semiconductor, to achieve relatively high-mobility and high-uniformity performance for large-area applications. We analyzed the behavior of fluorescent particles in droplets and applied the results to determining a method of controlling the behavior of TIPS-pentacene molecules. The grain morphology of TIPS-pentacene varied depending on the temperature applied to the droplets during drying. We were able to obtain large and uniform grains at 46 degrees C without any "coffee stain". The process was applied to a large-size organic thin-film transistor (OTFT) backplane for an electrophoretic display panel containing 192 x 150 pixels on a 6-in.-sized substrate. The average of mobilities of 36 OTFTs, which were taken from different locations of the backplane, was 0.44 +/- 0.08 cm2.V-1.s-1, with a small deviation of 20%, over a 6-in.-size area comprising 28,800 OTFTs. This process providing high mobility and high uniformity can be achieved by simply maintaining the whole area of the substrate at a specific temperature (46 degrees C in this case) during drying of the droplets.

Relative Expression Levels Rather Than Specific Activity Plays the Major Role in Determining In Vivo AKT Isoform Substrate Specificity

PubMed Central

Lee, Rachel S.; House, Colin M.; Cristiano, Briony E.; Hannan, Ross D.; Pearson, Richard B.; Hannan, Katherine M.

2011-01-01

The AKT protooncogene mediates many cellular processes involved in normal development and disease states such as cancer. The three structurally similar isoforms: AKT1, AKT2, and AKT3 exhibit both functional redundancy and isoform-specific functions; however the basis for their differential signalling remains unclear. Here we show that in vitro, purified AKT3 is ∼47-fold more active than AKT1 at phosphorylating peptide and protein substrates. Despite these marked variations in specific activity between the individual isoforms, a comprehensive analysis of phosphorylation of validated AKT substrates indicated only subtle differences in signalling via individual isoforms in vivo. Therefore, we hypothesise, at least in this model system, that relative tissue/cellular abundance, rather than specific activity, plays the dominant role in determining AKT substrate specificity in situ. PMID:21869924

Involvement of arginine 878 together with Ca2+ in mouse aminopeptidase A substrate specificity for N-terminal acidic amino-acid residues

PubMed Central

Couvineau, Pierre; de Almeida, Hugo; Maigret, Bernard; Llorens-Cortes, Catherine

2017-01-01

Aminopeptidase A (APA) is a membrane-bound zinc metalloprotease cleaving, in the brain, the N-terminal aspartyl residue of angiotensin II to generate angiotensin III, which exerts a tonic stimulatory effect on the control of blood pressure in hypertensive animals. Using a refined APA structure derived from the human APA crystal structure, we docked the specific and selective APA inhibitor, EC33 in the presence of Ca2+. We report the presence in the S1 subsite of Arg-887 (Arg-878 in mouse APA), the guanidinium moiety of which established an interaction with the electronegative sulfonate group of EC33. Mutagenic replacement of Arg-878 with an alanine or a lysine residue decreased the affinity of the recombinant enzymes for the acidic substrate, α-L-glutamyl-β-naphthylamide, with a slight decrease in substrate hydrolysis velocity either with or without Ca2+. In the absence of Ca2+, the mutations modified the substrate specificity of APA for the acidic substrate, the mutated enzymes hydrolyzing more efficiently basic and neutral substrates, although the addition of Ca2+ partially restored the acidic substrate specificity. The analysis of the 3D models of the Arg-878 mutated APAs revealed a change in the volume of the S1 subsite, which may impair the binding and/or the optimal positioning of the substrate in the active site as well as its hydrolysis. These findings demonstrate the key role of Arg-878 together with Ca2 + in APA substrate specificity for N-terminal acidic amino acid residues by ensuring the optimal positioning of acidic substrates during catalysis. PMID:28877217

Vertical-Substrate MPCVD Epitaxial Nanodiamond Growth

DOE PAGES

Tzeng, Yan-Kai; Zhang, Jingyuan Linda; Lu, Haiyu; ...

2017-02-09

Color center-containing nanodiamonds have many applications in quantum technologies and biology. Diamondoids, molecular-sized diamonds have been used as seeds in chemical vapor deposition (CVD) growth. However, optimizing growth conditions to produce high crystal quality nanodiamonds with color centers requires varying growth conditions that often leads to ad-hoc and time-consuming, one-at-a-time testing of reaction conditions. In order to rapidly explore parameter space, we developed a microwave plasma CVD technique using a vertical, rather than horizontally oriented stage-substrate geometry. With this configuration, temperature, plasma density, and atomic hydrogen density vary continuously along the vertical axis of the substrate. Finally, this variation allowedmore » rapid identification of growth parameters that yield single crystal diamonds down to 10 nm in size and 75 nm diameter optically active center silicon-vacancy (Si-V) nanoparticles. Furthermore, this method may provide a means of incorporating a wide variety of dopants in nanodiamonds without ion irradiation damage.« less

YAP-dependent mechanotransduction is required for proliferation and migration on native-like substrate topography.

PubMed

Mascharak, Shamik; Benitez, Patrick L; Proctor, Amy C; Madl, Christopher M; Hu, Kenneth H; Dewi, Ruby E; Butte, Manish J; Heilshorn, Sarah C

2017-01-01

Native vascular extracellular matrices (vECM) consist of elastic fibers that impart varied topographical properties, yet most in vitro models designed to study the effects of topography on cell behavior are not representative of native architecture. Here, we engineer an electrospun elastin-like protein (ELP) system with independently tunable, vECM-mimetic topography and demonstrate that increasing topographical variation causes loss of endothelial cell-cell junction organization. This loss of VE-cadherin signaling and increased cytoskeletal contractility on more topographically varied ELP substrates in turn promote YAP activation and nuclear translocation, resulting in significantly increased endothelial cell migration and proliferation. Our findings identify YAP as a required signaling factor through which fibrous substrate topography influences cell behavior and highlights topography as a key design parameter for engineered biomaterials. Copyright © 2016 Elsevier Ltd. All rights reserved.

Conferring specificity in redox pathways by enzymatic thiol/disulfide exchange reactions.

PubMed

Netto, Luis Eduardo S; de Oliveira, Marcos Antonio; Tairum, Carlos A; da Silva Neto, José Freire

2016-01-01

Thiol-disulfide exchange reactions are highly reversible, displaying nucleophilic substitutions mechanism (S(N)2 type). For aliphatic, low molecular thiols, these reactions are slow, but can attain million times faster rates in enzymatic processes. Thioredoxin (Trx) proteins were the first enzymes described to accelerate thiol-disulfide exchange reactions and their high reactivity is related to the high nucleophilicity of the attacking thiol. Substrate specificity in Trx is achieved by several factors, including polar, hydrophobic, and topological interactions through a groove in the active site. Glutaredoxin (Grx) enzymes also contain the Trx fold, but they do not share amino acid sequence similarity with Trx. A conserved glutathione binding site is a typical feature of Grx that can reduce substrates by two mechanisms (mono and dithiol). The high reactivity of Grx enzymes is related to the very acid pK(a) values of reactive Cys that plays roles as good leaving groups. Therefore, although distinct oxidoreductases catalyze similar thiol–disulfide exchange reactions, their enzymatic mechanisms vary. PDI and DsbA are two other oxidoreductases, but they are involved in disulfide bond formation, instead of disulfide reduction, which is related to the oxidative environment where they are found. PDI enzymes and DsbC are endowed with disulfide isomerase activity, which is related with their tetra-domain architecture. As illustrative description of specificity in thiol-disulfide exchange, redox aspects of transcription activation in bacteria, yeast, and mammals are presented in an evolutionary perspective. Therefore, thiol-disulfide exchange reactions play important roles in conferring specificity to pathways, a required feature for signaling.

Process Of Bonding Copper And Tungsten

DOEpatents

Slattery, Kevin T.; Driemeyer, Daniel E.

1999-11-23

Process for bonding a copper substrate to a tungsten substrate by providing a thin metallic adhesion promoting film bonded to a tungsten substrate and a functionally graded material (FGM) interlayer bonding the thin metallic adhesion promoting film to the copper substrate. The FGM interlayer is formed by thermal plasma spraying mixtures of copper powder and tungsten powder in a varied blending ratio such that the blending ratio of the copper powder and the tungsten powder that is fed to a plasma torch is intermittently adjusted to provide progressively higher copper content/tungsten content, by volume, ratio values in the interlayer in a lineal direction extending from the tungsten substrate towards the copper substrate. The resulting copper to tungsten joint well accommodates the difference in the coefficient of thermal expansion of the materials.

Evaluating Factor XIII Specificity for Glutamine-Containing Substrates Using a MALDI-TOF Mass Spectrometry Assay

PubMed Central

Doiphode, Prakash G.; Malovichko, Marina V.; Mouapi, Kelly Njine; Maurer, Muriel C.

2014-01-01

Activated Factor XIII (FXIIIa) catalyzes the formation of γ-glutamyl-ε-lysyl cross-links within the fibrin blood clot network. Although several cross-linking targets have been identified, the characteristic features that define FXIIIa substrate specificity are not well understood. To learn more about how FXIIIa selects its targets, a matrix-assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF MS) based assay was developed that could directly follow the consumption of a glutamine-containing substrate and the formation of a cross-linked product with glycine ethylester. This FXIIIa kinetics assay is no longer reliant on a secondary coupled reaction, on substrate labeling, or on detecting the final deacylation portion of the transglutaminase reaction. With the MALDI-TOF MS assay, glutamine-containing peptides derived from α2-antiplasmin, S. Aureus fibronectin binding protein A, and thrombin activatable fibrinolysis inhibitor were examined directly. Results suggest that the FXIIIa active site surface responds to changes in substrate residues following the reactive glutamine. The P-1 substrate position is sensitive to charge character and the P-2 and P-3 to the broad FXIIIa substrate specificity pockets. The more distant P-8 to P-11 region serves as a secondary substrate anchoring point. New knowledge on FXIIIa specificity may be used to design better substrates or inhibitors of this transglutaminase. PMID:24751466

Structural Basis of Substrate Specificity and Regiochemistry in the MycF/TylF Family of Sugar O -Methyltransferases.

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

Bernard, Steffen M.; Akey, David L.; Tripathi, Ashootosh

Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6'-deoxyallose substituent occurs in a stepwise manner first at the 2'- and then the 3'-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C-H functionalization reactions mediated by the P450 monooxygenase MycG. The structural basis of pathway ordering and substrate specificity is unknown. A series of crystal structures of MycF, the 3'-O-methyltransferase, including the free enzyme and complexes with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates,more » show that SAM binding induces substantial ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single amino acid substitution relaxed the 2'-methoxy specificity but retained regiospecificity. The engineered variant produced a new mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the modeled substrate complex of a 4'-specific homolog, active site residues were identified that correlate with the 3'- or 4'- specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer. Lastly, this classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.« less

Structural Basis of Substrate Specificity and Regiochemistry in the MycF/TylF Family of Sugar O -Methyltransferases.

DOE PAGES

Bernard, Steffen M.; Akey, David L.; Tripathi, Ashootosh; ...

2015-02-18

Sugar moieties in natural products are frequently modified by O-methylation. In the biosynthesis of the macrolide antibiotic mycinamicin, methylation of a 6'-deoxyallose substituent occurs in a stepwise manner first at the 2'- and then the 3'-hydroxyl groups to produce the mycinose moiety in the final product. The timing and placement of the O-methylations impact final stage C-H functionalization reactions mediated by the P450 monooxygenase MycG. The structural basis of pathway ordering and substrate specificity is unknown. A series of crystal structures of MycF, the 3'-O-methyltransferase, including the free enzyme and complexes with S-adenosyl homocysteine (SAH), substrate, product, and unnatural substrates,more » show that SAM binding induces substantial ordering that creates the binding site for the natural substrate, and a bound metal ion positions the substrate for catalysis. A single amino acid substitution relaxed the 2'-methoxy specificity but retained regiospecificity. The engineered variant produced a new mycinamicin analog, demonstrating the utility of structural information to facilitate bioengineering approaches for the chemoenzymatic synthesis of complex small molecules containing modified sugars. Using the MycF substrate complex and the modeled substrate complex of a 4'-specific homolog, active site residues were identified that correlate with the 3'- or 4'- specificity of MycF family members and define the protein and substrate features that direct the regiochemistry of methyltransfer. Lastly, this classification scheme will be useful in the annotation of new secondary metabolite pathways that utilize this family of enzymes.« less

Mannanase hydrolysis of spruce galactoglucomannan focusing on the influence of acetylation on enzymatic mannan degradation.

PubMed

Arnling Bååth, Jenny; Martínez-Abad, Antonio; Berglund, Jennie; Larsbrink, Johan; Vilaplana, Francisco; Olsson, Lisbeth

2018-01-01

Galactoglucomannan (GGM) is the most abundant hemicellulose in softwood, and consists of a backbone of mannose and glucose units, decorated with galactose and acetyl moieties. GGM can be hydrolyzed into fermentable sugars, or used as a polymer in films, gels, and food additives. Endo -β-mannanases, which can be found in the glycoside hydrolase families 5 and 26, specifically cleave the mannan backbone of GGM into shorter oligosaccharides. Information on the activity and specificity of different mannanases on complex and acetylated substrates is still lacking. The aim of this work was to evaluate and compare the modes of action of two mannanases from Cellvibrio japonicus ( Cj Man5A and Cj Man26A) on a variety of mannan substrates, naturally and chemically acetylated to varying degrees, including naturally acetylated spruce GGM. Both enzymes were evaluated in terms of cleavage patterns and their ability to accommodate acetyl substitutions. Cj Man5A and Cj Man26A demonstrated different substrate preferences on mannan substrates with distinct backbone and decoration structures. Cj Man5A action resulted in higher amounts of mannotriose and mannotetraose than that of Cj Man26A, which mainly generated mannose and mannobiose as end products. Mass spectrometric analysis of products from the enzymatic hydrolysis of spruce GGM revealed that an acetylated hexotriose was the shortest acetylated oligosaccharide produced by Cj Man5A, whereas Cj Man26A generated acetylated hexobiose as well as diacetylated oligosaccharides. A low degree of native acetylation did not significantly inhibit the enzymatic action. However, a high degree of chemical acetylation resulted in decreased hydrolyzability of mannan substrates, where reduced substrate solubility seemed to reduce enzyme activity. Our findings demonstrate that the two mannanases from C. japonicus have different cleavage patterns on linear and decorated mannan polysaccharides, including the abundant and industrially important resource spruce GGM. Cj Man26A released higher amounts of fermentable sugars suitable for biofuel production, while Cj Man5A, producing higher amounts of oligosaccharides, could be a good candidate for the production of oligomeric platform chemicals and food additives. Furthermore, chemical acetylation of mannan polymers was found to be a potential strategy for limiting the biodegradation of mannan-containing materials.

Is the molecular diversity of marine dissolved organic matter already imprinted in the exometabolome of single strains?

NASA Astrophysics Data System (ADS)

Noriega-Ortega, B. E.; Wienhausen, G.; Dittmar, T.; Simon, M.; Niggemann, J.

2016-02-01

Dissolved organic matter (DOM) in the ocean, the marine geometabolome, is an extremely complex mixture composed of a wide variety of compounds. The molecular chemodiversity affects the function and turnover rate of DOM in the ocean. We hypothesize that the active microbial community essentially contributes to the complexity of the DOM pool through uptake and excretion of compounds. We tested this hypothesis in culture experiments with fully-sequenced strains of the Roseobacter clade. Bacteria of the Roseobacter clade are among the most abundant microbial players in the ocean. We studied the exometabolome of two representatives of the Roseobacter clade, Phaeobacter inhibens DSM 17395 and Dinoroseobacter shibae. The organisms were grown separately in cultures on defined single model substrates (acetate, succinate, glutamate, glucose). We used a non-targeted analytical approach via Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) to characterize the exometabolome at the molecular level, complemented by compound-specific analyses of free and combined amino acids and carbohydrates. The exometabolome composition varied between the tested strains, which released a different suite of compounds depending on the growth phase as well as on growth conditions (substrate). Both organisms exhibited a core exometabolome with compounds released when growing on either substrate and at all growth phases, and a variable exometabolome specific for different substrates and growth phases. However, only a small fraction of the exometabolites detected by FT-ICR-MS could be directly linked to the genome or transcriptome. We interpret these findings as evidence for the excretion of molecularly highly-diverse metabolic waste, whose composition is dependent on the metabolic state and genetic repertoire of the organisms. The molecular diversity of compounds excreted by a single strain is extraordinary and is likely the reason for the molecular diversity of natural DOM in the ocean.

Fabrication of wedged multilayer Laue lenses

DOE PAGES

Prasciolu, M.; Leontowich, A. F. G.; Krzywinski, J.; ...

2015-01-01

We present a new method to fabricate wedged multilayer Laue lenses, in which the angle of diffracting layers smoothly varies in the lens to achieve optimum diffracting efficiency across the entire pupil of the lens. This was achieved by depositing a multilayer onto a flat substrate placed in the penumbra of a straight-edge mask. The distance between the mask and the substrate was calibrated and the multilayer Laue lens was cut in a position where the varying layer thickness and the varying layer tilt simultaneously satisfy the Fresnel zone plate condition and Bragg’s law for all layers in the stack.more » This method can be used to extend the achievable numerical aperture of multilayer Laue lenses to reach considerably smaller focal spot sizes than achievable with lenses composed of parallel layers.« less

Copper-phthalocyanine based metal-organic interfaces: the effect of fluorination, the substrate, and its symmetry.

PubMed

de Oteyza, D G; El-Sayed, A; Garcia-Lastra, J M; Goiri, E; Krauss, T N; Turak, A; Barrena, E; Dosch, H; Zegenhagen, J; Rubio, A; Wakayama, Y; Ortega, J E

2010-12-07

Metal-organic interfaces based on copper-phthalocyanine monolayers are studied in dependence of the metal substrate (Au versus Cu), of its symmetry [hexagonal (111) surfaces versus fourfold (100) surfaces], as well as of the donor or acceptor semiconducting character associated with the nonfluorinated or perfluorinated molecules, respectively. Comparison of the properties of these systematically varied metal-organic interfaces provides new insight into the effect of each of the previously mentioned parameters on the molecule-substrate interactions.

Low-Temperature Growth of Amorphous Silicon Films and Direct Fabrication of Solar Cells on Flexible Polyimide and Photo-Paper Substrates

NASA Astrophysics Data System (ADS)

Madaka, Ramakrishna; Kanneboina, Venkanna; Agarwal, Pratima

2018-05-01

Direct deposition of hydrogenated amorphous silicon (a-Si:H) thin films and fabrication of solar cells on polyimide (PI) and photo-paper (PP) substrates using a rf-plasma-enhanced chemical vapor deposition technique is reported. Intrinsic amorphous silicon films were deposited on PI and PP substrates by varying the substrate temperature (T s) over 70-150°C to optimize the deposition parameters for best quality films. The films deposited on both PI and PP substrates at a temperature as low as 70°C showed a photosensitivity (σ ph/σ d) of nearly 4 orders of magnitude which increased to 5-6 orders of magnitude when the substrate temperature was increased to 130-150°C. The increase in σ ph/σ d is due to the presence of a few nanometer-sized crystallites embedded in the film. Solar cells (n-i-p) were fabricated directly on PI, PP and Corning 1737 glass (Corning) at 150°C for different thicknesses of an intrinsic amorphous silicon layer (i-layer). With the increase in i-layer thickness from 330 nm to 700 nm, the solar cell efficiency was found to increase from 3.81% to 5.02% on the Corning substrate whereas on the flexible PI substrate an increase from 3.38% to 4.38% was observed. On the other hand, in the case of cells on PP, the i-layer thickness was varied from 200 nm to 700 nm and the best cell efficiency 1.54% was obtained for the 200-nm-thick i-layer. The fabrication of a-Si (n-i-p) solar cells on photo-paper is presented for the first time.

Light-induced V{sub oc} increase and decrease in high-efficiency amorphous silicon solar cells

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

Stuckelberger, M., E-mail: michael.stuckelberger@epfl.ch; Riesen, Y.; Despeisse, M.

High-efficiency amorphous silicon (a-Si:H) solar cells were deposited with different thicknesses of the p-type amorphous silicon carbide layer on substrates of varying roughness. We observed a light-induced open-circuit voltage (V{sub oc}) increase upon light soaking for thin p-layers, but a decrease for thick p-layers. Further, the V{sub oc} increase is enhanced with increasing substrate roughness. After correction of the p-layer thickness for the increased surface area of rough substrates, we can exclude varying the effective p-layer thickness as the cause of the substrate roughness dependence. Instead, we explain the observations by an increase of the dangling-bond density in both themore » p-layer—causing a V{sub oc} increase—and in the intrinsic absorber layer, causing a V{sub oc} decrease. We present a mechanism for the light-induced increase and decrease, justified by the investigation of light-induced changes of the p-layer and supported by Advanced Semiconductor Analysis simulation. We conclude that a shift of the electron quasi-Fermi level towards the conduction band is the reason for the observed V{sub oc} enhancements, and poor amorphous silicon quality on rough substrates enhances this effect.« less

Projection parameters for zirconia-alumina-ceria coatings made by flame spraying from results of numerical simulation

NASA Astrophysics Data System (ADS)

Rodríguez, L.; Ferrer, M.; Vargas, F.; Peña, G.

2017-12-01

A numerical simulation was performed with the software Jets et Poudres, the results let choose the parameters to deposit zirconia-alumina-ceria coatings of different composition on substrates of red clay, by thermal spraying with the oxyacetylene flame to obtain homogeneous coatings with good adhesion to the substrate. The effect of the projection distance (7, 10 and 12cm) between the substrate and the torch, the fusion percentage of particles and the K-Sommerfeld number was determined. This number is dimensionless and is affected by the projection distance and by the chemical composition of the particles. For a projection distance of 9cm, the fusion percentage of the particles varies between 83.8% and 100%, and the K-Sommerfeld number between 47.3 and 50 for the different compounds. This makes possible to obtain uniform coatings with good wettability, therefore, good adhesion to the substrate, while for the distance of 7cm the fusion percentage varies between 22% and 38%, due to the short time of the particles in the flame which causes low adhesion, when the projection distance is 12cm the particles do not have sufficient kinetic energy to reach the substrate and therefore the coating is not deposited.

Density controlled carbon nanotube array electrodes

DOEpatents

Ren, Zhifeng F [Newton, MA; Tu, Yi [Belmont, MA

2008-12-16

CNT materials comprising aligned carbon nanotubes (CNTs) with pre-determined site densities, catalyst substrate materials for obtaining them and methods for forming aligned CNTs with controllable densities on such catalyst substrate materials are described. The fabrication of films comprising site-density controlled vertically aligned CNT arrays of the invention with variable field emission characteristics, whereby the field emission properties of the films are controlled by independently varying the length of CNTs in the aligned array within the film or by independently varying inter-tubule spacing of the CNTs within the array (site density) are disclosed. The fabrication of microelectrode arrays (MEAs) formed utilizing the carbon nanotube material of the invention is also described.

Self-assembly of silica nanoparticles by tuning substrate-adsorbate interaction

NASA Astrophysics Data System (ADS)

Utsav, Khanna, Sakshum; Mukhopadhayay, Indrajit; Banerjee, Rupak

2018-05-01

We report on self-assembled nanodisc formations of silica nanoparticles on a surface modified silicon substrate using modified Langmuir-Schafer deposition technique (stamping). The size, inter-particle separation as well as the packing of the silica nanoparticles within the nanodiscs formed spontaneously can be tuned by the surface pressure applied on the water surface. We obtain self-assembled nanodiscs of silica nanoparticle arranged in a hexagonal symmetry. We also observe that by varying the surface pressure of deposition at the water-molecule-air interface we obtain such 2D disc-shaped structure with varying sizes and a packing ratio of the silica nanoparticle.

Comparison of inkjet-printed silver conductors on different microsystem substrates

NASA Astrophysics Data System (ADS)

Kruger, Jené; Bezuidenhout, Petroné H.; Joubert, Trudi-Heleen

2016-02-01

Applications for diagnostic and environmental point-of-need require processes and building blocks to add smart features to disposable biosensors on low-cost substrates. A novel method for producing such biosensors is printing electronics using additive technologies. This work contributes to the toolbox of processes, materials and components for printed electronics manufacturing - as well as rapid prototyping - of circuits. Printing protocols were developed to facilitate successful inkjet printing of nanosilver ink (Harima NPS-JL) onto different microsystem substrates using a functional printer (Dimatix DMP-3281). Photo paper is a standard inkjet substrate, which were compared with glass, polycarbonate (PC), plastic projector transparency foil, and polydimethylsiloxane (PDMS). Comparison attributes include physical and electrical properties. The layout design comprised dogbone elements of 8 mm length, and widths varying between 100 μm and 2 mm. All printed features were thermally cured for 1 hour at 120 °C. The physical characteristics were measured with a laser scanning microscope (Zeiss LSM-5) to determine the width, thickness and surface roughness of the printed features. An LCR meter (GW-Instek 8110) was used to measure the printed structures' electrical characteristics (resistance, capacitance and inductance). A lumped element model and layout design rules were extracted to assist in standardized design procedures. The model incorporates prediction of the bandwidth attainable with these structures. The layer thickness on all substrates is larger than the 1 μm on photo paper, and varies between 1.6 μm (PC) and 7 μm (PDMS). The spreading for PDMS is similar to photo paper, but since for the other substrates it is between 5 (glass) and 10 (PC) times larger than for photo paper, the layout design rules require large spacing, leading to larger area networks. Electrical probing on the PDMS is not consistent and results are inconclusive. For the other substrates, the comparative dogbone resistance (100 μm width) is significantly larger than the 2 Ω standard, varying from 12.6 Ω (PC) to 19.3 Ω (glass). The bandwidth relative to photo paper is smaller by a factor of between 6 (PC) and 9.5 (glass).

Substrate-specific regulation of ubiquitination by the anaphase-promoting complex

PubMed Central

Song, Ling

2011-01-01

By orchestrating the sequential degradation of a large number of cell cycle regulators, the ubiquitin ligase anaphase-promoting complex (APC/C) is essential for proliferation in all eukaryotes. The correct timing of APC/C-dependent substrate degradation, a critical feature of progression through mitosis, was long known to be controlled by mechanisms targeting the core APC/C-machinery. Recent experiments, however have revealed an important contribution of substrate-specific regulation of the APC/C to achieve accurate cell division. In this perspective, we describe different mechanisms of substrate-specific APC/C-regulation and discuss their importance for cell division. PMID:21191176

Exploring the specific features of interfacial enzymology based on lipase studies.

PubMed

Aloulou, Ahmed; Rodriguez, Jorge A; Fernandez, Sylvie; van Oosterhout, Dirk; Puccinelli, Delphine; Carrière, Frédéric

2006-09-01

Many enzymes are active at interfaces in the living world (such as in the signaling processes at the surface of cell membranes, digestion of dietary lipids, starch and cellulose degradation, etc.), but fundamental enzymology remains largely focused on the interactions between enzymes and soluble substrates. The biochemical and kinetic characterization of lipolytic enzymes has opened up new paths of research in the field of interfacial enzymology. Lipases are water-soluble enzymes hydrolyzing insoluble triglyceride substrates, and studies on these enzymes have led to the development of specific interfacial kinetic models. Structure-function studies on lipases have thrown light on the interfacial recognition sites present in the molecular structure of these enzymes, the conformational changes occurring in the presence of lipids and amphiphiles, and the stability of the enzymes present at interfaces. The pH-dependent activity, substrate specificity and inhibition of these enzymes can all result from both "classical" interactions between a substrate or inhibitor and the active site, as well as from the adsorption of the enzymes at the surface of aggregated substrate particles such as oil drops, lipid bilayers or monomolecular lipid films. The adsorption step can provide an alternative target for improving substrate specificity and developing specific enzyme inhibitors. Several data obtained with gastric lipase, classical pancreatic lipase, pancreatic lipase-related protein 2 and phosphatidylserine-specific phospholipase A1 were chosen here to illustrate these specific features of interfacial enzymology.

Activation of the proteasomes of sand dollar eggs at fertilization depends on the intracellular pH rise.

PubMed

Chiba, K; Alderton, J M; Hoshi, M; Steinhardt, R A

1999-05-01

The mechanism of the activation of intracellular proteasomes at fertilization was measured in living sand dollar eggs using the membrane-impermeant fluorogenic substrate, succinyl-Phe-Leu-Arg-coumarylamido-4-methanesulfonic acid. When the substrate was microinjected into unfertilized eggs, the initial velocity of hydrolysis of the substrate (V0) was low. V0 measured 5 to 10 min after fertilization was five to nine times the prefertilization level and remained high throughout the first cell cycle. Hydrolysis of the substrate was inhibited by clasto-lactacystin beta-lactone, a specific inhibitor of the proteasome. There has been in vitro evidence that calcium may be involved in regulation of proteasome activity to either inhibit the increase in peptidase activity associated with PA 28 binding to the 20S proteasome or stimulate activity of the PA 700-proteasome complex. Since both intracellular free Ca2+ concentration ([Ca2+]i) and intracellular pH (pHi) increase after fertilization, hydrolysis of the proteasome substrate was measured under conditions in which [Ca2+]i and pHi were varied independently during activation. When the pHi of unfertilized eggs was elevated by exposure to 15 mM ammonium chloride in pH 9 seawater, V0 increased to a level comparable to that measured after fertilization. In contrast, [Ca2+]i elevation without pHi change, induced by calcium ionophore in sodium-free seawater, had no effect on V0 in the unfertilized egg. Moreover, when unfertilized eggs were microinjected with buffers modulating pHi, V0 increased in a pH-dependent manner. These results indicate that the pHi rise at fertilization is the necessary prerequisite for activation of the proteasome, an essential component in the regulation of the cell cycle. Copyright 1999 Academic Press.

Bacterial Enzymes and Antibiotic Resistance- Oral Presentation

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

Maltz, Lauren

By using protein crystallography and X-ray diffraction, structures of bacterial enzymes were solved to gain a better understanding of how enzymatic modification acts as an antibacterial resistance mechanism. Aminoglycoside phosphotransferases (APHs) are one of three aminoglycoside modifying enzymes that confer resistance to the aminoglycoside antibiotics via enzymatic modification, rendering many drugs obsolete. Specifically, the APH(2”) family vary in their substrate specificities and also in their preference for the phosphate donor (ADP versus GDP). By solving the structures of members of the APH(2”) family of enzymes, we can see how domain movements are important to their substrate specificity. Our structure ofmore » the ternary complex of APH(2”)-IIIa with GDP and kanamycin, when compared to the known structures of APH(2”)-IVa, reveals that there are real physical differences between these two enzymes, a structural finding that explains why the two enzymes differ in their preferences for certain aminoglycosides. Another important group of bacterial resistance enzymes are the Class D β-lactamases. Oxacillinase carbapenemases (OXAs) are part of this enzyme class and have begun to confer resistance to ‘last resort’ drugs, most notably carbapenems. Our structure of OXA-143 shows that the conformational flexibility of a conserved hydrophobic residue in the active site (Val130) serves to control the entry of a transient water molecule responsible for a key step in the enzyme’s mechanism. Our results provide insight into the structural mechanisms of these two different enzymes.« less

Deciphering kinase-substrate relationships by analysis of domain-specific phosphorylation network.

PubMed

Damle, Nikhil Prakash; Mohanty, Debasisa

2014-06-15

In silico prediction of site-specific kinase-substrate relationships (ssKSRs) is crucial for deciphering phosphorylation networks by linking kinomes to phosphoproteomes. However, currently available predictors for ssKSRs give rise to a large number of false-positive results because they use only a short sequence stretch around phosphosite as determinants of kinase specificity and do not consider the biological context of kinase-substrate recognition. Based on the analysis of domain-specific kinase-substrate relationships, we have constructed a domain-level phosphorylation network that implicitly incorporates various contextual factors. It reveals preferential phosphorylation of specific domains by certain kinases. These novel correlations have been implemented in PhosNetConstruct, an automated program for predicting target kinases for a substrate protein. PhosNetConstruct distinguishes cognate kinase-substrate pairs from a large number of non-cognate combinations. Benchmarking on independent datasets using various statistical measures demonstrates the superior performance of PhosNetConstruct over ssKSR-based predictors. PhosNetConstruct is freely available at http://www.nii.ac.in/phosnetconstruct.html. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease*

PubMed Central

Galiullina, Raisa A.; Kasperkiewicz, Paulina; Chichkova, Nina V.; Szalek, Aleksandra; Serebryakova, Marina V.; Poreba, Marcin; Drag, Marcin; Vartapetian, Andrey B.

2015-01-01

Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones. PMID:26283788

Substrate Sorting by a Supercharged Nanoreactor

PubMed Central

2017-01-01

Compartmentalization of proteases enables spatially and temporally controlled protein degradation in cells. Here we show that an engineered lumazine synthase protein cage, which possesses a negatively supercharged lumen, can exploit electrostatic effects to sort substrates for an encapsulated protease. This proteasome-like nanoreactor preferentially cleaves positively charged polypeptides over both anionic and zwitterionic substrates, inverting the inherent substrate specificity of the guest enzyme approximately 480 fold. Our results suggest that supercharged nanochambers could provide a simple and potentially general means of conferring substrate specificity to diverse encapsulated catalysts. PMID:29278496

Allosteric regulation of rhomboid intramembrane proteolysis.

PubMed

Arutyunova, Elena; Panwar, Pankaj; Skiba, Pauline M; Gale, Nicola; Mak, Michelle W; Lemieux, M Joanne

2014-09-01

Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases. © 2014 The Authors.

Allosteric regulation of rhomboid intramembrane proteolysis

PubMed Central

Arutyunova, Elena; Panwar, Pankaj; Skiba, Pauline M; Gale, Nicola; Mak, Michelle W; Lemieux, M Joanne

2014-01-01

Proteolysis within the lipid bilayer is poorly understood, in particular the regulation of substrate cleavage. Rhomboids are a family of ubiquitous intramembrane serine proteases that harbour a buried active site and are known to cleave transmembrane substrates with broad specificity. In vitro gel and Förster resonance energy transfer (FRET)-based kinetic assays were developed to analyse cleavage of the transmembrane substrate psTatA (TatA from Providencia stuartii). We demonstrate significant differences in catalytic efficiency (kcat/K0.5) values for transmembrane substrate psTatA (TatA from Providencia stuartii) cleavage for three rhomboids: AarA from P. stuartii, ecGlpG from Escherichia coli and hiGlpG from Haemophilus influenzae demonstrating that rhomboids specifically recognize this substrate. Furthermore, binding of psTatA occurs with positive cooperativity. Competitive binding studies reveal an exosite-mediated mode of substrate binding, indicating allostery plays a role in substrate catalysis. We reveal that exosite formation is dependent on the oligomeric state of rhomboids, and when dimers are dissociated, allosteric substrate activation is not observed. We present a novel mechanism for specific substrate cleavage involving several dynamic processes including positive cooperativity and homotropic allostery for this interesting class of intramembrane proteases. PMID:25009246

Method of making coherent multilayer crystals

DOEpatents

Schuller, Ivan K.; Falco, Charles M.

1984-01-01

A new material consisting of a coherent multilayer crystal of two or more elements where each layer is composed of a single element. Each layer may vary in thickness from about 2 .ANG. to 2500 .ANG.. The multilayer crystals are prepared by sputter deposition under conditions which slow the sputtered atoms to near substrate temperatures before they contact the substrate.

Suitability of sphagnum moss, coir, and douglas fir bark as soilless substrates for container production of highbush blueberry

USDA-ARS?s Scientific Manuscript database

The purpose of the present study was to investigate the suitability of different soilless substrates for container production of highbush blueberry (Vaccinium sp.). Young plants of ‘Snowchaser’ blueberry were grown in 4.4-liter pots filled with media containing 10% perlite and varying proportions of...

Evolutionary dynamics of enzymes.

PubMed

Demetrius, L

1995-08-01

This paper codifies and rationalizes the large diversity in reaction rates and substrate specificity of enzymes in terms of a model which postulates that the kinetic properties of present-day enzymes are the consequence of the evolutionary force of mutation and selection acting on a class of primordial enzymes with poor catalytic activity and broad substrate specificity. Enzymes are classified in terms of their thermodynamic parameters, activation enthalpy delta H* and activation entropy delta S*, in their kinetically significant transition states as follows: type 1, delta H* > 0, delta S* < 0; type 2, delta H* < or = 0, delta S* < or = 0; type 3, delta H* > 0, delta S* > 0. We study the evolutionary dynamics of these three classes of enzymes subject to mutation, which acts at the level of the gene which codes for the enzyme and selection, which acts on the organism that contains the enzyme. Our model predicts the following evolutionary trends in the reaction rate and binding specificity for the three classes of molecules. In type 1 enzymes, evolution results in random, non-directional changes in the reaction rate and binding specificity. In type 2 and 3 enzymes, evolution results in a unidirectional increase in both the reaction rate and binding specificity. We exploit these results in order to codify the diversity in functional properties of present-day enzymes. Type 1 molecules will be described by intermediate reaction rates and broad substrate specificity. Type 2 enzymes will be characterized by diffusion-controlled rates and absolute substrate specificity. The type 3 catalysts can be further subdivided in terms of their activation enthalpy into two classes: type 3a (delta H* small) and type 3b (delta H* large). We show that type 3a will be represented by the same functional properties that identify type 2, namely, diffusion-controlled rates and absolute substrate specificity, whereas type 3b will be characterized by non-diffusion-controlled rates and absolute substrate specificity. We infer from this depiction of the three classes of enzymes, a general relation between the two functional properties, reaction rate and substrate specificity, namely, enzymes with diffusion-controlled rates have absolute substrate specificity. By appealing to energetic considerations, we furthermore show that enzymes with diffusion-controlled rates (types 2 and 3a) form a small subset of the class of all enzymes. This codification of present-day enzymes derived from an evolutionary model, essentially relates the structural properties of enzymes, as described by their thermodynamic parameters, to their functional properties, as represented by the reaction rate and substrate specificity.

Engineering the Substrate Specificity of the DhbE Adenylation Domain by Yeast Cell Surface Display

PubMed Central

Zhang, Keya; Nelson, Kathryn M.; Bhuripanyo, Karan; Grimes, Kimberly D.; Zhao, Bo; Aldrich, Courtney C.; Yin, Jun

2013-01-01

SUMMARY The adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) activate aryl acids or amino acids to launch their transfer through the NRPS assembly line for the biosynthesis of many medicinally important natural products. In order to expand the substrate pool of NRPSs, we developed a method based on yeast cell surface display to engineer the substrate specificities of the A-domains. We acquired A-domain mutants of DhbE that have 11- and 6-fold increases in kcat/Km with nonnative substrates 3-hydroxybenzoic acid and 2-aminobenzoic acid, respectively and corresponding 3- and 33-fold decreases in kcat/Km values with the native substrate 2,3-dihydroxybenzoic acid, resulting in a dramatic switch in substrate specificity of up to 200-fold. Our study demonstrates that yeast display can be used as a high throughput selection platform to reprogram the “nonribosomal code” of A-domains. PMID:23352143

Cold spraying of aluminum bronze on profiled submillimeter cermet structures formed by laser cladding

NASA Astrophysics Data System (ADS)

Ryashin, N. S.; Malikov, A. G.; Shikalov, V. S.; Gulyaev, I. P.; Kuchumov, B. M.; Klinkov, S. V.; Kosarev, V. F.; Orishich, A. M.

2017-10-01

The paper presents results of the cold spraying of aluminum bronze coatings on substrates profiled with WC/Ni tracks obtained by laser cladding. Reinforcing cermet frames shaped as grids with varied mesh sizes were clad on stainless steel substrates using a CO2 laser machine "Siberia" (ITAM SB RAS, Russia). As a result, surfaces/substrates with heterogeneous shape, composition, and mechanical properties were obtained. Aluminum bronze coatings were deposited from 5lF-NS powder (Oerlikon Metco, Switzerland) on those substrates using cold spraying equipment (ITAM SB RAS). Data of profiling, microstructure diagnostics, EDS analysis, and mechanical tests of obtained composites is reported. Surface relief of the sprayed coatings dependence on substrate structure has been demonstrated.

Calcium-dependent protein kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates.

PubMed

Curran, Amy; Chang, Ing-Feng; Chang, Chia-Lun; Garg, Shilpi; Miguel, Rodriguez Milla; Barron, Yoshimi D; Li, Ying; Romanowsky, Shawn; Cushman, John C; Gribskov, Michael; Harmon, Alice C; Harper, Jeffrey F

2011-01-01

The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca(2+)-dependent protein kinases (CPKs). While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16, and 34). Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with K(M) ∼70 μM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites). Of these, 74 (27%) were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies.

Calcium-Dependent Protein Kinases from Arabidopsis Show Substrate Specificity Differences in an Analysis of 103 Substrates

PubMed Central

Curran, Amy; Chang, Ing-Feng; Chang, Chia-Lun; Garg, Shilpi; Miguel, Rodriguez Milla; Barron, Yoshimi D.; Li, Ying; Romanowsky, Shawn; Cushman, John C.; Gribskov, Michael; Harmon, Alice C.; Harper, Jeffrey F.

2011-01-01

The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca2+-dependent protein kinases (CPKs). While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16, and 34). Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with KM ∼70 μM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites). Of these, 74 (27%) were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies. PMID:22645532

A method for the quantitative site-specific study of the biochemistry within dental plaque biofilms formed in vivo.

PubMed

Robinson, C; Kirkham, J; Percival, R; Shore, R C; Bonass, W A; Brookes, S J; Kusa, L; Nakagaki, H; Kato, K; Nattress, B

1997-01-01

The study of plaque biofilms in the oral cavity is difficult as plaque removal inevitably disrupts biofilm integrity precluding kinetic studies involving the penetration of components and metabolism of substrates in situ. A method is described here in which plaque is formed in vivo under normal (or experimental) conditions using a collection device which can be removed from the mouth after a specified time without physical disturbance to the plaque biofilm, permitting site-specific analysis or exposure of the undisturbed plaque to experimental conditions in vitro. Microbiological analysis revealed plaque flora which was similar to that reported from many natural sources. Analytical data can be related to plaque volume rather than weight. Using this device, plaque fluoride concentrations have been shown to vary with plaque depth and in vitro short-term exposure to radiolabelled components may be carried out, permitting important conclusions to be drawn regarding the site-specific composition and dynamics of dental plaque.

Sugar microarray via click chemistry: molecular recognition with lectins and amyloid β (1-42)

NASA Astrophysics Data System (ADS)

Matsumoto, Erino; Yamauchi, Takahiro; Fukuda, Tomohiro; Miura, Yoshiko

2009-06-01

Sugar microarrays were fabricated on various substrates via click chemistry. Acetylene-terminated substrates were prepared by forming self-assembled monolayers (SAMs) on a gold substrate with alkyl-disulfide and on silicon, quartz and glass substrates with a silane-coupling reagent. The gold substrates were subjected to surface plasmon resonance measurements, and the quartz and glass substrates were subjected to spectroscopy measurements and optical microscopy observation. The saccharide-immobilized substrate on the gold substrate showed specific interaction with the corresponding lectin, and the saccharides showed inert surface properties to other proteins with a high signal-to-noise ratio. We also focused on the saccharide-protein interaction on protein amyloidosis of Alzheimer amyloid β. Amyloid β peptide showed conformation transition on the saccharide-immobilization substrate into a β-sheet, and fibril formation and amyloid aggregates were found on the specific saccharides.

Structure of the carboxypeptidase B complex with N-sulfamoyl-L-phenylalanine - a transition state analog of non-specific substrate.

PubMed

Akparov, Valery; Timofeev, Vladimir; Khaliullin, Ilyas; Švedas, Vytas; Kuranova, Inna

2018-03-01

Carboxypeptidase B (EC 3.4.17.2) (CPB) is commonly used in the industrial insulin production and as a template for drug design. However, its ability to discriminate substrates with hydrophobic, hydrophilic, and charged side chains is not well understood. We report structure of CPB complex with a transition state analog N-sulfamoyl-L-phenylalanine solved at 1.74Å. The study provided an insight into structural basis of CPB substrate specificity. Ligand binding is affected by structure-depended conformational changes of Asp255 in S1'-subsite, interactions with Asn144 and Arg145 in C-terminal binding subsite, and Glu270 in the catalytic center. Side chain of the non-specific substrate analog SPhe in comparison with that of specific substrate analog SArg (reported earlier) not only loses favorable electrostatic interactions and two hydrogen bonds with Asp255 and three fixed water molecules, but is forced to be in the unfavorable hydrophilic environment. Thus, Ser207, Gly253, Tyr248, and Asp255 residues play major role in the substrate recognition by S1'-subsite.

Novel α-L-arabinofuranosidase from Cellulomonas fimi ATCC 484 and its substrate-specificity analysis with the aid of computer.

PubMed

Yang, Ying; Zhang, Lujia; Guo, Mingrong; Sun, Jiaqi; Matsukawa, Shingo; Xie, Jingli; Wei, Dongzhi

2015-04-15

In the process of gene mining for novel α-L-arabinofuranosidases (AFs), the gene Celf_3321 from Cellulomonas fimi ATCC 484 encodes an AF, termed as AbfCelf, with potent activity, 19.4 U/mg under the optimum condition, pH 6.0 and 40 °C. AbfCelf can hydrolyze α-1,5-linked oligosaccharides, sugar beet arabinan, linear 1,5-α-arabinan, and wheat flour arabinoxylan, which is partly different from some previously well-characterized GH 51 AFs. The traditional substrate-specificity analysis for AFs is labor-consuming and money costing, because the substrates include over 30 kinds of various 4-nitrophenol (PNP)-glycosides, oligosaccharides, and polysaccharides. Hence, a preliminary structure and mechanism based method was applied for substrate-specificity analysis. The binding energy (ΔG, kcal/mol) obtained by docking suggested the reaction possibility and coincided with the experimental results. AbfA crystal 1QW9 was used to test the rationality of docking method in simulating the interaction between enzyme and substrate, as well the credibility of the substrate-specificity analysis method in silico.

In silico design, synthesis, and assays of specific substrates for proteinase 3: influence of fluorogenic and charged groups.

PubMed

Narawane, Shailesh; Budnjo, Adnan; Grauffel, Cédric; Haug, Bengt Erik; Reuter, Nathalie

2014-02-13

Neutrophil serine proteases are specific regulators of the immune response, and proteinase 3 is a major target antigen in antineutrophil cytoplasmic antibody-associated vasculitis. FRET peptides containing 2-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp) as fluorophore and quencher groups, respectively, have been widely used to probe proteases specificity. Using in silico design followed by enzymatic assays, we show that Abz and EDDnp significantly contribute to substrate hydrolysis by PR3. We also propose a new substrate specific for PR3.

Fungal Taxa Target Different Carbon Substrates in Harvard Forest Soils

NASA Astrophysics Data System (ADS)

Hanson, C. A.; Allison, S. D.; Wallenstein, M. D.; Mellilo, J. M.; Treseder, K. K.

2006-12-01

The mineralization of soil organic carbon is a major component of the global carbon cycle and is largely controlled by soil microbial communities. However, little is known about the functional roles of soil microbes or whether different microbial taxa target different carbon substrates under natural conditions. To examine this possibility, we assessed the community composition of active fungi by using a novel nucleotide analog technique in soils from the Harvard Forest. We hypothesized that fungal community composition would shift in response to the addition of different substrates and that specific fungal taxa would respond differentially to particular carbon sources. To test this hypothesis, we added a nucleotide analog probe directly to soils in conjunction with one of five carbon compounds of increasing recalcitrance: glycine, sucrose, cellulose, tannin-protein complex, and lignin. During 48 hour incubations, the nucleotide analog was incorporated into newly replicated DNA of soil organisms that proliferated following the addition of the substrates. In this way, we labeled the DNA of microbes that respond to a particular carbon source. Labeled DNA was isolated and fungal Internal Transcribed Spacer (ITS) regions of ribosomal DNA (rDNA) were sequenced and analyzed to identify active fungi to near-species resolution. Diversity analyses at the ≥97% sequence similarity level indicated that taxonomic richness was greater under cellulose (Shannon Index: 3.23 ± 0.11 with ± 95% CI) and lignin (2.87 ± 0.15) additions than the other treatments (2.34 ± 0.16 to 2.64 ± 0.13). In addition, community composition of active fungi shifted under glycine, sucrose, and cellulose additions. Specifically, the community under glycine was significantly different from communities under control, cellulose, and tannin-protein (P<0.05). Additionally, the sucrose and cellulose communities were marginally different from the control community (P = 0.059 and 0.054, respectively) and each other (P = 0.058). Together these results support our hypothesis that fungal communities change in response to different carbon sources. We found 11 fungal operational taxonomic units (OTUs) whose relative abundances differed at least marginally significantly among substrates. One OTU related to Mortierella increased in abundance under cellulose, but was absent or rare under the other substrates. Another OTU related to an unidentified Basidiomycete was only present under lignin addition, while yet another OTU closely related to Mortierella macrocystis greatly increased in abundance under tannin-protein and slightly increased in response to lignin and sucrose. This confirms our hypothesis that particular taxa respond differently to specific carbon substrates and suggests that some fungal taxa may specialize in the break-down of particular carbon sources in soils. Overall, our results imply that microbes have varying roles in the mineralization of soil carbon, and thus microbial community composition may be an important control over ecosystem carbon dynamics and storage, especially in relation to global change.

Mutational analysis of the active site flap (20s loop) of mandelate racemase.

PubMed

Bourque, Jennifer R; Bearne, Stephen L

2008-01-15

Mandelate racemase from Pseudomonas putida catalyzes the Mg2+-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate. Residues of the 20s and 50s loops determine, in part, the topology and polarity of the active site and hence the substrate specificity. Previously, we proposed that, during racemization, the phenyl ring of mandelate moves between an S-pocket comprised of residues from the 50s loop and an R-pocket comprised of residues from the 20s loop [Siddiqi, F., Bourque, J. R., Jiang, H., Gardner, M., St. Maurice, M., Blouin, C., and Bearne, S. L. (2005) Biochemistry 44, 9013-9021]. The 20s loop constitutes a mobile beta-meander flap that covers the active site cavity shielding it from solvent and controlling entry and egress of ligands. To understand the role of the 20s loop in catalysis and substrate specificity, we constructed a series of mutants (V22A, V22I, V22F, T24S, A25V, V26A, V26L, V26F, V29A, V29L, V29F, V26A/V29L, and V22I/V29L) in which the sizes of hydrophobic side chains of the loop residues were varied. Catalytic efficiencies (kcat/Km) for all mutants were reduced between 6- and 40-fold with the exception of those of V22I, V26A, V29L, and V22I/V29L which had near wild-type efficiencies with mandelate. Thr 24 and Ala 25, located at the tip of the 20s loop, were particularly sensitive to minor alterations in the size of their hydrophobic side chains; however, most mutations were tolerated quite well, suggesting that flap mobility could compensate for increases in the steric bulk of hydrophobic side chains. With the exception of V29L, with mandelate as the substrate, and V22F and V26A/V29L, with 2-naphthylglycolate (2-NG) as the substrate, the values of kcat and Km were not altered in a manner consistent with steric obstruction of the R-pocket, perhaps due to flap mobility compensating for the increased size of the hydrophobic side chains. Surprisingly, V22I and V29L catalyzed the racemization of the bulkier substrate 2-NG with kcat/Km values approximately 2-fold greater than those observed for wild-type mandelate racemase. Although minor changes in substrate specificity were achieved through alterations of the active site flap of mandelate racemase, our results suggest that hydrophobic residues that reside on a flexible flap and define the topology of an active site through their van der Waals contacts with the substrate are quite tolerant of a variety of steric substitutions.

Substrate Specificities and Conformational Flexibility of 3-Ketosteroid 9α-Hydroxylases*

PubMed Central

Penfield, Jonathan S.; Worrall, Liam J.; Strynadka, Natalie C.; Eltis, Lindsay D.

2014-01-01

KshA is the oxygenase component of 3-ketosteroid 9α-hydroxylase, a Rieske oxygenase involved in the bacterial degradation of steroids. Consistent with its role in bile acid catabolism, KshA1 from Rhodococcus rhodochrous DSM43269 had the highest apparent specificity (kcat/Km) for steroids with an isopropyl side chain at C17, such as 3-oxo-23,24-bisnorcholesta-1,4-diene-22-oate (1,4-BNC). By contrast, the KshA5 homolog had the highest apparent specificity for substrates with no C17 side chain (kcat/Km >105 s−1 m−1 for 4-estrendione, 5α-androstandione, and testosterone). Unexpectedly, substrates such as 4-androstene-3,17-dione (ADD) and 4-BNC displayed strong substrate inhibition (KiS ∼100 μm). By comparison, the cholesterol-degrading KshAMtb from Mycobacterium tuberculosis had the highest specificity for CoA-thioesterified substrates. These specificities are consistent with differences in the catabolism of cholesterol and bile acids, respectively, in actinobacteria. X-ray crystallographic structures of the KshAMtb·ADD, KshA1·1,4-BNC-CoA, KshA5·ADD, and KshA5·1,4-BNC-CoA complexes revealed that the enzymes have very similar steroid-binding pockets with the substrate's C17 oriented toward the active site opening. Comparisons suggest Tyr-245 and Phe-297 are determinants of KshA1 specificity. All enzymes have a flexible 16-residue “mouth loop,” which in some structures completely occluded the substrate-binding pocket from the bulk solvent. Remarkably, the catalytic iron and α-helices harboring its ligands were displaced up to 4.4 Å in the KshA5·substrate complexes as compared with substrate-free KshA, suggesting that Rieske oxygenases may have a dynamic nature similar to cytochrome P450. PMID:25049233

Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries

PubMed Central

Harris, Jennifer L.; Backes, Bradley J.; Leonetti, Francesco; Mahrus, Sami; Ellman, Jonathan A.; Craik, Charles S.

2000-01-01

A method is presented for the preparation and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of proteases. The substrates contain the fluorogenic leaving group 7-amino-4-carbamoylmethylcoumarin (ACC). Substrates incorporating the ACC leaving group show kinetic profiles comparable to those with the traditionally used 7-amino-4-methylcoumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries by using 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase synthesis techniques. The approximately 3-fold-increased quantum yield of ACC over AMC permits reduction in enzyme and substrate concentrations. As a consequence, a greater number of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Soluble positional protease substrate libraries of 137,180 and 6,859 members, possessing amino acid diversity at the P4-P3-P2-P1 and P4-P3-P2 positions, respectively, were constructed. Employing this screening method, we profiled the substrate specificities of a diverse array of proteases, including the serine proteases thrombin, plasmin, factor Xa, urokinase-type plasminogen activator, tissue plasminogen activator, granzyme B, trypsin, chymotrypsin, human neutrophil elastase, and the cysteine proteases papain and cruzain. The resulting profiles create a pharmacophoric portrayal of the proteases to aid in the design of selective substrates and potent inhibitors. PMID:10869434

All-dielectric metasurface for wavefront control at terahertz frequencies

NASA Astrophysics Data System (ADS)

Dharmavarapu, Raghu; Hock Ng, Soon; Bhattacharya, Shanti; Juodkazis, Saulius

2018-01-01

Recently, metasurfaces have gained popularity due to their ability to offer a spatially varying phase response, low intrinsic losses and high transmittance. Here, we demonstrate numerically and experimentally a silicon meta-surface at THz frequencies that converts a Gaussian beam into a Vortex beam independent of the polarization of the incident beam. The metasurface consists of an array of sub-wavelength silicon cross resonators made of a high refractive index material on substrates such as sapphire and CaF2 that are transparent at IR-THz spectral range. With these substrates, it is possible to create phase elements for a specific spectral range including at the molecular finger printing around 10 μm as well as at longer THz wavelengths where secondary molecular structures can be revealed. This device offers high transmittance and a phase coverage of 0 to 2π. The transmittance phase is tuned by varying the dimensions of the meta-atoms. To demonstrate wavefront engineering, we used a discretized spiraling phase profile to convert the incident Gaussian beam to vortex beam. To realize this, we divided the metasurface surface into eight angular sectors and chose eight different dimensions for the crosses providing successive phase shifts spaced by π/4 radians for each of these sectors. Photolithography and reactive ion etching (RIE) were used to fabricate these silicon crosses as the dimensions of these cylinders range up to few hundreds of micrometers. Large 1-cm-diameter optical elements were successfully fabricated and characterised by optical profilometry.

Cleavage-site specificity of prolyl endopeptidase FAP investigated with a full-length protein substrate.

PubMed

Huang, Chih-Hsiang; Suen, Ching-Shu; Lin, Ching-Ting; Chien, Chia-Hui; Lee, Hsin-Ying; Chung, Kuei-Min; Tsai, Ting-Yueh; Jiaang, Weir-Tong; Hwang, Ming-Jing; Chen, Xin

2011-06-01

Fibroblast activation protein (FAP) is a prolyl-cleaving endopeptidase proposed as an anti-cancer drug target. It is necessary to define its cleavage-site specificity to facilitate the identification of its in vivo substrates and to understand its biological functions. We found that the previously identified substrate of FAP, α(2)-anti-plasmin, is not a robust substrate in vitro. Instead, an intracellular protein, SPRY2, is cleavable by FAP and more suitable for investigation of its substrate specificity in the context of the full-length globular protein. FAP prefers uncharged residues, including small or bulky hydrophobic amino acids, but not charged amino acids, especially acidic residue at P1', P3 and P4 sites. Molecular modelling analysis shows that the substrate-binding site of FAP is surrounded by multiple tyrosine residues and some negatively charged residues, which may exert least preference for substrates with acidic residues. This provides an explanation why FAP cannot cleave interleukins, which have a glutamate at either P4 or P2', despite their P3-P2-P1 sites being identical to SPRY2 or α-AP. Our study provided new information on FAP cleavage-site specificity, which differs from the data obtained by profiling with a peptide library or with the denatured protein, gelatin, as the substrate. Furthermore, our study suggests that negatively charged residues should be avoided when designing FAP inhibitors.

Interference effects in the sum frequency generation spectra of thin organic films. II: Applications to different thin-film systems.

PubMed

Tong, Yujin; Zhao, Yanbao; Li, Na; Ma, Yunsheng; Osawa, Masatoshi; Davies, Paul B; Ye, Shen

2010-07-21

In this paper, the results of the modeling calculations carried out for predicting the interference effects expected in the sum frequency generation (SFG) spectra of a specific thin-layer system, described in the accompanying paper, are tested by comparing them with the experimental spectra obtained for a real thin-layer film comprising an organic monolayer/variable thickness dielectric layer/gold substrate. In this system, two contributions to the SFG spectra arise, a resonant contribution from the organic film and a nonresonant contribution from the gold substrate. The modeling calculations are in excellent agreement with the experimental spectra over a wide range of thicknesses and for different polarization combinations. The introduction of another resonant monolayer adjacent to the gold substrate and with the molecules having a reverse orientation has a significant affect on the spectral shapes which is predicted. If a dielectric substrate such as CaF(2) is used instead of a gold substrate, only the spectral intensities vary with the film thickness but not the spectral shapes. The counterpropagating beam geometry will change both the thickness dependent spectral shapes and the intensity of different vibrational modes in comparison with a copropagating geometry. The influences of these experimental factors, i.e., the molecular orientational structure in the thin film, the nature of the substrate, and the selected incident beam geometry, on the experimental SFG spectra are quantitatively predicted by the calculations. The thickness effects on the signals from a SFG active monolayer contained in a thin liquid-layer cell of the type frequently used for in situ electrochemical measurements is also discussed. The modeling calculation is also valid for application to other thin-film systems comprising more than two resonant SFG active interfaces by appropriate choice of optical geometries and relevant optical properties.

Dynamic substrate preferences predict metabolic properties of a simple microbial consortium

DOE PAGES

Erbilgin, Onur; Bowen, Benjamin P.; Kosina, Suzanne M.; ...

2017-01-23

Mixed cultures of different microbial species are increasingly being used to carry out a specific biochemical function in lieu of engineering a single microbe to do the same t ask. However, knowing how different species' metabolisms will integrate to reach a desired outcome is a difficult problem that has been studied in great detail using steady-state models. However, many biotechnological processes, as well as natural habitats, represent a more dynamic system. Examining how individual species use resources in their growth medium or environment (exometabolomics) over time in batch culture conditions can provide rich phenotypic data that encompasses regulation and transporters,more » creating an opportunity to integrate the data into a predictive model of resource use by a mixed community. Here we use exometabolomic profiling to examine the time-varying substrate depletion from a mixture of 19 amino acids and glucose by two Pseudomonas and one Bacillus species isolated from ground water. Contrary to studies in model organisms, we found surprisingly few correlations between resource preferences and maximal growth rate or biomass composition. We then modeled patterns of substrate depletion, and used these models to examine if substrate usage preferences and substrate depletion kinetics of individual isolates can be used to predict the metabolism of a co-culture of the isolates. We found that most of the substrates fit the model predictions, except for glucose and histidine, which were depleted more slowly than predicted, and proline, glycine, glutamate, lysine and arginine, which were all consumed significantly faster. Our results indicate that a significant portion of a model community's overall metabolism can be predicted based on the metabolism of the individuals. Based on the nature of our model, the resources that significantly deviate from the prediction highlight potential metabolic pathways affected by species-species interactions, which when further studied can potentially be used to modulate microbial community structure and/or function.« less

Layer-dependent supercapacitance of graphene films grown by chemical vapor deposition on nickel foam

NASA Astrophysics Data System (ADS)

Chen, Wei; Fan, Zhongli; Zeng, Gaofeng; Lai, Zhiping

2013-03-01

High-quality, large-area graphene films with few layers are synthesized on commercial nickel foams under optimal chemical vapor deposition conditions. The number of graphene layers is adjusted by varying the rate of the cooling process. It is found that the capacitive properties of graphene films are related to the number of graphene layers. Owing to the close attachment of graphene films on the nickel substrate and the low charge-transfer resistance, the specific capacitance of thinner graphene films is almost twice that of the thicker ones and remains stable up to 1000 cycles. These results illustrate the potential for developing high-performance graphene-based electrical energy storage devices.

Chemical pretreatment of lignocellulosic agroindustrial waste for methane production.

PubMed

Pellera, Frantseska-Maria; Gidarakos, Evangelos

2018-01-01

This study investigates the effect of different chemical pretreatments on the solubilization and the degradability of different solid agroindustrial waste, namely winery waste, cotton gin waste, olive pomace and juice industry waste. Eight different reagents were investigated, i.e. sodium hydroxide (NaOH), sodium bicarbonate (NaHCO 3 ), sodium chloride (NaCl), citric acid (H 3 Cit), acetic acid (AcOH), hydrogen peroxide (H 2 O 2 ), acetone (Me 2 CO) and ethanol (EtOH), under three condition sets resulting in treatments of varying intensity, depending on process duration, reagent dosage and temperature. Results indicated that chemical pretreatment under more severe conditions is more effective on the solubilization of lignocellulosic substrates, such as those of the present study and among the investigated reagents, H 3 Cit, H 2 O 2 and EtOH appeared to be the most effective to this regard. At the same time, although chemical pretreatment in general did not improve the methane potential of the substrates, moderate to high severity conditions were found to generally be the most satisfactory in terms of methane production from pretreated materials. In fact, moderate severity treatments using EtOH for winery waste, H 3 Cit for olive pomace and H 2 O 2 for juice industry waste and a high severity treatment with EtOH for cotton gin waste, resulted in maximum specific methane yield values. Ultimately, the impact of pretreatment parameters on the different substrates seems to be dependent on their characteristics, in combination with the specific mode of action of each reagent. The overall energy balance of such a system could probably be improved by using lower operating powers and higher solid to liquid ratios. Copyright © 2017 Elsevier Ltd. All rights reserved.

Crystal structures of mammalian glutamine synthetases illustrate substrate-induced conformational changes and provide opportunities for drug and herbicide design.

PubMed

Krajewski, Wojciech W; Collins, Ruairi; Holmberg-Schiavone, Lovisa; Jones, T Alwyn; Karlberg, Tobias; Mowbray, Sherry L

2008-01-04

Glutamine synthetase (GS) catalyzes the ligation of glutamate and ammonia to form glutamine, with concomitant hydrolysis of ATP. In mammals, the activity eliminates cytotoxic ammonia, at the same time converting neurotoxic glutamate to harmless glutamine; there are a number of links between changes in GS activity and neurodegenerative disorders, such as Alzheimer's disease. In plants, because of its importance in the assimilation and re-assimilation of ammonia, the enzyme is a target of some herbicides. GS is also a central component of bacterial nitrogen metabolism and a potential drug target. Previous studies had investigated the structures of bacterial and plant GSs. In the present publication, we report the first structures of mammalian GSs. The apo form of the canine enzyme was solved by molecular replacement and refined at a resolution of 3 A. Two structures of human glutamine synthetase represent complexes with: a) phosphate, ADP, and manganese, and b) a phosphorylated form of the inhibitor methionine sulfoximine, ADP and manganese; these structures were refined to resolutions of 2.05 A and 2.6 A, respectively. Loop movements near the active site generate more closed forms of the eukaryotic enzymes when substrates are bound; the largest changes are associated with the binding of the nucleotide. Comparisons with earlier structures provide a basis for the design of drugs that are specifically directed at either human or bacterial enzymes. The site of binding the amino acid substrate is highly conserved in bacterial and eukaryotic GSs, whereas the nucleotide binding site varies to a much larger degree. Thus, the latter site offers the best target for specific drug design. Differences between mammalian and plant enzymes are much more subtle, suggesting that herbicides targeting GS must be designed with caution.

Kinetics and methane gas yields of selected C1 to C5 organic acids in anaerobic digestion.

PubMed

Yang, Yu; Chen, Qian; Guo, Jialiang; Hu, Zhiqiang

2015-12-15

Volatile fatty acids (VFAs) and other short-chain organic acids such as lactic and pyruvic acids are intermediates in anaerobic organic degradation. In this study, anaerobic degradation of seven organic acids in salt form was investigated, including formate (C1), acetate (C2), propionate (C3), pyruvate (C3), lactate (C3), butyrate (C4), and valerate (C5). Microbial growth kinetics on these organic acids were determined individually at 37 °C through batch anaerobic digestion tests by varying substrate concentrations from 250 to 4000 mg COD/L. The cumulative methane generation volume was determined real-time by respirometry coupled with gas chromatographic analysis while methane yield and related kinetics were calculated. The methane gas yields (fe, mg CH4 COD/mg substrate COD) from anaerobic degradation of formate, acetate, propionate, pyruvate, lactate, butyrate, and valerate were 0.44 ± 0.27, 0.58 ± 0.05, 0.53 ± 0.18, 0.24 ± 0.05, 0.17 ± 0.05, 0.43 ± 0.15, 0.49 ± 0.11, respectively. Anaerobic degradation of formate showed self-substrate inhibition at the concentrations above 3250 mg COD/L. Acetate, propionate, pyruvate, butyrate, lactate, and valerate did not inhibit methane production at the highest concentrations tested (i.e., 4000 mg COD/L). Microbes growing on acetate had the highest overall specific growth rate (0.30 d(-1)) in methane production. For comparison, the specific microbial growth rates on formate, propionate, pyruvate, butyrate, lactate, and valerate for methane production were 0.10, 0.06, 0.08, 0.07, 0.05, 0.15 d(-1), respectively. Copyright © 2015 Elsevier Ltd. All rights reserved.

Attention for speaking: domain-general control from the anterior cingulate cortex in spoken word production

PubMed Central

Piai, Vitória; Roelofs, Ardi; Acheson, Daniel J.; Takashima, Atsuko

2013-01-01

Accumulating evidence suggests that some degree of attentional control is required to regulate and monitor processes underlying speaking. Although progress has been made in delineating the neural substrates of the core language processes involved in speaking, substrates associated with regulatory and monitoring processes have remained relatively underspecified. We report the results of an fMRI study examining the neural substrates related to performance in three attention-demanding tasks varying in the amount of linguistic processing: vocal picture naming while ignoring distractors (picture-word interference, PWI); vocal color naming while ignoring distractors (Stroop); and manual object discrimination while ignoring spatial position (Simon task). All three tasks had congruent and incongruent stimuli, while PWI and Stroop also had neutral stimuli. Analyses focusing on common activation across tasks identified a portion of the dorsal anterior cingulate cortex (ACC) that was active in incongruent trials for all three tasks, suggesting that this region subserves a domain-general attentional control function. In the language tasks, this area showed increased activity for incongruent relative to congruent stimuli, consistent with the involvement of domain-general mechanisms of attentional control in word production. The two language tasks also showed activity in anterior-superior temporal gyrus (STG). Activity increased for neutral PWI stimuli (picture and word did not share the same semantic category) relative to incongruent (categorically related) and congruent stimuli. This finding is consistent with the involvement of language-specific areas in word production, possibly related to retrieval of lexical-semantic information from memory. The current results thus suggest that in addition to engaging language-specific areas for core linguistic processes, speaking also engages the ACC, a region that is likely implementing domain-general attentional control. PMID:24368899

Overlapping protective roles for glutathione transferase gene family members in chemical and oxidative stress response in Agrobacterium tumefaciens.

PubMed

Skopelitou, Katholiki; Muleta, Abdi W; Pavli, Ourania; Skaracis, Georgios N; Flemetakis, Emmanouil; Papageorgiou, Anastassios C; Labrou, Nikolaos E

2012-03-01

In the present work, we describe the characterisation of the glutathione transferase (GST) gene family from Agrobacterium tumefaciens C58. A genome survey revealed the presence of eight GST-like proteins in A. tumefaciens (AtuGSTs). Comparison by multiple sequence alignment generated a dendrogram revealing the phylogenetic relationships of AtuGSTs-like proteins. The beta and theta classes identified in other bacterial species are represented by five members in A. tumefaciens C58. In addition, there are three "orphan" sequences that do not fit into any previously recognised GST classes. The eight GST-like genes were cloned, expressed in Escherichia coli and their substrate specificity was determined towards 17 different substrates. The results showed that AtuGSTs catalyse a broad range of reactions, with different members of the family exhibiting quite varied substrate specificity. The 3D structures of AtuGSTs were predicted using molecular modelling. The use of comparative sequence and structural analysis of the AtuGST isoenzymes allowed us to identify local sequence and structural characteristics between different GST isoenzymes and classes. Gene expression profiling was conducted under normal culture conditions as well as under abiotic stress conditions (addition of xenobiotics, osmotic stress and cold and heat shock) to induce and monitor early stress-response mechanisms. The results reveal the constitutive expression of GSTs in A. tumefaciens and a modulation of GST activity after treatments, indicating that AtuGSTs presumably participate in a wide range of functions, many of which are important in counteracting stress conditions. These functions may be relevant to maintaining cellular homeostasis as well as in the direct detoxification of toxic compounds.

Temperature dependent localized surface plasmon resonance properties of supported gold nanoparticles

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

Laha, Ranjit; Ranjan, Pranay

2016-05-23

The well known localized surface plasmon resonance (LSPR) of gold nanoparticles (AuNPs) supported on a dielectric substrate depends on the particle shape, size and type of dielectric material. The particle size and shape mainly vary with the method of preparation and the parameters involved there in. In this report, we show preparation of AuNPs supported on quartz substrate by direct current sputtering followed by thermal annealing at an optimized temperature of 400 °C. The samples were characterized using optical absorption spectra, scanning electron microscopy (SEM) and the energy dispersive x-ray spectrum. The LSPR position could be tuned by varying annealingmore » temperature. The LSPR was found to be blue shifted up to 10 nm with annealing temperature varying from 400 °C to 800 °C. The change in LSPR was ascribed to the morphology of AuNPs over quartz.« less

Temperature dependent localized surface plasmon resonance properties of supported gold nanoparticles

NASA Astrophysics Data System (ADS)

Laha, Ranjit; Ranjan, Pranay

2016-05-01

The well known localized surface plasmon resonance (LSPR) of gold nanoparticles (AuNPs) supported on a dielectric substrate depends on the particle shape, size and type of dielectric material. The particle size and shape mainly vary with the method of preparation and the parameters involved there in. In this report, we show preparation of AuNPs supported on quartz substrate by direct current sputtering followed by thermal annealing at an optimized temperature of 400 °C. The samples were characterized using optical absorption spectra, scanning electron microscopy (SEM) and the energy dispersive x-ray spectrum. The LSPR position could be tuned by varying annealing temperature. The LSPR was found to be blue shifted up to 10 nm with annealing temperature varying from 400 °C to 800 °C. The change in LSPR was ascribed to the morphology of AuNPs over quartz.

Bauhinia proteinase inhibitor-based synthetic fluorogenic substrates for enzymes isolated from insect midgut and caterpillar bristles.

PubMed

Andrade, Sonia A; Santomauro-Vaz, Eugênio M; Lopes, Adriana R; Chudzinski-Tavassi, Ana M; Juliano, Maria A; Terra, Walter R; Sampaio, Misako U; Sampaio, Claudio A M; Oliva, Maria Luiza V

2003-03-01

Bauhinia ungulata factor Xa inhibitor (BuXI) inactivates factor Xa and LOPAP, a prothrombin activator proteinase isolated from the venom of Lonomia obliqua caterpillar bristles. The reactive site of the enzyme-inhibitor interaction was explored to design specific substrates for both enzymes. Methionine is crucial for LOPAP and factor Xa substrate interaction, since the change of both Met residues in the substrates abolished the hydrolysis. Synthetic substrates containing the sequence around the reactive site of BbKI, a plasma kallikrein inhibitor, were shown to be specific for trypsin hydrolysis. Therefore, these substrates may be an alternative in studies aiming at a characterization of trypsin-like enzyme activities, especially non-mammalian enzymes.

fMRI Brain-Computer Interface: A Tool for Neuroscientific Research and Treatment

PubMed Central

Sitaram, Ranganatha; Caria, Andrea; Veit, Ralf; Gaber, Tilman; Rota, Giuseppina; Kuebler, Andrea; Birbaumer, Niels

2007-01-01

Brain-computer interfaces based on functional magnetic resonance imaging (fMRI-BCI) allow volitional control of anatomically specific regions of the brain. Technological advancement in higher field MRI scanners, fast data acquisition sequences, preprocessing algorithms, and robust statistical analysis are anticipated to make fMRI-BCI more widely available and applicable. This noninvasive technique could potentially complement the traditional neuroscientific experimental methods by varying the activity of the neural substrates of a region of interest as an independent variable to study its effects on behavior. If the neurobiological basis of a disorder (e.g., chronic pain, motor diseases, psychopathy, social phobia, depression) is known in terms of abnormal activity in certain regions of the brain, fMRI-BCI can be targeted to modify activity in those regions with high specificity for treatment. In this paper, we review recent results of the application of fMRI-BCI to neuroscientific research and psychophysiological treatment. PMID:18274615

Insight into the substrate specificity change caused by the Y227H mutation of α-glucosidase III from the European honeybee (Apis mellifera) through molecular dynamics simulations.

PubMed

Na Ayutthaya, Pratchaya Pramoj; Chanchao, Chanpen; Chunsrivirot, Surasak

2018-01-01

Honey from the European honeybee, Apis mellifera, is produced by α-glucosidases (HBGases) and is widely used in food, pharmaceutical, and cosmetic industries. Categorized by their substrate specificities, HBGases have three isoforms: HBGase I, II and III. Previous experimental investigations showed that wild-type HBGase III from Apis mellifera (WT) preferred sucrose to maltose as a substrate, while the Y227H mutant (MT) preferred maltose to sucrose. This mutant can potentially be used for malt hydrolysis because it can efficiently hydrolyze maltose. In this work, to elucidate important factors contributing to substrate specificity of this enzyme and gain insight into how the Y227H mutation causes substrate specificity change, WT and MT homology models were constructed, and sucrose/maltose was docked into active sites of the WT and MT. AMBER14 was employed to perform three independent molecular dynamics runs for these four complexes. Based on the relative binding free energies calculated by the MM-GBSA method, sucrose is better than maltose for WT binding, while maltose is better than sucrose for MT binding. These rankings support the experimentally observed substrate specificity that WT preferred sucrose to maltose as a substrate, while MT preferred maltose to sucrose, suggesting the importance of binding affinity for substrate specificity. We also found that the Y227H mutation caused changes in the proximities between the atoms necessary for sucrose/maltose hydrolysis that may affect enzyme efficiency in the hydrolysis of sucrose/maltose. Moreover, the per-residue binding free energy decomposition results show that Y227/H227 may be a key residue for preference binding of sucrose/maltose in the WT/MT active site. Our study provides important and novel insight into the binding of sucrose/maltose in the active site of Apis mellifera HBGase III and into how the Y227H mutation leads to the substrate specificity change at the molecular level. This knowledge could be beneficial in the design of this enzyme for increased production of desired products.

Long-range electrostatic complementarity governs substrate recognition by human chymotrypsin C, a key regulator of digestive enzyme activation.

PubMed

Batra, Jyotica; Szabó, András; Caulfield, Thomas R; Soares, Alexei S; Sahin-Tóth, Miklós; Radisky, Evette S

2013-04-05

Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5' subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2' positions of CTRC, although acidic P2' residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels.

Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease.

PubMed

Galiullina, Raisa A; Kasperkiewicz, Paulina; Chichkova, Nina V; Szalek, Aleksandra; Serebryakova, Marina V; Poreba, Marcin; Drag, Marcin; Vartapetian, Andrey B

2015-10-09

Plants lack aspartate-specific cell death proteases homologous to animal caspases. Instead, a subtilisin-like serine-dependent plant protease named phytaspase shown to be involved in the accomplishment of programmed death of plant cells is able to hydrolyze a number of peptide-based caspase substrates. Here, we determined the substrate specificity of rice (Oryza sativa) phytaspase by using the positional scanning substrate combinatorial library approach. Phytaspase was shown to display an absolute specificity of hydrolysis after an aspartic acid residue. The preceding amino acid residues, however, significantly influence the efficiency of hydrolysis. Efficient phytaspase substrates demonstrated a remarkable preference for an aromatic amino acid residue in the P3 position. The deduced optimum phytaspase recognition motif has the sequence IWLD and is strikingly hydrophobic. The established pattern was confirmed through synthesis and kinetic analysis of cleavage of a set of optimized peptide substrates. An amino acid motif similar to the phytaspase cleavage site is shared by the human gastrointestinal peptide hormones gastrin and cholecystokinin. In agreement with the established enzyme specificity, phytaspase was shown to hydrolyze gastrin-1 and cholecystokinin at the predicted sites in vitro, thus destroying the active moieties of the hormones. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural and Kinetic Basis for Substrate Selectivity in Populus tremuloides Sinapyl Alcohol Dehydrogenase

PubMed Central

Bomati, Erin K.; Noel, Joseph P.

2005-01-01

We describe the three-dimensional structure of sinapyl alcohol dehydrogenase (SAD) from Populus tremuloides (aspen), a member of the NADP(H)-dependent dehydrogenase family that catalyzes the last reductive step in the formation of monolignols. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde. We also report substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes. Although SAD and classical cinnamyl alcohol dehydrogenases (CADs) catalyze the same reaction and share some sequence identity, the active site topology of SAD is strikingly different from that predicted for classical CADs. Kinetic analyses of wild-type SAD and several active site mutants demonstrate the complexity of defining determinants of substrate specificity in these enzymes. These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. We used the SAD three-dimensional structure to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, we describe a correlation between substrate specificity and amino acid substitution patterns in their active sites. The SAD structure thus provides a framework for understanding substrate specificity in this family of enzymes and for engineering new enzyme specificities. PMID:15829607

Structural and kinetic basis for substrate selectivity in Populus tremuloides sinapyl alcohol dehydrogenase.

PubMed

Bomati, Erin K; Noel, Joseph P

2005-05-01

We describe the three-dimensional structure of sinapyl alcohol dehydrogenase (SAD) from Populus tremuloides (aspen), a member of the NADP(H)-dependent dehydrogenase family that catalyzes the last reductive step in the formation of monolignols. The active site topology revealed by the crystal structure substantiates kinetic results indicating that SAD maintains highest specificity for the substrate sinapaldehyde. We also report substantial substrate inhibition kinetics for the SAD-catalyzed reduction of hydroxycinnamaldehydes. Although SAD and classical cinnamyl alcohol dehydrogenases (CADs) catalyze the same reaction and share some sequence identity, the active site topology of SAD is strikingly different from that predicted for classical CADs. Kinetic analyses of wild-type SAD and several active site mutants demonstrate the complexity of defining determinants of substrate specificity in these enzymes. These results, along with a phylogenetic analysis, support the inclusion of SAD in a plant alcohol dehydrogenase subfamily that includes cinnamaldehyde and benzaldehyde dehydrogenases. We used the SAD three-dimensional structure to model several of these SAD-like enzymes, and although their active site topologies largely mirror that of SAD, we describe a correlation between substrate specificity and amino acid substitution patterns in their active sites. The SAD structure thus provides a framework for understanding substrate specificity in this family of enzymes and for engineering new enzyme specificities.

Coherent multilayer crystals and method of making

DOEpatents

Schuller, I.K.; Falco, C.M.

1980-10-30

A new material is described consisting of a coherent multilayer crystal of two or more elements where each layer is composed of a single element. Each layer may vary in thickness from about 2 A to 2500 A. The multilayer crystals are prepared by sputter deposition under conditions which slow the sputtered atoms to near substrate temperatures before they contact the substrate.

DNA curtains for high-throughput single-molecule optical imaging.

PubMed

Greene, Eric C; Wind, Shalom; Fazio, Teresa; Gorman, Jason; Visnapuu, Mari-Liis

2010-01-01

Single-molecule approaches provide a valuable tool in the arsenal of the modern biologist, and new discoveries continue to be made possible through the use of these state-of-the-art technologies. However, it can be inherently difficult to obtain statistically relevant data from experimental approaches specifically designed to probe individual reactions. This problem is compounded with more complex biochemical reactions, heterogeneous systems, and/or reactions requiring the use of long DNA substrates. Here we give an overview of a technology developed in our laboratory, which relies upon simple micro- or nanofabricated structures in combination with "bio-friendly" lipid bilayers, to align thousands of long DNA molecules into defined patterns on the surface of a microfluidic sample chamber. We call these "DNA curtains," and we have developed several different versions varying in complexity and DNA substrate configuration, which are designed to meet different experimental needs. This novel approach to single-molecule imaging provides a powerful experimental platform that offers the potential for concurrent observation of hundreds or even thousands of protein-DNA interactions in real time. Copyright 2010 Elsevier Inc. All rights reserved.

Biosynthetic multitasking facilitates thalassospiramide structural diversity in marine bacteria.

PubMed

Ross, Avena C; Xu, Ying; Lu, Liang; Kersten, Roland D; Shao, Zongze; Al-Suwailem, Abdulaziz M; Dorrestein, Pieter C; Qian, Pei-Yuan; Moore, Bradley S

2013-01-23

Thalassospiramides A and B are immunosuppressant cyclic lipopeptides first reported from the marine α-proteobacterium Thalassospira sp. CNJ-328. We describe here the discovery and characterization of an extended family of 14 new analogues from four Tistrella and Thalassospira isolates. These potent calpain 1 protease inhibitors belong to six structure classes in which the length and composition of the acylpeptide side chain varies extensively. Genomic sequence analysis of the thalassospiramide-producing microbes revealed related, genus-specific biosynthetic loci encoding hybrid nonribosomal peptide synthetase/polyketide synthases consistent with thalassospiramide assembly. The bioinformatics analysis of the gene clusters suggests that structural diversity, which ranges from the 803.4 Da thalassospiramide C to the 1291.7 Da thalassospiramide F, results from a complex sequence of reactions involving amino acid substrate channeling and enzymatic multimodule skipping and iteration. Preliminary biochemical analysis of the N-terminal nonribosomal peptide synthetase module from the Thalassospira TtcA megasynthase supports a biosynthetic model in which in cis amino acid activation competes with in trans activation to increase the range of amino acid substrates incorporated at the N terminus.

Biosynthetic Multitasking Facilitates Thalassospiramide Structural Diversity in Marine Bacteria

PubMed Central

Ross, Avena C.; Xu, Ying; Lu, Liang; Kersten, Roland D.; Shao, Zongze; Al-Suwailem, Abdulaziz M.; Dorrestein, Pieter C.; Qian, Pei-Yuan; Moore, Bradley S.

2013-01-01

Thalassospiramides A and B are immunosuppressant cyclic lipopeptides first reported from the marine α-proteobacterium Thalassospira sp. CNJ-328. We describe here the discovery and characterization of an extended family of 14 new analogues from four Tistrella and Thalassospira isolates. These potent calpain 1 protease inhibitors belong to six structure classes in which the length and composition of the acylpeptide side chain varies extensively. Genomic sequence analysis of the thalassospiramide-producing microbes revealed related, genus-specific biosynthetic loci encoding hybrid nonribosomal peptide synthetase/polyketide synthases consistent with thalassospiramide assembly. The bioinformatics analysis of the gene clusters suggests that structural diversity, which ranges from the 803.4 Da thalassospiramide C to the 1291.7 Da thalassospiramide F, results from a complex sequence of reactions involving amino acid substrate channeling and enzymatic multi-module skipping and iteration. Preliminary biochemical analysis of the N-terminal NRPS module from the Thalassospira TtcA megasynthase supports a biosynthetic model in which in cis amino acid activation competes with in trans activation to increase the range of amino acid substrates incorporated at the N-terminus. PMID:23270364

Spontaneous Droplet Motion on a Periodically Compliant Substrate.

PubMed

Liu, Tianshu; Nadermann, Nichole; He, Zhenping; Strogatz, Steven H; Hui, Chung-Yuen; Jagota, Anand

2017-05-23

Droplet motion arises in many natural phenomena, ranging from the familiar gravity-driven slip and arrest of raindrops on windows to the directed transport of droplets for water harvesting by plants and animals under dry conditions. Deliberate transportation and manipulation of droplets are also important in many technological applications, including droplet-based microfluidic chemical reactors and for thermal management. Droplet motion usually requires gradients of surface energy or temperature or external vibration to overcome contact angle hysteresis. Here, we report a new phenomenon in which a drying droplet placed on a periodically compliant surface undergoes spontaneous, erratic motion in the absence of surface energy gradients and external stimuli such as vibration. By modeling the droplet as a mass-spring system on a substrate with periodically varying compliance, we show that the stability of equilibrium depends on the size of the droplet. Specifically, if the center of mass of the drop lies at a stable equilibrium point of the system, it will stay there until evaporation reduces its size and this fixed point becomes unstable; with any small perturbation, the droplet then moves to one of its neighboring fixed points.

Hide and seek: How do DNA glycosylases locate oxidatively damaged DNA bases amidst a sea of undamaged bases?

PubMed

Lee, Andrea J; Wallace, Susan S

2017-06-01

The first step of the base excision repair (BER) pathway responsible for removing oxidative DNA damage utilizes DNA glycosylases to find and remove the damaged DNA base. How glycosylases find the damaged base amidst a sea of undamaged bases has long been a question in the BER field. Single molecule total internal reflection fluorescence microscopy (SM TIRFM) experiments have allowed for an exciting look into this search mechanism and have found that DNA glycosylases scan along the DNA backbone in a bidirectional and random fashion. By comparing the search behavior of bacterial glycosylases from different structural families and with varying substrate specificities, it was found that glycosylases search for damage by periodically inserting a wedge residue into the DNA stack as they redundantly search tracks of DNA that are 450-600bp in length. These studies open up a wealth of possibilities for further study in real time of the interactions of DNA glycosylases and other BER enzymes with various DNA substrates. Copyright © 2016 Elsevier Inc. All rights reserved.

Wireless Open-Circuit In-Plane Strain and Displacement Sensor Requiring No Electrical Connections

NASA Technical Reports Server (NTRS)

Woodard, Stanley E. (Inventor)

2014-01-01

A wireless in-plane strain and displacement sensor includes an electrical conductor fixedly coupled to a substrate subject to strain conditions. The electrical conductor is shaped between its ends for storage of an electric field and a magnetic field, and remains electrically unconnected to define an unconnected open-circuit having inductance and capacitance. In the presence of a time-varying magnetic field, the electrical conductor so-shaped resonates to generate harmonic electric and magnetic field responses. The sensor also includes at least one electrically unconnected electrode having an end and a free portion extending from the end thereof. The end of each electrode is fixedly coupled to the substrate and the free portion thereof remains unencumbered and spaced apart from a portion of the electrical conductor so-shaped. More specifically, at least some of the free portion is disposed at a location lying within the magnetic field response generated by the electrical conductor. A motion guidance structure is slidingly engaged with each electrode's free portion in order to maintain each free portion parallel to the electrical conductor so-shaped.

Substrate specificity and pH dependence of homogeneous wheat germ acid phosphatase.

PubMed

Van Etten, R L; Waymack, P P

1991-08-01

The broad substrate specificity of a homogeneous isoenzyme of wheat germ acid phosphatase (WGAP) was extensively investigated by chromatographic, electrophoretic, NMR, and kinetic procedures. WGAP exhibited no divalent metal ion requirement and was unaffected upon incubation with EDTA or o-phenanthroline. A comparison of two catalytically homogeneous isoenzymes revealed little difference in substrate specificity. The specificity of WGAP was established by determining the Michaelis constants for a wide variety of substrates. p-Nitrophenyl phosphate, pyrophosphate, tripolyphosphate, and ATP were preferred substrates while lesser activities were seen toward sugar phosphates, trimetaphosphate, phosphoproteins, and (much less) phosphodiesters. An extensive table of Km and Vmax values is given. The pathway for the hydrolysis of trimetaphosphate was examined by colorimetric and 31P NMR methods and it was found that linear tripolyphosphate is not a free intermediate in the enzymatic reaction. In contrast to literature reports, homogeneous wheat germ acid phosphatase exhibits no measurable carboxylesterase activity, nor does it hydrolyze phenyl phosphonothioate esters or phytic acid at significant rates.

Properties of Silica-Based Aerogel Substrates and Application to C-Band Circular Patch Antenna

NASA Astrophysics Data System (ADS)

Abdel-Rahman, Mohamed; Haraz, Osama M.; Ashraf, Nadeem; Zia, Muhammad Fakhar; Khaled, Usama; Elsahfiey, Ibrahim; Alshebeili, Saleh; Sebak, Abdel Razik

2018-03-01

Silica aerogel is a lightweight and low-permittivity dielectric material that possesses attractive features for use as an antenna substrate. In this paper, we characterize the radio frequency and microwave dielectric permittivity properties of substrates composed of silica aerogel encapsulated in polymer aerogel in the frequency range from 10 MHz to 8.5 GHz. Characterized silica-based aerogel substrates show relative permittivity values varying between 1.055 and 1.25 and loss tangent values ranging from 5.08 × 10-4 to 0.0206. Silica-based aerogel substrates thus have the potential of use in designing antennas with high gain and large bandwidth. Validation is presented by characterizing the performance of a manufactured C-band circular patch antenna on silica-based aerogel substrate. The performance is also compared to a design that uses Rogers Duroid RT5880 substrate. The results reveal that the silica aerogel substrate antenna at 7.2 GHz provides 1.5 dB increase in gain, 88% enhancement in bandwidth and 68.5% reduction in mass, in comparison with the antenna on RT5880 substrate.

Optimization of process parameters for RF sputter deposition of tin-nitride thin-films

NASA Astrophysics Data System (ADS)

Jangid, Teena; Rao, G. Mohan

2018-05-01

Radio frequency Magnetron sputtering technique was employed to deposit Tin-nitride thin films on Si and glass substrate at different process parameters. Influence of varying parameters like substrate temperature, target-substrate distance and RF power is studied in detail. X-ray diffraction method is used as a key technique for analyzing the changes in the stoichiometric and structural properties of the deposited films. Depending on the combination of deposition parameters, crystalline as well as amorphous films were obtained. Pure tin-nitride thin films were deposited at 15W RF power and 600°C substrate temperature with target-substrate distance fixed at 10cm. Bandgap value of 1.6 eV calculated for the film deposited at optimum process conditions matches well with reported values.

Synthesis of ceria based superhydrophobic coating on Ni20Cr substrate via cathodic electrodeposition.

PubMed

Pedraza, F; Mahadik, S A; Bouchaud, B

2015-12-21

In this work, superhydrophobic cerium oxide coating surface (111) with dual scale texture on Ni20Cr substrate is obtained by combination of electropolishing the substrate and subsequent cathodic electrodeposition and long-term UVH surface relaxation. To form hierarchical structures of CeO2 is controllable by varying the substrate roughness, and electropolishing period. The results indicated that at the optimal condition, the surface of the cerium oxide coating showed a superhydrophobicity with a great water contact angle (151.0 ± 1.4°) with Gecko state. An interface model for electropolishing of substrate surface in cerium nitrate medium is proposed. We expect that this facile process can be readily and widely adopted for the design of superhydrophobic coating on engineering materials.

Strontium source and depth of uptake shifts with substrate age in semiarid ecosystems

NASA Astrophysics Data System (ADS)

Coble, Ashley A.; Hart, Stephen C.; Ketterer, Michael E.; Newman, Gregory S.; Kowler, Andrew L.

2015-06-01

Without exogenous rock-derived nutrient sources, terrestrial ecosystems may eventually regress or reach a terminal steady state, but the degree to which exogenous nutrient sources buffer or slow to a theoretical terminal steady state remains unclear. We used strontium isotope ratios (87Sr/86Sr) as a tracer and measured 87Sr/86Sr values in aeolian dust, soils, and vegetation across a well-constrained 3 Myr semiarid substrate age gradient to determine (1) whether the contribution of atmospheric sources of rock-derived nutrients to soil and vegetation pools varied with substrate age and (2) to determine if the depth of uptake varied with substrate age. We found that aeolian-derived nutrients became increasingly important, contributing as much as 71% to plant-available soil pools and tree (Pinus edulis) growth during the latter stages of ecosystem development in a semiarid climate. The depth of nutrient uptake increased on older substrates, demonstrating that trees in arid regions can acquire nutrients from greater depths as ecosystem development progresses presumably in response to nutrient depletion in the more weathered surface soils. Our results demonstrate that global and regional aeolian transport of nutrients to local ecosystems is a vital process for ecosystem development in arid regions. Furthermore, these aeolian nutrient inputs contribute to deep soil nutrient pools, which become increasingly important for maintaining plant productivity over long time scales.

Computer Simulations Reveal Substrate Specificity of Glycosidic Bond Cleavage in Native and Mutant Human Purine Nucleoside Phosphorylase.

PubMed

Isaksen, Geir Villy; Hopmann, Kathrin Helen; Åqvist, Johan; Brandsdal, Bjørn Olav

2016-04-12

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribonucleosides and 2'-deoxyribonucleosides, yielding the purine base and (2'-deoxy)ribose 1-phosphate as products. While this enzyme has been extensively studied, several questions with respect to the catalytic mechanism have remained largely unanswered. The role of the phosphate and key amino acid residues in the catalytic reaction as well as the purine ring protonation state is elucidated using density functional theory calculations and extensive empirical valence bond (EVB) simulations. Free energy surfaces for adenosine, inosine, and guanosine are fitted to ab initio data and yield quantitative agreement with experimental data when the surfaces are used to model the corresponding enzymatic reactions. The cognate substrates 6-aminopurines (inosine and guanosine) interact with PNP through extensive hydrogen bonding, but the substrate specificity is found to be a direct result of the electrostatic preorganization energy along the reaction coordinate. Asn243 has previously been identified as a key residue providing substrate specificity. Mutation of Asn243 to Asp has dramatic effects on the substrate specificity, making 6-amino- and 6-oxopurines equally good as substrates. The principal effect of this particular mutation is the change in the electrostatic preorganization energy between the native enzyme and the Asn243Asp mutant, clearly favoring adenosine over inosine and guanosine. Thus, the EVB simulations show that this particular mutation affects the electrostatic preorganization of the active site, which in turn can explain the substrate specificity.

Soil functional diversity analysis of a bauxite-mined restoration chronosequence.

PubMed

Lewis, Dawn E; White, John R; Wafula, Denis; Athar, Rana; Dickerson, Tamar; Williams, Henry N; Chauhan, Ashvini

2010-05-01

Soil microorganisms are sensitive to environmental perturbations such that changes in microbial community structure and function can provide early signs of anthropogenic disturbances and even predict restoration success. We evaluated the bacterial functional diversity of un-mined and three chronosequence sites at various stages of rehabilitation (0, 10, and 20 years old) located in the Mocho Mountains of Jamaica. Samples were collected during the dry and wet seasons and analyzed for metal concentrations, microbial biomass carbon, bacterial numbers, and functional responses of soil microbiota using community-level physiological profile (CLPP) assays. Regardless of the season, un-mined soils consisted of higher microbial biomass and numbers than any of the rehabilitated sites. Additionally, the number and rate of substrates utilized and substrate evenness (the distribution of color development between the substrates) were significantly greater in the un-mined soils with carbohydrates being preferentially utilized than amino acids, polymers, carboxylic acids, and esters. To some extent, functional responses varied with the seasons but the least physiological activity was shown by the site rehabilitated in 1987 indicating long-term perturbation to this ecosystem. Small subunit ribosomal DNA (SSUrDNA)-denaturing gradient-gel electrophoresis analyses on the microbiota collected from the most preferred CLPP substrates followed by taxonomic analyses showed Proteobacteria, specifically the gamma-proteobacteria, as the most functionally active phyla, indicating a propensity of this phyla to out-compete other groups under the prevailing conditions. Additionally, multivariate statistical analyses, Shannon's diversity, and evenness indices, principal component analysis, biplot and un-weighted-pair-group method with arithmetic averages dendrograms further confirmed that un-mined sites were distinctly different from the rehabilitated soils.

Intraspecific Colour Variation among Lizards in Distinct Island Environments Enhances Local Camouflage

PubMed Central

Marshall, Kate L. A.; Philpot, Kate E.; Damas-Moreira, Isabel; Stevens, Martin

2015-01-01

Within-species colour variation is widespread among animals. Understanding how this arises can elucidate evolutionary mechanisms, such as those underlying reproductive isolation and speciation. Here, we investigated whether five island populations of Aegean wall lizards (Podarcis erhardii) have more effective camouflage against their own (local) island substrates than against other (non-local) island substrates to avian predators, and whether this was linked to island differences in substrate appearance. We also investigated whether degree of local substrate matching varied among island populations and between sexes. In most populations, both sexes were better matched against local backgrounds than against non-local backgrounds, particularly in terms of luminance (perceived lightness), which usually occurred when local and non-local backgrounds were different in appearance. This was found even between island populations that historically had a land connection and in populations that have been isolated relatively recently, suggesting that isolation in these distinct island environments has been sufficient to cause enhanced local background matching, sometimes on a rapid evolutionary time-scale. However, heightened local matching was poorer in populations inhabiting more variable and unstable environments with a prolonged history of volcanic activity. Overall, these results show that lizard coloration is tuned to provide camouflage in local environments, either due to genetic adaptation or changes during development. Yet, the occurrence and extent of selection for local matching may depend on specific conditions associated with local ecology and biogeographic history. These results emphasize how anti-predator adaptations to different environments can drive divergence within a species, which may contribute to reproductive isolation among populations and lead to ecological speciation. PMID:26372454

Intraspecific Colour Variation among Lizards in Distinct Island Environments Enhances Local Camouflage.

PubMed

Marshall, Kate L A; Philpot, Kate E; Damas-Moreira, Isabel; Stevens, Martin

2015-01-01

Within-species colour variation is widespread among animals. Understanding how this arises can elucidate evolutionary mechanisms, such as those underlying reproductive isolation and speciation. Here, we investigated whether five island populations of Aegean wall lizards (Podarcis erhardii) have more effective camouflage against their own (local) island substrates than against other (non-local) island substrates to avian predators, and whether this was linked to island differences in substrate appearance. We also investigated whether degree of local substrate matching varied among island populations and between sexes. In most populations, both sexes were better matched against local backgrounds than against non-local backgrounds, particularly in terms of luminance (perceived lightness), which usually occurred when local and non-local backgrounds were different in appearance. This was found even between island populations that historically had a land connection and in populations that have been isolated relatively recently, suggesting that isolation in these distinct island environments has been sufficient to cause enhanced local background matching, sometimes on a rapid evolutionary time-scale. However, heightened local matching was poorer in populations inhabiting more variable and unstable environments with a prolonged history of volcanic activity. Overall, these results show that lizard coloration is tuned to provide camouflage in local environments, either due to genetic adaptation or changes during development. Yet, the occurrence and extent of selection for local matching may depend on specific conditions associated with local ecology and biogeographic history. These results emphasize how anti-predator adaptations to different environments can drive divergence within a species, which may contribute to reproductive isolation among populations and lead to ecological speciation.

A magnetically actuated cellular strain assessment tool for quantitative analysis of strain induced cellular reorientation and actin alignment

NASA Astrophysics Data System (ADS)

Khademolhosseini, F.; Liu, C.-C.; Lim, C. J.; Chiao, M.

2016-08-01

Commercially available cell strain tools, such as pneumatically actuated elastomer substrates, require special culture plates, pumps, and incubator setups. In this work, we present a magnetically actuated cellular strain assessment tool (MACSAT) that can be implemented using off-the-shelf components and conventional incubators. We determine the strain field on the MACSAT elastomer substrate using numerical models and experimental measurements and show that a specific region of the elastomer substrate undergoes a quasi-uniaxial 2D stretch, and that cells confined to this region of the MACSAT elastomer substrate undergo tensile, compressive, or zero axial strain depending on their angle of orientation. Using the MACSAT to apply cyclic strain on endothelial cells, we demonstrate that actin filaments within the cells reorient away from the stretching direction, towards the directions of minimum axial strain. We show that the final actin orientation angles in strained cells are spread over a region of compressive axial strain, confirming previous findings on the existence of a varied pre-tension in the actin filaments of the cytoskeleton. We also demonstrate that strained cells exhibit distinctly different values of actin alignment coherency compared to unstrained cells and therefore propose that this parameter, i.e., the coherency of actin alignment, can be used as a new readout to determine the occurrence/extent of actin alignment in cell strain experiments. The tools and methods demonstrated in this study are simple and accessible and can be easily replicated by other researchers to study the strain response of other adherent cells.

Germination and seedling establishment in orchids: a complex of requirements.

PubMed

Rasmussen, Hanne N; Dixon, Kingsley W; Jersáková, Jana; Těšitelová, Tamara

2015-09-01

Seedling recruitment is essential to the sustainability of any plant population. Due to the minute nature of seeds and early-stage seedlings, orchid germination in situ was for a long time practically impossible to observe, creating an obstacle towards understanding seedling site requirements and fluctuations in orchid populations. The introduction of seed packet techniques for sowing and retrieval in natural sites has brought with it important insights, but many aspects of orchid seed and germination biology remain largely unexplored. The germination niche for orchids is extremely complex, because it is defined by requirements not only for seed lodging and germination, but also for presence of a fungal host and its substrate. A mycobiont that the seedling can parasitize is considered an essential element, and a great diversity of Basidiomycota and Ascomycota have now been identified for their role in orchid seed germination, with fungi identifiable as imperfect Rhizoctonia species predominating. Specificity patterns vary from orchid species employing a single fungal lineage to species associating individually with a limited selection of distantly related fungi. A suitable organic carbon source for the mycobiont constitutes another key requirement. Orchid germination also relies on factors that generally influence the success of plant seeds, both abiotic, such as light/shade, moisture, substrate chemistry and texture, and biotic, such as competitors and antagonists. Complexity is furthermore increased when these factors influence seeds/seedling, fungi and fungal substrate differentially. A better understanding of germination and seedling establishment is needed for conservation of orchid populations. Due to the obligate association with a mycobiont, the germination niches in orchid species are extremely complex and varied. Microsites suitable for germination can be small and transient, and direct observation is difficult. An experimental approach using several levels of environmental manipulation/control is recommended. © The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Study of Enzymatic Hydrolysis of Fructans from Agave salmiana Characterization and Kinetic Assessment

PubMed Central

Michel-Cuello, Christian; Ortiz-Cerda, Imelda; Moreno-Vilet, Lorena; Grajales-Lagunes, Alicia; Moscosa-Santillán, Mario; Bonnin, Johanne; González-Chávez, Marco Martín; Ruiz-Cabrera, Miguel

2012-01-01

Fructans were extracted from Agave salmiana juice, characterized and subjected to hydrolysis process using a commercial inulinase preparation acting freely. To compare the performance of the enzymatic preparation, a batch of experiments were also conducted with chicory inulin (reference). Hydrolysis was performed for 6 h at two temperatures (50, 60°C) and two substrate concentrations (40, 60 mg/ml). Hydrolysis process was monitored by measuring the sugars released and residual substrate by HPLC. A mathematical model which describes the kinetics of substrate degradation as well as fructose production was proposed to analyze the hydrolysis assessment. It was found that kinetics were significantly influenced by temperature, substrate concentration, and type of substrate (P < 0.01). The extent of substrate hydrolysis varied from 82 to 99%. Hydrolysis product was mainly constituted of fructose, obtaining from 77 to 96.4% of total reducing sugars. PMID:22629216

Growth of porous anodized alumina on the sputtered aluminum films with 2D-3D morphology for high specific surface area

NASA Astrophysics Data System (ADS)

Liao, M. W.; Chung, C. K.

2014-08-01

The porous anodic aluminum oxide (AAO) with high-aspect-ratio pore channels is widely used as a template for fabricating nanowires or other one-dimensional (1D) nanostructures. The high specific surface area of AAO can also be applied to the super capacitor and the supporting substrate for catalysis. The rough surface could be helpful to enhance specific surface area but it generally results in electrical field concentration even to ruin AAO. In this article, the aluminum (Al) films with the varied 2D-3D morphology on Si substrates were prepared using magnetron sputtering at a power of 50 W-185 W for 1 h at a working pressure of 2.5 × 10-1 Pa. Then, AAO was fabricated from the different Al films by means of one-step hybrid pulse anodizing (HPA) between the positive 40 V and the negative -2 V (1 s:1 s) for 3 min in 0.3 M oxalic acid at a room temperature. The microstructure and morphology of Al films were characterized by X-ray diffraction, scanning electron microscope and atomic force microscope, respectively. Some hillocks formed at the high target power could be attributed to the grain texture growth in the normal orientation of Al(1 1 1). The 3D porous AAO structure which is different from the conventional 2D planar one has been successfully demonstrated using HPA on the film with greatly rough hillock-surface formed at the highest power of 185 W. It offers a potential application of the new 3D AAO to high specific surface area devices.

Resveratrol serves as a protein-substrate interaction stabilizer in human SIRT1 activation

NASA Astrophysics Data System (ADS)

Hou, Xuben; Rooklin, David; Fang, Hao; Zhang, Yingkai

2016-11-01

Resveratrol is a natural compound found in red wine that has been suggested to exert its potential health benefit through the activation of SIRT1, a crucial member of the mammalian NAD+-dependent deacetylases. SIRT1 has emerged as an attractive therapeutic target for many aging related diseases, however, how its activity can only be activated toward some specific substrates by resveratrol has been poorly understood. Herein, by employing extensive molecular dynamics simulations as well as fragment-centric topographical mapping of binding interfaces, we have clarified current controversies in the literature and elucidated that resveratrol plays an important activation role by stabilizing SIRT1/peptide interactions in a substrate-specific manner. This new mechanism highlights the importance of the N-terminal domain in substrate recognition, explains the activity restoration role of resveratrol toward some “loose-binding” substrates of SIRT1, and has significant implications for the rational design of new substrate-specific SIRT1 modulators.

Measuring the Global Substrate Specificity of Mycobacterial Serine Hydrolases Using a Library of Fluorogenic Ester Substrates.

PubMed

Bassett, Braden; Waibel, Brent; White, Alex; Hansen, Heather; Stephens, Dominique; Koelper, Andrew; Larsen, Erik M; Kim, Charles; Glanzer, Adam; Lavis, Luke D; Hoops, Geoffrey C; Johnson, R Jeremy

2018-04-16

Among the proteins required for lipid metabolism in Mycobacterium tuberculosis are a significant number of uncharacterized serine hydrolases, especially lipases and esterases. Using a streamlined synthetic method, a library of immolative fluorogenic ester substrates was expanded to better represent the natural lipidomic diversity of Mycobacterium. This expanded fluorogenic library was then used to rapidly characterize the global structure activity relationship (SAR) of mycobacterial serine hydrolases in M. smegmatis under different growth conditions. Confirmation of fluorogenic substrate activation by mycobacterial serine hydrolases was performed using nonspecific serine hydrolase inhibitors and reinforced the biological significance of the SAR. The hydrolases responsible for the global SAR were then assigned using gel-resolved activity measurements, and these assignments were used to rapidly identify the relative substrate specificity of previously uncharacterized mycobacterial hydrolases. These measurements provide a global SAR of mycobacterial hydrolase activity, a picture of cycling hydrolase activity, and a detailed substrate specificity profile for previously uncharacterized hydrolases.

Resveratrol serves as a protein-substrate interaction stabilizer in human SIRT1 activation.

PubMed

Hou, Xuben; Rooklin, David; Fang, Hao; Zhang, Yingkai

2016-11-30

Resveratrol is a natural compound found in red wine that has been suggested to exert its potential health benefit through the activation of SIRT1, a crucial member of the mammalian NAD + -dependent deacetylases. SIRT1 has emerged as an attractive therapeutic target for many aging related diseases, however, how its activity can only be activated toward some specific substrates by resveratrol has been poorly understood. Herein, by employing extensive molecular dynamics simulations as well as fragment-centric topographical mapping of binding interfaces, we have clarified current controversies in the literature and elucidated that resveratrol plays an important activation role by stabilizing SIRT1/peptide interactions in a substrate-specific manner. This new mechanism highlights the importance of the N-terminal domain in substrate recognition, explains the activity restoration role of resveratrol toward some "loose-binding" substrates of SIRT1, and has significant implications for the rational design of new substrate-specific SIRT1 modulators.

Engineering the substrate specificity of the DhbE adenylation domain by yeast cell surface display.

PubMed

Zhang, Keya; Nelson, Kathryn M; Bhuripanyo, Karan; Grimes, Kimberly D; Zhao, Bo; Aldrich, Courtney C; Yin, Jun

2013-01-24

The adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) activate aryl acids or amino acids to launch their transfer through the NRPS assembly line for the biosynthesis of many medicinally important natural products. In order to expand the substrate pool of NRPSs, we developed a method based on yeast cell surface display to engineer the substrate specificities of the A-domains. We acquired A-domain mutants of DhbE that have 11- and 6-fold increases in k(cat)/K(m) with nonnative substrates 3-hydroxybenzoic acid and 2-aminobenzoic acid, respectively and corresponding 3- and 33-fold decreases in k(cat)/K(m) values with the native substrate 2,3-dihydroxybenzoic acid, resulting in a dramatic switch in substrate specificity of up to 200-fold. Our study demonstrates that yeast display can be used as a high throughput selection platform to reprogram the "nonribosomal code" of A-domains. Copyright © 2013 Elsevier Ltd. All rights reserved.

Magnetic coupling between liquid 3He and a solid state substrate: a new approach

NASA Astrophysics Data System (ADS)

Klochkov, Alexander V.; Naletov, Vladimir V.; Tayurskii, Dmitrii A.; Tagirov, Murat S.; Suzuki, Haruhiko

2000-07-01

We suggest a new approach for solving the long-standing problem of a magnetic coupling between liquid 3He and a solid state substrate at temperatures above the Fermi temperature. The approach is based on our previous careful investigations of the physical state of a solid substrate by means of several experimental methods (EPR, NMR, conductometry, and magnetization measurements). The developed approach allows, first, to get more detailed information about the magnetic coupling phenomenon by varying the repetition time in pulse NMR investigations of liquid 3He in contact with the solid state substrate and, second, to compare the obtained dependences and the data of NMR-cryoporometry and AFM-microscopy.

Substrate-Driven Mapping of the Degradome by Comparison of Sequence Logos

PubMed Central

Fuchs, Julian E.; von Grafenstein, Susanne; Huber, Roland G.; Kramer, Christian; Liedl, Klaus R.

2013-01-01

Sequence logos are frequently used to illustrate substrate preferences and specificity of proteases. Here, we employed the compiled substrates of the MEROPS database to introduce a novel metric for comparison of protease substrate preferences. The constructed similarity matrix of 62 proteases can be used to intuitively visualize similarities in protease substrate readout via principal component analysis and construction of protease specificity trees. Since our new metric is solely based on substrate data, we can engraft the protease tree including proteolytic enzymes of different evolutionary origin. Thereby, our analyses confirm pronounced overlaps in substrate recognition not only between proteases closely related on sequence basis but also between proteolytic enzymes of different evolutionary origin and catalytic type. To illustrate the applicability of our approach we analyze the distribution of targets of small molecules from the ChEMBL database in our substrate-based protease specificity trees. We observe a striking clustering of annotated targets in tree branches even though these grouped targets do not necessarily share similarity on protein sequence level. This highlights the value and applicability of knowledge acquired from peptide substrates in drug design of small molecules, e.g., for the prediction of off-target effects or drug repurposing. Consequently, our similarity metric allows to map the degradome and its associated drug target network via comparison of known substrate peptides. The substrate-driven view of protein-protein interfaces is not limited to the field of proteases but can be applied to any target class where a sufficient amount of known substrate data is available. PMID:24244149

In situ SERS spectroelectrochemical analysis of antioxidants deposited on copper substrates: What is the effect of applied potential on sorption behavior?

NASA Astrophysics Data System (ADS)

Dendisova-Vyskovska, Marcela; Broncova, Gabriela; Clupek, Martin; Prokopec, Vadym; Matejka, Pavel

2012-12-01

The detection of p-coumaric acid and ferulic acid using a combined in situ electrochemical and surface-enhanced Raman scattering spectroscopic technique in specially made electrode cell is described. New in situ spectroelectrochemical cell was designed as the three-electrode arrangement connected via positioning device to fiber-optic probe of Raman spectrometer Dimension P2 (excitation wavelength 785 nm). In situ SERS spectra of p-coumaric acid and ferulic acid were recorded at varying applied negative potentials to copper substrates. The spectral intensities and shapes of bands as well as spatial orientation of molecules on the surface depend significantly on varying values of the applied electrode potential. The change of electrode potential influences analyte adsorption/desorption behavior on the surface of copper substrates, affecting the reversibility of the whole process and overall spectral enhancement level. Principal component analysis is used to distinguish several stages of spectral variations on potential changes.

Quantitative time-resolved analysis reveals intricate, differential regulation of standard- and immuno-proteasomes

PubMed Central

Liepe, Juliane; Holzhütter, Hermann-Georg; Bellavista, Elena; Kloetzel, Peter M; Stumpf, Michael PH; Mishto, Michele

2015-01-01

Proteasomal protein degradation is a key determinant of protein half-life and hence of cellular processes ranging from basic metabolism to a host of immunological processes. Despite its importance the mechanisms regulating proteasome activity are only incompletely understood. Here we use an iterative and tightly integrated experimental and modelling approach to develop, explore and validate mechanistic models of proteasomal peptide-hydrolysis dynamics. The 20S proteasome is a dynamic enzyme and its activity varies over time because of interactions between substrates and products and the proteolytic and regulatory sites; the locations of these sites and the interactions between them are predicted by the model, and experimentally supported. The analysis suggests that the rate-limiting step of hydrolysis is the transport of the substrates into the proteasome. The transport efficiency varies between human standard- and immuno-proteasomes thereby impinging upon total degradation rate and substrate cleavage-site usage. DOI: http://dx.doi.org/10.7554/eLife.07545.001 PMID:26393687

Effect of nanoparticle size on sessile droplet contact angle

NASA Astrophysics Data System (ADS)

Munshi, A. M.; Singh, V. N.; Kumar, Mukesh; Singh, J. P.

2008-04-01

We report a significant variation in the static contact angle measured on indium oxide (IO) nanoparticle coated Si substrates that have different nanoparticle sizes. These IO nanoparticles, which have well defined shape and sizes, were synthesized by chemical vapor deposition in a horizontal alumina tube furnace. The size of the IO nanoparticles was varied by changing the source material, substrate temperature, and the deposition time. A sessile droplet method was used to determine the macroscopic contact angle on these IO nanoparticle covered Si substrate using two different liquids: de-ionized water and diethylene glycol (DEG). It was observed that contact angle depends strongly on the nanoparticle size. The contact angle was found to vary from 24° to 67° for de-ionized water droplet and from 15° to 60° for DEG droplet, for the nanoparticle sizes varying from 14 to 620 nm. The contact angle decreases with a decrease in the particles size. We have performed a theoretical analysis to determine the dependence of contact angle on the nanoparticle size. This formulation qualitatively shows a similar trend of decrease in the contact angle with a decrease in nanoparticle size. Providing a rough estimate of nanoparticle size by sessile droplet contact angle measurement is the novelty in this work.

Lightweight Electrode For Nickel/Hydrogen Cell

NASA Technical Reports Server (NTRS)

Britton, Doris L.

1994-01-01

Improved substrate for nickel electrode increases specific energy of nickel/hydrogen cell. Consists of 50 percent by weight nickel fiber, 35 percent nickel powder, and 15 percent cobalt powder. Porosity and thickness of nickel electrodes affect specific energy, initial performance, and cycle life of cell. Substrate easily manufactured with much larger porosities than those of heavy-sintered state-of-art nickel substrate.

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

Crosby, Heidi A; Pelletier, Dale A; Hurst, Gregory

Background: Protein acetylation is widespread in prokaryotes. Results: Six new acyl-CoA synthetases whose activities are controlled by acetylation were identified, and their substrate preference established. A new protein acetyltransferase was also identified and its substrate specificity determined. Conclusion: Protein acetyltransferases acetylate a conserved lysine residue in protein substrates. Significance: The R. palustris Pat enzyme specifically acetylates AMP-forming acyl-CoA synthetases and regulates fatty acid metabolism.

Substrate-Versatile Approach to Robust Antireflective and Superhydrophobic Coatings with Excellent Self-Cleaning Property in Varied Environments.

PubMed

Ren, Tingting; He, Junhui

2017-10-04

Robust antireflective and superhydrophobic coatings are highly desired in wide applications, such as optical devices, solar cell panels, architectural and automotive glasses, lab-on chip systems, and windows for electronic devices. Meanwhile, simple, low-cost, and substrate-versatile fabrication is also essential toward real applications of such coatings. Herein, we developed a substrate-versatile strategy to fabricate robust antireflective and superhydrophobic coatings with excellent self-cleaning property in varied environments, including air and oil and after oil contamination. A mixed ethanol suspension, which consists of 1H,1H,2H,2H-perfluorooctyltriethoxysilane modified dual-sized silica nanoparticles and acid-catalyzed silica precursor, was first synthesized. The acid-catalyzed silica precursor could help to form a highly cross-linked silica network by connecting the silica nanoparticles, thus significantly enhancing the robustness of coatings. The as-prepared coatings were able to withstand a water drop impact test, sand abrasion test, tape adhesion test, and knife and pencil scratching tests. More importantly, it was also found that the wettability and self-cleaning property of coatings after oil contamination were surprisingly different from those in air and oil. These observations are explainable by the alteration of interface; i.e., the alteration of interface has significant effects on the functional properties of coatings. Additionally, the mixed suspension could be sprayed onto various hard and soft substrates including glass, polyethylene terephthalate (PET), polycarbonate (PC), and poly(methyl methacrylate) (PMMA), opening up a feasible route toward varied practical applications in solar cell panels, optical devices, architectural and automotive glasses, droplet manipulators, and fluid control.

Physical activity as a metabolic stressor.

PubMed

Coyle, E F

2000-08-01

Both physical activity and diet stimulate processes that, over time, alter the morphologic composition and biochemical function of the body. Physical activity provides stimuli that promote very specific and varied adaptations according to the type, intensity, and duration of exercise performed. There is further interest in the extent to which diet or supplementation can enhance the positive stimuli. Prolonged walking at low intensity presents little metabolic, hormonal, or cardiovascular stress, and the greatest perturbation from rest appears to be from increased fat oxidation and plasma free fatty acid mobilization resulting from a combination of increased lipolysis and decreased reesterification. More intense jogging or running largely stimulates increased oxidation of glycogen and triacylglycerol, both of which are stored directly within the muscle fibers. Furthermore, these intramuscular stores of carbohydrate and fat appear to be the primary substrates for the enhanced oxidative and performance ability derived from endurance training-induced increases in muscle mitochondrial density. Weightlifting that produces fatigue in brief periods (ie, in 15-90 s and after 15 repetitive contractions) elicits a high degree of motor unit recruitment and muscle fiber stimulation. This is a remarkably potent stimulus for altering protein synthesis in muscle and increasing neuromuscular function. The metabolic stress of physical activity can be measured by substrate turnover and depletion, cardiovascular response, hormonal perturbation, accumulation of metabolites, or even the extent to which the synthesis and degradation of specific proteins are altered, either acutely or by chronic exercise training.

Induction of multixenobiotic defense mechanisms in resistant Daphnia magna clones as a general cellular response to stress.

PubMed

Jordão, Rita; Campos, Bruno; Lemos, Marco F L; Soares, Amadeu M V M; Tauler, Romà; Barata, Carlos

2016-06-01

Multixenobiotic resistance mechanisms (MXR) were recently identified in Daphnia magna. Previous results characterized gene transcripts of genes encoding and efflux activities of four putative ABCB1 and ABCC transporters that were chemically induced but showed low specificity against model transporter substrates and inhibitors, thus preventing us from distinguishing between activities of different efflux transporter types. In this study we report on the specificity of induction of ABC transporters and of the stress protein hsp70 in clones selected to be genetically resistant to ABCB1 chemical substrates. Clones resistant to mitoxantrone, ivermectin and pentachlorophenol showed distinctive transcriptional responses of transporter protein coding genes and of putative transporter dye activities. Expression of hsp70 proteins also varied across resistant clones. Clones resistant to mitoxantrone and pentachlorophenol showed high constitutive levels of hsp70. Transcriptional levels of the abcb1 gene transporter and of putative dye transporter activity were also induced to a greater extent in the pentachlorophenol resistant clone. Observed higher dye transporter activities in individuals from clones resistant to mitoxantrone and ivermectin were unrelated with transcriptional levels of the studied four abcc and abcb1 transporter genes. These findings suggest that Abcb1 induction in D. magna may be a part of a general cellular stress response. Copyright © 2016 Elsevier B.V. All rights reserved.

Expression of cholinesterase gene(s) in human brain tissues: translational evidence for multiple mRNA species.

PubMed Central

Soreq, H; Zevin-Sonkin, D; Razon, N

1984-01-01

To resolve the origin(s) of the molecular heterogeneity of human nervous system cholinesterases (ChEs), we used Xenopus oocytes, which produce biologically active ChE when microinjected with unfractionated brain mRNA. The RNA was prepared from primary gliomas, meningiomas and embryonic brain, each of which expresses ChE activity with distinct substrate specificities and molecular forms. Sucrose gradient fractionation of DMSO-denatured mRNA from these sources revealed three size classes of ChE-inducing mRNAs, sedimenting at approximately 32S, 20S and 9S. The amounts of these different classes of ChE-inducing mRNAs varied between the three tissue sources examined. To distinguish between ChEs produced in oocytes and having different substrate specificities, their activity was determined in the presence of selective inhibitors. Both 'true' (acetylcholine hydrolase, EC 3.1.1.7) and 'pseudo' (acylcholine acylhydrolase, EC 3.1.1.8) multimeric cholinesterase activities were found in the mRNA-injected oocytes. Moreover, human brain mRNAs inducing 'true' and 'pseudo' ChE activities had different size distribution, indicating that different mRNAs might be translated into various types of ChEs. These findings imply that the heterogeneity of ChEs in the human nervous system is not limited to the post-translational level, but extends to the level of mRNA. PMID:6745236

Substrate-Specific Development of Thermophilic Bacterial Consortia by Using Chemically Pretreated Switchgrass.

PubMed

Eichorst, Stephanie A; Joshua, Chijioke; Sathitsuksanoh, Noppadon; Singh, Seema; Simmons, Blake A; Singer, Steven W

2014-12-01

Microbial communities that deconstruct plant biomass have broad relevance in biofuel production and global carbon cycling. Biomass pretreatments reduce plant biomass recalcitrance for increased efficiency of enzymatic hydrolysis. We exploited these chemical pretreatments to study how thermophilic bacterial consortia adapt to deconstruct switchgrass (SG) biomass of various compositions. Microbial communities were adapted to untreated, ammonium fiber expansion (AFEX)-pretreated, and ionic-liquid (IL)-pretreated SG under aerobic, thermophilic conditions using green waste compost as the inoculum to study biomass deconstruction by microbial consortia. After microbial cultivation, gravimetric analysis of the residual biomass demonstrated that both AFEX and IL pretreatment enhanced the deconstruction of the SG biomass approximately 2-fold. Two-dimensional nuclear magnetic resonance (2D-NMR) experiments and acetyl bromide-reactive-lignin analysis indicated that polysaccharide hydrolysis was the dominant process occurring during microbial biomass deconstruction, and lignin remaining in the residual biomass was largely unmodified. Small-subunit (SSU) rRNA gene amplicon libraries revealed that although the dominant taxa across these chemical pretreatments were consistently represented by members of the Firmicutes, the Bacteroidetes, and Deinococcus-Thermus, the abundance of selected operational taxonomic units (OTUs) varied, suggesting adaptations to the different substrates. Combining the observations of differences in the community structure and the chemical and physical structure of the biomass, we hypothesize specific roles for individual community members in biomass deconstruction. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Thermally Dried Ink-Jet Process for 6,13-Bis(triisopropylsilylethynyl)-Pentacene for High Mobility and High Uniformity on a Large Area Substrate

NASA Astrophysics Data System (ADS)

Ryu, Gi Seong; Lee, Myung Won; Jeong, Seung Hyeon; Song, Chung Kun

2012-05-01

In this study we developed a simple ink-jet process for 6,13-bis(triisopropylsilylethynyl)-pentacene (TIPS-pentacene), which is known as a high-mobility soluble organic semiconductor, to achieve relatively high-mobility and high-uniformity performance for large-area applications. We analyzed the behavior of fluorescent particles in droplets and applied the results to determining a method of controlling the behavior of TIPS-pentacene molecules. The grain morphology of TIPS-pentacene varied depending on the temperature applied to the droplets during drying. We were able to obtain large and uniform grains at 46 °C without any “coffee stain”. The process was applied to a large-size organic thin-film transistor (OTFT) backplane for an electrophoretic display panel containing 192×150 pixels on a 6-in.-sized substrate. The average of mobilities of 36 OTFTs, which were taken from different locations of the backplane, was 0.44±0.08 cm2·V-1·s-1, with a small deviation of 20%, over a 6-in.-size area comprising 28,800 OTFTs. This process providing high mobility and high uniformity can be achieved by simply maintaining the whole area of the substrate at a specific temperature (46 °C in this case) during drying of the droplets.

Macro controlling of copper oxide deposition processes and spray mode by using home-made fully computerized spray pyrolysis system

NASA Astrophysics Data System (ADS)

Essa, Mohammed Sh.; Chiad, Bahaa T.; Shafeeq, Omer Sh.

2017-09-01

Thin Films of Copper Oxide (CuO) absorption layer have been deposited using home-made Fully Computerized Spray Pyrolysis Deposition system FCSPD on glass substrates, at the nozzle to substrate distance equal to 20,35 cm, and computerized spray mode (continues spray, macro-control spray). The substrate temperature has been kept at 450 °c with the optional user can enter temperature tolerance values ± 5 °C. Also that fixed molar concentration of 0.1 M, and 2D platform speed or deposition platform speed of 4mm/s. more than 1000 instruction program code, and specific design of graphical user interface GUI to fully control the deposition process and real-time monitoring and controlling the deposition temperature at every 200 ms. The changing in the temperature has been recorded during deposition processes, in addition to all deposition parameters. The films have been characterized to evaluate the thermal distribution over the X, Y movable hot plate, the structure and optical energy gap, thermal and temperature distribution exhibited a good and uniform distribution over 20 cm2 hot plate area, X-ray diffraction (XRD) measurement revealed that the films are polycrystalline in nature and can be assigned to monoclinic CuO structure. Optical band gap varies from 1.5-1.66 eV depending on deposition parameter.

The characterization of cytosolic glutathione transferase from four species of sea turtles: loggerhead (Caretta caretta), green (Chelonia mydas), olive ridley (Lepidochelys olivacea), and hawksbill (Eretmochelys imbricata).

PubMed

Richardson, Kristine L; Gold-Bouchot, Gerardo; Schlenk, Daniel

2009-08-01

Glutathione s-transferases (GST) play a critical role in the detoxification of exogenous and endogenous electrophiles, as well as the products of oxidative stress. As compared to mammals, GST activity has not been extensively characterized in reptiles. Throughout the globe, most sea turtle populations face the risk of extinction. Of the natural and anthropogenic threats to sea turtles, the effects of environmental chemicals and related biochemical mechanisms, such as GST catalyzed detoxification, are probably the least understood. In the present study, GST activity was characterized in four species of sea turtles with varied life histories and feeding strategies: loggerhead (Caretta caretta), green (Chelonia mydas), olive ridley (Lepidochelys olivacea), and hawksbill (Eretmochelys imbricata). Although similar GST kinetics was observed between species, rates of catalytic activities using class-specific substrates show inter- and intra-species variation. GST from the spongivorous hawksbill sea turtle shows 3-4.5 fold higher activity with the substrate 4-nitrobenzylchloride than the other 3 species. GST from the herbivorous green sea turtle shows 3 fold higher activity with the substrate ethacrynic acid than the carnivorous olive ridley sea turtle. The results of this study may provide insight into differences in biotransformation potential in the four species of sea turtles and the possible health impacts of contaminant biotransformation by sea turtles.

Colorless polyimide/organoclay nanocomposite substrates for flexible organic light-emitting devices.

PubMed

Kim, Jin-Hoe; Choi, Myeon-Chon; Kim, Hwajeong; Kim, Youngkyoo; Chang, Jin-Hae; Han, Mijeong; Kim, Il; Ha, Chang-Sik

2010-01-01

We report the preparation and application of indium tin oxide (ITO) coated fluorine-containing polyimide/organoclay nanocomposite substrate. Fluorine-containing polyimide/organoclay nanocomposite films were prepared through thermal imidization of poly(amic acid)/organoclay mixture films, whilst on which ITO thin films were coated on the films using a radio-frequency planar magnetron sputtering by varying the substrate temperature and the ITO thickness. Finally the ITO coated fluorine-containing polyimide/organoclay nanocomposite substrate was employed to make flexible organic light-emitting devices (OLED). Results showed that the lower sheet resistance was achieved when the substrate temperature was high and the ITO film was thick even though the optical transmittance was slightly lowered as the thickness increased. approximately 10 nm width ITO nanorods were found for all samples but the size of clusters with the nanorods was generally increased with the substrate temperature and the thickness. The flexible OLED made using the present substrate was quite stable even when the device was extremely bended.

Identification of amino acids important for substrate specificity in sucrose transporters using gene shuffling.

PubMed

Reinders, Anke; Sun, Ye; Karvonen, Kayla L; Ward, John M

2012-08-31

Plant sucrose transporters (SUTs) are H(+)-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and β-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin β-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general.

Identification of Amino Acids Important for Substrate Specificity in Sucrose Transporters Using Gene Shuffling*

PubMed Central

Reinders, Anke; Sun, Ye; Karvonen, Kayla L.; Ward, John M.

2012-01-01

Plant sucrose transporters (SUTs) are H+-coupled uptake transporters. Type I and II (SUTs) are phylogenetically related but have different substrate specificities. Type I SUTs transport sucrose, maltose, and a wide range of natural and synthetic α- and β-glucosides. Type II SUTs are more selective for sucrose and maltose. Here, we investigated the structural basis for this difference in substrate specificity. We used a novel gene shuffling method called synthetic template shuffling to introduce 62 differentially conserved amino acid residues from type I SUTs into OsSUT1, a type II SUT from rice. The OsSUT1 variants were tested for their ability to transport the fluorescent coumarin β-glucoside esculin when expressed in yeast. Fluorescent yeast cells were selected using fluorescence-activated cell sorting (FACS). Substitution of five amino acids present in type I SUTs in OsSUT1 was found to be sufficient to confer esculin uptake activity. The changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was almost identical to that of type I SUTs. Corresponding changes in the sugarcane type II transporter ShSUT1 also changed substrate specificity, indicating that these residues contribute to substrate specificity in type II SUTs in general. PMID:22807445

Substrate specificities of the ntg1 and ntg2 proteins of Saccharomyces cerevisiae for oxidized DNA bases are not identical.

PubMed Central

Sentürker, S; Auffret van der Kemp, P; You, H J; Doetsch, P W; Dizdaroglu, M; Boiteux, S

1998-01-01

Two genes of Saccharomyces cerevisiae, NTG1 and NTG2, encode proteins with a significant sequence homology to the endonuclease III of Escherichia coli. The Ntg1 and Ntg2 proteins were overexpressed in E.coli and purified to apparent homogeneity. The substrate specificity of Ntg1 and Ntg2 proteins for modified bases in oxidatively damaged DNA was investigated using gas chromatography/isotope-dilution mass spectrometry. The substrate used was calf-thymus DNA exposed to gamma-radiation in N2O-saturated aqueous solution. The results reveal excision by Ntg1 and Ntg2 proteins of six pyrimidine-derived lesions, 5-hydroxy-6-hydrothymine, 5-hydroxy-6-hydrouracil, 5-hydroxy-5-methylhydantoin, 5-hydroxyuracil, 5-hydroxycytosine and thymine glycol, and two purine-derived lesions, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine from gamma-irradiated DNA. In contrast, Ntg1 and Ntg2 proteins do not release 8-hydroxyguanine or 8-hydroxyadenine from gamma-irradiated DNA. The Ntg1 and Ntg2 proteins also release 2, 6-diamino-4-hydroxy-5-N-methylformamido-pyrimidine from damaged poly(dG-dC).poly(dG-dC). Excision was measured as a function of enzyme concentration and time. Furthermore, kinetic parameters were determined for each lesion. The results show that kinetic constants varied among the different lesions for the same enzyme. We also investigated the capacity of the Ntg1 and Ntg2 proteins to cleave 34mer DNA duplexes containing a single 8-OH-Gua residue mispaired with each of the four DNA bases. The results show that the Ntg1 protein preferentially cleaves a DNA duplex containing 8-OH-Gua mispaired with a guanine. Moreover, the Ntg1 protein releases free 8-OH-Gua from 8-OH-Gua/Gua duplex but not from duplexes containing 8-OH-Gua mispaired with adenine, thymine or cytosine. In contrast, the Ntg2 protein does not incise duplexes containing 8-OH-Gua mispaired with any of the four DNA bases. These results demonstrate that substrate specificities of the Ntg1 and Ntg2 proteins are similar but not identical and clearly different from that of the endonuclease III of E.coli and its homologues in Schizosaccharomyces pombe or human cells. PMID:9826748

Specificity of hammerhead ribozyme cleavage.

PubMed Central

Hertel, K J; Herschlag, D; Uhlenbeck, O C

1996-01-01

To be effective in gene inactivation, the hammerhead ribozyme must cleave a complementary RNA target without deleterious effects from cleaving non-target RNAs that contain mismatches and shorter stretches of complementarity. The specificity of hammerhead cleavage was evaluated using HH16, a well-characterized ribozyme designed to cleave a target of 17 residues. Under standard reaction conditions, HH16 is unable to discriminate between its full-length substrate and 3'-truncated substrates, even when six fewer base pairs are formed between HH16 and the substrate. This striking lack of specificity arises because all the substrates bind to the ribozyme with sufficient affinity so that cleavage occurs before their affinity differences are manifested. In contrast, HH16 does exhibit high specificity towards certain 3'-truncated versions of altered substrates that either also contain a single base mismatch or are shortened at the 5' end. In addition, the specificity of HH16 is improved in the presence of p7 nucleocapsid protein from human immunodeficiency virus (HIV)-1, which accelerates the association and dissociation of RNA helices. These results support the view that the hammerhead has an intrinsic ability to discriminate against incorrect bases, but emphasizes that the high specificity is only observed in a certain range of helix lengths. Images PMID:8670879

Long-range Electrostatic Complementarity Governs Substrate Recognition by Human Chymotrypsin C, a Key Regulator of Digestive Enzyme Activation*

PubMed Central

Batra, Jyotica; Szabó, András; Caulfield, Thomas R.; Soares, Alexei S.; Sahin-Tóth, Miklós; Radisky, Evette S.

2013-01-01

Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5′ subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2′ positions of CTRC, although acidic P2′ residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels. PMID:23430245

Substrate specificity of xenobiotic metabolizing esterases in the liver of two catfish species

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

Jaiswal, R.G.; Huang, T.L.; Obih, P.O.

1994-12-31

The preliminary studies were conducted on the characterization of substrate specificity in the liver microsomes and cytosol of two catfish species, Ictalurus punctatus and Ictalurus natalie. A series of five esters of p-nitrophenol were used as calorimetric substrates to assay the carboxylesterases. The substrate specificity of liver microsomal and cytosolic carboxylesterases were remarkably different from each other. The valerate ester of p-nitrophenol was most rapidly hydrolyzed by the microsomal carboxylesterases, whereas the prioponate ester was the best substrate for cytosolic carboxylesterases. The Ictalurus natalie catfish species were obtained from the Devil Swamp site of the Mississippi River Basin which ismore » known to be heavily contaminated with toxic and hazardous industrial wastes. These results will be discussed in relation to the responses of xenobiotic metabolizing esterases to environmental pollutants and their possible use as biomarkers.« less

Analysis of substrate specificity of human DHHC protein acyltransferases using a yeast expression system

PubMed Central

Ohno, Yusuke; Kashio, Atsushi; Ogata, Ren; Ishitomi, Akihiro; Yamazaki, Yuki; Kihara, Akio

2012-01-01

Palmitoylation plays important roles in the regulation of protein localization, stability, and activity. The protein acyltransferases (PATs) have a common DHHC Cys-rich domain. Twenty-three DHHC proteins have been identified in humans. However, it is unclear whether all of these DHHC proteins function as PATs. In addition, their substrate specificities remain largely unknown. Here we develop a useful method to examine substrate specificities of PATs using a yeast expression system with six distinct model substrates. We identify 17 human DHHC proteins as PATs. Moreover, we classify 11 human and 5 yeast DHHC proteins into three classes (I, II, and III), based on the cellular localization of their respective substrates (class I, soluble proteins; class II, integral membrane proteins; class III, lipidated proteins). Our results may provide an important clue for understanding the function of individual DHHC proteins. PMID:23034182

Dissecting substrate specificities of the mitochondrial AFG3L2 protease.

PubMed

Ding, Bojian; Martin, Dwight W; Rampello, Anthony J; Glynn, Steven E

2018-06-22

Human AFG3L2 is a compartmental AAA+ protease that performs ATP-fueled degradation at the matrix face of the inner mitochondrial membrane. Identifying how AFG3L2 selects substrates from the diverse complement of matrix-localized proteins is essential for understanding mitochondrial protein biogenesis and quality control. Here, we create solubilized forms of AFG3L2 to examine the enzyme's substrate specificity mechanisms. We show that conserved residues within the pre-sequence of the mitochondrial ribosomal protein, MrpL32, target the subunit to the protease for processing into a mature form. Moreover, these residues can act as a degron, delivering diverse model proteins to AFG3L2 for degradation. By determining the sequence of degra-dation products from multiple substrates using mass spectrometry, we construct a peptidase specificity pro-file that displays constrained product lengths and is dominated by the identity of the residue at the P1' posi-tion, with a strong preference for hydrophobic and small polar residues. This specificity profile is validated by examining the cleavage of both fluorogenic reporter peptides and full polypeptide substrates bearing different P1' residues. Together, these results demonstrate that AFG3L2 contains multiple modes of specificity, dis-criminating between potential substrates by recognizing accessible degron sequences, and performing peptide bond cleavage at preferred patterns of residues within the compartmental chamber.

Critical Amino Acids in the Active Site of Meprin Metalloproteinases for Substrate and Peptide Bond Specificity*

PubMed Central

Villa, James P.; Bertenshaw, Greg P.; Bond, Judith S.

2008-01-01

SUMMARY The protease domains of the evolutionarily-related α and ß subunits of meprin metalloproteases are approximately 55% identical at the amino acid level, however, their substrate and peptide bond specificities differ markedly. The meprin ß subunit favors acidic residues proximal to the scissile bond, while the α subunit prefers small or aromatic amino acids flanking the scissile bond. Thus gastrin, a peptide that contains a string of five Glu residues, is an excellent substrate for meprin ß while it is not hydrolyzed by meprin α. Work herein aimed to identify critical amino acids in the meprin active sites that determine the substrate specificity differences. Sequence alignments and homology models, based on the crystal structure of the crayfish astacin, showed electrostatic differences within the meprin active sites. Site-directed mutagenesis of active site residues demonstrated that replacement of a hydrophobic residue by a basic amino acid enabled the meprin α protease to cleave gastrin. The meprin αY199K mutant was most effective; the corresponding mutation of meprin ßK185Y resulted in decreased activity toward gastrin. Peptide cleavage site determinations and kinetic analyses using a variety of peptides extended evidence that meprin αTyr199/ßLys185 are substrate specificity determinants in meprin active sites. These studies shed light on the molecular basis for the substrate specificity differences of astacin metalloproteinases. PMID:12888571

Computational Study on Substrate Specificity of a Novel Cysteine Protease 1 Precursor from Zea mays

PubMed Central

Liu, Huimin; Chen, Liangcheng; Li, Quan; Zheng, Mingzhu; Liu, Jingsheng

2014-01-01

Cysteine protease 1 precursor from Zea mays (zmCP1) is classified as a member of the C1A family of peptidases (papain-like cysteine protease) in MEROPS (the Peptidase Database). The 3D structure and substrate specificity of the zmCP1 is still unknown. This study is the first one to build the 3D structure of zmCP1 by computer-assisted homology modeling. In order to determine the substrate specificity of zmCP1, docking study is used for rapid and convenient analysis of large populations of ligand–enzyme complexes. Docking results show that zmCP1 has preference for P1 position and P2 position for Arg and a large hydrophobic residue (such as Phe). Gly147, Gly191, Cys189, and Asp190 are predicted to function as active residues at the S1 subsite, and the S2 subsite contains Leu283, Leu193, Ala259, Met194, and Ala286. SIFt results indicate that Gly144, Arg268, Trp308, and Ser311 play important roles in substrate binding. Then Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) method was used to explain the substrate specificity for P1 position of zmCp1. This study provides insights into the molecular basis of zmCP1 activity and substrate specificity. PMID:24921705

The role of added feed enzymes in promoting gut health in swine and poultry.

PubMed

Kiarie, Elijah; Romero, Luis F; Nyachoti, Charles M

2013-06-01

The value of added feed enzymes (FE) in promoting growth and efficiency of nutrient utilisation is well recognised in single-stomached animal production. However, the effects of FE on the microbiome of the gastrointestinal tract (GIT) are largely unrecognised. A critical role in host nutrition, health, performance and quality of the products produced is played by the intestinal microbiota. FE can make an impact on GIT microbial ecology by reducing undigested substrates and anti-nutritive factors and producing oligosaccharides in situ from dietary NSP with potential prebiotic effects. Investigations with molecular microbiology techniques have demonstrated FE-mediated responses on energy utilisation in broiler chickens that were associated with certain clusters of GIT bacteria. Furthermore, investigations using specific enteric pathogen challenge models have demonstrated the efficacy of FE in modulating gut health. Because FE probably change the substrate characteristics along the GIT, subsequent microbiota responses will vary according to the populations present at the time of administration and their reaction to such changes. Therefore, the microbiota responses to FE administration, rather than being absolute, are a continuum or a population of responses. However, recognition that FE can make an impact on the gut microbiota and thus gut health will probably stimulate development of FE capable of modulating gut microbiota to the benefit of host health under specific production conditions. The present review brings to light opportunities and challenges for the role of major FE (carbohydrases and phytase) on the gut health of poultry and swine species with a specific focus on the impact on GIT microbiota.

Reactivity of Toluate Dioxygenase with Substituted Benzoates and Dioxygen

PubMed Central

Ge, Yong; Vaillancourt, Frédéric H.; Agar, Nathalie Y. R.; Eltis, Lindsay D.

2002-01-01

Toluate dioxygenase (TADO) of Pseudomonas putida mt-2 catalyzes the dihydroxylation of a broad range of substituted benzoates. The two components of this enzyme were hyperexpressed and anaerobically purified. Reconstituted TADO had a specific activity of 3.8 U/mg with m-toluate, and each component had a full complement of their respective Fe2S2 centers. Steady-state kinetics data obtained by using an oxygraph assay and by varying the toluate and dioxygen concentrations were analyzed by a compulsory order ternary complex mechanism. TADO had greatest specificity for m-toluate, displaying apparent parameters of KmA = 9 ± 1 μM, kcat = 3.9 ± 0.2 s−1, and KmO2 = 16 ± 2 μM (100 mM sodium phosphate, pH 7.0; 25°C), where KmO2 represents the Km for O2 and KmA represents the Km for the aromatic substrate. The enzyme utilized benzoates in the following order of specificity: m-toluate > benzoate ≃ 3-chlorobenzoate > p-toluate ≃ 4-chlorobenzoate ≫ o-toluate ≃ 2-chlorobenzoate. The transformation of each of the first five compounds was well coupled to O2 utilization and yielded the corresponding 1,2-cis-dihydrodiol. In contrast, the transformation of ortho-substituted benzoates was poorly coupled to O2 utilization, with >10 times more O2 being consumed than benzoate. However, the apparent Km of TADO for these benzoates was >100 μM, indicating that they do not effectively inhibit the turnover of good substrates. PMID:12107126

Structural Studies of Bacterial Enzymes and their Relation to Antibiotic Resistance Mechanisms - Final Paper

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

Maltz, Lauren

By using protein crystallography and X-ray diffraction, structures of bacterial enzymes were solved to gain a better understanding of how enzymatic modification acts as an antibacterial resistance mechanism. Aminoglycoside phosphotransferases (APHs) are one of three aminoglycoside modifying enzymes that confer resistance to the aminoglycoside antibiotics via enzymatic modification, rendering many drugs obsolete. Specifically, the APH(2”) family vary in their substrate specificities and also in their preference for the phosphate donor (ADP versus GDP). By solving the structures of members of the APH(2”) family of enzymes, we can see how domain movements are important to their substrate specificity. Our structure ofmore » the ternary complex of APH(2”)-IIIa with GDP and kanamycin, when compared to the known structures of APH(2”)-IVa, reveals that there are real physical differences between these two enzymes, a structural finding that explains why the two enzymes differ in their preferences for certain aminoglycosides. Another important group of bacterial resistance enzymes are the Class D β- lactamases. Oxacillinase carbapenemases (OXAs) are part of this enzyme class and have begun to confer resistance to ‘last resort’ drugs, most notably carbapenems. Our structure of OXA-143 shows that the conformational flexibility of a conserved hydrophobic residue in the active site (Val130) serves to control the entry of a transient water molecule responsible for a key step in the enzyme’s mechanism. Our results provide insight into the structural mechanisms of these two different enzymes« less

A multiwell format assay for heparanase.

PubMed

Behzad, Farhad; Brenchley, Paul E C

2003-09-15

This assay employs a biotinylated heparan sulfate glycosaminoglycan (HSGAG) substrate that is covalently linked to the surface of 96-well immunoassay plates. The ratio of biotin:HSGAG and the coating concentration of substrate bound to the wells have been optimized and allow removal of biotin HSGAG within 60 min of incubation at 37 degrees C in assay buffer with a standard dilution of bacterial heparitinase or platelet heparanase. Loss of biotin signal from the well surface is detected on incubation with peroxidase-streptavidin followed by color development using 3,3',5,5'-tetramethylbenzidine as the peroxidase substrate. The new assay allows specific detection of heparanase activity in multiple samples in a total time of 3 h including a 1-h substrate digestion step and is a significant improvement with regard to sensitivity, specificity, and ease of handling of multiple samples compared to other described assays. Heparanase specifically degrades the biotinylated HSGAG substrate, when used with an optimized assay buffer. A range of enzymes including collagenase, trypsin, plasmin, pepsin, chondroitinases, hyaluronidase, and neuraminidase show no effect on the substrate under optimized assay conditions. The covalent linkage of the substrate to the well prevents leaching of substrate and allows preparation and long-term storage of substrate-coated plates. The assay can be used to detect heparanase levels in clinical samples and cell culture supernatants and is ideal as a screening method for antagonists of enzyme activity.

A method to screen and evaluate tissue adhesives for joint repair applications

PubMed Central

2012-01-01

Background Tissue adhesives are useful means for various medical procedures. Since varying requirements cause that a single adhesive cannot meet all needs, bond strength testing remains one of the key applications used to screen for new products and study the influence of experimental variables. This study was conducted to develop an easy to use method to screen and evaluate tissue adhesives for tissue engineering applications. Method Tissue grips were designed to facilitate the reproducible production of substrate tissue and adhesive strength measurements in universal testing machines. Porcine femoral condyles were used to generate osteochondral test tissue cylinders (substrates) of different shapes. Viability of substrates was tested using PI/FDA staining. Self-bonding properties were determined to examine reusability of substrates (n = 3). Serial measurements (n = 5) in different operation modes (OM) were performed to analyze the bonding strength of tissue adhesives in bone (OM-1) and cartilage tissue either in isolation (OM-2) or under specific requirements in joint repair such as filling cartilage defects with clinical applied fibrin/PLGA-cell-transplants (OM-3) or tissues (OM-4). The efficiency of the method was determined on the basis of adhesive properties of fibrin glue for different assembly times (30 s, 60 s). Seven randomly generated collagen formulations were analyzed to examine the potential of method to identify new tissue adhesives. Results Viability analysis of test tissue cylinders revealed vital cells (>80%) in cartilage components even 48 h post preparation. Reuse (n = 10) of test substrate did not significantly change adhesive characteristics. Adhesive strength of fibrin varied in different test settings (OM-1: 7.1 kPa, OM-2: 2.6 kPa, OM-3: 32.7 kPa, OM-4: 30.1 kPa) and was increasing with assembly time on average (2.4-fold). The screening of the different collagen formulations revealed a substance with significant higher adhesive strength on cartilage (14.8 kPa) and bone tissue (11.8 kPa) compared to fibrin and also considerable adhesive properties when filling defects with cartilage tissue (23.2 kPa). Conclusion The method confirmed adhesive properties of fibrin and demonstrated the dependence of adhesive properties and applied settings. Furthermore the method was suitable to screen for potential adhesives and to identify a promising candidate for cartilage and bone applications. The method can offer simple, replicable and efficient evaluation of adhesive properties in ex vivo specimens and may be a useful supplement to existing methods in clinical relevant settings. PMID:22984926

Nanoengineered Polystyrene Surfaces with Nanopore Array Pattern Alters Cytoskeleton Organization and Enhances Induction of Neural Differentiation of Human Adipose-Derived Stem Cells.

PubMed

Jung, Ae Ryang; Kim, Richard Y; Kim, Hyung Woo; Shrestha, Kshitiz Raj; Jeon, Seung Hwan; Cha, Kyoung Je; Park, Yong Hyun; Kim, Dong Sung; Lee, Ji Youl

2015-07-01

Human adipose-derived stem cells (hADSCs) can differentiate into various cell types depending on chemical and topographical cues. One topographical cue recently noted to be successful in inducing differentiation is the nanoengineered polystyrene surface containing nanopore array-patterned substrate (NP substrate), which is designed to mimic the nanoscale topographical features of the extracellular matrix. In this study, efficacies of NP and flat substrates in inducing neural differentiation of hADSCs were examined by comparing their substrate-cell adhesion rates, filopodia growth, nuclei elongation, and expression of neural-specific markers. The polystyrene nano Petri dishes containing NP substrates were fabricated by a nano injection molding process using a nickel electroformed nano-mold insert (Diameter: 200 nm. Depth of pore: 500 nm. Center-to-center distance: 500 nm). Cytoskeleton and filopodia structures were observed by scanning electron microscopy and F-actin staining, while cell adhesion was tested by vinculin staining after 24 and 48 h of seeding. Expression of neural specific markers was examined by real-time quantitative polymerase chain reaction and immunocytochemistry. Results showed that NP substrates lead to greater substrate-cell adhesion, filopodia growth, nuclei elongation, and expression of neural specific markers compared to flat substrates. These results not only show the advantages of NP substrates, but they also suggest that further study into cell-substrate interactions may yield great benefits for biomaterial engineering.

Wettability of partially suspended graphene

DOE PAGES

Ondarçuhu, Thierry; Thomas, Vincent; Nuñez, Marc; ...

2016-04-13

Dependence on the wettability of graphene on the nature of the underlying substrate remains only partially understood. We systematically investigate the role of liquid-substrate interactions on the wettability of graphene by varying the area fraction of suspended graphene from 0 to 95% by means of nanotextured substrates. We find that completely suspended graphene exhibits the highest water contact angle (85° ± 5°) compared to partially suspended or supported graphene, regardless of the hydrophobicity (hydrophilicity) of the substrate. Moreover, 80% of the long-range water-substrate interactions are screened by the graphene monolayer, the wettability of which is primarily determined by short-range graphene-liquidmore » interactions. By its well-defined chemical and geometrical properties, supported graphene therefore provides a model system to elucidate the relative contribution of short and long range interactions to the macroscopic contact angle.« less

Hybrid intelligent control of substrate feeding for industrial fed-batch chlortetracycline fermentation process.

PubMed

Jin, Huaiping; Chen, Xiangguang; Yang, Jianwen; Wu, Lei; Wang, Li

2014-11-01

The lack of accurate process models and reliable online sensors for substrate measurements poses significant challenges for controlling substrate feeding accurately, automatically and optimally in fed-batch fermentation industries. It is still a common practice to regulate the feeding rate based upon manual operations. To address this issue, a hybrid intelligent control method is proposed to enable automatic substrate feeding. The resulting control system consists of three modules: a presetting module for providing initial set-points; a predictive module for estimating substrate concentration online based on a new time interval-varying soft sensing algorithm; and a feedback compensator using expert rules. The effectiveness of the proposed approach is demonstrated through its successful applications to the industrial fed-batch chlortetracycline fermentation process. Copyright © 2014 ISA. Published by Elsevier Ltd. All rights reserved.

Substrate specificity effects of lipoxygenase products and inhibitors on soybean lipoxygenase-1.

PubMed

Wecksler, Aaron T; Garcia, Natalie K; Holman, Theodore R

2009-09-15

Recently, it has been shown that lipoxygenase (LO) products affect the substrate specificity of human 15-LO. In the current paper, we demonstrate that soybean LO-1 (sLO-1) is not affected by its own products, however, inhibitors which bind the allosteric site, oleyl sulfate (OS) and palmitoleyl sulfate (PS), not only lower catalytic activity, but also change the substrate specificity, by increasing the arachidonic acid (AA)/linoleic acid (LA) ratio to 4.8 and 4.0, respectively. The fact that LO inhibitors can lower activity and also change the LO product ratio is a new concept in lipoxygenase inhibition, where the goal is to not only reduce the catalytic activity but also alter substrate selectivity towards a physiologically beneficial product.

Identification of ribozymes within a ribozyme library that efficiently cleave a long substrate RNA.

PubMed Central

Campbell, T B; Cech, T R

1995-01-01

Positions 2-6 of the substrate-binding internal guide sequence (IGS) of the L-21 Sca I form of the Tetrahymena thermophila intron were mutagenized to produce a GN5 IGS library. Ribozymes within the GN5 library capable of efficient cleavage of an 818-nt human immunodeficiency virus type 1 vif-vpr RNA, at 37 degrees C, were identified by ribozyme-catalyzed guanosine addition to the 3' cleavage product. Three ribozymes (IGS = GGGGCU, GGCUCC, and GUGGCU) within the GN5 library that actively cleaved the long substrate were characterized kinetically and compared to the wild-type ribozyme (GGAGGG) and two control ribozymes (GGAGUC and GGAGAU). The two control ribozymes have specific sites within the long substrate, but were not identified during screening of the library. Under single-turnover conditions, ribozymes GGGGCU, GGCUCC, and GUGGCU cleaved the 818-nt substrate 4- to 200-fold faster than control ribozymes. Short cognate substrates, which should be structureless and therefore accessible to ribozyme binding, were cleaved at similar rates by all ribozymes except GGGGCU, which showed a fourfold rate enhancement. The rate of cleavage of long relative to short substrate under single-turnover conditions suggests that GGCUCC and GUGGCU were identified because of accessibility to their specific cleavage sites within the long substrate (substrate-specific effects), whereas GGGGCU was identified because of an enhanced rate of substrate binding despite a less accessible site in the long substrate. Even though screening was performed with 100-fold excess substrate (relative to total ribozyme), the rate of multiple-turnover catalysis did not contribute to identification of trans-cleaving ribozymes in the GN5 library. PMID:7489519

Influence of the deposition conditions on radiofrequency magnetron sputtered MoS2 films

NASA Technical Reports Server (NTRS)

Steinmann, Pierre A.; Spalvins, Talivaldis

1990-01-01

By varying the radiofrequency (RF) power, the Ar pressure, and the potential on the substrates, MoS(x) films of various stoichiometry, density, adhesion, and morphology were produced. An increase of RF power increased the deposition rate and density of the MoS2 films as well as improved adhesion. However, the stoichiometry remained constant. An increase of Ar pressure increased the deposition rate but decreased the density, wheras both stoichiometry and adhesion were maximized at around 20 mtorr Ar pressure. Furthermore, a transition from compact film growth to columnar film growth was observed when the pressure was varied from 5 to 15 mtorr. Substoichiometric films were grown when a negative (bias) voltage was applied to the substrates.

Microbial Degradation of Asphalt1

PubMed Central

Phillips, U. A.; Traxler, R. W.

1963-01-01

Organisms of the genera Pseudomonas, Chromobacterium, and Bacillus capable of degrading asphalt were isolated by enrichment cultures. The asphalt degradation by these organisms varied from 3 to 25% after incubation for 1 week. The effects of temperature, pH, and atmosphere of incubation on asphalt degradation were investigated and were shown to vary with different organisms on the same substrate. PMID:16349633

Activation energy for diamond growth from the carbon-hydrogen gas system at low substrate temperatures

NASA Astrophysics Data System (ADS)

Stiegler, J.; Lang, T.; von Kaenel, Y.; Michler, J.; Blank, E.

1997-01-01

The growth kinetics of diamond films deposited at low substrate temperatures (600-400 °C) from the carbon-hydrogen gas system have been studied. When the substrate temperature alone was varied, independently of all other process parameters in the microwave plasma reactor, an activation energy in the order of 7 kcal/mol was observed. This value did not change with different carbon concentrations in hydrogen. It is supposed that growth kinetics in this temperature range are controlled by a single chemical reaction, probably the abstraction of surface bonded hydrogen by gas phase atomic hydrogen.

Bulk and surface loss in superconducting transmon qubits

NASA Astrophysics Data System (ADS)

Dial, Oliver; McClure, Douglas T.; Poletto, Stefano; Keefe, G. A.; Rothwell, Mary Beth; Gambetta, Jay M.; Abraham, David W.; Chow, Jerry M.; Steffen, Matthias

2016-04-01

Decoherence of superconducting transmon qubits is purported to be consistent with surface loss from two-level systems on the substrate surface. Here, we present a study of surface loss in transmon devices, explicitly designed to have varying sensitivities to different surface loss contributors. Our experiments also encompass two particular different sapphire substrates, which reveal the onset of a yet unknown additional loss mechanism outside of surface loss for one of the substrates. Tests across different wafers and devices demonstrate substantial variation, and we emphasize the importance of testing large numbers of devices for disentangling different sources of decoherence.

Effect of oxidation of the non-catalytic β-propeller domain on the substrate specificity of prolyl oligopeptidase from Pleurotus eryngii.

PubMed

Tokai, Shota; Bito, Tomohiro; Shimizu, Katsuhiko; Arima, Jiro

2017-05-27

Enzymes belonging to the S9 family of prolyl oligopeptidases are of interest because of their pharmacological importance and have a non-catalytic β-propeller domain. In this study, we found that the oxidation of Met203, which lies on surface of the β-propeller domain, leads to change in the substrate specificity of eryngase, an enzyme from Pleurotus eryngii and a member of the S9 family of prolyl oligopeptidases. The activity of eryngase for L-Phe-p-nitroanilide was maintained following hydrogen peroxide treatment but was dramatically reduced for other p-nitroanilide substrates. MALDI-TOF MS analysis using tryptic peptides of eryngase indicated that the change in substrate specificity was triggered by oxidizing Met203 to methionine sulfoxide. In addition, mutations of Met203 to smaller residues provided specificities similar to those observed following oxidation of the wild-type enzyme. Substitution of Met203 with Phe significantly decreased activity, indicating that Met203 may be involved in substrate gating. Copyright © 2017 Elsevier Inc. All rights reserved.

Design of ultrasensitive probes for human neutrophil elastase through hybrid combinatorial substrate library profiling

PubMed Central

Kasperkiewicz, Paulina; Poreba, Marcin; Snipas, Scott J.; Parker, Heather; Winterbourn, Christine C.; Salvesen, Guy S.; Drag, Marcin

2014-01-01

The exploration of protease substrate specificity is generally restricted to naturally occurring amino acids, limiting the degree of conformational space that can be surveyed. We substantially enhanced this by incorporating 102 unnatural amino acids to explore the S1–S4 pockets of human neutrophil elastase. This approach provides hybrid natural and unnatural amino acid sequences, and thus we termed it the Hybrid Combinatorial Substrate Library. Library results were validated by the synthesis of individual tetrapeptide substrates, with the optimal substrate demonstrating more than three orders of magnitude higher catalytic efficiency than commonly used substrates of elastase. This optimal substrate was converted to an activity-based probe that demonstrated high selectivity and revealed the specific presence of active elastase during the process of neutrophil extracellular trap formation. We propose that this approach can be successfully used for any type of endopeptidase to deliver high activity and selectivity in substrates and probes. PMID:24550277

Neurotransmitter and psychostimulant recognition by the dopamine transporter

PubMed Central

Wang, Kevin H.; Penmatsa, Aravind; Gouaux, Eric

2015-01-01

Na+/Cl−-coupled biogenic amine transporters are the primary targets of therapeutic and abused drugs, ranging from antidepressants to the psychostimulants cocaine and amphetamines, and to their cognate substrates. Here we determine x-ray crystal structures of the Drosophila melanogaster dopamine transporter (dDAT) bound to its substrate dopamine (DA), a substrate analogue 3,4-dichlorophenethylamine, the psychostimulants D-amphetamine, methamphetamine, or to cocaine and cocaine analogues. All ligands bind to the central binding site, located approximately halfway across the membrane bilayer, in close proximity to bound sodium and chloride ions. The central binding site recognizes three chemically distinct classes of ligands via conformational changes that accommodate varying sizes and shapes, thus illustrating molecular principles that distinguish substrates from inhibitors in biogenic amine transporters. PMID:25970245

The influence of the surrounding gas on drop impact onto a wet substrate

NASA Astrophysics Data System (ADS)

Deegan, Robert; Zhang, Li; Toole, Jameson

2011-11-01

The impact of a droplet with a wet or solid substrate creates a spray of secondary droplets. The effect of the surrounding gas on this process was widely neglected prior to the work of Xu, Zhang, & Nagel which showed that lowering the gas pressure suppresses splashing for impact with a dry solid substrate. Here we present the results of our experimental investigation of the effect of the surrounding gas on the evolution of splashes from a wet substrate. We varied the density and pressure of the surrounding gas. We find quantitative changes to the onset thresholds of splashing and on the size distribution of, but no qualitative changes. The effects are most pronounced on the evolution of the ejecta sheet.

Figure correction of multilayer coated optics

DOEpatents

Chapman; Henry N. , Taylor; John S.

2010-02-16

A process is provided for producing near-perfect optical surfaces, for EUV and soft-x-ray optics. The method involves polishing or otherwise figuring the multilayer coating that has been deposited on an optical substrate, in order to correct for errors in the figure of the substrate and coating. A method such as ion-beam milling is used to remove material from the multilayer coating by an amount that varies in a specified way across the substrate. The phase of the EUV light that is reflected from the multilayer will be affected by the amount of multilayer material removed, but this effect will be reduced by a factor of 1-n as compared with height variations of the substrate, where n is the average refractive index of the multilayer.

Hydrophobic fluorescent probes introduce artifacts into single molecule tracking experiments due to non-specific binding.

PubMed

Zanetti-Domingues, Laura C; Tynan, Christopher J; Rolfe, Daniel J; Clarke, David T; Martin-Fernandez, Marisa

2013-01-01

Single-molecule techniques are powerful tools to investigate the structure and dynamics of macromolecular complexes; however, data quality can suffer because of weak specific signal, background noise and dye bleaching and blinking. It is less well-known, but equally important, that non-specific binding of probe to substrates results in a large number of immobile fluorescent molecules, introducing significant artifacts in live cell experiments. Following from our previous work in which we investigated glass coating substrates and demonstrated that the main contribution to this non-specific probe adhesion comes from the dye, we carried out a systematic investigation of how different dye chemistries influence the behaviour of spectrally similar fluorescent probes. Single-molecule brightness, bleaching and probe mobility on the surface of live breast cancer cells cultured on a non-adhesive substrate were assessed for anti-EGFR affibody conjugates with 14 different dyes from 5 different manufacturers, belonging to 3 spectrally homogeneous bands (491 nm, 561 nm and 638 nm laser lines excitation). Our results indicate that, as well as influencing their photophysical properties, dye chemistry has a strong influence on the propensity of dye-protein conjugates to adhere non-specifically to the substrate. In particular, hydrophobicity has a strong influence on interactions with the substrate, with hydrophobic dyes showing much greater levels of binding. Crucially, high levels of non-specific substrate binding result in calculated diffusion coefficients significantly lower than the true values. We conclude that the physic-chemical properties of the dyes should be considered carefully when planning single-molecule experiments. Favourable dye characteristics such as photostability and brightness can be offset by the propensity of a conjugate for non-specific adhesion.

Hydrophobic Fluorescent Probes Introduce Artifacts into Single Molecule Tracking Experiments Due to Non-Specific Binding

PubMed Central

Rolfe, Daniel J.; Clarke, David T.; Martin-Fernandez, Marisa

2013-01-01

Single-molecule techniques are powerful tools to investigate the structure and dynamics of macromolecular complexes; however, data quality can suffer because of weak specific signal, background noise and dye bleaching and blinking. It is less well-known, but equally important, that non-specific binding of probe to substrates results in a large number of immobile fluorescent molecules, introducing significant artifacts in live cell experiments. Following from our previous work in which we investigated glass coating substrates and demonstrated that the main contribution to this non-specific probe adhesion comes from the dye, we carried out a systematic investigation of how different dye chemistries influence the behaviour of spectrally similar fluorescent probes. Single-molecule brightness, bleaching and probe mobility on the surface of live breast cancer cells cultured on a non-adhesive substrate were assessed for anti-EGFR affibody conjugates with 14 different dyes from 5 different manufacturers, belonging to 3 spectrally homogeneous bands (491 nm, 561 nm and 638 nm laser lines excitation). Our results indicate that, as well as influencing their photophysical properties, dye chemistry has a strong influence on the propensity of dye-protein conjugates to adhere non-specifically to the substrate. In particular, hydrophobicity has a strong influence on interactions with the substrate, with hydrophobic dyes showing much greater levels of binding. Crucially, high levels of non-specific substrate binding result in calculated diffusion coefficients significantly lower than the true values. We conclude that the physic-chemical properties of the dyes should be considered carefully when planning single-molecule experiments. Favourable dye characteristics such as photostability and brightness can be offset by the propensity of a conjugate for non-specific adhesion. PMID:24066121

Interaction of Substrate Mechanics with Dental Pulp Stem Cells (DPSCs) differentiation to generate a scaffold for Bone regeneration

NASA Astrophysics Data System (ADS)

Rafailovich, Miriam; Bhatnagar, Divya; Bherwani, Aneel; Simon, Marcia

2012-02-01

This work investigates the interaction of the substrate mechanics with the differentiation in the absence of chemical induction and only resulting from the stimuli of the substrate mechanics and chemistry. We chose enzymatically cross-linked gelatin hydrogels substrates of different stiffness varying from 8KPa to 100Pa. DPSCs were cultured and differentiated on the substrates for 7, 14 and 21 days with and without dexamethasone induction media. SEM and EDX analysis after 21 days indicate that cells produced a sheet of biomineralized deposits, several tenths of mm thick on the hard substrate irrespective of chemical induction. Modulli of the cells was independent of the induction and stiffness of the hydrogels. RT-PCR assays indicated that cells expressed more osteocalcin when cultured in non-induction media and harder substrate. The shape of the deposits was more uniform and in close packing on the harder substrate with a higher Ca:P ratio. On soft substrate the deposits were more flat with less Ca:P ratio. Further experiments indicated that conformational change due to the crosslinking of gelatin could be the reason for biomineralization.

Substrate-biasing during plasma-assisted atomic layer deposition to tailor metal-oxide thin film growth

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

Profijt, H. B.; Sanden, M. C. M. van de; Kessels, W. M. M.

2013-01-15

Two substrate-biasing techniques, i.e., substrate-tuned biasing and RF biasing, have been implemented in a remote plasma configuration, enabling control of the ion energy during plasma-assisted atomic layer deposition (ALD). With both techniques, substrate bias voltages up to -200 V have been reached, which allowed for ion energies up to 272 eV. Besides the bias voltage, the ion energy and the ion flux, also the electron temperature, the electron density, and the optical emission of the plasma have been measured. The effects of substrate biasing during plasma-assisted ALD have been investigated for Al{sub 2}O{sub 3}, Co{sub 3}O{sub 4}, and TiO{sub 2}more » thin films. The growth per cycle, the mass density, and the crystallinity have been investigated, and it was found that these process and material properties can be tailored using substrate biasing. Additionally, the residual stress in substrates coated with Al{sub 2}O{sub 3} films varied with the substrate bias voltage. The results reported in this article demonstrate that substrate biasing is a promising technique to tailor the material properties of thin films synthesized by plasma-assisted ALD.« less

Echinococcus granulosus: specificity of amino acid transport systems in protoscoleces.

PubMed

Jeffs, S A; Arme, C

1987-08-01

Protoscoleces of Echinococcus granulosus absorb the L-amino acids proline, methionine, leucine, alanine, serine, phenylalanine, lysine and glutamic acid by a combination of mediated transport and diffusion. All eight amino acids were accumulated against a concentration gradient. Comparison of Kt and Vmax values suggests that a low affinity for a particular compound is compensated for by a relatively larger number of transport sites for that compound. Four systems serve for the transport of the eight substrates studied: 2 for neutral (EgN1, EgN2) and 1 each for acidic (EgA) and basic (EgB) amino acids. All eight amino acids are incorporated into protein to varying degrees and substantial portions of absorbed L-alanine and L-methionine are metabolized into other compounds.

A Bottom-Up Proteomic Approach to Identify Substrate Specificity of Outer-Membrane Protease OmpT.

PubMed

Wood, Sarah E; Sinsinbar, Gaurav; Gudlur, Sushanth; Nallani, Madhavan; Huang, Che-Fan; Liedberg, Bo; Mrksich, Milan

2017-12-22

Identifying peptide substrates that are efficiently cleaved by proteases gives insights into substrate recognition and specificity, guides development of inhibitors, and improves assay sensitivity. Peptide arrays and SAMDI mass spectrometry were used to identify a tetrapeptide substrate exhibiting high activity for the bacterial outer-membrane protease (OmpT). Analysis of protease activity for the preferred residues at the cleavage site (P1, P1') and nearest-neighbor positions (P2, P2') and their positional interdependence revealed FRRV as the optimal peptide with the highest OmpT activity. Substituting FRRV into a fragment of LL37, a natural substrate of OmpT, led to a greater than 400-fold improvement in OmpT catalytic efficiency, with a k cat /K m value of 6.1×10 6  L mol -1  s -1 . Wild-type and mutant OmpT displayed significant differences in their substrate specificities, demonstrating that even modest mutants may not be suitable substitutes for the native enzyme. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Two distinct domains contribute to the substrate acyl chain length selectivity of plant acyl-ACP thioesterase.

PubMed

Jing, Fuyuan; Zhao, Le; Yandeau-Nelson, Marna D; Nikolau, Basil J

2018-02-28

The substrate specificity of acyl-ACP thioesterase (TE) plays an essential role in controlling the fatty acid profile produced by type II fatty acid synthases. Here we identify two groups of residues that synergistically determine different substrate specificities of two acyl-ACP TEs from Cuphea viscosissima (CvFatB1 and CvFatB2). One group (V194, V217, N223, R226, R227, and I268 in CvFatB2) is critical in determining the structure and depth of a hydrophobic cavity in the N-terminal hotdog domain that binds the substrate's acyl moiety. The other group (255-RKLSKI-260 and 285-RKLPKL-289 in CvFatB2) defines positively charged surface patches that may facilitate binding of the ACP moiety. Mutagenesis of residues within these two groups results in distinct synthetic acyl-ACP TEs that efficiently hydrolyze substrates with even shorter chains (C4- to C8-ACPs). These insights into structural determinants of acyl-ACP TE substrate specificity are useful in modifying this enzyme for tailored fatty acid production in engineered organisms.

[Substrate specificities of bile salt hydrolase 1 and its mutants from Lactobacillus salivarius].

PubMed

Bi, Jie; Fang, Fang; Qiu, Yuying; Yang, Qingli; Chen, Jian

2014-03-01

In order to analyze the correlation between critical residues in the catalytic centre of BSH and the enzyme substrate specificity, seven mutants of Lactobacillus salivarius bile salt hydrolase (BSH1) were constructed by using the Escherichia coli pET-20b(+) gene expression system, rational design and site-directed mutagenesis. These BSH1 mutants exhibited different hydrolytic activities against various conjugated bile salts through substrate specificities comparison. Among the residues being tested, Cys2 and Thr264 were deduced as key sites for BSH1 to catalyze taurocholic acid and glycocholic acid, respectively. Moreover, Cys2 and Thr264 were important for keeping the catalytic activity of BSH1. The high conservative Cys2 was not the only active site, other mutant amino acid sites were possibly involved in substrate binding. These mutant residues might influence the space and shape of the substrate-binding pockets or the channel size for substrate passing through and entering active site of BSH1, thus, the hydrolytic activity of BSH1 was changed to different conjugated bile salt.

Unnatural amino acids increase activity and specificity of synthetic substrates for human and malarial cathepsin C.

PubMed

Poreba, Marcin; Mihelic, Marko; Krai, Priscilla; Rajkovic, Jelena; Krezel, Artur; Pawelczak, Malgorzata; Klemba, Michael; Turk, Dusan; Turk, Boris; Latajka, Rafal; Drag, Marcin

2014-04-01

Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes.

Varied effects of Pyrococcus furiosus prefoldin and P. furiosus chaperonin on the refolding reactions of substrate proteins.

PubMed

Hongo, Kunihiro; Itai, Hiroshi; Mizobata, Tomohiro; Kawata, Yasushi

2012-04-01

Prefoldin is a molecular chaperone found in the archaeal and eukaryotic cytosol. Prefoldin can stabilize tentatively nascent polypeptide chains or non-native forms of mainly cytoskeletal proteins, which are subsequently delivered to group II chaperonin to accomplish their precise folding. However, the detailed mechanism is not well known, especially with regard to endogenous substrate proteins. Here, we report the effects of Pyrococcus furiosus prefoldin (PfuPFD) on the refolding reactions of Pyrococcus furiosus citrate synthase (PfuCS) and Aequorea enhanced green fluorescence protein (GFPuv) in the presence or absence of Pyrococcus furiosus chaperonin (PfuCPN). We confirmed that both PfuPFD and PfuCPN interacted with PfuCS and GFPuv refolding intermediates. However, the interactions between chaperone and substrate were different for each case, as was the final effect on the refolding reaction. Effects on the refolding reaction varied from passive effects such as ATP-dependent binding and release (PfuCPN towards GFPuv) and binding which leads to folding arrest (PfuPFD towards GFPuv), to active effects such as net increase in thermal stability (PfuCPN towards PfuCS) to an active improvement in refolding yield (PfuPFD towards PfuCS). We postulate that differences in molecular interactions between substrate and chaperone lead to these differences in chaperoning effects.

Method and system for producing sputtered thin films with sub-angstrom thickness uniformity or custom thickness gradients

DOEpatents

Folta, James A.; Montcalm, Claude; Walton, Christopher

2003-01-01

A method and system for producing a thin film with highly uniform (or highly accurate custom graded) thickness on a flat or graded substrate (such as concave or convex optics), by sweeping the substrate across a vapor deposition source with controlled (and generally, time-varying) velocity. In preferred embodiments, the method includes the steps of measuring the source flux distribution (using a test piece that is held stationary while exposed to the source), calculating a set of predicted film thickness profiles, each film thickness profile assuming the measured flux distribution and a different one of a set of sweep velocity modulation recipes, and determining from the predicted film thickness profiles a sweep velocity modulation recipe which is adequate to achieve a predetermined thickness profile. Aspects of the invention include a practical method of accurately measuring source flux distribution, and a computer-implemented method employing a graphical user interface to facilitate convenient selection of an optimal or nearly optimal sweep velocity modulation recipe to achieve a desired thickness profile on a substrate. Preferably, the computer implements an algorithm in which many sweep velocity function parameters (for example, the speed at which each substrate spins about its center as it sweeps across the source) can be varied or set to zero.

Structural and optical properties of tin disulphide thin films grown by flash evaporation

NASA Astrophysics Data System (ADS)

Banotra, Arun; Padha, Naresh

2018-04-01

Tin Disulphide thin films were deposited by Flash Evaporation method on corning Glass Substrate at different substrate temperatures. The deposited films were undertaken for Structural, Optical and compositional characterizations. Compositional analysis of the films exhibited decrease in the sulphur content enabling S/Sn ratio to vary from 2.05 to 1.32 with increasing substrate temperature. X-ray diffraction reveals amorphous nature of the as-deposited films with varying substrate temperatures. Optical measurements estimated from absorbance spectra suggest higher absorbance at λ≤500nm and higher transmission at λ≥500nm with bandgap changes from 2.45eV to 2.09eV. The 323K as-deposited films were undertaken for annealing which transforms the films into crystalline form corresponding to hexagonal SnS2 phase at 423K and above. However, the optical response for the annealed samples shows a higher transmission of 70% in the visible region which increases further in the Infrared region of the spectrum achieving maximum transmission upto 98%. This higher transmission in the Visible to Infrared region of the solar spectrum in amorphous as well as crystalline form makes the film suitable for their use as a window layer in the Solar Cell Design.

Action mechanism of tyrosinase on meta- and para-hydroxylated monophenols.

PubMed

Fenoll, L G; Rodríguez-López, J N; Varón, R; García-Ruiz, P A; García-Cánovas, F; Tudela, J

2000-04-01

The relationship between the structure and activity of meta- and para-hydroxylated monophenols was studied during their tyrosinase-catalysed hydroxylation and the rate-limiting steps of the reaction mechanism were identified. The para-hydroxylated substrates permit us to study the effect of a substituent (R) in the carbon-1 position (C-1) of the benzene ring on the nucleophilic attack step, while the meta group permits a similar study of the effect on the electrophilic attack step. Substrates with a -OCH3 group on C-1, as p-hydroxyanisol (4HA) and m-hydroxyanisol (3HA), or with a -CH2OH group, as p-hydroxybenzylalcohol (4HBA) and m-hydroxybenzylalcohol (3HBA), were used because the effect of the substituent (R) size was assumed to be similar. However, the electron-donating effect of the -OCH3 group means that the carbon-4 position (C-4) is favoured for nucleophilic attack (para-hydroxylated substrates) or for electrophilic attack (meta-hydroxylated substrates). The electron-attracting effect of the -CH2OH group has the opposite effect, hindering nucleophilic (para) or electrophilic (meta) attack of C-4. The experimental data point to differences between the maximum steady-state rate (V(M)Max) of the different substrates, the value of this parameter depends on the nucleophilic and electrophilic attack. However, differences are greatest in the Michaelis constants (K(M)m), with the meta-hydroxylated substrates having very large values. The catalytic efficiency k(M)cat/K(M)m is much greater for thepara-hydroxylated substrates although it varies greatly between one substrate and the other. However, it varies much less in the meta-hydroxylated substrates since this parameter describes the power of the nucleophilic attack, which is weaker in the meta OH. The large increase in the K(M)m of the meta-hydroxylated substrates might suggest that the phenolic OH takes part in substrate binding. Since this is a weaker nucleophil than the para-hydroxylated substrates, the binding constant decreases, leading to an increase in K(M)m. The catalytic efficiency of tyrosinase on a monophenol (para or meta) is directly related to the nucleophilic power of the oxygen of the phenolic OH. The oxidation step is not limiting since if this were the case, the para and meta substrates would have the same V(M)max. The small difference between the absolute values of V(M)max suggests that the rate constants of the nucleophilic and electrophilic attacks are on the same order of magnitude.

Impact of Experimental Conditions on the Evaluation of Interactions between Multidrug and Toxin Extrusion Proteins and Candidate Drugs.

PubMed

Lechner, Christian; Ishiguro, Naoki; Fukuhara, Ayano; Shimizu, Hidetada; Ohtsu, Naoko; Takatani, Masahito; Nishiyama, Kotaro; Washio, Ikumi; Yamamura, Norio; Kusuhara, Hiroyuki

2016-08-01

Multidrug and toxin extrusion transporters (MATEs) have a determining influence on the pharmacokinetic profiles of many drugs and are involved in several clinical drug-drug interactions (DDIs). Cellular uptake assays with recombinant cells expressing human MATE1 or MATE2-K are widely used to investigate MATE-mediated transport for DDI assessment; however, the experimental conditions and used test substrates vary among laboratories. We therefore initially examined the impact of three assay conditions that have been applied for MATE substrate and inhibitor profiling in the literature. One of the tested conditions resulted in significantly higher uptake rates of the three test substrates, [(14)C]metformin, [(3)H]thiamine, and [(3)H]1-methyl-4-phenylpyridinium (MPP(+)), but IC50 values of four tested MATE inhibitors varied only slightly among the three conditions (<2.5-fold difference). Subsequently, we investigated the uptake characteristics of the five MATE substrates: [(14)C]metformin, [(3)H]thiamine, [(3)H]MPP(+), [(3)H]estrone-3-sulfate (E3S), and rhodamine 123, as well as the impact of the used test substrate on the inhibition profiles of 10 MATE inhibitors at one selected assay condition. [(3)H]E3S showed atypical uptake characteristics compared with those observed with the other four substrates. IC50 values of the tested inhibitors were in a similar range (<4-fold difference) when [(14)C]metformin, [(3)H]thiamine, [(3)H]MPP(+), or [(3)H]E3S were used as substrates but were considerably higher with rhodamine 123 (9.8-fold and 4.1-fold differences compared with [(14)C]metformin with MATE1 and MATE2-K, respectively). This study demonstrated for the first time that the impact of assay conditions on IC50 determination is negligible, that kinetic characteristics differ among used test substrates, and that substrate-dependent inhibition exists for MATE1 and MATE2-K, giving valuable insight into the assessment of clinically relevant MATE-mediated DDIs in vitro. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Coupled biotic-abiotic oxidation of organic matter by biogenic MnO_{2}

NASA Astrophysics Data System (ADS)

Gonzalez, Julia; Peña, Jasquelin

2016-04-01

Some reactive soil minerals are strongly implicated in stabilising organic matter. However, others can play an active role in the oxidation of organic molecules. In natural systems, layer-type manganese oxide minerals (MnO2) typically occur as biomineral assemblages consisting of mineral particles and microbial biomass. Both the mineral and biological fractions of the assemblage can be powerful oxidants of organic C. The biological compartment relies on a set of enzymes to drive oxidative transformations of reduced C-substrates, whereas MnO2 minerals are strong, less specific abiotic oxidants that are assumed to rely on interfacial interactions between C-substrates and the mineral surface. This project aims to understand the coupling between microbial C mineralization and abiotic C oxidation mediated by MnO2 in bacterial-MnO2 assemblages. Specifically, under conditions of high C turnover, microbial respiration can significantly alter local pH, dissolved oxygen and pool of available reductants, which may modify rates and mechanism of C oxidation by biotic and abiotic components. We first investigated changes in the solution chemistry of Pseudomonas putida suspensions exposed to varying concentrations of glucose, chosen to represent readily bioavailable substrates in soils. Glucose concentrations tested ranged between 0 and 5.5mM and changes in pH, dissolved oxygen and dissolved organic and inorganic carbon were tracked over 48h. We then combined literature review and wet-chemical experiments to compile the pH dependence of rates of organic substrate oxidation by MnO2, including glucose. Our results demonstrate a strong pH dependence for these abiotic reactions. In assemblages of P. putida - MnO2, kinetic limitations for abiotic C oxidation by MnO2 are overcome by changes in biogeochemical conditions that result from bacterial C metabolism. When extrapolated to a soil solution confronted to an input of fresh dissolved organic matter, bacterial C metabolism of the labile fraction may lower solution pH into a regime that favours abiotic oxidation of recalcitrant C by MnO2. This project demonstrates that the co-occurrence of mineral particles with metabolically active cells provides a direct link between the C and Mn cycles.

Self-organization in complex oxide thin films: from 2D to 0D nanostructures of SrRuO3 and CoCr2O4

NASA Astrophysics Data System (ADS)

Sánchez, F.; Lüders, U.; Herranz, G.; Infante, I. C.; Fontcuberta, J.; García-Cuenca, M. V.; Ferrater, C.; Varela, M.

2005-05-01

We report here on the controlled fabrication of nanostructures of varied dimensionality by self-organization processes in the heteroepitaxial growth of SrRuO3 (SRO) and CoCr2O4 (CCO) films. The surface of SRO films on SrTiO3(001) substrates can show extremely smooth terraces (2D objects) separated by atomic steps, a structure of faceted islands (0D objects), a cross-hatch morphology (1D objects), an array of finger-like units (1D objects), or an array of giant bunched steps (1D objects). The surface can be tailored to a particular structure by controlling the vicinality of the substrate and the growth rate and nominal thickness of the film. In the case of CCO films, grown on (001)-oriented MgAl2O4 or MgO substrates, high aspect ratio {111}-faceted pyramids and hut clusters (0D objects), highly oriented and having a similar size, appear above a critical thickness. The size and spatial density can be tuned by varying deposition temperature, nominal thickness, and substrate. This dependence allows the fabrication of surfaces being fully faceted (2D objects), or having arrays of dislocated pyramids of up to micrometric size, or small coherently lattice strained pyramids having a nanometric size. We discuss the driving forces that originate the peculiar SRO and CCO nanostructures. The findings illustrate that the growth of complex oxides can promote a variety of novel self-organized morphologies, and suggest original strategies to fabricate templates or hybrid structures of oxides combining varied functionalities.

Combining affinity proteomics and network context to identify new phosphatase substrates and adapters in growth pathways

PubMed Central

Sacco, Francesca; Boldt, Karsten; Calderone, Alberto; Panni, Simona; Paoluzi, Serena; Castagnoli, Luisa; Ueffing, Marius; Cesareni, Gianni

2014-01-01

Protein phosphorylation homoeostasis is tightly controlled and pathological conditions are caused by subtle alterations of the cell phosphorylation profile. Altered levels of kinase activities have already been associated to specific diseases. Less is known about the impact of phosphatases, the enzymes that down-regulate phosphorylation by removing the phosphate groups. This is partly due to our poor understanding of the phosphatase-substrate network. Much of phosphatase substrate specificity is not based on intrinsic enzyme specificity with the catalytic pocket recognizing the sequence/structure context of the phosphorylated residue. In addition many phosphatase catalytic subunits do not form a stable complex with their substrates. This makes the inference and validation of phosphatase substrates a non-trivial task. Here, we present a novel approach that builds on the observation that much of phosphatase substrate selection is based on the network of physical interactions linking the phosphatase to the substrate. We first used affinity proteomics coupled to quantitative mass spectrometry to saturate the interactome of eight phosphatases whose down regulations was shown to affect the activation of the RAS-PI3K pathway. By integrating information from functional siRNA with protein interaction information, we develop a strategy that aims at inferring phosphatase physiological substrates. Graph analysis is used to identify protein scaffolds that may link the catalytic subunits to their substrates. By this approach we rediscover several previously described phosphatase substrate interactions and characterize two new protein scaffolds that promote the dephosphorylation of PTPN11 and ERK by DUSP18 and DUSP26, respectively. PMID:24847354

Impact of substrate on structure and electrical properties in lead-based ferroelectric thin films

NASA Astrophysics Data System (ADS)

Valanoor, Nagarajan Venkatasubramanian

Current trends in semiconductor technology demand that ferroelectric materials be used in thin film form, rather than bulk, for integration and scaling purposes. An inevitable consequence of integration is substrate induced constraint and stress. Sources of this stress are the lattice and thermal mismatch between film and substrate, structural phase transformation which leads to spontaneous strains, and dislocation cores at the film substrate interface. In addition to classical stress relaxation mechanisms all highly tetragonal ferroelectrics relax internal stress via formation of polydomain (90° domains and not 180° domains) structures below the phase transformation, which brings about a change in the microstructure of the film. Hence it is possible to control the resultant microstructure by controlling the degree of polydomain relaxation. Obviously this affects the electrical and electro-mechanical properties and in turn the device performance. The goal of this research is to study this structure-property relationship of ferroelectric thin films where in the structure has been systematically modified by changing the substrate-induced effect. To investigate the effect of the substrate, epitaxial films of PbZr 0.2Ti0.8O3 were grown by pulsed laser deposition (PLD). Epitaxial films reduce the complexities introduced grain boundaries and multiple domain orientations. By systematically changing the thickness the spontaneous strain or c/a ratio can be varied. As a consequence polydomain formation varies as a function of film thickness. Thus this is an effective yet simple method to fully understand the impact of stress on structure-property inter-relationships. The theoretical background for these experiments is first laid out by a thermodynamic analysis of the polydomain formation. It leads to the construction of a domain stability map and indicates a presence of a critical thickness for polydomain formation. This is followed by an investigation of the impact of polydomain formation on quasi-static and dynamic polarization switching. To correlate the material microstructure to switching, an activation field, alpha, is introduced. It is shown theoretically that alpha ∝ (c/a-1)3.5 and a good experimental fit can be obtained. However it is observed that polydomain formation does not impact the electromechanical and dielectric response significantly. It is shown experimentally and theoretically that stress-induced polarization varies only by 10%. Therefore to study the impact of in plane stresses induced by substrate on piezoelectric and dielectric response we chose a "soft" relaxor ferroelectric (RFE) wherein the Curie temperature is close to room temperature. In this case even a small application of stress can change the properties significantly. The relaxor composition chosen was PbMg1/3Nb 2/3O3(90%)-PbTiO3(10%). By systematically changing the substrate and the thickness, stresses in the film the electromechanical constants is varied. High-resolution electron microscopy revealed a distinct change in the microstructure as a function of thickness, and a probable answer as to why thin films show inferior properties compared to bulk materials is proposed. The last part of this thesis focuses on the effect of micro stresses. Two examples are demonstrated where the mechanical forces of interaction between the film and substrate are manipulated on a very local scale. We show that by inducing stresses at local regions one can induce polydomains in film thinner than previously calculated critical thickness, while by removing constraint at local regions we can enhance the d33 co-efficient to values higher than those shown by bulk ceramics.

Biochemistry Students' Ideas about How an Enzyme Interacts with a Substrate

ERIC Educational Resources Information Center

Linenberger, Kimberly J.; Bretz, Stacey Lowery

2015-01-01

Enzyme-substrate interactions are a fundamental concept of biochemistry that is built upon throughout multiple biochemistry courses. Central to understanding enzyme-substrate interactions is specific knowledge of exactly how an enzyme and substrate interact. Within this narrower topic, students must understand the various binding sites on an…

Heparin/heparan sulfate 6-O-sulfatase from Flavobacterium heparinum: integrated structural and biochemical investigation of enzyme active site and substrate specificity.

PubMed

Myette, James R; Soundararajan, Venkataramanan; Shriver, Zachary; Raman, Rahul; Sasisekharan, Ram

2009-12-11

Heparin and heparan sulfate glycosaminoglycans (HSGAGs) comprise a chemically heterogeneous class of sulfated polysaccharides. The development of structure-activity relationships for this class of polysaccharides requires the identification and characterization of degrading enzymes with defined substrate specificity and enzymatic activity. Toward this end, we report here the molecular cloning and extensive structure-function analysis of a 6-O-sulfatase from the Gram-negative bacterium Flavobacterium heparinum. In addition, we report the recombinant expression of this enzyme in Escherichia coli in a soluble, active form and identify it as a specific HSGAG sulfatase. We further define the mechanism of action of the enzyme through biochemical and structural studies. Through the use of defined substrates, we investigate the kinetic properties of the enzyme. This analysis was complemented by homology-based molecular modeling studies that sought to rationalize the substrate specificity of the enzyme and mode of action through an analysis of the active-site topology of the enzyme including identifying key enzyme-substrate interactions and assigning key amino acids within the active site of the enzyme. Taken together, our structural and biochemical studies indicate that 6-O-sulfatase is a predominantly exolytic enzyme that specifically acts on N-sulfated or N-acetylated 6-O-sulfated glucosamines present at the non-reducing end of HSGAG oligosaccharide substrates. This requirement for the N-acetyl or N-sulfo groups on the glucosamine substrate can be explained through eliciting favorable interactions with key residues within the active site of the enzyme. These findings provide a framework that enables the use of 6-O-sulfatase as a tool for HSGAG structure-activity studies as well as expand our biochemical and structural understanding of this important class of enzymes.

Nucleation and growth of single layer graphene on electrodeposited Cu by cold wall chemical vapor deposition

NASA Astrophysics Data System (ADS)

Das, Shantanu; Drucker, Jeff

2017-03-01

The nucleation density and average size of graphene crystallites grown using cold wall chemical vapor deposition (CVD) on 4 μm thick Cu films electrodeposited on W substrates can be tuned by varying growth parameters. Growth at a fixed substrate temperature of 1000 °C and total pressure of 700 Torr using Ar, H2 and CH4 mixtures enabled the contribution of total flow rate, CH4:H2 ratio and dilution of the CH4/H2 mixture by Ar to be identified. The largest variation in nucleation density was obtained by varying the CH4:H2 ratio. The observed morphological changes are analogous to those that would be expected if the deposition rate were varied at fixed substrate temperature for physical deposition using thermal evaporation. The graphene crystallite boundary morphology progresses from irregular/jagged through convex hexagonal to regular hexagonal as the effective C deposition rate decreases. This observation suggests that edge diffusion of C atoms along the crystallite boundaries, in addition to H2 etching, may contribute to shape evolution of the graphene crystallites. These results demonstrate that graphene grown using cold wall CVD follows a nucleation and growth mechanism similar to hot wall CVD. As a consequence, the vast knowledge base relevant to hot wall CVD may be exploited for graphene synthesis by the industrially preferable cold wall method.

Transfer of monolayer TMD WS2 and Raman study of substrate effects

PubMed Central

Mlack, Jerome T.; Masih Das, Paul; Danda, Gopinath; Chou, Yung-Chien; Naylor, Carl H.; Lin, Zhong; López, Néstor Perea; Zhang, Tianyi; Terrones, Mauricio; Johnson, A. T. Charlie; Drndić, Marija

2017-01-01

A facile transfer process for transition metal dichalcogenide WS2 flakes is reported and the effect of the underlying substrate on the flake properties is investigated using Raman spectroscopy. The flakes are transferred from their growth substrate using polymethyl methacrylate (PMMA) and a wet etch to allow the user to transfer the flakes to a final substrate using a microscope and micromanipulator combined with semi-transparent Kapton tape. The substrates used range from insulators such as industry standard high-k dielectric HfO2 and “green polymer” parylene-C, to conducting chemical vapor deposition (CVD) grown graphene. Raman spectroscopy is used first to confirm the material quality of the transferred flakes to the substrates and subsequently to analyze and separate the effects arising from material transfer from those arising from interactions with the substrate. We observe changes in the Raman spectra associated with the interactions between the substrates in the flakes. These interactions affect both in-plane and out-of-plane modes in different ways depending on their sources, for example strain or surface charge. These changes vary with final substrate, with the strongest effects being observed for WS2 transferred onto graphene and HfO2, demonstrating the importance of understanding substrate interaction for fabrication of future devices. PMID:28220852

The trimethylammonium headgroup of choline is a major determinant for substrate binding and specificity in choline oxidase.

PubMed

Gadda, Giovanni; Powell, Nichole L N; Menon, Prashanthi

2004-10-15

Choline oxidase catalyzes the oxidation of choline to glycine betaine via two sequential flavin-linked transfers of hydride equivalents to molecular oxygen and formation of a betaine aldehyde intermediate. In the present study, choline and glycine betaine analogs were used as substrates and inhibitors for the enzyme to investigate the structural determinants that are relevant for substrate recognition and specificity. Competitive inhibition patterns with respect to choline were determined for a number of substituted amines at pH 6.5 and 25 degrees C. The Kis values for the carboxylate-containing ligands glycine betaine, N,N-dimethylglycine, and N-methylglycine increased monotonically with decreasing number of methyl groups, consistent with the trimethylammonium portion of the ligand being important for binding. In contrast, the acetate portion of glycine betaine did not contribute to binding, as suggested by lack of changes in the Kis values upon substituting glycine betaine with inhibitors containing methyl, ethyl, allyl, and 2-amino-ethyl side chains. In agreement with the inhibition data, the specificity of the enzyme for the organic substrate (kcat/Km value) decreased when N,N-dimethylethanolamine, N-methylethanolamine, and the isosteric substrate 3,3-dimethyl-1-butanol were used as substrate instead of choline; a contribution of approximately 7 kcal mol(-1) toward substrate discrimination was estimated for the interaction of the trimethylammonium portion of the substrate with the active site of choline oxidase.

Influence of Rearing Substrates and Nontarget Hosts on the Bionomics of the Tachinid Parasitoid Nemorilla maculosa (Diptera: Tachinidae).

PubMed

Agbessenou, Ayaovi; Tounou, Agbéko Kodjo; Dannon, Elie Ayitondji; Datinon, Benjamin; Agboton, Cyriaque; Srinivasan, Ramasamy; Pittendrigh, Barry Robert; Tamò, Manuele

2018-04-05

The tachinid Nemorilla maculosa Meigen (Diptera: Tachinidae) was introduced from Taiwan to Benin for evaluating its potential as a biocontrol candidate against the cowpea pest Maruca vitrata (Fabricius) (Lepidoptera: Crambidae). To optimize its rearing, we assessed the influence of M. vitrata larval age and rearing substrate-cowpea germinating grains and peabush leaves-on its life table parameters, while its host specificity was investigated with regard to nontarget effects. Parasitism rates were higher when older larvae (10- and 14-d old) were offered to females of N. maculosa compared to the younger (2-, 4-, and 6-d old) host larvae. Regardless of the rearing substrate, development time was longer for females than males, and females lived longer than males irrespective of the age of the host. Sex ratio did not vary significantly with host ages or rearing substrate. The average number of eggs laid by a female reared from M. vitrata larvae feeding on cowpea germinating grains or peabush leaves was 94.2 ± 4.38 and 71.9 ± 1.70 eggs, respectively. The host suitability of N. maculosa was assessed by testing four nontarget Lepidoptera species: Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae), Sesamia calamistis Hampson (Lepidoptera: Noctuidae), Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae), and Eldana saccharina Walker (Lepidoptera: Pyralidae). Larvae of S. littoralis and C. cephalonica were successfully parasitized while N. maculosa did not develop in the larvae of E. saccharina and S. calamistis although they were parasitized. Despite the potential of N. maculosa as a biological control agent against the pod borer M. vitrata, more detailed nontarget studies, extending to other native Crambidae species, are needed before making decisions on field releases.

Vulnerability Assessment of Mangrove Habitat to the Variables of the Oceanography Using CVI Method (Coastal Vulnerability Index) in Trimulyo Mangrove Area, Genuk District, Semarang

NASA Astrophysics Data System (ADS)

Ahmad, Rifandi Raditya; Fuad, Muhammad

2018-02-01

Some functions of mangrove areas in coastal ecosystems as a green belt, because mangrove serves as a protector of the beach from the sea waves, as a good habitat for coastal biota and for nutrition supply. Decreased condition or degradation of mangrove habitat caused by several oceanographic factors. Mangrove habitats have some specific characteristics such as salinity, tides, and muddy substrates. Considering the role of mangrove area is very important, it is necessary to study about the potential of mangrove habitat so that the habitat level of mangrove habitat in the east coast of Semarang city is known. The purpose of this research is to obtain an index and condition of habitat of mangrove habitat at location of research based on tidal, salinity, substrate type, coastline change. Observation by using purposive method and calculation of habitat index value of mangrove habitat using CVI (Coastal Vulnerability Index) method with scores divided into 3 groups namely low, medium and high. The results showed that there is a zone of research belonging to the medium vulnerability category with the most influential variables is because there is abrasion that sweeps the mangrove substrate. Trimulyo mangrove habitat has high vulnerable variable of tidal frequency, then based on value variable Salinity is categorized as low vulnerability, whereas for mangrove habitat vulnerability based on variable type of substrate belong to low and medium vulnerability category. The CVI values of mangrove habitats divided into zones 1; 2; and 3 were found to varying values of 1.54; 3.79; 1.09, it indicates that there is a zone with the vulnerability of mangrove habitat at the study site belonging to low and medium vulnerability category.

High Resolution Structures of the Human ABO(H) Blood Group Enzymes in Complex with Donor Analogs Reveal That the Enzymes Utilize Multiple Donor Conformations to Bind Substrates in a Stepwise Manner*

PubMed Central

Gagnon, Susannah M. L.; Meloncelli, Peter J.; Zheng, Ruixiang B.; Haji-Ghassemi, Omid; Johal, Asha R.; Borisova, Svetlana N.; Lowary, Todd L.; Evans, Stephen V.

2015-01-01

Homologous glycosyltransferases α-(1→3)-N-acetylgalactosaminyltransferase (GTA) and α-(1→3)-galactosyltransferase (GTB) catalyze the final step in ABO(H) blood group A and B antigen synthesis through sugar transfer from activated donor to the H antigen acceptor. These enzymes have a GT-A fold type with characteristic mobile polypeptide loops that cover the active site upon substrate binding and, despite intense investigation, many aspects of substrate specificity and catalysis remain unclear. The structures of GTA, GTB, and their chimeras have been determined to between 1.55 and 1.39 Å resolution in complex with natural donors UDP-Gal, UDP-Glc and, in an attempt to overcome one of the common problems associated with three-dimensional studies, the non-hydrolyzable donor analog UDP-phosphono-galactose (UDP-C-Gal). Whereas the uracil moieties of the donors are observed to maintain a constant location, the sugar moieties lie in four distinct conformations, varying from extended to the “tucked under” conformation associated with catalysis, each stabilized by different hydrogen bonding partners with the enzyme. Further, several structures show clear evidence that the donor sugar is disordered over two of the observed conformations and so provide evidence for stepwise insertion into the active site. Although the natural donors can both assume the tucked under conformation in complex with enzyme, UDP-C-Gal cannot. Whereas UDP-C-Gal was designed to be “isosteric” with natural donor, the small differences in structure imposed by changing the epimeric oxygen atom to carbon appear to render the enzyme incapable of binding the analog in the active conformation and so preclude its use as a substrate mimic in GTA and GTB. PMID:26374898

Peptide microarray analysis of substrate specificity of the transmembrane Ser/Thr kinase KPI-2 reveals reactivity with cystic fibrosis transmembrane conductance regulator and phosphorylase.

PubMed

Wang, Hong; Brautigan, David L

2006-11-01

Human lemur (Lmr) kinases are predicted to be Tyr kinases based on sequences and are related to neurotrophin receptor Trk kinases. This study used homogeneous recombinant KPI-2 (Lmr2, LMTK2, Cprk, brain-enriched protein kinase) kinase domain and a library of 1,154 peptides on a microarray to analyze substrate specificity. We found that KPI-2 is strictly a Ser/Thr kinase that reacts with Ser either preceded by or followed by Pro residues but unlike other Pro-directed kinases does not strictly require an adjacent Pro residue. The most reactive peptide in the library corresponds to Ser-737 of cystic fibrosis transmembrane conductance regulator, and the recombinant R domain of cystic fibrosis transmembrane conductance regulator was a preferred substrate. Furthermore the KPI-2 kinase phosphorylated peptides corresponding to the single site in phosphorylase and purified phosphorylase b, making this only the second known phosphorylase b kinase. Phosphorylase was used as a specific substrate to show that KPI-2 is inhibited in living cells by addition of nerve growth factor or serum. The results demonstrate the utility of the peptide library to probe specificity and discover kinase substrates and offer a specific assay that reveals hormonal regulation of the activity of this unusual transmembrane kinase.

Microstructural studies by TEM of diamond films grown by combustion flame

NASA Astrophysics Data System (ADS)

Ma, G.-H. M.; Hirose, Y.; Amanuma, S.; McClure, M.; Prater, J. T.; Glass, J. T.

Microstructures of diamond films grown in an oxygen-acetylene combustion flame were studied by TEM. The O2/C2H2 gas ratio was fixed and the substrate materials and temperature were varied. High quality diamond films were grown by this method at high growth rates of about 30 micron/hr. A rough surface and high density of secondary nucleation sites and microtwins were observed in the diamond grains grown on molybdenum (Mo) at a substrate temperature of 500 C. When the substrate temperature wass raised to between 500 and 870 C, the defect density was greatly reduced, revealing a low density of stacking faults and dislocations. Diamond films grown on Si substrates did not show the same substrate temperature dependence on defect density, at least not over the same temperature range. However, the same correlation between defect density, secondary nucleation, and surface morphology was observed.

Hysteresis in the Cell Response to Time-Dependent Substrate Stiffness

PubMed Central

Besser, Achim; Schwarz, Ulrich S.

2010-01-01

Abstract Mechanical cues like the rigidity of the substrate are main determinants for the decision-making of adherent cells. Here we use a mechano-chemical model to predict the cellular response to varying substrate stiffnesses. The model equations combine the mechanics of contractile actin filament bundles with a model for the Rho-signaling pathway triggered by forces at cell-matrix contacts. A bifurcation analysis of cellular contractility as a function of substrate stiffness reveals a bistable response, thus defining a lower threshold of stiffness, below which cells are not able to build up contractile forces, and an upper threshold of stiffness, above which cells are always in a strongly contracted state. Using the full dynamical model, we predict that rate-dependent hysteresis will occur in the cellular traction forces when cells are exposed to substrates of time-dependent stiffness. PMID:20655823

Ultra-low roughness magneto-rheological finishing for EUV mask substrates

NASA Astrophysics Data System (ADS)

Dumas, Paul; Jenkins, Richard; McFee, Chuck; Kadaksham, Arun J.; Balachandran, Dave K.; Teki, Ranganath

2013-09-01

EUV mask substrates, made of titania-doped fused silica, ideally require sub-Angstrom surface roughness, sub-30 nm flatness, and no bumps/pits larger than 1 nm in height/depth. To achieve the above specifications, substrates must undergo iterative global and local polishing processes. Magnetorheological finishing (MRF) is a local polishing technique which can accurately and deterministically correct substrate figure, but typically results in a higher surface roughness than the current requirements for EUV substrates. We describe a new super-fine MRF® polishing fluid whichis able to meet both flatness and roughness specifications for EUV mask blanks. This eases the burden on the subsequent global polishing process by decreasing the polishing time, and hence the defectivity and extent of figure distortion.

QM/MM Investigation of Substrate and Product Specificities of Suv4-20h2: How Does This Enzyme Generate Dimethylated H4K20 from Monomethylated Substrate?

PubMed

Qian, Ping; Guo, Haobo; Wang, Liang; Guo, Hong

2017-06-13

Protein lysine methyltransferases (PKMTs) catalyze the methylation of lysine residues on histone proteins in the regulation of chromatin structure and gene expression. In contrast to many other PKMTs for which unmodified lysine is the methylation target, the enzymes in the Suv4-20 family are able to generate dimethylated product (H4K20me2) based exclusively on the monomethylated H4K20 substrate (H4K20me1). The origin of such substrate/product specificity is still not clear. Here, molecular dynamics (MD) and free energy (potential of mean force) simulations are undertaken using quantum mechanical/molecular mechanical (QM/MM) potentials to understand the substrate/product specificities of Suv4-20h2, a member of the Suv4-20 family. The free energy barriers for mono-, di-, and trimethylation in Suv4-20h2 obtained from the simulations are found to be well correlated with the specificities observed experimentally with the allowed dimethylation based on the H4K20me1 substrate and prohibited monomethylation and trimethylation based on H4K20 and H4K20me2, respectively. It is demonstrated that the reason for the relatively efficient dimethylation is an effective transition state (TS) stabilization through strengthening the CH···O interactions as well as the presence of a cation-π interaction at the transition state. The simulations also show that the failures of Suv4-20h2 to catalyze monomethylation and trimethylation are due, respectively, to a less effective TS stabilization and inability of the reactant complex containing H4K20me2 to adopt a reactive (near attack) configuration for methyl transfer. The results suggest that care must be exercised in the prediction of the substrate specificity based only on the existence of near attack configurations in substrate complexes.

Influence of Substrate Biasing on (Ba,Sr)TiO3 Films Prepared by Electron Cyclotron Resonance Plasma Sputtering

NASA Astrophysics Data System (ADS)

Matsumoto, Takeshi; Niino, Atsushi; Ohtsu, Yasunori; Misawa, Tatsuya; Yonesu, Akira; Fujita, Hiroharu; Miyake, Shoji

2004-03-01

(Ba,Sr)TiO3 (BST) films were deposited by electron cyclotron resonance (ECR) plasma sputtering with mirror confinement. DC bias voltage was applied to Pt/Ti/SiO2/Si substrates during deposition to vary the intensity of bombardment of energetic ions and to modify film properties. BST films deposited on the substrates at floating potential (approximately +20 V) were found to be amorphous, while films deposited on +40 V-biased substrates were crystalline in spite of a low substrate temperature below 648 K. In addition, atomic diffusion, which causes deterioration in the electrical properties of the films, was hardly observed in the crystallized films deposited with +40 V bias perhaps due to the low substrate temperature. Plasma diagnoses revealed that application of a positive bias to the substrate reduced the energy of ion bombardment and increased the density of excited neutral particles, which was assumed to result in the promotion of chemical reactions during deposition and the crystallization of BST films at a low temperature.

Cell-Imprinted Substrates Modulate Differentiation, Redifferentiation, and Transdifferentiation.

PubMed

Bonakdar, Shahin; Mahmoudi, Morteza; Montazeri, Leila; Taghipoor, Mojtaba; Bertsch, Arnaud; Shokrgozar, Mohammad Ali; Sharifi, Shahriar; Majidi, Mohammad; Mashinchian, Omid; Hamrang Sekachaei, Mohammad; Zolfaghari, Pegah; Renaud, Philippe

2016-06-08

Differentiation of stem cells into mature cells through the use of physical approaches is of great interest. Here, we prepared smart nanoenvironments by cell-imprinted substrates based on chondrocytes, tenocytes, and semifibroblasts as templates and demonstrated their potential for differentiation, redifferentiation, and transdifferentiation. Analysis of shape and upregulation/downregulation of specific genes of stem cells, which were seeded on these cell-imprinted substrates, confirmed that imprinted substrates have the capability to induce specific shapes and molecular characteristics of the cell types that were used as templates for cell-imprinting. Interestingly, immunofluorescent staining of a specific protein in chondrocytes (i.e., collagen type II) confirmed that adipose-derived stem cells, semifibroblasts, and tenocytes can acquire the chondrocyte phenotype after a 14 day culture on chondrocyte-imprinted substrates. In summary, we propose that common polystyrene tissue culture plates can be replaced by this imprinting technique as an effective and promising way to regulate any cell phenotype in vitro with significant potential applications in regenerative medicine and cell-based therapies.

Insights into the Specificity of Lysine Acetyltransferases

DOE PAGES

Tucker, Alex C.; Taylor, Keenan C.; Rank, Katherine C.; ...

2014-11-07

Reversible lysine acetylation by protein acetyltransferases is a conserved regulatory mechanism that controls diverse cellular pathways. Gcn5-related N-acetyltransferases (GNATs), named after their founding member, are found in all domains of life. GNATs are known for their role as histone acetyltransferases, but non-histone bacterial protein acetytransferases have been identified. Only structures of GNAT complexes with short histone peptide substrates are available in databases. Given the biological importance of this modification and the abundance of lysine in polypeptides, how specificity is attained for larger protein substrates is central to understanding acetyl-lysine-regulated networks. In this paper, we report the structure of a GNATmore » in complex with a globular protein substrate solved to 1.9 Å. GNAT binds the protein substrate with extensive surface interactions distinct from those reported for GNAT-peptide complexes. Finally, our data reveal determinants needed for the recognition of a protein substrate and provide insight into the specificity of GNATs.« less

Acquisition and evolution of plant pathogenesis-associated gene clusters and candidate determinants of tissue-specificity in xanthomonas.

PubMed

Lu, Hong; Patil, Prabhu; Van Sluys, Marie-Anne; White, Frank F; Ryan, Robert P; Dow, J Maxwell; Rabinowicz, Pablo; Salzberg, Steven L; Leach, Jan E; Sonti, Ramesh; Brendel, Volker; Bogdanove, Adam J

2008-01-01

Xanthomonas is a large genus of plant-associated and plant-pathogenic bacteria. Collectively, members cause diseases on over 392 plant species. Individually, they exhibit marked host- and tissue-specificity. The determinants of this specificity are unknown. To assess potential contributions to host- and tissue-specificity, pathogenesis-associated gene clusters were compared across genomes of eight Xanthomonas strains representing vascular or non-vascular pathogens of rice, brassicas, pepper and tomato, and citrus. The gum cluster for extracellular polysaccharide is conserved except for gumN and sequences downstream. The xcs and xps clusters for type II secretion are conserved, except in the rice pathogens, in which xcs is missing. In the otherwise conserved hrp cluster, sequences flanking the core genes for type III secretion vary with respect to insertion sequence element and putative effector gene content. Variation at the rpf (regulation of pathogenicity factors) cluster is more pronounced, though genes with established functional relevance are conserved. A cluster for synthesis of lipopolysaccharide varies highly, suggesting multiple horizontal gene transfers and reassortments, but this variation does not correlate with host- or tissue-specificity. Phylogenetic trees based on amino acid alignments of gum, xps, xcs, hrp, and rpf cluster products generally reflect strain phylogeny. However, amino acid residues at four positions correlate with tissue specificity, revealing hpaA and xpsD as candidate determinants. Examination of genome sequences of xanthomonads Xylella fastidiosa and Stenotrophomonas maltophilia revealed that the hrp, gum, and xcs clusters are recent acquisitions in the Xanthomonas lineage. Our results provide insight into the ancestral Xanthomonas genome and indicate that differentiation with respect to host- and tissue-specificity involved not major modifications or wholesale exchange of clusters, but subtle changes in a small number of genes or in non-coding sequences, and/or differences outside the clusters, potentially among regulatory targets or secretory substrates.

Substrate-specific modifications on magnetic iron oxide nanoparticles as an artificial peroxidase for improving sensitivity in glucose detection.

PubMed

Liu, Yanping; Yu, Faquan

2011-04-08

Magnetic iron oxide nanoparticles (MION) were recently found to act as a peroxidase with intrinsic advantages over natural counterparts. Their limited affinity toward catalysis substrates, however, dramatically reduces their utility. In this paper, some effective groups were screened out and conjugated on MION as substrate-specific modifications for improving MION's affinity to substrates and hence utility. Nanoparticles of four different superficial structures were synthesized and characterized by TEM, size, zeta potential and SQUID, and assayed for peroxidase activity. Glucose detection was selected as an application model system to evaluate the bonus thereof. Catalysis was found to follow Michaelis-Menten kinetics. Sulfhydryl groups incorporated on MION (SH-MION) notably improve the affinity toward a substrate (hydrogen peroxide) and so do amino groups (NH₂-MION) toward another substrate, proved by variation in the determined kinetic parameters. A synergistically positive effect was observed and an apparently elevated detection sensitivity and a significantly lowered detection limit of glucose were achieved when integrated with both sulfhydryl and amino groups (SH-NH₂-MION). Our findings suggest that substrate-specific surface modifications are a straightforward and robust strategy to improve MION peroxidase-like activity. The high activity extends magnetic nanoparticles to wide applications other than glucose detection.

Low energy electron catalyst: the electronic origin of catalytic strategies.

PubMed

Davis, Daly; Sajeev, Y

2016-10-12

Using a low energy electron (LEE) as a catalyst, the electronic origin of the catalytic strategies corresponding to substrate selectivity, reaction specificity and reaction rate enhancement is investigated for a reversible unimolecular elementary reaction. An electronic energy complementarity between the catalyst and the substrate molecule is the origin of substrate selectivity and reaction specificity. The electronic energy complementarity is induced by tuning the electronic energy of the catalyst. The energy complementarity maximizes the binding forces between the catalyst and the molecule. Consequently, a new electronically metastable high-energy reactant state and a corresponding new low barrier reaction path are resonantly created for a specific reaction of the substrate through the formation of a catalyst-substrate transient adduct. The LEE catalysis also reveals a fundamental structure-energy correspondence in the formation of the catalyst-substrate transient adduct. Since the energy complementarities corresponding to the substrate molecules of the forward and the backward steps of the reversible reactions are not the same due to their structural differences, the LEE catalyst exhibits a unique one-way catalytic strategy, i.e., the LEE catalyst favors the reversible reaction more effectively in one direction. A characteristic stronger binding of the catalyst to the transition state of the reaction than in the initial reactant state and the final product state is the molecular origin of barrier lowering.

Polyelectrolyte brushes on dielectric surfaces

NASA Astrophysics Data System (ADS)

Antila, Hanne; Luijten, Erik

When chains of charged polymers are grafted to a solid surface, a polyelectrolyte (PE) brush results. These types of PE assemblies have a wide range of applications ranging from fuel cells and switchable electrodes to drug delivery. Many of these applications stem from the ability of PE brushes to respond to external stimuli: the brush properties can be tuned, for example, by varying electric field, PE grafting density, pH, salt concentration or salt valency. Accordingly, deciphering the brush behavior under different conditions has been a subject of considerable experimental, theoretical, and computational research efforts. However, the effect of the dielectric properties of the substrate on the PE brush has received much less attention. We use coarse-grained molecular dynamics simulations to show how varying the dielectric mismatch between the solvent and the substrate can significantly affect the brush. We demonstrate how tuning this mismatch can either diminish or enhance the effects of other control parameters, such as pH, on the brush properties. Furthermore, we investigate how dielectric properties of the substrate affect the brush, and the ion distribution and mobility within the brush, when the brush is exposed to an electric field.

Effect of film thickness on soft magnetic behavior of Fe2CoSi Heusler alloy for spin transfer torque device applications

NASA Astrophysics Data System (ADS)

Asvini, V.; Saravanan, G.; Kalaiezhily, R. K.; Raja, M. Manivel; Ravichandran, K.

2018-04-01

Fe2CoSi based Heusler alloy thin films were deposited on Si (111) wafer (substrate) of varying thickness using ultra high vacuum DC magnetron sputtering. The structural behavior was observed and found to be hold the L21 structure. The deposited thin films were characterized magnetic properties using vibrating sample magnetometer; the result shows a very high saturated magnetization (Ms), lowest coercivity (Hc), high curie transition temperature (Tc) and low hysteresis loss. Thin film thickness of 75 nm Fe2CoSi sample maintained at substrate temperature 450°C shows the lowest coercivity (Hc=7 Oe). In general, Fe2CoSi Heusler alloys curie transition temperature is very high, due to strong exchange interaction between the Fe and Co atoms. The substrate temperature was kept constant at 450°C for varying thickness (e.g. 5, 20, 50, 75 and 100 nm) of thin film sample. The 75 nm thickness thin film sample shows well crystallanity and good magnetic properties, further squareness ratio in B-H loop increases with the increase in film thickness.

Structural and kinetic studies of a novel nerol dehydrogenase from Persicaria minor, a nerol-specific enzyme for citral biosynthesis.

PubMed

Tan, Cheng Seng; Hassan, Maizom; Mohamed Hussein, Zeti Azura; Ismail, Ismanizan; Ho, Kok Lian; Ng, Chyan Leong; Zainal, Zamri

2018-02-01

Geraniol degradation pathway has long been elucidated in microorganisms through bioconversion studies, yet weakly characterised in plants; enzyme with specific nerol-oxidising activity has not been reported. A novel cDNA encodes nerol dehydrogenase (PmNeDH) was isolated from Persicaria minor. The recombinant PmNeDH (rPmNeDH) is a homodimeric enzyme that belongs to MDR (medium-chain dehydrogenases/reductases) superfamily that catalyses the first oxidative step of geraniol degradation pathway in citral biosynthesis. Kinetic analysis revealed that rPmNeDH has a high specificity for allylic primary alcohols with backbone ≤10 carbons. rPmNeDH has ∼3 fold higher affinity towards nerol (cis-3,7-dimethyl-2,6-octadien-1-ol) than its trans-isomer, geraniol. To our knowledge, this is the first alcohol dehydrogenase with higher preference towards nerol, suggesting that nerol can be effective substrate for citral biosynthesis in P. minor. The rPmNeDH crystal structure (1.54 Å) showed high similarity with enzyme structures from MDR superfamily. Structure guided mutation was conducted to describe the relationships between substrate specificity and residue substitutions in the active site. Kinetics analyses of wild-type rPmNeDH and several active site mutants demonstrated that the substrate specificity of rPmNeDH can be altered by changing any selected active site residues (Asp 280 , Leu 294 and Ala 303 ). Interestingly, the L294F, A303F and A303G mutants were able to revamp the substrate preference towards geraniol. Furthermore, mutant that exhibited a broader substrate range was also obtained. This study demonstrates that P. minor may have evolved to contain enzyme that optimally recognise cis-configured nerol as substrate. rPmNeDH structure provides new insights into the substrate specificity and active site plasticity in MDR superfamily. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Comparison of the tribological properties at 25 C of seven different polyimide films bonded to 301 stainless steel

NASA Technical Reports Server (NTRS)

Fusaro, R. L.

1980-01-01

A pin-on-disk type of friction and wear apparatus was used to study the tribological properties of seven different polyimide films bonded to AISI 301 stainless steel disks at 25 C. It was found that the substrate material was extremely influential in determining the lubricating ability of the polyimide films. All seven films spalled in less than 1000 cycles of sliding. This was believed to be caused by poor adherence to the 301 stainless steel or the inability of the films to withstand the high localized tensile stresses imparted by the deformation of the soft substrate under sliding conditions. The friction coefficients obtained for six of the polyimides varied between 0.21 to 0.32 while one varied between 0.32 to 0.39.

Selecting the correct cellular model for assessing of the biological response of collagen-based biomaterials.

PubMed

Davidenko, Natalia; Hamaia, Samir; Bax, Daniel V; Malcor, Jean-Daniel; Schuster, Carlos F; Gullberg, Donald; Farndale, Richard W; Best, Serena M; Cameron, Ruth E

2018-01-01

Accurate evaluation of the biological performance of biomaterials requires the correct assessment of their native-like cell ligation properties. However, cell attachment studies often overlook the details of the substrate-cell binding mechanisms, be they integrin-mediated or non-specific, and ignore the class- and species-specificities of the cell adhesion receptor involved. In this work we have used different collagen (Col) substrates (fibrillar collagens I, II and III and network-forming Col IV), containing different affinity cell-recognition motifs, to establish the influence of the receptor identity and species-specificity on collagen-cell interactive properties. Receptor expression was varied by using cells of different origin, or transfecting collagen-binding integrins into integrin-null cells. These include mouse C2C12 myoblasts transfected with human α1, α2, α10 or α11; human fibrosarcoma HT1080 cells which constitutively express only human α2β1, and rat glioma Rugli cells, with only rat α1β1. Using these lines, the nature of integrin binding sites was studied in order to delineate the bioactivity of different collagen substrates. Integrin ligation was studied on collagen coatings alongside synthetic (GFOGER/GLOGEN) and Toolkit (Col II-28/Col III-7) triple-helical peptides to evaluate (1) their affinity towards different integrins and (2) to confirm the activity of the inserted integrin in the transfected cells. Thin films of dermal and tendon Col I were used to evaluate the influence of the carbodiimide (EDC)-based treatment on the cellular response on Col of different origin. The results showed that the binding properties of transfected C2C12 cells to collagens depend on the identity of inserted integrin. Similar ligation characteristics were observed using α1+ and α10+ cells, but these were distinct from the similar binding features of α2+ and α11+ cells. Recombinant human and rat-α1 I domain binding to collagens and peptides correlated with the cell adhesion results, showing receptor class- and species-specificities. The understanding of the physiologically relevant cell anchorage characteristics of bio-constructs may assist in the selection of (1) the optimum collagen source for cellular supports and (2) the correct cellular model for their biological assessment. This, in turn, may allow reliable prediction of the biological performance of bio-scaffolds in vivo for specific TE applications. Integrins play a vital role in cellular responses to environmental cues during early-stage cell-substrate interaction. We describe physiologically relevant cell anchorage to collagen substrates that present different affinity cell-recognition motifs, to provide experimental tools to assist in understanding integrin binding. Using different cell types and recombinant integrin α1-I-domains, we found that cellular response was highly dependent on collagen type, origin and EDC-crosslinking status, as well as on the integrin class and species of origin. This comprehensive study establishes selectivity amongst the four collagen-binding integrins and species-specific properties that together may influence choice of cell type and receptor in different experimental settings. This work offers key guidance in selecting of the correct cellular model for the biological testing of collagen-based biomaterials. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity.

PubMed

Jing, Fuyuan; Cantu, David C; Tvaruzkova, Jarmila; Chipman, Jay P; Nikolau, Basil J; Yandeau-Nelson, Marna D; Reilly, Peter J

2011-08-10

Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids. To explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their in vivo activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (Elaeis guineensis), coconut (Cocos nucifera) and Cuphea viscosissima, organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of the acyl-ACP TEs were determined in E. coli. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family. These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids.

Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity

PubMed Central

2011-01-01

Background Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids. Results To explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their in vivo activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (Elaeis guineensis), coconut (Cocos nucifera) and Cuphea viscosissima, organisms that produce medium-chain and short-chain fatty acids in their seeds. The in vivo substrate specificities of the acyl-ACP TEs were determined in E. coli. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family. Conclusion These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids. PMID:21831316

A universal small molecule, inorganic phosphate, restricts the substrate specificity of Dicer-2 in small RNA biogenesis

PubMed Central

Fukunaga, Ryuya; Zamore, Phillip D

2014-01-01

The enzyme Dicer is central to the production of small silencing RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs). Like other insects, Drosophila melanogaster uses different Dicers to make siRNAs and miRNAs: Dicer-1 produces miRNAs from pre-miRNAs, whereas Dicer-2 generates siRNAs from long double-stranded RNA (dsRNA). How do the 2 Dicers achieve their substrate specificity? Here, we review recent findings that inorganic phosphate restricts the substrate specificity of Dicer-2 to long dsRNA. Inorganic phosphate inhibits Dicer-2 from binding and cleaving pre-miRNAs, without affecting the processing of long dsRNA. Crystal structures of a fragment of human Dicer in complex with an RNA duplex identify a phosphate-binding pocket that recognizes both the 5′-monophosphate of a substrate RNA and inorganic phosphate. We propose that inorganic phosphate occupies the phosphate-binding pocket in the fly Dicer-2, blocking binding of pre-miRNA and restricting pre-miRNA processing to Dicer-1. Thus, a small molecule can alter the substrate specificity of a nucleic acid-processing enzyme. PMID:24787225

Mammalian Protein Arginine Methyltransferase 7 (PRMT7) Specifically Targets RXR Sites in Lysine- and Arginine-rich Regions*

PubMed Central

Feng, You; Maity, Ranjan; Whitelegge, Julian P.; Hadjikyriacou, Andrea; Li, Ziwei; Zurita-Lopez, Cecilia; Al-Hadid, Qais; Clark, Amander T.; Bedford, Mark T.; Masson, Jean-Yves; Clarke, Steven G.

2013-01-01

The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylarginine residues, and we confirm its activity as the prototype type III protein arginine methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine- and arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7. PMID:24247247

Mammalian protein arginine methyltransferase 7 (PRMT7) specifically targets RXR sites in lysine- and arginine-rich regions.

PubMed

Feng, You; Maity, Ranjan; Whitelegge, Julian P; Hadjikyriacou, Andrea; Li, Ziwei; Zurita-Lopez, Cecilia; Al-Hadid, Qais; Clark, Amander T; Bedford, Mark T; Masson, Jean-Yves; Clarke, Steven G

2013-12-27

The mammalian protein arginine methyltransferase 7 (PRMT7) has been implicated in roles of transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation, and metastasis. However, the type of reaction that it catalyzes and its substrate specificity remain controversial. In this study, we purified a recombinant mouse PRMT7 expressed in insect cells that demonstrates a robust methyltransferase activity. Using a variety of substrates, we demonstrate that the enzyme only catalyzes the formation of ω-monomethylarginine residues, and we confirm its activity as the prototype type III protein arginine methyltransferase. This enzyme is active on all recombinant human core histones, but histone H2B is a highly preferred substrate. Analysis of the specific methylation sites within intact histone H2B and within H2B and H4 peptides revealed novel post-translational modification sites and a unique specificity of PRMT7 for methylating arginine residues in lysine- and arginine-rich regions. We demonstrate that a prominent substrate recognition motif consists of a pair of arginine residues separated by one residue (RXR motif). These findings will significantly accelerate substrate profile analysis, biological function study, and inhibitor discovery for PRMT7.

Starbursts and Wispy Drops : Surfactants Spreading on Gel Substrates

NASA Astrophysics Data System (ADS)

Mukhopadhyay, Shomeek; Daniels, Karen; Behringer, Robert

2005-11-01

We report a phase diagram for a novel instability seen in drops of nonionic surfactant solution (Triton X-305) spreading on viscoelastic agar gel substrate . This system allows us to examine the effect of varying the effective fluidity/stiffness of aqueous substrates. The morphology is strongly affected by the substrate fluidity, ranging from spreading starbursts of arms on weak gels, to wispy drops on intermediate strength gels, to circular drops on stiff gels. We analyze the dynamics of spreading in the starburst phase, where the arm length grows as t ^3/4 at early times, independent of the gel strength and surfactant concentration. The number of arms is proportional to the surfactant concentration and inversely proportional to the gel strength. Ongoing work is exploring the effects of changing the drop volume.

Using oriented peptide array libraries to evaluate methylarginine-specific antibodies and arginine methyltransferase substrate motifs

PubMed Central

Gayatri, Sitaram; Cowles, Martis W.; Vemulapalli, Vidyasiri; Cheng, Donghang; Sun, Zu-Wen; Bedford, Mark T.

2016-01-01

Signal transduction in response to stimuli relies on the generation of cascades of posttranslational modifications that promote protein-protein interactions and facilitate the assembly of distinct signaling complexes. Arginine methylation is one such modification, which is catalyzed by a family of nine protein arginine methyltransferases, or PRMTs. Elucidating the substrate specificity of each PRMT will promote a better understanding of which signaling networks these enzymes contribute to. Although many PRMT substrates have been identified, and their methylation sites mapped, the optimal target motif for each of the nine PRMTs has not been systematically addressed. Here we describe the use of Oriented Peptide Array Libraries (OPALs) to methodically dissect the preferred methylation motifs for three of these enzymes – PRMT1, CARM1 and PRMT9. In parallel, we show that an OPAL platform with a fixed methylarginine residue can be used to validate the methyl-specific and sequence-specific properties of antibodies that have been generated against different PRMT substrates, and can also be used to confirm the pan nature of some methylarginine-specific antibodies. PMID:27338245

Design and isolation of ribozyme-substrate pairs using RNase P-based ribozymes containing altered substrate binding sites.

PubMed Central

Mobley, E M; Pan, T

1999-01-01

Substrate recognition and cleavage by the bacterial RNase P RNA requires two domains, a specificity domain, or S-domain, and a catalytic domain, or C-domain. The S-domain binds the T stem-loop region in a pre-tRNA substrate to confer specificity for tRNA substrates. In this work, the entire S-domain of the Bacillus subtilis RNase P RNA is replaced with an artificial substrate binding module. New RNA substrates are isolated by in vitro selection using two libraries containing random regions of 60 nt. At the end of the selection, the cleavage rates of the substrate library are approximately 0.7 min(-1)in 10 mM MgCl(2)at 37 degrees C, approximately 4-fold better than the cleavage of a pre-tRNA substrate by the wild-type RNase P RNA under the same conditions. The contribution of the S-domain replacement to the catalytic efficiency is from 6- to 22 000-fold. Chemical and nuclease mapping of two ribozyme-product complexes shows that this contribution correlates with direct interactions between the S-domain replacement and the selected substrate. These results demonstrate the feasibility of design and isolation of RNase P-based, matching ribozyme-substrate pairs without prior knowledge of the sequence or structure of the interactive modules in the ribozyme or substrate. PMID:10518624

Biochemistry students' ideas about how an enzyme interacts with a substrate.

PubMed

Linenberger, Kimberly J; Bretz, Stacey Lowery

2015-01-01

Enzyme-substrate interactions are a fundamental concept of biochemistry that is built upon throughout multiple biochemistry courses. Central to understanding enzyme-substrate interactions is specific knowledge of exactly how an enzyme and substrate interact. Within this narrower topic, students must understand the various binding sites on an enzyme and be able to reason from simplistic lock and key or induced fit models to the more complex energetics model of transition state theory. Learning to understand these many facets of enzyme-substrate interactions and reasoning from multiple models present challenges where students incorrectly make connections between concepts or make no connection at all. This study investigated biochemistry students' understanding of enzyme-substrate interactions through the use of clinical interviews and a national administration (N = 707) of the Enzyme-Substrate Interactions Concept Inventory. Findings include misconceptions regarding the nature of enzyme-substrate interactions, naïve ideas about the active site, a lack of energetically driven interactions, and an incomplete understanding of the specificity pocket. © 2015 by the International Union of Biochemistry and Molecular Biology.

Structural insights into the difference in substrate recognition of two mannoside phosphorylases from two GH130 subfamilies.

PubMed

Ye, Yuxin; Saburi, Wataru; Odaka, Rei; Kato, Koji; Sakurai, Naofumi; Komoda, Keisuke; Nishimoto, Mamoru; Kitaoka, Motomitsu; Mori, Haruhide; Yao, Min

2016-03-01

In Ruminococcus albus, 4-O-β-D-mannosyl-D-glucose phosphorylase (RaMP1) and β-(1,4)-mannooligosaccharide phosphorylase (RaMP2) belong to two subfamilies of glycoside hydrolase family 130. The two enzymes phosphorolyze β-mannosidic linkages at the nonreducing ends of their substrates, and have substantially diverse substrate specificity. The differences in their mechanism of substrate binding have not yet been fully clarified. In the present study, we report the crystal structures of RaMP1 with/without 4-O-β-D-mannosyl-d-glucose and RaMP2 with/without β-(1→4)-mannobiose. The structures of the two enzymes differ at the +1 subsite of the substrate-binding pocket. Three loops are proposed to determine the different substrate specificities. One of these loops is contributed from the adjacent molecule of the oligomer structure. In RaMP1, His245 of loop 3 forms a hydrogen-bond network with the substrate through a water molecule, and is indispensible for substrate binding. © 2016 Federation of European Biochemical Societies.

Gold-silicon nanofiber synthesized by femtosecond laser radiation for enhanced light absorptance.

PubMed

Mahmood, Abdul Salam; Venkatakrishnan, Krishnan; Tan, Bo

2014-01-01

In this study, we devised a new concept for the precise nanofabrication of Au-Si fibrous nanostructures using megahertz femtosecond laser irradiation in air and atmospheric pressure conditions. The weblike fibrous nanostructures of Au thin layer on silicon substrate, which are proposed for the application of solar cells, exhibit a specific improvement of the optical properties in visible wavelength. Varying numbers of laser interaction pulses were used to control the synthesis of the nanofibrous structures. Electron microscopy analysis revealed that the nanostructures are formed due to the aggregation of polycrystalline nanoparticles of the respective constituent materials with diameters varying between 30 and 90 nm. Measurement of the reflectance through a spectroradiometer showed that the coupling of incident electromagnetic irradiation was greatly improved over the broadband wavelength range. Lower reflectance intensity was obtained with a higher number of laser pulses due to the bulk of gold nanoparticles being agglomerated by the mechanism of fusion. This forms interweaving fibrous nanostructures which reveal a certain degree of assembly. 81.05.Zx; 81.07.-b.

Generative Representations for Evolving Families of Designs

NASA Technical Reports Server (NTRS)

Hornby, Gregory S.

2003-01-01

Since typical evolutionary design systems encode only a single artifact with each individual, each time the objective changes a new set of individuals must be evolved. When this objective varies in a way that can be parameterized, a more general method is to use a representation in which a single individual encodes an entire class of artifacts. In addition to saving time by preventing the need for multiple evolutionary runs, the evolution of parameter-controlled designs can create families of artifacts with the same style and a reuse of parts between members of the family. In this paper an evolutionary design system is described which uses a generative representation to encode families of designs. Because a generative representation is an algorithmic encoding of a design, its input parameters are a way to control aspects of the design it generates. By evaluating individuals multiple times with different input parameters the evolutionary design system creates individuals in which the input parameter controls specific aspects of a design. This system is demonstrated on two design substrates: neural-networks which solve the 3/5/7-parity problem and three-dimensional tables of varying heights.

Gold-silicon nanofiber synthesized by femtosecond laser radiation for enhanced light absorptance

PubMed Central

2014-01-01

In this study, we devised a new concept for the precise nanofabrication of Au-Si fibrous nanostructures using megahertz femtosecond laser irradiation in air and atmospheric pressure conditions. The weblike fibrous nanostructures of Au thin layer on silicon substrate, which are proposed for the application of solar cells, exhibit a specific improvement of the optical properties in visible wavelength. Varying numbers of laser interaction pulses were used to control the synthesis of the nanofibrous structures. Electron microscopy analysis revealed that the nanostructures are formed due to the aggregation of polycrystalline nanoparticles of the respective constituent materials with diameters varying between 30 and 90 nm. Measurement of the reflectance through a spectroradiometer showed that the coupling of incident electromagnetic irradiation was greatly improved over the broadband wavelength range. Lower reflectance intensity was obtained with a higher number of laser pulses due to the bulk of gold nanoparticles being agglomerated by the mechanism of fusion. This forms interweaving fibrous nanostructures which reveal a certain degree of assembly. PACS 81.05.Zx; 81.07.-b PMID:24940179

Atomic ordering in GaAsP

NASA Astrophysics Data System (ADS)

Chen, G. S.; Jaw, D. H.; Stringfellow, G. B.

1991-04-01

CuPt type ordering, which consists of a monolayer compositional modulation along one of the 4 <111> directions in the lattice, was studied using transmission electron microscopy for GaAs1-xPx with values of x extending from 0.25 to 0.85. The samples were grown by organometallic vapor phase epitaxy on nominal (001) GaAs substrates that were misoriented by varying amounts in three directions. No CuPt type ordering was observed for GaAs1-xPx with x ≤0.35, while ordering was found to occur for 0.4≤x≤0.85. The direction of substrate misorientation has a major effect on the determination of which of the four possible CuPt variants are formed for 0.4≤x≤0.85. Two variants, with ordering on the (1¯11) and (11¯1) planes, appear for epilayers grown on substrates oriented exactly on the (001) plane and for substrates misoriented by 6° towards the [110] direction. Only one variant, with ordering on the (1¯11) plane, appears for epilayers grown on substrates misoriented by 6° towards [1¯10]. These ordering-induced spots observed in transmission electron diffraction (TED) patterns for GaAsP occur only for the [110] cross section. From TED studies of GaInP grown on similar substrates, we conclude that the CuPt variants in GaAsP are exactly the same as for GaInP. Further evidence supporting this conclusion was obtained by growing first a layer of GaInP followed by a layer of GaAsP. High-resolution dark field electron micrographs show domains of the same variants in both layers. A mechanism describing the formation of the specific ordered variant for both GaAsP and GaInP is proposed. From studies of ordering in a strain-layer superlattice, the strain due to lattice mismatch was found to play no significant role in the propagation of ordered domains. Microtwins, also generated due to lattice mismatch, can act as domain boundaries and prevent the propagation of the ordered domains.

Wild-Type Phosphoribosylpyrophosphate Synthase (PRS) from Mycobacterium tuberculosis: A Bacterial Class II PRS?

PubMed Central

Breda, Ardala; Martinelli, Leonardo K. B.; Bizarro, Cristiano V.; Rosado, Leonardo A.; Borges, Caroline B.; Santos, Diógenes S.; Basso, Luiz A.

2012-01-01

The 5-phospho-α-D-ribose 1-diphosphate (PRPP) metabolite plays essential roles in several biosynthetic pathways, including histidine, tryptophan, nucleotides, and, in mycobacteria, cell wall precursors. PRPP is synthesized from α-D-ribose 5-phosphate (R5P) and ATP by the Mycobacterium tuberculosis prsA gene product, phosphoribosylpyrophosphate synthase (MtPRS). Here, we report amplification, cloning, expression and purification of wild-type MtPRS. Glutaraldehyde cross-linking results suggest that MtPRS predominates as a hexamer, presenting varied oligomeric states due to distinct ligand binding. MtPRS activity measurements were carried out by a novel coupled continuous spectrophotometric assay. MtPRS enzyme activity could be detected in the absence of Pi. ADP, GDP and UMP inhibit MtPRS activity. Steady-state kinetics results indicate that MtPRS has broad substrate specificity, being able to accept ATP, GTP, CTP, and UTP as diphosphoryl group donors. Fluorescence spectroscopy data suggest that the enzyme mechanism for purine diphosphoryl donors follows a random order of substrate addition, and for pyrimidine diphosphoryl donors follows an ordered mechanism of substrate addition in which R5P binds first to free enzyme. An ordered mechanism for product dissociation is followed by MtPRS, in which PRPP is the first product to be released followed by the nucleoside monophosphate products to yield free enzyme for the next round of catalysis. The broad specificity for diphosphoryl group donors and detection of enzyme activity in the absence of Pi would suggest that MtPRS belongs to Class II PRS proteins. On the other hand, the hexameric quaternary structure and allosteric ADP inhibition would place MtPRS in Class I PRSs. Further data are needed to classify MtPRS as belonging to a particular family of PRS proteins. The data here presented should help augment our understanding of MtPRS mode of action. Current efforts are toward experimental structure determination of MtPRS to provide a solid foundation for the rational design of specific inhibitors of this enzyme. PMID:22745722

Identification and substrate prediction of new Fragaria x ananassa aquaporins and expression in different tissues and during strawberry fruit development.

PubMed

Merlaen, Britt; De Keyser, Ellen; Van Labeke, Marie-Christine

2018-01-01

The newly identified aquaporin coding sequences presented here pave the way for further insights into the plant-water relations in the commercial strawberry ( Fragaria x ananassa ). Aquaporins are water channel proteins that allow water to cross (intra)cellular membranes. In Fragaria x ananassa , few of them have been identified hitherto, hampering the exploration of the water transport regulation at cellular level. Here, we present new aquaporin coding sequences belonging to different subclasses: plasma membrane intrinsic proteins subtype 1 and subtype 2 (PIP1 and PIP2) and tonoplast intrinsic proteins (TIP). The classification is based on phylogenetic analysis and is confirmed by the presence of conserved residues. Substrate-specific signature sequences (SSSSs) and specificity-determining positions (SDPs) predict the substrate specificity of each new aquaporin. Expression profiling in leaves, petioles and developing fruits reveals distinct patterns, even within the same (sub)class. Expression profiles range from leaf-specific expression over constitutive expression to fruit-specific expression. Both upregulation and downregulation during fruit ripening occur. Substrate specificity and expression profiles suggest that functional specialization exists among aquaporins belonging to a different but also to the same (sub)class.

Trends in water monomer adsorption and dissociation on flat insulating surfaces.

PubMed

Hu, Xiao Liang; Carrasco, Javier; Klimeš, Jiří; Michaelides, Angelos

2011-07-21

The interaction of water with solid surfaces is key to a wide variety of industrial and natural processes. However, the basic principles that dictate how stable and in which state (intact or dissociated) water will be on a given surface are not fully understood. Towards this end, we have used density functional theory to examine water monomer adsorption on the (001) surfaces of a broad range of alkaline earth oxides, alkaline earth sulfides, alkali fluorides, and alkali chlorides. Some interesting general conclusions are arrived at: (i) on all the surfaces considered only a few specific adsorption structures are favoured; (ii) water becomes more stable upon descending the oxide and fluoride series but does not vary much upon going down the chloride and sulfide series; (iii) water is stabilised both by an increase in the lattice constant, which facilitates hydrogen bonding to the substrate, and by the flexibility of the substrate. These are also factors that favour water dissociation. We hope that this study is of some value in better understanding the surface science of water in general, and in assisting in the interpretation and design of future experiments. This journal is © the Owner Societies 2011

Flexible and conformable broadband metamaterial absorber with wide-angle and polarization stability for radar application

NASA Astrophysics Data System (ADS)

Chen, Huijie; Yang, Xiaoqing; Wu, Shiyue; Zhang, Di; Xiao, Hui; Huang, Kama; Zhu, Zhanxia; Yuan, Jianping

2018-01-01

In this work, a type of flexible, broadband electromagnetic microwave absorber is designed, fabricated and experimentally characterized. The absorber is composed of lumped resistors loaded frequency selective surface which is mounted on flexible substrate using silicone rubber and in turn backed by copper film. The simulated results show that an effective absorption (over 90%) bandwidth spans from 7.6 to 18.3 GHz, which covers both X (8-12 GHz) and Ku (12-18 GHz) bands, namely a 82.6% fraction bandwidth. And the bandwidth performs a good absorption response by varying the incident angle up to 60° for both TE and TM polarization. Moreover, the flexibility of the substrate enables the absorber conformably to bend and attach to cylinders of various radius without breakdown of the absorber. The designed structure has been fabricated and measured for both planar and conformable cases, and absorption responses show a good agreement of the broadband absorption feature with the simulated ones. This work has demonstrated specifically that proposed structure provides polarization-insensitive, wide-angle, flexible and conformable wideband absorption, which extends the absorber’s application to practical radar cross section reductions for radars and warships.

Persistent atrial fibrillation vs paroxysmal atrial fibrillation: differences in management.

PubMed

Margulescu, Andrei D; Mont, Lluis

2017-08-01

Atrial fibrillation (AF) is the most common human arrhythmia. AF is a progressive disease, initially being nonsustained and induced by trigger activity, and progressing towards persistent AF through alteration of the atrial myocardial substrate. Treatment of AF aims to decrease the risk of stroke and improve the quality of life, by preventing recurrences (rhythm control) or controlling the heart rate during AF (rate control). In the last 20 years, catheter-based and, less frequently, surgical and hybrid ablation techniques have proven more successful compared with drug therapy in achieving rhythm control in patients with AF. However, the efficiency of ablation techniques varies greatly, being highest in paroxysmal and lowest in long-term persistent AF. Areas covered: In this review, we discuss the fundamental differences between paroxysmal and persistent AF and the potential impact of those differences on patient management, emphasizing the available therapeutic strategies to achieve rhythm control. Expert commentary: Treatment to prevent AF recurrences is suboptimal, particularly in patients with persistent AF. Emerging technologies, such as documentation of atrial fibrosis using magnetic resonance imaging and documentation of electrical substrate using advanced electrocardiographic imaging techniques are likely to provide valuable insights about patient-specific tailoring of treatments.

In vitro kinetics of soybean lipoxygenase with combinatorial fatty substrates and its functional significance in off flavour development.

PubMed

Mandal, Somnath; Dahuja, Anil; Kar, Abhijit; Santha, I M

2014-03-01

Lipoxygenase (Lox) mediated oxidation of polyunsaturated fatty acids (PUFA) in mature soya seeds results in objectionable flavour. In the present study, Lox isozymes were purified to near homogeneity (107-fold). Lox-2 and 3 displayed remarkable kinetic preference (1.7 and 1.5-fold, respectively) for low PUFA ratios (LA/LeA) (PRs) among the selected PUFA combinations. Lox-1 displayed no specific preference. Pure Lox-1 displayed unbiased response towards substrates with marginal preference (1.2-fold) for linoleic acid at its optimum pH. Volatile compounds profiling showed a direct relationship between PRs and hexanal to trans-2-hexenal (1.47, 2.24 and 18.90 for 2, 7 and 15 PRs, respectively) ratio. The off-flavour determining parameters like TBA value, carbonyl value and lipid hydroperoxides (LHPODs) exhibited significant negative correlation (0.76, 0.74, 0.72; p<0.0001) in selected soya genotypes displaying varied PRs and significant positive correlation (0.89, 0.81. 0.89; p<0.0001) with ratio of PI (polyene index) to PRs - suggesting the plausible significance of PUFA ratios in biological lipid peroxidation. Copyright © 2013 Elsevier Ltd. All rights reserved.

Visual and semantic processing of living things and artifacts: an FMRI study.

PubMed

Zannino, Gian Daniele; Buccione, Ivana; Perri, Roberta; Macaluso, Emiliano; Lo Gerfo, Emanuele; Caltagirone, Carlo; Carlesimo, Giovanni A

2010-03-01

We carried out an fMRI study with a twofold purpose: to investigate the relationship between networks dedicated to semantic and visual processing and to address the issue of whether semantic memory is subserved by a unique network or by different subsystems, according to semantic category or feature type. To achieve our goals, we administered a word-picture matching task, with within-category foils, to 15 healthy subjects during scanning. Semantic distance between the target and the foil and semantic domain of the target-foil pairs were varied orthogonally. Our results suggest that an amodal, undifferentiated network for the semantic processing of living things and artifacts is located in the anterolateral aspects of the temporal lobes; in fact, activity in this substrate was driven by semantic distance, not by semantic category. By contrast, activity in ventral occipito-temporal cortex was driven by category, not by semantic distance. We interpret the latter finding as the effect exerted by systematic differences between living things and artifacts at the level of their structural representations and possibly of their lower-level visual features. Finally, we attempt to reconcile contrasting data in the neuropsychological and functional imaging literature on semantic substrate and category specificity.

Plasmonic behaviour of sputtered Au nanoisland arrays

NASA Astrophysics Data System (ADS)

Tvarožek, V.; Szabó, O.; Novotný, I.; Kováčová, S.; Škriniarová, J.; Šutta, P.

2017-02-01

The specificity of the formation of Au sputtered nanoisland arrays (NIA) on a glass substrate or on a ZnO thin film doped by Ga is demonstrated. Statistical analysis of morphology images (SEM, AFM) exhibited the Log-normal distribution of the size (area) of nanoislands-their modus AM varied from 8 to 328 nm2 depending on the sputtering power density, which determined the nominal thicknesses in the range of 2-8 nm. Preferential polycrystalline texture (111) of Au NIA increased with the power density and after annealing. Transverse localised surface plasmonic resonance (LSPR; evaluated by transmission UV-vis spectroscopy) showed the red shift of the extinction peaks (Δl ≤ 100 nm) with an increase of the nominal thickness, and the blue shift (Δλ ≤ -65 nm) after annealing of Au NIA. The plasmonic behaviour of Au NIA was described by modification of a size-scaling universal model using the nominal thin film thickness as a technological scaling parameter. Sputtering of a Ti intermediate adhesive ultrathin film between the glass substrate and gold improves the adhesion of Au nanoislands as well as supporting the formation of more defined Au NIA structures of smaller dimensions.

The Structure of Lombricine Kinase

PubMed Central

Bush, D. Jeffrey; Kirillova, Olga; Clark, Shawn A.; Davulcu, Omar; Fabiola, Felcy; Xie, Qing; Somasundaram, Thayumanasamy; Ellington, W. Ross; Chapman, Michael S.

2011-01-01

Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309–317, which is disordered in substrate-free structures of homologs and is known from the NMR of arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His178. Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates. PMID:21212263

Catalytic properties of IgMs with amylolytic activity isolated from patients with multiple sclerosis.

PubMed

Ivanen, Dina R; Kulminskaya, Anna A; Shabalin, Konstantin A; Isaeva-Ivanova, Luydmila V; Ershova, Nadezhda A; Saveliev, Andrew N; Nevinsky, Gregory A; Neustroev, Kirill N

2004-08-01

Recently, amylolytic activity was detected in IgMs isolated from the sera of the patients with multiple sclerosis. All purified samples of IgM were electrophoretically homogenous and did not contain any co-purified a-amylase and a-glucosidase activities, in accordance with a set of criteria developed for abzymes. The amylolytic activity of abzymes was studied in the hydrolysis of p-nitrophenyl a-D-maltooligosaccharides with different degrees of polymerization from 1 to 8 by TLC and reverse-phase HPLC techniques. All IgM samples isolated from 54 patients with clinically definite multiple sclerosis demonstrated hydrolytic activity towards the above artificial substrates. The Michaelis constant values (Km) in the hydrolysis of p-nitrophenyl a-D-maltoheptaoside were in the range of 10 p-nitrophenyl or p-nitrophenyl a-D-glucosides, thus indicating the presence of an a-D-glucosidase activity. For a number of the investigated samples, specific amylolytic activity increased depending on the length of substrates (from p-nitrophenyl maltopentaoside to p-nitrophenyl maltohexaoside); for other IgMs, the opposite dependence was observed. All IgMs studied did not exhibit any other glycoside hydrolase activities toward p-nitrophenyl glycoside substrates. Abzyme fractions from different donors demonstrated catalytic heterogeneity in Michaelis-Menten parameters and different modes of action in the hydrolysis of p-nitrophenyl maltooligosaccharides. Enzymatic properties of the IgMs tested varied from human a-amylases. All investigated abzyme samples did not show transglycosylating ability.

Black, green, and red abalones. Species profiles: life histories and environmental requirements of coastal fishes and invertebrates (Pacific Southwest. )

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

Ault, J.S.

1985-03-01

Black, green, and red abalones (Haliotis cracherodii, H. fulgens, and H. rufescens, respectivley) are of commercial and ecological importance and are distributed widely along the California coast. The abalones are morphologically similar; species are distinguished by particular shell sculpture, color, and body characteristics. Their latitudinal and bathymetric distribution is stratified and most closely related to temperature. Small juveniles eat mainly microflora; adults eat primarily drift macro-algae, preferring specific brown or red algae, when available. Spawning occurs during summer; gonad ripening depends on food quality and quantity and water temperature. Larvae are lecithotrophic and remain planktonic for periods of 5 tomore » 14 days after hatching; settling is substrate specific. Postlarvae and adults require hard substrate for attachment. Juveniles are cryptic, adults usually more exposed. Growth rates are similar, although maximum size varies with species. Increases in shell length and body weight correlate positively with food abundance and temperature. Below depths of 6 m, sea urchins are major competitors for food and space. Predation by invertebrates is low. Decreased abalone production from central California is associated with range expansion and increased predation by sea otters, the major source of abalone mortality. General declines in California landings are due to mortality from improper picking and replacement, habitat degradation, and perhaps overfishing. Commercial and sport diving efforts have increased sharply, whereas annual landings of abalones declined from 1965 to 1982.« less

Biochemical profiling in silico--predicting substrate specificities of large enzyme families.

PubMed

Tyagi, Sadhna; Pleiss, Juergen

2006-06-25

A general high-throughput method for in silico biochemical profiling of enzyme families has been developed based on covalent docking of potential substrates into the binding sites of target enzymes. The method has been tested by systematically docking transition state--analogous intermediates of 12 substrates into the binding sites of 20 alpha/beta hydrolases from 15 homologous families. To evaluate the effect of side chain orientations to the docking results, 137 crystal structures were included in the analysis. A good substrate must fulfil two criteria: it must bind in a productive geometry with four hydrogen bonds between the substrate and the catalytic histidine and the oxyanion hole, and a high affinity of the enzyme-substrate complex as predicted by a high docking score. The modelling results in general reproduce experimental data on substrate specificity and stereoselectivity: the differences in substrate specificity of cholinesterases toward acetyl- and butyrylcholine, the changes of activity of lipases and esterases upon the size of the acid moieties, activity of lipases and esterases toward tertiary alcohols, and the stereopreference of lipases and esterases toward chiral secondary alcohols. Rigidity of the docking procedure was the major reason for false positive and false negative predictions, as the geometry of the complex and docking score may sensitively depend on the orientation of individual side chains. Therefore, appropriate structures have to be identified. In silico biochemical profiling provides a time efficient and cost saving protocol for virtual screening to identify the potential substrates of the members of large enzyme family from a library of molecules.

Microbial enzymes with special characteristics for biotechnological applications.

PubMed

Nigam, Poonam Singh

2013-08-23

This article overviews the enzymes produced by microorganisms, which have been extensively studied worldwide for their isolation, purification and characterization of their specific properties. Researchers have isolated specific microorganisms from extreme sources under extreme culture conditions, with the objective that such isolated microbes would possess the capability to bio-synthesize special enzymes. Various Bio-industries require enzymes possessing special characteristics for their applications in processing of substrates and raw materials. The microbial enzymes act as bio-catalysts to perform reactions in bio-processes in an economical and environmentally-friendly way as opposed to the use of chemical catalysts. The special characteristics of enzymes are exploited for their commercial interest and industrial applications, which include: thermotolerance, thermophilic nature, tolerance to a varied range of pH, stability of enzyme activity over a range of temperature and pH, and other harsh reaction conditions. Such enzymes have proven their utility in bio-industries such as food, leather, textiles, animal feed, and in bio-conversions and bio-remediations.

High sequence variability among hemocyte-specific Kazal-type proteinase inhibitors in decapod crustaceans.

PubMed

Cerenius, Lage; Liu, Haipeng; Zhang, Yanjiao; Rimphanitchayakit, Vichien; Tassanakajon, Anchalee; Gunnar Andersson, M; Söderhäll, Kenneth; Söderhäll, Irene

2010-01-01

Crustacean hemocytes were found to produce a large number of transcripts coding for Kazal-type proteinase inhibitors (KPIs). A detailed study performed with the crayfish Pacifastacus leniusculus and the shrimp Penaeus monodon revealed the presence of at least 26 and 20 different Kazal domains from the hemocyte KPIs, respectively. Comparisons with KPIs from other taxa indicate that the sequences of these domains evolve rapidly. A few conserved positions, e.g. six invariant cysteines were present in all domain sequences whereas the position of P1 amino acid, a determinant for substrate specificity, varied highly. A study with a single crayfish animal suggested that even at the individual level considerable sequence variability among hemocyte KPIs produced exist. Expression analysis of four crayfish KPI transcripts in hematopoietic tissue cells and different hemocyte types suggest that some of these KPIs are likely to be involved in hematopoiesis or hemocyte release as they were produced in particular hemocyte types or maturation stages only.

Decoding the patterns of ubiquitin recognition by ubiquitin-associated domains from free energy simulations.

PubMed

Bouvier, Benjamin

2014-01-07

Ubiquitin is a highly conserved, highly represented protein acting as a regulating signal in numerous cellular processes. It leverages a single hydrophobic binding patch to recognize and bind a large variety of protein domains with remarkable specificity, but can also self-assemble into chains of poly-diubiquitin units in which these interfaces are sequestered, profoundly altering the individual monomers' recognition characteristics. Despite numerous studies, the origins of this varied specificity and the competition between substrates for the binding of the ubiquitin interface patch remain under heated debate. This study uses enhanced sampling all-atom molecular dynamics to simulate the unbinding of complexes of mono- or K48-linked diubiquitin bound to several ubiquitin-associated domains, providing insights into the mechanism and free energetics of ubiquitin recognition and binding. The implications for the subtle tradeoff between the stability of the polyubiquitin signal and its easy recognition by target protein assemblies are discussed, as is the enhanced affinity of the latter for long polyubiquitin chains compared to isolated mono- or diubiquitin.

Monolayers of derivatized poly(l-lysine)-grafted poly(ethylene glycol) on metal oxides as a class of biomolecular interfaces

PubMed Central

Ruiz-Taylor, L. A.; Martin, T. L.; Zaugg, F. G.; Witte, K.; Indermuhle, P.; Nock, S.; Wagner, P.

2001-01-01

We report on the design and characterization of a class of biomolecular interfaces based on derivatized poly(l-lysine)-grafted poly(ethylene glycol) copolymers adsorbed on negatively charged surfaces. As a model system, we synthesized biotin-derivatized poly(l-lysine)-grafted poly(ethylene glycol) copolymers, PLL-g-[(PEGm)(1−x) (PEG-biotin)x], where x varies from 0 to 1. Monolayers were produced on titanium dioxide substrates and characterized by x-ray photoelectron spectroscopy. The specific biorecognition properties of these biotinylated surfaces were investigated with the use of radiolabeled streptavidin alone and within complex protein mixtures. The PLL-g-PEG-biotin monolayers specifically capture streptavidin, even from a complex protein mixture, while still preventing nonspecific adsorption of other proteins. This streptavidin layer can subsequently capture biotinylated proteins. Finally, with the use of microfluidic networks and protein arraying, we demonstrate the potential of this class of biomolecular interfaces for applications based on protein patterning. PMID:11158560

Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates

PubMed Central

Xue, Liang; Wang, Wen-Horng; Iliuk, Anton; Hu, Lianghai; Galan, Jacob A.; Yu, Shuai; Hans, Michael; Geahlen, Robert L.; Tao, W. Andy

2012-01-01

Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity. PMID:22451900

Interactions between Casein Kinase Iε (CKIε) and Two Substrates from Disparate Signaling Pathways Reveal Mechanisms for Substrate-Kinase Specificity

PubMed Central

Dahlberg, Caroline Lund; Nguyen, Elizabeth Z.; Goodlett, David; Kimelman, David

2009-01-01

Background Members of the Casein Kinase I (CKI) family of serine/threonine kinases regulate diverse biological pathways. The seven mammalian CKI isoforms contain a highly conserved kinase domain and divergent amino- and carboxy-termini. Although they share a preferred target recognition sequence and have overlapping expression patterns, individual isoforms often have specific substrates. In an effort to determine how substrates recognize differences between CKI isoforms, we have examined the interaction between CKIε and two substrates from different signaling pathways. Methodology/Principal Findings CKIε, but not CKIα, binds to and phosphorylates two proteins: Period, a transcriptional regulator of the circadian rhythms pathway, and Disheveled, an activator of the planar cell polarity pathway. We use GST-pull-down assays data to show that two key residues in CKIα's kinase domain prevent Disheveled and Period from binding. We also show that the unique C-terminus of CKIε does not determine Dishevelled's and Period's preference for CKIε nor is it essential for binding, but instead plays an auxillary role in stabilizing the interactions of CKIε with its substrates. We demonstrate that autophosphorylation of CKIε's C-terminal tail prevents substrate binding, and use mass spectrometry and chemical crosslinking to reveal how a phosphorylation-dependent interaction between the C-terminal tail and the kinase domain prevents substrate phosphorylation and binding. Conclusions/Significance The biochemical interactions between CKIε and Disheveled, Period, and its own C-terminus lead to models that explain CKIε's specificity and regulation. PMID:19274088

A Haloalkane Dehalogenase from a Marine Microbial Consortium Possessing Exceptionally Broad Substrate Specificity.

PubMed

Buryska, Tomas; Babkova, Petra; Vavra, Ondrej; Damborsky, Jiri; Prokop, Zbynek

2018-01-15

The haloalkane dehalogenase enzyme DmmA was identified by marine metagenomic screening. Determination of its crystal structure revealed an unusually large active site compared to those of previously characterized haloalkane dehalogenases. Here we present a biochemical characterization of this interesting enzyme with emphasis on its structure-function relationships. DmmA exhibited an exceptionally broad substrate specificity and degraded several halogenated environmental pollutants that are resistant to other members of this enzyme family. In addition to having this unique substrate specificity, the enzyme was highly tolerant to organic cosolvents such as dimethyl sulfoxide, methanol, and acetone. Its broad substrate specificity, high overexpression yield (200 mg of protein per liter of cultivation medium; 50% of total protein), good tolerance to organic cosolvents, and a broad pH range make DmmA an attractive biocatalyst for various biotechnological applications. IMPORTANCE We present a thorough biochemical characterization of the haloalkane dehalogenase DmmA from a marine metagenome. This enzyme with an unusually large active site shows remarkably broad substrate specificity, high overexpression, significant tolerance to organic cosolvents, and activity under a broad range of pH conditions. DmmA is an attractive catalyst for sustainable biotechnology applications, e.g., biocatalysis, biosensing, and biodegradation of halogenated pollutants. We also report its ability to convert multiple halogenated compounds to corresponding polyalcohols. Copyright © 2018 American Society for Microbiology.

Structural tuning of residual conductivity in highly mismatched III-V layers

DOEpatents

Han, Jung; Figiel, Jeffrey J.

2002-01-01

A new process to control the electrical conductivity of gallium nitride layers grown on a sapphire substrate has been developed. This process is based on initially coating the sapphire substrate with a thin layer of aluminum nitride, then depositing the gallium nitride thereon. This process allows one to controllably produce gallium nitride layers with resistivity varying over as much as 10 orders of magnitude, without requiring the introduction and activation of suitable dopants.

UFD4 lacking the proteasome-binding region catalyses ubiquitination but is impaired in proteolysis.

PubMed

Xie, Youming; Varshavsky, Alexander

2002-12-01

The ubiquitin system recognizes degradation signals of protein substrates through E3-E2 ubiquitin ligases, which produce a substrate-linked multi-ubiquitin chain. Ubiquitinated substrates are degraded by the 26S proteasome, which consists of the 20S protease and two 19S particles. We previously showed that UBR1 and UFD4, two E3 ligases of the yeast Saccharomyces cerevisiae, interact with specific proteasomal subunits. Here we advance this analysis for UFD4 and show that it interacts with RPT4 and RPT6, two subunits of the 19S particle. The 201-residue amino-terminal region of UFD4 is essential for its binding to RPT4 and RPT6. UFD4(DeltaN), which lacks this N-terminal region, adds ubiquitin to test substrates with apparently wild-type activity, but is impaired in conferring short half-lives on these substrates. We propose that interaction of a targeted substrate with the 26S proteasome involves contacts of specific proteasomal subunits with the substrate-bound ubiquitin ligase, with the substrate-linked multi-ubiquitin chain and with the substrate itself. This multiple-site binding may function to slow down dissociation of the substrate from the proteasome and to facilitate the unfolding of substrate through ATP-dependent movements of the chaperone subunits of the 19S particle.

Process for the deposition of high temperature stress and oxidation resistant coatings on silicon-based substrates

DOEpatents

Sarin, V.K.

1991-07-30

A process is disclosed for depositing a high temperature stress and oxidation resistant coating on a silicon nitride- or silicon carbide-based substrate body. A gas mixture is passed over the substrate at about 900--1500 C and about 1 torr to about ambient pressure. The gas mixture includes one or more halide vapors with other suitable reactant gases. The partial pressure ratios, flow rates, and process times are sufficient to deposit a continuous, fully dense, adherent coating. The halide and other reactant gases are gradually varied during deposition so that the coating is a graded coating of at least two layers. Each layer is a graded layer changing in composition from the material over which it is deposited to the material of the layer and further to the material, if any, deposited thereon, so that no clearly defined compositional interfaces exist. The gases and their partial pressures are varied according to a predetermined time schedule and the halide and other reactant gases are selected so that the layers include (a) an adherent, continuous intermediate layer about 0.5-20 microns thick of an aluminum nitride or an aluminum oxynitride material, over and chemically bonded to the substrate body, and (b) an adherent, continuous first outer layer about 0.5-900 microns thick including an oxide of aluminum or zirconium over and chemically bonded to the intermediate layer.

Process for the deposition of high temperature stress and oxidation resistant coatings on silicon-based substrates

DOEpatents

Sarin, Vinod K.

1991-01-01

A process for depositing a high temperature stress and oxidation resistant coating on a silicon nitride- or silicon carbide-based substrate body. A gas mixture is passed over the substrate at about 900.degree.-1500.degree. C. and about 1 torr to about ambient pressure. The gas mixture includes one or more halide vapors with other suitable reactant gases. The partial pressure ratios, flow rates, and process times are sufficient to deposit a continuous, fully dense, adherent coating. The halide and other reactant gases are gradually varied during deposition so that the coating is a graded coating of at least two layers. Each layer is a graded layer changing in composition from the material over which it is deposited to the material of the layer and further to the material, if any, deposited thereon, so that no clearly defined compositional interfaces exist. The gases and their partial pressures are varied according to a predetermined time schedule and the halide and other reactant gases are selected so that the layers include (a) an adherent, continuous intermediate layer about 0.5-20 microns thick of an aluminum nitride or an aluminum oxynitride material, over and chemically bonded to the substrate body, and (b) an adherent, continuous first outer layer about 0.5-900 microns thick including an oxide of aluminum or zirconium over and chemically bonded to the intermediate layer.

Varying hydric conditions during incubation influence egg water exchange and hatchling phenotype in the red-eared slider turtle.

PubMed

Delmas, Virginie; Bonnet, Xavier; Girondot, Marc; Prévot-Julliard, Anne-Caroline

2008-01-01

Environmental conditions within the nest, notably temperature and moisture of substrate, exert a powerful influence during embryogenesis in oviparous reptiles. The influence of fluctuating nest temperatures has been experimentally examined in different reptile species; however, similar experiments using moisture as the key variable are lacking. In this article, we examine the effect of various substrate moisture regimes during incubation on different traits (egg mass, incubation length, and hatchling mass) in a chelonian species with flexible-shelled eggs, the red-eared slider turtle (Trachemys scripta elegans). Our results show that the rate of water uptake by the eggs was higher in wet than in dry substrate and varied across development. More important, during the first third of development, the egg mass changes were relatively independent of the soil moisture level; they became very sensitive to moisture levels during the other two-thirds. Moreover, hydric conditions exerted a strong influence on the eggs' long-term sensitivity to the moisture of the substrate. Even short-term episodes of high or low levels of moisture modified permanently their water sensitivity, notably through modification of eggshell shape and volume, and in turn entailed significant effects on hatchling mass (and hence offspring quality). Such complex influences of fluctuating moisture levels at various incubation stages on hatchling phenotype better reflect the natural situation, compared to experiments based on stable, albeit different, moisture levels.

Exploration of CIGAS Alloy System for Thin-Film Photovoltaics on Novel Lightweight and Flexible Substrates

NASA Technical Reports Server (NTRS)

Woods, Lawrence M.; Kalla, Ajay; Ribelin, Rosine

2007-01-01

Thin-film photovoltaics (TFPV) on lightweight and flexible substrates offer the potential for very high solar array specific power (W/kg). ITN Energy Systems, Inc. (ITN) is developing flexible TFPV blanket technology that has potential for specific power greater than 2000 W/kg (including space coatings) that could result in solar array specific power between 150 and 500 W/kg, depending on array size, when mated with mechanical support structures specifically designed to take advantage of the lightweight and flexible substrates.(1) This level of specific power would far exceed the current state of the art for spacecraft PV power generation, and meet the needs for future spacecraft missions.(2) Furthermore the high specific power would also enable unmanned aircraft applications and balloon or high-altitude airship (HAA) applications, in addition to modular and quick deploying tents for surface assets or lunar base power, as a result of the high power density (W/sq m) and ability to be integrated into the balloon, HAA or tent fabric. ITN plans to achieve the high specific power by developing single-junction and two-terminal monolithic tandem-junction PV cells using thin-films of high-efficiency and radiation resistant CuInSe2 (CIS) partnered with bandgap-tunable CIS-alloys with Ga (CIGS) or Al (CIAS) on novel lightweight and flexible substrates. Of the various thin-film technologies, single-junction and radiation resistant CIS and associated alloys with gallium, aluminum and sulfur have achieved the highest levels of TFPV device performance, with the best efficiency reaching 19.5% under AM1.5 illumination conditions and on thick glass substrates.(3) Thus, it is anticipated that single- and tandem-junction devices with flexible substrates and based on CIS and related alloys will achieve the highest levels of thin-film space and HAA solar array performance.

Experimental and analytical parametric study of single-crystal unimorph beams for vibration energy harvesting.

PubMed

Karami, M Amin; Bilgen, Onur; Inman, Daniel J; Friswell, Michael I

2011-07-01

This research presents an experimental and theoretical energy harvesting characterization of beam-like, uniform cross-section, unimorph structures employing single-crystal piezoelectrics. Different piezoelectric materials, substrates, and configurations are examined to identify the best design configuration for lightweight energy harvesting devices for low-power applications. Three types of piezoelectrics (singlecrystal PMN-PZT, polycrystalline PZT-5A, and PZT-5H-type monolithic ceramics) are evaluated in a unimorph cantilevered beam configuration. The devices have been excited by harmonic base acceleration. All of the experimental characteristics have been used to validate an exact electromechanical model of the harvester. The study shows the optimum choice of substrate material for single-crystal piezoelectric energy harvesting. Comparison of energy scavengers with stainless steel substrates reveals that single-crystal harvesters produce superior power compared with polycrystalline devices. To further optimize the power harvesting, we study the relation between the thickness of the substrate and the power output for different substrate materials. The relation between power and substrate thickness profoundly varies among different substrate materials. The variation is understood by examining the change of mechanical transmissibility and the variations of the coupling figure of merit of the harvesters with thickness ratio. The investigation identifies the optimal thickness of the substrate for different substrate materials. The study also shows that the densities of the substrates and their mechanical damping coefficients have significant effects on the power output.

Engineering Nucleotide Specificity of Succinyl-CoA Synthetase in Blastocystis: The Emerging Role of Gatekeeper Residues.

PubMed

Vashisht, Kapil; Verma, Sonia; Gupta, Sunita; Lynn, Andrew M; Dixit, Rajnikant; Mishra, Neelima; Valecha, Neena; Hamblin, Karleigh A; Maytum, Robin; Pandey, Kailash C; van der Giezen, Mark

2017-01-24

Charged, solvent-exposed residues at the entrance to the substrate binding site (gatekeeper residues) produce electrostatic dipole interactions with approaching substrates, and control their access by a novel mechanism called "electrostatic gatekeeper effect". This proof-of-concept study demonstrates that the nucleotide specificity can be engineered by altering the electrostatic properties of the gatekeeper residues outside the binding site. Using Blastocystis succinyl-CoA synthetase (SCS, EC 6.2.1.5), we demonstrated that the gatekeeper mutant (ED) resulted in ATP-specific SCS to show high GTP specificity. Moreover, nucleotide binding site mutant (LF) had no effect on GTP specificity and remained ATP-specific. However, via combination of the gatekeeper mutant with the nucleotide binding site mutant (ED+LF), a complete reversal of nucleotide specificity was obtained with GTP, but no detectable activity was obtained with ATP. This striking result of the combined mutant (ED+LF) was due to two changes; negatively charged gatekeeper residues (ED) favored GTP access, and nucleotide binding site residues (LF) altered ATP binding, which was consistent with the hypothesis of the "electrostatic gatekeeper effect". These results were further supported by molecular modeling and simulation studies. Hence, it is imperative to extend the strategy of the gatekeeper effect in a different range of crucial enzymes (synthetases, kinases, and transferases) to engineer substrate specificity for various industrial applications and substrate-based drug design.

Reflective photoluminescence fiber temperature probe based on the CdSe/ZnS quantum dot thin film

NASA Astrophysics Data System (ADS)

Wang, Helin; Yang, Aijun; Chen, Zhongshi; Geng, Yan

2014-08-01

A reflective fiber temperature sensor based on the optical temperature dependent characteristics of a quantum dots (QDs) thin film is developed by depositing the CdSe/ZnS core/shell quantum dots on the SiO2 glass substrates. As the temperature is changed from 30 to 200°C, the peak wavelengths of PL spectra from the sensing head increase linearly with the temperature, while the peak intensity and the full width at half maximum (FWHM) of PL spectra vary exponentially according to the specific physical law. Using the obtained temperature-dependent peak-wavelength shift, the average resolution of the designed fiber temperature sensor can reach 0.12 nm/°C, while it reaches 0.056 nm/°C according to the FWHM of PL spectrum.

Kinetic modeling of lactic acid production from batch submerged fermentation of cheese whey

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

Tango, M.S.A.; Ghaly, A.E.

1999-12-01

A kinetic model for the production of lactic acid through batch submerged fermentation of cheese whey using Lactobacillus helveticus was developed. The model accounts for the effect of substrate limitation, substrate inhibition, lactic acid inhibition, maintenance energy and cell death on the cell growth, substrate utilization, and lactic acid production during the fermentation process. The model was evaluated using experimental data from Tango and Ghaly (1999). The predicted results obtained from the model compared well with experimental (R{sup 2} = 0.92--0.98). The model was also used to investigate the effect of the initial substrate concentration on the lag period, fermentationmore » time, specific growth rate, and cell productivity during batch fermentation. The maximum specific growth rate ({micro}{sub m}), the saturation constant (K{sub S}), the substrate inhibition constant (K{sub IS}), and the lactic acid inhibition constant (K{sub IP}) were found to be 0.25h{sup {minus}1}, 0.9 g/L, 250.0 g/L, and 60.0 g/L, respectively. High initial lactose concentration in cheese whey reduced both the specific growth rate and substrate utilization rate due to the substrate inhibition phenomenon. The maximum lactic acid production occurred at about 100 g/L initial lactose concentration after 40 h of fermentation. The maximum lactic acid concentration above which Lactobacillus helveticus did not grow was found to be 80.0 g/L.« less

Biological and physical conditions of macroinvertebrates in reference lowland streams

NASA Astrophysics Data System (ADS)

de Brouwer, Jan; Eekhout, Joris; Verdonschot, Piet

2016-04-01

Channelisation measures taken halfway the 20th century have had destructive consequences for the diversity of the ecology in the majority of the lowland streams in countries such as the Netherlands. Currently, stream restoration measures are being implemented in these degraded lowland streams, where design principles are often based on outdated relationships between biological and physical conditions. Little is known about the reference conditions in these streams. Therefore, the aim of this research is to quantify the relationships between biological and physical conditions of macroinvertebrates in reference lowland streams. The research was conducted in four near-natural lowland streams in Central Poland. Field data were obtained during a field campaign in 2011. The following data were obtained in a 50-m reach in each of the four streams: macroinvertebrate sampling, spatial habitat patterns, bathymetry, and flow-velocity. Furthermore, water level, light sensitivity and temperature sensors were installed to obtain the temporal dynamic of these streams. Macroinvertebrates were sampled in 9 different habitat types, i.e. sand, gravel, fine organic matter, stones, branches, leaves, silt, vegetation, and wood. Macroinvertebrates were determined to the highest taxonomic level possible. Data from the bathymetrical surveys were interpolated on a grid and bathymetrical metrics were determined. Flow velocity measurements were related to habitats and flow velocity metrics were determined. Analysis of the data shows that flow conditions vary among the different habitat, with a gradient from hard substrates towards soft substrates. Furthermore, the data show that stream as a unit best explains species composition, but also specific habitat conditions, such as substrate type and flow velocity, correlate with species composition. More specific, the data shows a strong effect of wood on species composition. These findings may have implications for stream restoration design, which mainly focus on large-scale reconstruction of channel planform, whereas this study shows that improvement of stream ecology should focus on the smaller habitat scale.

Molecular Basis for the Relative Substrate Specificity of Human Immunodeficiency Virus Type 1 and Feline Immunodeficiency Virus Proteases

PubMed Central

Beck, Zachary Q.; Lin, Ying-Chuan; Elder, John H.

2001-01-01

We have used a random hexamer phage library to delineate similarities and differences between the substrate specificities of human immunodeficiency virus type 1 (HIV-1) and feline immunodeficiency virus (FIV) proteases (PRs). Peptide sequences were identified that were specifically cleaved by each protease, as well as sequences cleaved equally well by both enzymes. Based on amino acid distinctions within the P3-P3′ region of substrates that appeared to correlate with these cleavage specificities, we prepared a series of synthetic peptides within the framework of a peptide sequence cleaved with essentially the same efficiency by both HIV-1 and FIV PRs, Ac-KSGVF↓VVNGLVK-NH2 (arrow denotes cleavage site). We used the resultant peptide set to assess the influence of specific amino acid substitutions on the cleavage characteristics of the two proteases. The findings show that when Asn is substituted for Val at the P2 position, HIV-1 PR cleaves the substrate at a much greater rate than does FIV PR. Likewise, Glu or Gln substituted for Val at the P2′ position also yields peptides specifically susceptible to HIV-1 PR. In contrast, when Ser is substituted for Val at P1′, FIV PR cleaves the substrate at a much higher rate than does HIV-1 PR. In addition, Asn or Gln at the P1 position, in combination with an appropriate P3 amino acid, Arg, also strongly favors cleavage by FIV PR over HIV PR. Structural analysis identified several protease residues likely to dictate the observed specificity differences. Interestingly, HIV PR Asp30 (Ile-35 in FIV PR), which influences specificity at the S2 and S2′ subsites, and HIV-1 PR Pro-81 and Val-82 (Ile-98 and Gln-99 in FIV PR), which influence specificity at the S1 and S1′ subsites, are residues which are often involved in development of drug resistance in HIV-1 protease. The peptide substrate KSGVF↓VVNGK, cleaved by both PRs, was used as a template for the design of a reduced amide inhibitor, Ac-GSGVFΨ(CH2NH)VVNGL-NH2. This compound inhibited both FIV and HIV-1 PRs with approximately equal efficiency. These findings establish a molecular basis for distinctions in substrate specificity between human and feline lentivirus PRs and offer a framework for development of efficient broad-based inhibitors. PMID:11533208

Effect of Process Variables on the Grain Size and Crystallographic Texture of Hot-Dip Galvanized Coatings

NASA Astrophysics Data System (ADS)

Kaboli, Shirin; McDermid, Joseph R.

2014-08-01

A galvanizing simulator was used to determine the effect of galvanizing bath antimony (Sb) content, substrate surface roughness, and cooling rate on the microstructural development of metallic zinc coatings. Substrate surface roughness was varied through the use of relatively rough hot-rolled and relatively smooth bright-rolled steels, cooling rates were varied from 0.1 to 10 K/s, and bulk bath Sb levels were varied from 0 to 0.1 wt pct. In general, it was found that increasing bath Sb content resulted in coatings with a larger grain size and strongly promoted the development of coatings with the close-packed {0002} basal plane parallel to the substrate surface. Increasing substrate surface roughness tended to decrease the coating grain size and promoted a more random coating crystallographic texture, except in the case of the highest Sb content bath (0.1 wt pct Sb), where substrate roughness had no significant effect on grain size except at higher cooling rates (10 K/s). Increased cooling rates tended to decrease the coating grain size and promote the {0002} basal orientation. Calculations showed that increasing the bath Sb content from 0 to 0.1 wt pct Sb increased the dendrite tip growth velocity from 0.06 to 0.11 cm/s by decreasing the solid-liquid interface surface energy from 0.77 to 0.45 J/m2. Increased dendrite tip velocity only partially explains the formation of larger zinc grains at higher Sb levels. It was also found that the classic nucleation theory cannot completely explain the present experimental observations, particularly the effect of increasing the bath Sb, where the classical theory predicts increased nucleation and a finer grain size. In this case, the "poisoning" theory of nucleation sites by segregated Sb may provide a partial explanation. However, any analysis is greatly hampered by the lack of fundamental thermodynamic information such as partition coefficients and surface energies and by a lack of fundamental structural studies. Overall, it was concluded that the fundamental mechanisms behind the microstructural development of solidified metallic zinc coatings have yet to be completely elucidated and require further investigation.

Purification and substrate specificities of a fructanase from Kluyveromyces marxianus isolated from the fermentation process of Mezcal.

PubMed

Arrizon, Javier; Morel, Sandrine; Gschaedler, Anne; Monsan, Pierre

2011-02-01

A fructanase, produced by a Kluyveromyces marxianus strain isolated during the fermentation step of the elaboration process of "Mezcal de Guerrero" was purified and biochemically characterized. The active protein was a glycosylated dimer with a molecular weight of approximately 250 kDa. The specific enzymatic activity of the protein was determined for different substrates: sucrose, inulin, Agave tequilana fructan, levan and Actilight® and compared with the activity of Fructozyme®. The hydrolysis profile of the different substrates analyzed by HPAEC-PAD showed that the enzyme has different affinities over the substrates tested with a sucrose/inulin enzymatic activity ratio (S/I) of 125. For the hydrolysis of Agave tequilana fructans, the enzyme also showed a higher enzymatic activity and specificity than Fructozyme®, which is important for its potential application in the tequila industry. Copyright © 2010 Elsevier Ltd. All rights reserved.

Chemical probing of the human sirtuin 5 active site reveals its substrate acyl specificity and peptide-based inhibitors.

PubMed

Roessler, Claudia; Nowak, Theresa; Pannek, Martin; Gertz, Melanie; Nguyen, Giang T T; Scharfe, Michael; Born, Ilona; Sippl, Wolfgang; Steegborn, Clemens; Schutkowski, Mike

2014-09-26

Sirtuins are NAD(+)-dependent deacetylases acting as sensors in metabolic pathways and stress response. In mammals there are seven isoforms. The mitochondrial sirtuin 5 is a weak deacetylase but a very efficient demalonylase and desuccinylase; however, its substrate acyl specificity has not been systematically analyzed. Herein, we investigated a carbamoyl phosphate synthetase 1 derived peptide substrate and modified the lysine side chain systematically to determine the acyl specificity of Sirt5. From that point we designed six potent peptide-based inhibitors that interact with the NAD(+) binding pocket. To characterize the interaction details causing the different substrate and inhibition properties we report several X-ray crystal structures of Sirt5 complexed with these peptides. Our results reveal the Sirt5 acyl selectivity and its molecular basis and enable the design of inhibitors for Sirt5. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

ATP-independent reversal of a membrane protein aggregate by a chloroplast SRP

PubMed Central

Jaru-Ampornpan, Peera; Shen, Kuang; Lam, Vinh Q.; Ali, Mona; Doniach, Sebastian; Jia, Tony Z.; Shan, Shu-ou

2010-01-01

Membrane proteins impose enormous challenges to cellular protein homeostasis during their post-translational targeting, and require chaperones to keep them soluble and translocation-competent. Here we show that a novel targeting factor in the chloroplast Signal Recognition Particle (cpSRP), cpSRP43, is a highly specific molecular chaperone that efficiently reverses the aggregation of its substrate proteins. In contrast to AAA+-chaperones, cpSRP43 utilizes specific binding interactions with its substrate to mediate its disaggregase activity. This ‘disaggregase’ capability can allow targeting machineries to more effectively capture their protein substrates, and emphasizes a close connection between protein folding and trafficking processes. Moreover, cpSRP43 provides the first example of an ATP-independent disaggregase, and demonstrates that efficient reversal of protein aggregation can be attained by specific binding interactions between a chaperone and its substrate. PMID:20424608

Development of a high-throughput liquid state assay for lipase activity using natural substrates and rhodamine B.

PubMed

Zottig, Ximena; Meddeb-Mouelhi, Fatma; Beauregard, Marc

2016-03-01

A fluorescence-based assay for the determination of lipase activity using rhodamine B as an indicator, and natural substrates such as olive oil, is described. It is based on the use of a rhodamine B-natural substrate emulsion in liquid state, which is advantageous over agar plate assays. This high-throughput method is simple and rapid and can be automated, making it suitable for screening and metagenomics application. Reaction conditions such as pH and temperature can be varied and controlled. Using triolein or olive oil as a natural substrate allows monitoring of lipase activity in reaction conditions that are closer to those used in industrial settings. The described method is sensitive over a wide range of product concentrations and offers good reproducibility. Copyright © 2015 Elsevier Inc. All rights reserved.

Substrate thermal conductivity controls the ability to manufacture microstructures via laser-induced direct write

NASA Astrophysics Data System (ADS)

Tomko, John A.; Olson, David H.; Braun, Jeffrey L.; Kelliher, Andrew P.; Kaehr, Bryan; Hopkins, Patrick E.

2018-01-01

In controlling the thermal properties of the surrounding environment, we provide insight into the underlying mechanisms driving the widely used laser direct write method for additive manufacturing. We find that the onset of silver nitrate reduction for the formation of direct write structures directly corresponds to the calculated steady-state temperature rises associated with both continuous wave and high-repetition rate, ultrafast pulsed laser systems. Furthermore, varying the geometry of the heat affected zone, which is controllable based on in-plane thermal diffusion in the substrate, and laser power, allows for control of the written geometries without any prior substrate preparation. These findings allow for the advance of rapid manufacturing of micro- and nanoscale structures with minimal material constraints through consideration of the laser-controllable thermal transport in ionic liquid/substrate media.

Characterization of the Serratia marcescens SdeCDE multidrug efflux pump studied via gene knockout mutagenesis.

PubMed

Begic, Sanela; Worobec, Elizabeth A

2008-05-01

Serratia marcescens is an important nosocomial agent having high antibiotic resistance. A major mechanism for S. marcescens antibiotic resistance is active efflux. To ascertain the substrate specificity of the S. marcescens SdeCDE efflux pump, we constructed pump gene deletion mutants. sdeCDE knockout strains showed no change in antibiotic susceptibility in comparison with the parental strains for any of the substrates, with the exception of novobiocin. In addition, novobiocin was the only antibiotic to be accumulated by sdeCDE-deficient strains. Based on the substrates used in our study, we conclude that SdeCDE is a Resistance-Nodulation-Cell Division family pump with limited substrate specificity.

Identification of Residues Involved in Substrate Specificity and Cytotoxicity of Two Closely Related Cutinases from Mycobacterium tuberculosis

PubMed Central

Dedieu, Luc; Serveau-Avesque, Carole; Canaan, Stéphane

2013-01-01

The enzymes belonging to the cutinase family are serine enzymes active on a large panel of substrates such as cutin, triacylglycerols, and phospholipids. In the M. tuberculosis H37Rv genome, seven genes coding for cutinase-like proteins have been identified with strong immunogenic properties suggesting a potential role as vaccine candidates. Two of these enzymes which are secreted and highly homologous, possess distinct substrates specificities. Cfp21 is a lipase and Cut4 is a phospholipase A2, which has cytotoxic effects on macrophages. Structural overlay of their three-dimensional models allowed us to identify three areas involved in the substrate binding process and to shed light on this substrate specificity. By site-directed mutagenesis, residues present in these Cfp21 areas were replaced by residues occurring in Cut4 at the same location. Three mutants acquired phospholipase A1 and A2 activities and the lipase activities of two mutants were 3 and 15 fold greater than the Cfp21 wild type enzyme. In addition, contrary to mutants with enhanced lipase activity, mutants that acquired phospholipase B activities induced macrophage lysis as efficiently as Cut4 which emphasizes the relationship between apparent phospholipase A2 activity and cytotoxicity. Modification of areas involved in substrate specificity, generate recombinant enzymes with higher activity, which may be more immunogenic than the wild type enzymes and could therefore constitute promising candidates for antituberculous vaccine production. PMID:23843969

Identification of residues involved in substrate specificity and cytotoxicity of two closely related cutinases from Mycobacterium tuberculosis.

PubMed

Dedieu, Luc; Serveau-Avesque, Carole; Canaan, Stéphane

2013-01-01

The enzymes belonging to the cutinase family are serine enzymes active on a large panel of substrates such as cutin, triacylglycerols, and phospholipids. In the M. tuberculosis H37Rv genome, seven genes coding for cutinase-like proteins have been identified with strong immunogenic properties suggesting a potential role as vaccine candidates. Two of these enzymes which are secreted and highly homologous, possess distinct substrates specificities. Cfp21 is a lipase and Cut4 is a phospholipase A2, which has cytotoxic effects on macrophages. Structural overlay of their three-dimensional models allowed us to identify three areas involved in the substrate binding process and to shed light on this substrate specificity. By site-directed mutagenesis, residues present in these Cfp21 areas were replaced by residues occurring in Cut4 at the same location. Three mutants acquired phospholipase A1 and A2 activities and the lipase activities of two mutants were 3 and 15 fold greater than the Cfp21 wild type enzyme. In addition, contrary to mutants with enhanced lipase activity, mutants that acquired phospholipase B activities induced macrophage lysis as efficiently as Cut4 which emphasizes the relationship between apparent phospholipase A2 activity and cytotoxicity. Modification of areas involved in substrate specificity, generate recombinant enzymes with higher activity, which may be more immunogenic than the wild type enzymes and could therefore constitute promising candidates for antituberculous vaccine production.

[Substrate-inhibitory analysis of monoamine oxidase from hepatopancreas of the octopus Bathypolypus arcticus].

PubMed

Basova, I N; Iagodina, O V

2012-01-01

Study of the substrate-inhibitory specificity of mitochondrial monoamine oxidase (MAO) of hepatopancreas of the octopus Bathypolypus arcticus revealed distinctive peculiarities of catalytic properties of this enzyme. The studied enzyme, on one hand, like the classic MAO of homoiothermal animals, is able to deaminate tyramine, serotonin, benzylamine, tryptamine, beta-phenylethylamine, while, on the other hand, deaminates histamine and does not deaminate putrescine--classic substrates of diamine oxidase (DAO). Results of the substrate-inhibitory analysis with use of chlorgiline and deprenyl are indirect proofs of the existence in the octopus hepatopancreas of one molecular MAO form. Semicarbazide and pyronine G turned out to be weak irreversible inhibitors, four derivatives of acridine--irreversible inhibitors of the intermediate effectiveness with respect to the octopus hepatopancreas MAO; specificity of action of inhibitors at deamination of different substrates was equal.

Friction measurements on InAs NWs by AFM manipulation

NASA Astrophysics Data System (ADS)

Pettersson, Hakan; Conache, Gabriela; Gray, Struan; Bordag, Michael; Ribayrol, Aline; Froberg, Linus; Samuelson, Lars; Montelius, Lars

2008-03-01

We discuss a new approach to measure the friction force between elastically deformed nanowires and a surface. The wires are bent, using an AFM, into an equilibrium shape determined by elastic restoring forces within the wire and friction between the wire and the surface. From measurements of the radius of curvature of the bent wires, elasticity theory allows the friction force per unit length to be calculated. We have studied friction properties of InAs nanowires deposited on SiO2, silanized SiO2 and Si3N4 substrates. The wires were typically from 0.5 to a few microns long, with diameters varying between 20 and 80 nm. Manipulation is done in a `Retrace Lift' mode, where feedback is turned off for the reverse scan and the tip follows a nominal path. The effective manipulation force during the reverse scan can be changed by varying an offset in the height of the tip over the surface. We will report on interesting static- and sliding friction experiments with nanowires on the different substrates, including how the friction force per unit length varies with the diameter of the wires.

Assessment of Alternative Substrates for Culturing Lumbriculus variegatus

USGS Publications Warehouse

Lasier, P.J.

2007-01-01

The freshwater oligochaete, Lumbriculus variegatus, is tank-cultured to provide organisms for aquatic-habitat assessments, regeneration research and as a clean source of live food for aquarium fishes. Shredded paper is the typical substrate in cultures used to rear L. variegatus for these purposes. However, the effort needed to separate large numbers from decomposing paper can be prohibitive. Burlap and nylon mesh material were compared to paper as potential alternatives that could reduce this effort. Oligochaete production and the amount of time needed to separate animals from substrate were compared for eight weeks among experimental cultures containing burlap, nylon mesh and paper. Cultures with paper substrate increased in number and weight two to three times faster than those with burlap or nylon mesh substrates. The time needed to separate animals from substrate was initially two to three times longer with paper substrate than with burlap or nylon mesh substrates, but this difference increased to between 10 and 40 times longer after six weeks as the paper substrate decomposed. Feeding rates varied by treatment and were based on average wet weight at the time of water replacement. Elevated ammonia and nitrite concentrations resulting from excess food may have reduced production in nylon mesh treatments and was lethal in paper treatments during the final phases of the study. The type of substrate recommended may depend on the desired production rate of oligochaetes, space available for cultures and the amount of effort available for substrate renewal and separating the animals from the cultures.

Hydrological performance of dual-substrate-layer green roofs using porous inert substrates with high sorption capacities.

PubMed

Wang, Xiaoou; Tian, Yimei; Zhao, Xinhua; Peng, Chenrui

2017-06-01

Given that the common medium in existing green roofs is a single layer composed of organic and inorganic substrates, seven pilot-scale dual-substrate-layer extensive green roofs (G1-G7), which include nutrition and adsorption substrate layers, were constructed in this study. The effectiveness of porous inert substrates (activated charcoal, zeolite, pumice, lava, vermiculite and expanded perlite) used as the adsorption substrate for stormwater retention was investigated. A single-substrate-layer green roof (G8) was built for comparison with G1-G7. Despite the larger total rainfall depth (mm) of six types of simulated rains (43.2, 54.6, 76.2, 87.0, 85.2 and 86.4, respectively), the total percent retention of G1-G7 varied between 14% and 82% with an average of 43%, exhibiting better runoff-retaining capacity than G8 based on the maximum potential rainfall storage depth per unit height of adsorption substrate. Regression analysis showed that there was a logarithmic relationship between cumulative rainfall depth with non-zero runoff and stormwater retention for G1-G4 and a linear relationship for G5-G8. To enhance the water retention capacity and extend the service life of dual-substrate-layer extensive green roofs, the mixture of activated charcoal and/or pumice with expanded perlite and/or vermiculite is more suitable as the adsorption substrate than the mixture containing lava and/or zeolite.

Effects of substrate voltage on noise characteristics and hole lifetime in SOI metal-oxide-semiconductor field-effect transistor photon detector.

PubMed

Putranto, Dedy Septono Catur; Priambodo, Purnomo Sidi; Hartanto, Djoko; Du, Wei; Satoh, Hiroaki; Ono, Atsushi; Inokawa, Hiroshi

2014-09-08

Low-frequency noise and hole lifetime in silicon-on-insulator (SOI) metal-oxide-semiconductor field-effect transistors (MOSFETs) are analyzed, considering their use in photon detection based on single-hole counting. The noise becomes minimum at around the transition point between front- and back-channel operations when the substrate voltage is varied, and increases largely on both negative and positive sides of the substrate voltage showing peculiar Lorentzian (generation-recombination) noise spectra. Hole lifetime is evaluated by the analysis of drain current histogram at different substrate voltages. It is found that the peaks in the histogram corresponding to the larger number of stored holes become higher as the substrate bias becomes larger. This can be attributed to the prolonged lifetime caused by the higher electric field inside the body of SOI MOSFET. It can be concluded that, once the inversion channel is induced for detection of the photo-generated holes, the small absolute substrate bias is favorable for short lifetime and low noise, leading to high-speed operation.

Elasticity modulated Electrowetting of a sessile liquid droplet

NASA Astrophysics Data System (ADS)

Kumar, Sumit; Subramanian, Sri Ganesh; Dasgupta, Sunando; Chakraborty, Suman

2017-11-01

The sessile liquid droplets on the elastic and soft deformable surface produce strong deformation near the three-phase contact line (TPCL). The capillary and elastic forces play an important role during this deformation, and deteriorate the wetting behaviour of a sessile drop. The present work combines the effects of liquid viscosity and substrate elasticity on the dynamics of EWOD. The influence of decreasing film elasticity and viscosity on the electrowetting response of a sessile drop is experimentally investigated by delineating the changes in equilibrium apparent contact angles on substrates with varying Young's modulus of elasticity. The increase in viscosity of the liquid leads to greater electrowetting for non-deformable substrates whereas; the dynamics are not greatly affected in case of soft substrates. Although the viscosity appears to be an influential factor, the dynamics are more skewed towards the substrate rigidity. The vertical component of Young's force creates a wetting ridge at the three-phase contact line, the height of which is a direct function of the substrate rigidity. The produced ridges reduce the overall wettability of the droplet.

Spectrophotometric determination of substrate-borne polyacrylamide.

PubMed

Lu, Jianhang; Wu, Laosheng

2002-08-28

Polyacrylamides (PAMs) have wide application in many industries and in agriculture. Scientific research and industrial applications manifested a need for a method that can quantify substrate-borne PAM. The N-bromination method (a PAM analytical technique based on N-bromination of amide groups and spectrophotometric determination of the formed starch-triiodide complex), which was originally developed for determining PAM in aqueous solutions, was modified to quantify substrate-borne PAM. In the modified method, the quantity of substrate-borne PAM was converted to a concentration of starch-triiodide complex in aqueous solution that was then measured by spectrophotometry. The method sensitivity varied with substrates due to sorption of reagents and reaction intermediates on the substrates. Therefore, separate calibration for each substrate was required. Results from PAM samples in sand, cellulose, organic matter burnt soils, and clay minerals showed that this method had good accuracy and reproducibility. The PAM recoveries ranged from 95.8% to 103.7%, and the relative standard deviations (n = 4) were <7.5% in all cases. The optimum range of PAM in each sample is 10-80 microg. The technique can serve as an effective tool in improving PAM application and facilitating PAM-related research.

The role of the substrate in micro-scale scratching of epoxy-polyester films

NASA Astrophysics Data System (ADS)

Barletta, M.; Gisario, A.

2011-02-01

The present investigation analyzes the deformation response of electrostatically sprayed epoxy-polyester powder coatings by 'in situ' micro-mechanical tests. The characterization of the performance of the coatings was carried out by micro-scale scratching, by varying the indenter type, the applied load and the sliding speed. The tests were carried out on polymeric coatings deposited on as-received, micro and macro-corrugated AISI 304 stainless steel substrates and 'rigidly adhered' to them. Further tests were performed on 'free-standing' coatings, that is, on the as-received metal substrates pre-coated with an intermediate layer of silicon-based heat curable release coating. Experimental data allow us to evaluate the influence of the contact conditions between substrate and indenter and the role of the loading conditions on the scratch and penetration resistance of the epoxy-polyester coatings. The different responses of the polymeric coatings when deposited on untreated or pre-treated substrates as well as on an intermediate layer of release coating, contribute to a better understanding of the intrinsic roles of the polymeric material and substrate as well as the influence of the interfacial adhesion between coating and substrate.

Structure and Activity Analyses of Escherichia coli K-12 NagD Provide Insight into the Evolution of Biochemical Function in the Haloakanoic Acid Dehlogenase Superfamily

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

Tremblay,L.; Dunaway-Mariano, D.; Allen, K.

2006-01-01

The HAD superfamily is a large superfamily of proteins which share a conserved core domain that provides those active site residues responsible for the chemistry common to all family members. The superfamily is further divided into the four subfamilies I, IIA, IIB, and III, based on the topology and insertion site of a cap domain that provides substrate specificity. This structural and functional division implies that members of a given HAD structural subclass may target substrates that have similar structural characteristics. To understand the structure/function relationships in all of the subfamilies, a type IIA subfamily member, NagD from Escherichia colimore » K-12, was selected (type I, IIB, and III members have been more extensively studied). The structure of the NagD protein was solved to 1.80 Angstroms with R{sub work} = 19.8% and R{sub free} = 21.8%. Substrate screening and kinetic analysis showed NagD to have high specificity for nucleotide monophosphates with kcat/Km = 3.12 x 10{sup 4} and 1.28 x 10{sup 4} {micro}M{sup -1} s{sup -1} for UMP and GMP, respectively. This specificity is consistent with the presence of analogues of NagD that exist as fusion proteins with a nucleotide pyrophosphatase from the Nudix family. Docking of the nucleoside substrate in the active site brings it in contact with conserved residues from the cap domain that can act as a substrate specificity loop (NagD residues 144-149) in the type IIA subfamily. NagD and other subfamily IIA and IIB members show the common trait that substrate specificity and catalytic efficiencies (k{sub cat}/K{sub m}) are low (1 x 10{sup 4} M{sup -1} s{sup -1}) and the boundaries defining physiological substrates are somewhat overlapping. The ability to catabolize other related secondary metabolites indicates that there is regulation at the genetic level.« less

DbPTM 3.0: an informative resource for investigating substrate site specificity and functional association of protein post-translational modifications.

PubMed

Lu, Cheng-Tsung; Huang, Kai-Yao; Su, Min-Gang; Lee, Tzong-Yi; Bretaña, Neil Arvin; Chang, Wen-Chi; Chen, Yi-Ju; Chen, Yu-Ju; Huang, Hsien-Da

2013-01-01

Protein modification is an extremely important post-translational regulation that adjusts the physical and chemical properties, conformation, stability and activity of a protein; thus altering protein function. Due to the high throughput of mass spectrometry (MS)-based methods in identifying site-specific post-translational modifications (PTMs), dbPTM (http://dbPTM.mbc.nctu.edu.tw/) is updated to integrate experimental PTMs obtained from public resources as well as manually curated MS/MS peptides associated with PTMs from research articles. Version 3.0 of dbPTM aims to be an informative resource for investigating the substrate specificity of PTM sites and functional association of PTMs between substrates and their interacting proteins. In order to investigate the substrate specificity for modification sites, a newly developed statistical method has been applied to identify the significant substrate motifs for each type of PTMs containing sufficient experimental data. According to the data statistics in dbPTM, >60% of PTM sites are located in the functional domains of proteins. It is known that most PTMs can create binding sites for specific protein-interaction domains that work together for cellular function. Thus, this update integrates protein-protein interaction and domain-domain interaction to determine the functional association of PTM sites located in protein-interacting domains. Additionally, the information of structural topologies on transmembrane (TM) proteins is integrated in dbPTM in order to delineate the structural correlation between the reported PTM sites and TM topologies. To facilitate the investigation of PTMs on TM proteins, the PTM substrate sites and the structural topology are graphically represented. Also, literature information related to PTMs, orthologous conservations and substrate motifs of PTMs are also provided in the resource. Finally, this version features an improved web interface to facilitate convenient access to the resource.

Design of N-acyl homoserine lactonase with high substrate specificity by a rational approach.

PubMed

Kyeong, Hyun-Ho; Kim, Jin-Hyun; Kim, Hak-Sung

2015-06-01

N-Acyl homoserine lactone (AHL) is a major quorum-sensing signaling molecule in many bacterial species. Quorum-quenching (QQ) enzymes, which degrade such signaling molecules, have attracted much attention as an approach to controlling and preventing bacterial virulence and pathogenesis. However, naturally occurring QQ enzymes show a broad substrate spectrum, raising the concern of unintentionally attenuating beneficial effects by symbiotic bacteria. Here we report the rational design of acyl homoserine lactonase with high substrate specificity. Through docking analysis, we identified three key residues which play a key role in the substrate preference of the enzyme. The key residues were changed in a way that increases hydrophobic contact with a substrate having a short acyl chain (C4-AHL) while generating steric clashes with that containing a long acyl chain (C12-AHL). The resulting mutants exhibited a significantly shifted preference toward a substrate with a short acyl chain. Molecular dynamics simulations suggested that the mutations affect the behavior of a flexible loop, allowing tighter binding of a substrate with a short acyl chain.

Targeting Apolipoproteins in Magnetic Resonance Imaging

NASA Astrophysics Data System (ADS)

Sriram, Renuka; Lagerstedt, Jens O.; Samardzic, Haris; Kreutzer, Ulrike; Petrolova, Jitka; Xie, Hongtao; Kaysen, George A.; Voss, John C.; Desreux, Jean F.; Jue, Thomas

Maintaining normal physiological homeostasis depends upon a coordinated metabolism of both water-soluble and -insoluble substrates. In humans the body derives these molecules — such as glucose, amino acids, and fatty acids — from complex food matter. Water-soluble substrates can circulate readily in blood, while water-insoluble molecules — such as fatty acid, triacylglycerol, and cholesterol — require ampiphathic carriers to transport them from the site of biosynthesis (liver and intestine) to the target tissue. For fatty acid, albumin serves as the major transporter. For triacylglycerol and cholesterol, however, macromolecular complexes aggregate the hydrophobic molecules into the core and cover the surface with amphiphatic proteins and phospholipids to solubilize the particles in the lymphatic and circulatory systems. These macromolecules belong to a class of proteins, plasma lipoproteins, with specific functions and cellular targets. In the clinic these lipoproteins prognosticate the risk of cardiovascular disease (CVD). Lipoproteins divide usually into five major types: chylomicron, very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL). Each lipoprotein type exhibits characteristic density, size, and composition. As implied in the name, the density varies from the low-density chylomicron (<0.95 g/ml) to the high-density HDL (1.2 g/ml). Size also varies. The chylomicron has the largest diameter (75-1,200 nm), and HDL has the smallest (5-12 nm). The physical property variation arises from each lipoprotein's distinct composition. In a chylomicron, cholesterol, triacylglycerol, and phospholipid predominate and constitute about 90% of the particle. Protein constitutes only about 10%. In contrast, the smaller HDL has less cholesterol, triacylglycerol, and phospholipid (65% of the particle) but more protein (over 30%).

The Structure of Lombricine Kinase: Implications for Phosphagen Conformational Changes

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

Bush, D. Jeffrey; Kirillova, Olga; Clark, Shawn A.

2012-05-29

Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309-317, which is disordered in substrate-free structures of homologs and is known from the NMR ofmore » arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His{sup 178}. Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates.« less

A Measure of the Broad Substrate Specificity of Enzymes Based on ‘Duplicate’ Catalytic Residues

PubMed Central

Chakraborty, Sandeep; Ásgeirsson, Bjarni; Rao, Basuthkar J.

2012-01-01

The ability of an enzyme to select and act upon a specific class of compounds with unerring precision and efficiency is an essential feature of life. Simultaneously, these enzymes often catalyze the reaction of a range of similar substrates of the same class, and also have promiscuous activities on unrelated substrates. Previously, we have established a methodology to quantify promiscuous activities in a wide range of proteins. In the current work, we quantitatively characterize the active site for the ability to catalyze distinct, yet related, substrates (BRASS). A protein with known structure and active site residues provides the framework for computing ‘duplicate’ residues, each of which results in slightly modified replicas of the active site scaffold. Such spatial congruence is supplemented by Finite difference Poisson Boltzmann analysis which filters out electrostatically unfavorable configurations. The congruent configurations are used to compute an index (BrassIndex), which reflects the broad substrate profile of the active site. We identify an acetylhydrolase and a methyltransferase as having the lowest and highest BrassIndex, respectively, from a set of non-homologous proteins extracted from the Catalytic Site Atlas. The acetylhydrolase, a regulatory enzyme, is known to be highly specific for platelet-activating factor. In the methyltransferase (PDB: 1QAM), various combinations of glycine (Gly38/40/42), asparagine (Asn101/11) and glutamic acid (Glu59/36) residues having similar spatial and electrostatic profiles with the specified scaffold (Gly38, Asn101 and Glu59) exemplifies the broad substrate profile such an active site may provide. ‘Duplicate’ residues identified by relaxing the spatial and/or electrostatic constraints can be the target of directed evolution methodologies, like saturation mutagenesis, for modulating the substrate specificity of proteins. PMID:23166637

QM/MM Free Energy Simulations of Salicylic Acid Methyltransferase: Effects of Stabilization of TS-like Structures on Substrate Specificity

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

Yao, Jianzhuang; Xu, Qin; Chen, Feng

2010-01-01

Salicylic acid methyltransferases (SAMTs) synthesize methyl salicylate (MeSA) using salicylate as the substrate. MeSA synthesized in plants may function as an airborne signal to activate the expression of defense-related genes and could also be a critical mobile signaling molecule that travels from the site of plant infection to establish systemic immunity in the induction of disease resistance. Here the results of QM/MM free energy simulations for the methyl transfer process in Clarkia breweri SAMT (CbSAMT) are reported to determine the origin of the substrate specificity of SAMTs. The free energy barrier for the methyl transfer from S-adenosyl-l-methionine (AdoMet) to 4-hydroxybenzoatemore » in CbSAMT is found to be about 5 kcal/mol higher than that from AdoMet to salicylate, consistent with the experimental observations. It is suggested that the relatively high efficiency for the methylation of salicylate compared to 4-hydroxybenzoate is due, at least in part, to the reason that a part of the stabilization of the transition state (TS) configuration is already reflected in the reactant complex, presumably, through the binding. The results seem to indicate that the creation of the substrate complex (e.g., through mutagenesis and substrate modifications) with its structure closely resembling TS might be fruitful for improving the catalytic efficiency for some enzymes. The results show that the computer simulations may provide important insights into the origin of the substrate specificity for the SABATH family and could be used to help experimental efforts in generating engineered enzymes with altered substrate specificity.« less

Substrate Specificities of MexAB-OprM, MexCD-OprJ, and MexXY-OprM Efflux Pumps in Pseudomonas aeruginosa

PubMed Central

Masuda, Nobuhisa; Sakagawa, Eiko; Ohya, Satoshi; Gotoh, Naomasa; Tsujimoto, Hideto; Nishino, Takeshi

2000-01-01

To find the exact substrate specificities of three species of tripartite efflux systems of Pseudomonas aeruginosa, MexAB-OprM, MexCD-OprJ, and MexXY-OprM, we constructed a series of isogenic mutants, each of which constitutively overproduced one of the three efflux systems and lacked the other two, and their isogenic mutants, which lacked all these systems. Comparison of the susceptibilities of the constructed mutants to 52 antimicrobial agents belonging to various groups suggested the following substrate specificities. All of the efflux systems extrude a wide variety of antimicrobial agent groups, i.e., quinolones, macrolides, tetracyclines, lincomycin, chloramphenicol, most penicillins (all but carbenicillin and sulbenicillin), most cephems (all but cefsulodin and ceftazidime), meropenem, and S-4661, but none of them extrude polymyxin B or imipenem. Extrusion of aminoglycosides is specific to MexXY-OprM, and extrusion of a group of the β-lactams, i.e., carbenicillin, sulbenicillin, ceftazidime, moxalactam, and aztreonam, is specific to MexAB-OprM. Moreover, MexAB-OprM and MexCD-OprJ extrude novobiocin, cefsulodin, and flomoxef, while MexXY-OprM does not. These substrate specificities are distinct from those reported previously. PMID:11083635

Reconstructed ancestral enzymes reveal that negative selection drove the evolution of substrate specificity in ADP-dependent kinases.

PubMed

Castro-Fernandez, Víctor; Herrera-Morande, Alejandra; Zamora, Ricardo; Merino, Felipe; Gonzalez-Ordenes, Felipe; Padilla-Salinas, Felipe; Pereira, Humberto M; Brandão-Neto, Jose; Garratt, Richard C; Guixe, Victoria

2017-09-22

One central goal in molecular evolution is to pinpoint the mechanisms and evolutionary forces that cause an enzyme to change its substrate specificity; however, these processes remain largely unexplored. Using the glycolytic ADP-dependent kinases of archaea, including the orders Thermococcales , Methanosarcinales , and Methanococcales , as a model and employing an approach involving paleoenzymology, evolutionary statistics, and protein structural analysis, we could track changes in substrate specificity during ADP-dependent kinase evolution along with the structural determinants of these changes. To do so, we studied five key resurrected ancestral enzymes as well as their extant counterparts. We found that a major shift in function from a bifunctional ancestor that could phosphorylate either glucose or fructose 6-phosphate (fructose-6-P) as a substrate to a fructose 6-P-specific enzyme was started by a single amino acid substitution resulting in negative selection with a ground-state mode against glucose and a subsequent 1,600-fold change in specificity of the ancestral protein. This change rendered the residual phosphorylation of glucose a promiscuous and physiologically irrelevant activity, highlighting how promiscuity may be an evolutionary vestige of ancestral enzyme activities, which have been eliminated over time. We also could reconstruct the evolutionary history of substrate utilization by using an evolutionary model of discrete binary characters, indicating that substrate uses can be discretely lost or acquired during enzyme evolution. These findings exemplify how negative selection and subtle enzyme changes can lead to major evolutionary shifts in function, which can subsequently generate important adaptive advantages, for example, in improving glycolytic efficiency in Thermococcales . © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

[Substrate specifity in Amoeba proteus].

PubMed

Sopina, V A

2006-01-01

Three different phosphatases ("slow", "middle" and "fast") were found in Amoeba proteus (strain B) after PAGE and a subsequent gel staining in 1-naphthyl phosphate containing incubation mixture (pH 9.0). Substrate specificity of these phosphatases was determined in supernatants of homogenates using inhibitors of phosphatase activity. All phosphatases showed a broad substrate specificity. Of 10 tested compounds, p-nitrophenyl phosphate was a preferable substrate for all 3 phosphatases. All phosphatases were able to hydrolyse bis-p-nitrophenyl phosphate and, hence, displayed phosphodiesterase activity. All phosphatases hydrolysed O-phospho-L-tyrosine to a greater or lesser degree. Only little differences in substrate specificity of phosphatases were noticed: 1) "fast" and "middle" phosphatases hydrolysed naphthyl phosphates and O-phospho-L-tyrosine less efficiently than did "slow" phosphatase; 2) "fast" and "middle" phosphatases hydrolysed 2- naphthyl phosphate to a lesser degree than 1-naphthyl phosphate 3) "fast" and "middle" phosphatases hydrolysed O-phospho-L-serine and O-phospho-L-threonine with lower intensity as compared with "slow" phosphatase; 4) as distinct from "middle" and "slow" phosphatases, the "fast" phosphatase hydrolysed glucose-6-phosphate very poorly. The revealed broad substrate specificity of "slow" phosphatase together with data of inhibitory analysis and results of experiments with reactivation of this phosphatase by Zn2+-ions after its inactivation by EDTA strongly suggest that only the "slow" phosphatase is a true alkaline phosphatase (EC 3.1.3.1). The alkaline phosphatase of A. proteus is secreted into culture medium where its activity is low. The enzyme displays both phosphomono- and phosphodiesterase activities, in addition to supposed protein phosphatase activity. It still remains unknown, to which particular phosphatase class the amoeban "middle" and "fast" phosphatases (pH 9.0) may be assigned.

On the levels of enzymatic substrate specificity: Implications for the early evolution of metabolic pathways

NASA Technical Reports Server (NTRS)

Lazcano, A.; Diaz-Villagomez, E.; Mills, T.; Oro, J.

1995-01-01

The most frequently invoked explanation for the origin of metabolic pathways is the retrograde evolution hypothesis. In contrast, according to the so-called 'patchwork' theory, metabolism evolved by the recruitment of relatively inefficient small enzymes of broad specificity that could react with a wide range of chemically related substrates. In this paper it is argued that both sequence comparisons and experimental results on enzyme substrate specificity support the patchwork assembly theory. The available evidence supports previous suggestions that gene duplication events followed by a gradual neoDarwinian accumulation of mutations and other minute genetic changes lead to the narrowing and modification of enzyme function in at least some primordial metabolic pathways.

MnO2-Based Electrochemical Supercapacitors on Flexible Carbon Substrates

NASA Astrophysics Data System (ADS)

Tadjer, Marko J.; Mastro, Michael A.; Rojo, José M.; Mojena, Alberto Boscá; Calle, Fernando; Kub, Francis J.; Eddy, Charles R.

2014-04-01

Manganese dioxide films were grown on large area flexible carbon aerogel substrates. Characterization by x-ray diffraction confirmed α-MnO2 growth. Three types of films were compared as a function of hexamethylenetetramine (HMTA) concentration during growth. The highest concentration of HM TA produced MnO2 flower-like films, as observed by scanning electron microscopy, whose thickness and surface coverage lead to both a higher specific capacitance and higher series resistance. Specific capacitance was measured to be 64 F/g using a galvanostatic setup, compared to the 47 F/g-specific capacitance of the carbon aerogel substrate. Such supercapacitor devices can be fabricated on large area sheets of carbon aerogel to achieve high total capacitance.

Structure and magnetic properties of Fe-Co-B alloy thin films prepared on cubic (001) single-crystal substrates

NASA Astrophysics Data System (ADS)

Ohtake, Mitsuru; Serizawa, Kana; Futamoto, Masaaki; Kirino, Fumiyoshi; Inaba, Nobuyuki

2018-04-01

Fe70Co30 and (Fe70Co30)0.95B5 (at. %) alloy films of 5 nm thickness are prepared by sputtering on cubic (001) oxide substrates at 200 °C. The lattice mismatch between film and substrate is varied from -4.2%, 0%, to +3.5% by employing MgO, MgAl2O4, and SrTiO3 substrates, respectively. Fe70Co30 and (Fe70Co30)0.95B5 single-crystal films with bcc structure grow epitaxially on all the substrates in the orientation relationship of (001)[110]film || (001)[100]substrate. The in-plane and out-of-plane lattice constants, a and c, are in agreement within small differences ranging between +1.1% and -0.9% with the value of bulk bcc-Fe70Co30 crystal, even though there exist the lattice mismatches of -4.2% and +3.5%. The result indicates that misfit dislocations are introduced around the film/substrate interface when films are deposited on MgO and SrTiO3 substrates. The single-crystal films show in-plane magnetic anisotropies with the easy magnetization direction of bcc[100], which are reflecting the magnetocrystalline anisotropy of bulk Fe70Co30 crystal.

Studying physical properties of CuInS2 absorber layers grown by spin coating method on different kinds of substrates

NASA Astrophysics Data System (ADS)

Amerioun, M. H.; Ghazi, M. E.; Izadifard, M.

2018-03-01

In this work, first the CuInS2 (CIS2) layers are deposited on Aluminum and polyethylene terephthalate (PET) as flexible substrates, and on glass and soda lime glass (SLG) as rigid substrates by the sol-gel method. Then the samples are analyzed by x-ray diffractomery (XRD) and atomic force microscope (AFM) to investigate the crystal structures and surface roughness of the samples. The I-V curve measurements and Seebeck effect setup are used to measure the electrical properties of the samples. The XRD data obtained for the CIS2 layers show that all the prepared samples have a single phase with a preferred orientation that is substrate-dependent. The samples grown on the rigid substrates had higher crystallite sizes. The results obtained for the optical measurements indicate the dependence of the band gap energy on the substrate type. The measured Seebeck coefficient showed that the carriers were of p-type in all the samples. According to the AFM images, the surface roughness also varied in the CIS2 layers with different substrates. In this regard, the type of substrate could be an important parameter for the final performance of the fabricated CIS2 cells.

Hydrolytic properties and substrate specificity of the foot-and-mouth disease leader protease.

PubMed

Santos, Jorge A N; Gouvea, Iuri E; Júdice, Wagner A S; Izidoro, Mario A; Alves, Fabiana M; Melo, Robson L; Juliano, Maria A; Skern, Tim; Juliano, Luiz

2009-08-25

Foot-and-mouth disease virus, a global animal pathogen, possesses a single-stranded RNA genome that, on release into the infected cell, is immediately translated into a single polyprotein. This polyprotein product is cleaved during synthesis by proteinases contained within it into the mature viral proteins. The first cleavage is performed by the leader protease (Lb(pro)) between its own C-terminus and the N-terminus of VP4. Lb(pro) also specifically cleaves the two homologues of cellular eukaryotic initiation factor (eIF) 4G, preventing translation of capped mRNA. Viral protein synthesis is initiated internally and is thus unaffected. We used a panel of specifically designed FRET peptides to examine the effects of pH and ionic strength on Lb(pro) activity and investigate the size of the substrate binding site and substrate specificity. Compared to the class prototypes, papain and the cathepsins, Lb(pro) possesses several unusual characteristics, including a high sensitivity to salt and a very specific substrate binding site extending up to P(7). Indeed, almost all substitutions investigated were detrimental to Lb(pro) activity. Analysis of structural data showed that Lb(pro) binds residues P(1)-P(3) in an extended conformation, whereas residues P(4)-P(7) are bound in a short 3(10) helix. The specificity of Lb(pro) as revealed by the substituted peptides could be explained for all positions except P(5). Strikingly, Lb(pro) residues L178 and L143 contribute to the architecture of more than one substrate binding pocket. The diverse functions of these two Lb(pro) residues explain why Lb(pro) is one of the smallest, but simultaneously most specific, papain-like enzymes.

Reactive ion etched substrates and methods of making and using

DOEpatents

Rucker, Victor C [San Francisco, CA; Shediac, Rene [Oakland, CA; Simmons, Blake A [San Francisco, CA; Havenstrite, Karen L [New York, NY

2007-08-07

Disclosed herein are substrates comprising reactive ion etched surfaces and specific binding agents immobilized thereon. The substrates may be used in methods and devices for assaying or isolating analytes in a sample. Also disclosed are methods of making the reactive ion etched surfaces.

Substrate Material for Holographic Emulsions Utilizing Fluorinated Polyimide Film

NASA Technical Reports Server (NTRS)

Gierow, Paul A. (Inventor); Clayton, William R. (Inventor); St.Clair, Anne K. (Inventor)

1999-01-01

A new holographic substrate utilizing flexible. optically transparent fluorinated polyimides. Said substrates have 0 extremely low birefringence which results in a high signal to noise ratio in subsequent holograms. Specific examples of said fluorinated polyimides include 6FDA+APB and 6FDA+4BDAF.

Synthesis and evaluation of fluorogenic triglycerides as lipase assay substrates.

PubMed

Andersen, Rokhsana J; Brask, Jesper

2016-06-01

Three racemic fluorogenic triglycerides are synthesized and evaluated as lipase assay substrates. The presented synthesis route goes through a key triglyceride intermediate which can be chemoselectively functionalized with a wide range of different probes. Hence the substrate can be tailor-made for a specific assay, or focus can be on low cost in larger scale for applications in high-throughput screening (HTS) assays. In the specific examples, TG-ED, TG-FD and TG-F2 are assembled with the Edans-Dabcyl or the fluorescein-Dabcyl FRET pair, or relying on fluorescein self-quenching, respectively. Proof-of-concept assays allowed determination of 1st order kinetic parameters (kcat/KM) of 460s(-1)M(-1), 59s(-1)M(-1) and 346s(-1)M(-1), respectively, for the three substrates. Commercially available EnzChek lipase substrate provided 204s(-1)M(-1). Substrate concentration was identified as a critical parameter, with measured reaction rates decreasing at higher concentrations when intermolecular quenching becomes significant. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Pigment-cellulose nanofibril composite and its application as a separator-substrate in printed supercapacitors

NASA Astrophysics Data System (ADS)

Torvinen, Katariina; Lehtimäki, Suvi; Keränen, Janne T.; Sievänen, Jenni; Vartiainen, Jari; Hellén, Erkki; Lupo, Donald; Tuukkanen, Sampo

2015-11-01

Pigment-cellulose nanofibril (PCN) composites were manufactured in a pilot line and used as a separator-substrate in printed graphene and carbon nanotube supercapacitors. The composites consisted typically of 80% pigment and 20% cellulose nanofibrils (CNF). This composition makes them a cost-effective alternative as a substrate for printed electronics at high temperatures that only very special plastic films can nowadays stand. The properties of these substrates can be varied within a relatively large range by the selection of raw materials and their relative proportions. A semi-industrial scale pilot line was successfully used to produce smooth, flexible, and nanoporous composites, and their performance was tested in a double functional separator-substrate element in supercapacitors. The nanostructural carbon films printed on the composite worked simultaneously as high surface area active electrodes and current collectors. Low-cost supercapacitors made from environmentally friendly materials have significant potential for use in flexible, wearable, and disposable low-end products. [Figure not available: see fulltext.

Ratchet Effects in Active Matter Systems

NASA Astrophysics Data System (ADS)

Reichhardt, C. J. Olson; Reichhardt, C.

2017-03-01

Ratchet effects can arise for single or collectively interacting Brownian particles on an asymmetric substrate when a net dc transport is produced by an externally applied ac driving force or by periodically flashing the substrate. Recently, a new class of active ratchet systems that do not require the application of external driving has been realized through the use of active matter; they are self-propelled units that can be biological or nonbiological in nature. When active materials such as swimming bacteria interact with an asymmetric substrate, a net dc directed motion can arise even without external driving, opening a wealth of possibilities such as sorting, cargo transport, or micromachine construction. We review the current status of active matter ratchets for swimming bacteria, cells, active colloids, and swarming models, focusing on the role of particle-substrate interactions. We describe ratchet reversals produced by collective effects and the use of active ratchets to transport passive particles. We discuss future directions including deformable substrates or particles, the role of different swimming modes, varied particle-particle interactions, and nondissipative effects.

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

Reichhardt, Cynthia Jane; Reichhardt, Charles

Ratchet effects can arise for single or collectively interacting Brownian particles on an asymmetric substrate when a net dc transport is produced by an externally applied ac driving force or by periodically flashing the substrate. Recently, a new class of active ratchet systems that do not require the application of external driving has been realized through the use of active matter; they are self-propelled units that can be biological or nonbiological in nature. When active materials such as swimming bacteria interact with an asymmetric substrate, a net dc directed motion can arise even without external driving, opening a wealth ofmore » possibilities such as sorting, cargo transport, or micromachine construction. We review the current status of active matter ratchets for swimming bacteria, cells, active colloids, and swarming models, focusing on the role of particle-substrate interactions. Here, we describe ratchet reversals produced by collective effects and the use of active ratchets to transport passive particles. We discuss future directions including deformable substrates or particles, the role of different swimming modes, varied particle–particle interactions, and nondissipative effects.« less

Guided growth of horizontal GaN nanowires on quartz and their transfer to other substrates.

PubMed

Goren-Ruck, Lior; Tsivion, David; Schvartzman, Mark; Popovitz-Biro, Ronit; Joselevich, Ernesto

2014-03-25

The guided growth of horizontal nanowires has so far been demonstrated on a limited number of substrates. In most cases, the nanowires are covalently bonded to the substrate where they grow and cannot be transferred to other substrates. Here we demonstrate the guided growth of well-aligned horizontal GaN nanowires on quartz and their subsequent transfer to silicon wafers by selective etching of the quartz while maintaining their alignment. The guided growth was observed on different planes of quartz with varying degrees of alignment. We characterized the crystallographic orientations of the nanowires and proposed a new mechanism of "dynamic graphoepitaxy" for their guided growth on quartz. The transfer of the guided nanowires enabled the fabrication of back-gated field-effect transistors from aligned nanowire arrays on oxidized silicon wafers and the production of crossbar arrays. The guided growth of transferrable nanowires opens up the possibility of massively parallel integration of nanowires into functional systems on virtually any desired substrate.

Metabolomics Reveal Optimal Grain Preprocessing (Milling) toward Rice Koji Fermentation.

PubMed

Lee, Sunmin; Lee, Da Eun; Singh, Digar; Lee, Choong Hwan

2018-03-21

A time-correlated mass spectrometry (MS)-based metabolic profiling was performed for rice koji made using the substrates with varying degrees of milling (DOM). Overall, 67 primary and secondary metabolites were observed as significantly discriminant among different samples. Notably, a higher abundance of carbohydrate (sugars, sugar alcohols, organic acids, and phenolic acids) and lipid (fatty acids and lysophospholipids) derived metabolites with enhanced hydrolytic enzyme activities were observed for koji made with DOM of 5-7 substrates at 36 h. The antioxidant secondary metabolites (flavonoids and phenolic acid) were relatively higher in koji with DOM of 0 substrates, followed by DOM of 5 > DOM of 7 > DOM of 9 and 11 at 96 h. Hence, we conjecture that the rice substrate preprocessing between DOM of 5 and 7 was potentially optimal toward koji fermentation, with the end product being rich in distinctive organoleptic, nutritional, and functional metabolites. The study rationalizes the substrate preprocessing steps vital for commercial koji making.

Hydrogen isotope fractionation during lipid biosynthesis by Haloarcula marismortui

NASA Astrophysics Data System (ADS)

Dirghangi, Sitindra S.; Pagani, Mark

2013-10-01

We studied the controls on the fractionation of hydrogen isotopes during lipid biosynthesis by Haloarcula marismortui, a halophilic archaea, in pure culture experiments by varying organic substrate, the hydrogen isotope composition (D/H) of water, temperature, and salinity. Cultures were grown on three substrates: succinate, pyruvate and glycerol with known hydrogen isotope compositions, and in water with different hydrogen isotopic compositions. All culture series grown on a particular substrate show strong correlations between δDarchaeol and δDwater. However, correlations are distinctly different for cultures grown on different substrates. Our results indicate that the metabolic pathway of substrate exerts a fundamental influence on the δD value of lipids, likely by influencing the D/H composition of NADPH (nicotinamide adenine dinucleotide phosphate), the reducing agent that contributes hydrogen to carbon atoms during lipid biosynthesis. Temperature and salinity have smaller, but similar effects on δDlipid, primarily due to the way temperature and salinity influence growth rate, as well as temperature effects on the activity of enzymes.

Substrate stiffness regulates cadherin-dependent collective migration through myosin-II contractility

PubMed Central

Ng, Mei Rosa; Besser, Achim

2012-01-01

The mechanical microenvironment is known to influence single-cell migration; however, the extent to which mechanical cues affect collective migration of adherent cells is not well understood. We measured the effects of varying substrate compliance on individual cell migratory properties in an epithelial wound-healing assay. Increasing substrate stiffness increased collective cell migration speed, persistence, and directionality as well as the coordination of cell movements. Dynamic analysis revealed that wounding initiated a wave of motion coordination from the wound edge into the sheet. This was accompanied by a front-to-back gradient of myosin-II activation and establishment of cell polarity. The propagation was faster and farther reaching on stiff substrates, indicating that substrate stiffness affects the transmission of directional cues. Manipulation of myosin-II activity and cadherin–catenin complexes revealed that this transmission is mediated by coupling of contractile forces between neighboring cells. Thus, our findings suggest that the mechanical environment integrates in a feedback with cell contractility and cell–cell adhesion to regulate collective migration. PMID:23091067

Investigation of the structural, optical and piezoelectric properties of ALD ZnO films on PEN substrates

NASA Astrophysics Data System (ADS)

Blagoev, B. S.; Aleksandrova, M.; Terziyska, P.; Tzvetkov, P.; Kovacheva, D.; Kolev, G.; Mehandzhiev, V.; Denishev, K.; Dimitrov, D.

2018-03-01

We present the results of studies on the structural, optical and piezoelectric properties of ZnO thin films deposited by ALD on flexible polyethylene naphthalate (PEN) substrates. Changes were observed in the optical transmission and crystal structures as the deposition temperature was varied. The electromechanical behavior, dielectric losses and voltage generated from ZnO flexible devices were investigated and discussed, in order to estimate their suitability for potential application as microgenerators activated by human motion.

Regulation of Proteolysis by Human Deubiquitinating Enzymes

PubMed Central

Eletr, Ziad M.; Wilkinson, Keith D.

2013-01-01

The post-translational attachment of one or several ubiquitin molecules to a protein generates a variety of targeting signals that are used in many different ways in the cell. Ubiquitination can alter the activity, localization, protein-protein interactions or stability of the targeted protein. Further, a very large number of proteins are subject to regulation by ubiquitin-dependent processes, meaning that virtually all cellular functions are impacted by these pathways. Nearly a hundred enzymes from five different gene families (the deubiquitinating enzymes or DUBs), reverse this modification by hydrolyzing the (iso)peptide bond tethering ubiquitin to itself or the target protein. Four of these families are thiol proteases and one is a metalloprotease. DUBs of the Ubiquitin C-terminal Hydrolase (UCH) family act on small molecule adducts of ubiquitin, process the ubiquitin proprotein, and trim ubiquitin from the distal end of a polyubiquitin chain. Ubiquitin Specific Proteases (USP) tend to recognize and encounter their substrates by interaction of the variable regions of their sequence with the substrate protein directly, or with scaffolds or substrate adapters in multiprotein complexes. Ovarian Tumor (OTU) domain DUBs show remarkable specificity for different Ub chain linkages and may have evolved to recognize substrates on the basis of those linkages. The Josephin family of DUBs may specialize in distinguishing between polyubiquitin chains of different lengths. Finally, the JAB1/MPN+/MOV34 (JAMM) domain metalloproteases cleave the isopeptide bond near the attachment point of polyubiquitin and substrate, as well as being highly specific for the K63 poly-Ub linkage. These DUBs regulate proteolysis by: directly interacting with and co-regulating E3 ligases; altering the level of substrate ubiquitination; hydrolyzing or remodeling ubiquitinated and poly-ubiquitinated substrates; acting in specific locations in the cell and altering the localization of the target protein; and acting on proteasome bound substrates to facilitate or inhibit proteolysis. Thus, the scope and regulation of the ubiquitin pathway is very similar to that of phosphorylation, with the DUBs serving the same functions as the phosphatase. PMID:23845989

Classical Swine Fever Virus Glycoprotein Erns Is an Endoribonuclease with an Unusual Base Specificity

PubMed Central

Hausmann, Yvonne; Roman-Sosa, Gleyder; Thiel, Heinz-Jürgen; Rümenapf, Till

2004-01-01

The glycoprotein Erns of pestiviruses is a virion-associated and -secreted RNase that is involved in virulence. The requirements at the cleavage site in heteropolymeric RNA substrates were studied for Erns. Limited digestion of heteropolymeric RNA substrates indicated a cleavage 5′ of uridine residues irrespective of the preceding nucleotide (Np/U). To further study specificity radiolabeled RNA, molecules of 45 to 56 nucleotides in length were synthesized that contained no or a single Np/U cleavage site. Cleavage was only observed in substrates containing an ApU, CpU, GpU, or UpU dinucleotide and occurred in two steps, an initial NpU-specific and a consecutive unspecific degradation. The NpU-specific cleavage was resistant to 7 M urea while the second-order cleavage was sensitive to denaturation. Kinetic analyses revealed that Erns is a highly active endoribonuclease (kcat/Km = 2 × 106 to 10 × 106 M−1 s−1) with a strong affinity to NpU containing single-stranded RNA substrates (Km = 85 to 260 nM). PMID:15113930

CNG site-specific and methyl-sensitive endonuclease WEN1 from wheat seedlings.

PubMed

Fedoreyeva, L I; Vanyushin, B F

2011-06-01

Endonuclease WEN1 with apparent molecular mass about 27 kDa isolated from cytoplasmic vesicular fraction of aging coleoptiles of wheat seedlings has expressed site specificity action. This is a first detection and isolation of a site-specific endonuclease from higher eukaryotes, in general, and higher plants, in particular. The enzyme hydrolyzes deoxyribooligonucleotides of different composition on CNG (N is G, A, C, or T) sites by splitting the phosphodiester bond between C and N nucleotide residues in CNG sequence independent from neighbor nucleotide context except for CCCG. WEN1 prefers to hydrolyze methylated λ phage DNA and double-stranded deoxyribooligonucleotides containing 5-methylcytosine sites (m(5)CAG, m(5)CTG) compared with unmethylated substrates. The enzyme is also able to hydrolyze single-stranded substrates, but in this case it splits unmethylated substrates predominantly. Detection in wheat seedlings of WEN1 endonuclease that is site specific, sensitive to the substrate methylation status, and modulated with S-adenosyl-L-methionine indicates that in higher plants restriction--modification systems or some of their elements, at least, may exist.

Mutant fatty acid desaturase

DOEpatents

Shanklin, John; Cahoon, Edgar B.

2004-02-03

The present invention relates to a method for producing mutants of a fatty acid desaturase having a substantially increased activity towards fatty acid substrates with chains containing fewer than 18 carbons relative to an unmutagenized precursor desaturase having an 18 carbon atom chain length substrate specificity. The method involves inducing one or more mutations in the nucleic acid sequence encoding the precursor desaturase, transforming the mutated sequence into an unsaturated fatty acid auxotroph cell such as MH13 E. coli, culturing the cells in the absence of supplemental unsaturated fatty acids, thereby selecting for recipient cells which have received and which express a mutant fatty acid desaturase with an elevated specificity for fatty acid substrates having chain lengths of less than 18 carbon atoms. A variety of mutants having 16 or fewer carbon atom chain length substrate specificities are produced by this method. Mutant desaturases produced by this method can be introduced via expression vectors into prokaryotic and eukaryotic cells and can also be used in the production of transgenic plants which may be used to produce specific fatty acid products.

Fertilizer effects on attaining vegetation requirements.

DOT National Transportation Integrated Search

2014-02-01

This project was developed to evaluate the effects of varying the substrate and fertilization regimes on the success of complex warm-season grass and forb seedings on recent roadside construction sites. Re-vegetating construction projects is required...

Arginine Kinase. Joint Crystallographic & NMR RDC Analyses link Substrate-Associated Motions to Intrinsic Flexibility

PubMed Central

Niu, Xiaogang; Brüschweiler-Li, Lei; Davulcu, Omar; Skalicky, Jack J.; Brüschweiler, Rafael; Chapman, Michael S.

2010-01-01

The phosphagen kinase family, including creatine and arginine kinases, catalyze the reversible transfer of a “high energy” phosphate between ATP and a phospho-guanidino substrate. They have become a model for the study of both substrate-induced conformational change and intrinsic protein dynamics. Prior crystallographic studies indicated large substrate-induced domain rotations, but differences among a recent set of arginine kinase structures was interpreted as a plastic deformation. Here, the structure of Limulus substrate-free arginine kinase is refined against high resolution crystallographic data and compared quantitatively with NMR chemical shifts and residual dipolar couplings (RDCs). This demonstrates the feasibility of this type of RDC analysis of proteins that are large by NMR standards (42 kDa), and illuminates the solution structure, free from crystal-packing constraints. Detailed comparison of the 1.7 Å resolution substrate-free crystal structure against the 1.2 Å transition state analog complex shows large substrate-induced domain motions which can be broken down into movements of smaller quasi-rigid bodies. The solution state structure of substrate-free arginine kinase is most consistent with an equilibrium of substrate-free and –bound structures, with the substrate-free form dominating, but with varying displacements of the quasi-rigid groups. Rigid-group rotations evident from the crystal structures are about axes previously associated with intrinsic millisecond dynamics using NMR relaxation dispersion. Thus, “substrate-induced” motions are along modes that are intrinsically flexible in the substrate-free enzyme, and likely involve some degree of conformational selection. PMID:21075117

Single molecule atomic force microscopy and force spectroscopy of chitosan.

PubMed

Kocun, Marta; Grandbois, Michel; Cuccia, Louis A

2011-02-01

Atomic force microscopy (AFM) and AFM-based force spectroscopy was used to study the desorption of individual chitosan polymer chains from substrates with varying chemical composition. AFM images of chitosan adsorbed onto a flat mica substrate show elongated single strands or aggregated bundles. The aggregated state of the polymer is consistent with the high level of flexibility and mobility expected for a highly positively charged polymer strand. Conversely, the visualization of elongated strands indicated the presence of stabilizing interactions with the substrate. Surfaces with varying chemical composition (glass, self-assembled monolayer of mercaptoundecanoic acid/decanethiol and polytetrafluoroethylene (PTFE)) were probed with chitosan modified AFM tips and the corresponding desorption energies, calculated from plateau-like features, were attributed to the desorption of individual polymer strands. Desorption energies of 2.0±0.3×10(-20)J, 1.8±0.3×10(-20)J and 3.5±0.3×10(-20)J were obtained for glass, SAM of mercaptoundecanoic/dodecanethiol and PTFE, respectively. These single molecule level results can be used as a basis for investigating chitosan and chitosan-based materials for biomaterial applications. Copyright © 2010 Elsevier B.V. All rights reserved.

Substrate Integrated Waveguide (SIW)-Based Wireless Temperature Sensor for Harsh Environments.

PubMed

Tan, Qiulin; Guo, Yanjie; Zhang, Lei; Lu, Fei; Dong, Helei; Xiong, Jijun

2018-05-03

This paper presents a new wireless sensor structure based on a substrate integrated circular waveguide (SICW) for the temperature test in harsh environments. The sensor substrate material is 99% alumina ceramic, and the SICW structure is composed of upper and lower metal plates and a series of metal cylindrical sidewall vias. A rectangular aperture antenna integrated on the surface of the SICW resonator is used for electromagnetic wave transmission between the sensor and the external antenna. The resonant frequency of the temperature sensor decreases when the temperature increases, because the relative permittivity of the alumina ceramic increases with temperature. The temperature sensor presented in this paper was tested four times at a range of 30⁻1200 °C, and a broad band coplanar waveguide (CPW)-fed antenna was used as an interrogation antenna during the test process. The resonant frequency changed from 2.371 to 2.141 GHz as the temperature varied from 30 to 1200 °C, leading to a sensitivity of 0.197 MHz/°C. The quality factor of the sensor changed from 3444.6 to 35.028 when the temperature varied from 30 to 1000 °C.

Growth and Characterization of the Evaporated Quaternary Absorber Cu2FeSnS4 for Solar Cell Applications

NASA Astrophysics Data System (ADS)

Oueslati, Hiba; Ben Rabeh, Mohamed; Kanzari, Mounir

2018-03-01

Cu2FeSnS4 (CFTS) was synthesized by direct fusion of high-purity elemental copper, iron, tin and sulfur. CFTS thin films were deposited on glass substrates heated by single source vacuum thermal evaporation, after which the obtained samples were annealed under a sulfur atmosphere in a sealed quartz tube at 400°C for 1 h in order to optimize the CFTS stannite phase. The substrate temperature was varied from room temperature to 200°C. The formation of a stannite structure with (112), (200) and (004) planes in the powder and thin films was confirmed using x-ray diffraction measurements and the crystallites were found to have a preferred orientation along the (112) direction. Optical measurements analysis showed that after the sulfurization process the layers have a relatively high absorption coefficient close to 105 cm-1 in the visible spectrum. The films show a direct optical band gap in the range 1.30-1.63 eV for substrate temperature varied from room temperature to 200°C. All samples revealed p-type conductivity as determined by the hot probe method.

Substrate Topography Induces a Crossover from 2D to 3D Behavior in Fibroblast Migration

PubMed Central

Ghibaudo, Marion; Trichet, Léa; Le Digabel, Jimmy; Richert, Alain; Hersen, Pascal; Ladoux, Benoît

2009-01-01

Abstract In a three-dimensional environment, cells migrate through complex topographical features. Using microstructured substrates, we investigate the role of substrate topography in cell adhesion and migration. To do so, fibroblasts are plated on chemically identical substrates composed of microfabricated pillars. When the dimensions of the pillars (i.e., the diameter, length, and spacing) are varied, migrating cells encounter alternating flat and rough surfaces that depend on the spacing between the pillars. Consequently, we show that substrate topography affects cell shape and migration by modifying cell-to-substrate interactions. Cells on micropillar substrates exhibit more elongated and branched shapes with fewer actin stress fibers compared with cells on flat surfaces. By analyzing the migration paths in various environments, we observe different mechanisms of cell migration, including a persistent type of migration, that depend on the organization of the topographical features. These responses can be attributed to a spatial reorganization of the actin cytoskeleton due to physical constraints and a preferential formation of focal adhesions on the micropillars, with an increased lifetime compared to that observed on flat surfaces. By changing myosin II activity, we show that actomyosin contractility is essential in the cellular response to micron-scale topographic signals. Finally, the analysis of cell movements at the frontier between flat and micropillar substrates shows that cell transmigration through the micropillar substrates depends on the spacing between the pillars. PMID:19580774

Stereoselectivity of Mucorales lipases toward triradylglycerols--a simple solution to a complex problem.

PubMed Central

Scheib, H.; Pleiss, J.; Kovac, A.; Paltauf, F.; Schmid, R. D.

1999-01-01

The lipases from Rhizopus and Rhizomucor are members of the family of Mucorales lipases. Although they display high sequence homology, their stereoselectivity toward triradylglycerols (sn-2 substituted triacylglycerols) varies. Four different triradylglycerols were investigated, which were classified into two groups: flexible substrates with rotatable O'-C1' ether or ester bonds adjacent to C2 of glycerol and rigid substrates with a rigid N'-C1' amide bond or a phenyl ring in sn-2. Although Rhizopus lipase shows opposite stereopreference for flexible and rigid substrates (hydrolysis in sn-1 and sn-3, respectively), Rhizomucor lipase hydrolyzes both groups of triradylglycerols preferably in sn-1. To explain these experimental observations, computer-aided molecular modeling was applied to study the molecular basis of stereoselectivity. A generalized model for both lipases of the Mucorales family highlights the residues mediating stereoselectivity: (1) L258, the C-terminal neighbor of the catalytic histidine, and (2) G266, which is located in a loop contacting the glycerol backbone of a bound substrate. Interactions with triradylglycerol substrates are dominated by van der Waals contacts. Stereoselectivity can be predicted by analyzing the value of a single substrate torsion angle that discriminates between sn-1 and sn-3 stereopreference for all substrates and lipases investigated here. This simple model can be easily applied in enzyme and substrate engineering to predict Mucorales lipase variants and synthetic substrates with desired stereoselectivity. PMID:10210199

The effect of substrate composition and storage time on urine specific gravity in dogs.

PubMed

Steinberg, E; Drobatz, K; Aronson, L

2009-10-01

The purpose of this study is to evaluate the effects of substrate composition and storage time on urine specific gravity in dogs. A descriptive cohort study of 15 dogs. The urine specific gravity of free catch urine samples was analysed during a 5-hour time period using three separate storage methods; a closed syringe, a diaper pad and non-absorbable cat litter. The urine specific gravity increased over time in all three substrates. The syringe sample had the least change from baseline and the diaper sample had the greatest change from baseline. The urine specific gravity for the litter and diaper samples had a statistically significant increase from the 1-hour to the 5-hour time point. The urine specific gravity from canine urine stored either on a diaper or in a non-absorbable litter increased over time. Although the change was found to be statistically significant over the 5-hour study period it is unlikely to be clinically significant.

Insight into the role of substrate-binding residues in conferring substrate specificity for the multifunctional polysaccharide lyase Smlt1473.

PubMed

MacDonald, Logan C; Berger, Bryan W

2014-06-27

Anionic polysaccharides are of growing interest in the biotechnology industry due to their potential pharmaceutical applications in drug delivery and wound treatment. Chemical composition and polymer length strongly influence the physical and biological properties of the polysaccharide and thus its potential industrial and medical applications. One promising approach to determining monomer composition and controlling the degree of polymerization involves the use of polysaccharide lyases, which catalyze the depolymerization of anionic polysaccharides via a β-elimination mechanism. Utilization of these enzymes for the production of custom-made oligosaccharides requires a high degree of control over substrate specificity. Previously, we characterized a polysaccharide lyase (Smlt1473) from Stenotrophomonas maltophilia k279a, which exhibited significant activity against hyaluronan (HA), poly-β-d-glucuronic acid (poly-GlcUA), and poly-β-d-mannuronic acid (poly-ManA) in a pH-regulated manner. Here, we utilize a sequence structure guided approach based on a homology model of Smlt1473 to identify nine putative substrate-binding residues and examine their effect on substrate specificity via site-directed mutagenesis. Interestingly, single point mutations H221F and R312L resulted in increased activity and specificity toward poly-ManA and poly-GlcUA, respectively. Furthermore, a W171A mutant nearly eliminated HA activity, while increasing poly-ManA and poly-GlcUA activity by at least 35%. The effect of these mutations was analyzed by comparison with the high resolution structure of Sphingomonas sp. A1-III alginate lyase in complex with poly-ManA tetrasaccharide and by taking into account the structural differences between HA, poly-GlcUA, and poly-ManA. Overall, our results demonstrate that even minor changes in active site architecture have a significant effect on the substrate specificity of Smlt1473, whose structural plasticity could be applied to the design of highly active and specific polysaccharide lyases. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Characterization and identification of ubiquitin conjugation sites with E3 ligase recognition specificities.

PubMed

Nguyen, Van-Nui; Huang, Kai-Yao; Huang, Chien-Hsun; Chang, Tzu-Hao; Bretaña, Neil; Lai, K; Weng, Julia; Lee, Tzong-Yi

2015-01-01

In eukaryotes, ubiquitin-conjugation is an important mechanism underlying proteasome-mediated degradation of proteins, and as such, plays an essential role in the regulation of many cellular processes. In the ubiquitin-proteasome pathway, E3 ligases play important roles by recognizing a specific protein substrate and catalyzing the attachment of ubiquitin to a lysine (K) residue. As more and more experimental data on ubiquitin conjugation sites become available, it becomes possible to develop prediction models that can be scaled to big data. However, no development that focuses on the investigation of ubiquitinated substrate specificities has existed. Herein, we present an approach that exploits an iteratively statistical method to identify ubiquitin conjugation sites with substrate site specificities. In this investigation, totally 6259 experimentally validated ubiquitinated proteins were obtained from dbPTM. After having filtered out homologous fragments with 40% sequence identity, the training data set contained 2658 ubiquitination sites (positive data) and 5532 non-ubiquitinated sites (negative data). Due to the difficulty in characterizing the substrate site specificities of E3 ligases by conventional sequence logo analysis, a recursively statistical method has been applied to obtain significant conserved motifs. The profile hidden Markov model (profile HMM) was adopted to construct the predictive models learned from the identified substrate motifs. A five-fold cross validation was then used to evaluate the predictive model, achieving sensitivity, specificity, and accuracy of 73.07%, 65.46%, and 67.93%, respectively. Additionally, an independent testing set, completely blind to the training data of the predictive model, was used to demonstrate that the proposed method could provide a promising accuracy (76.13%) and outperform other ubiquitination site prediction tool. A case study demonstrated the effectiveness of the characterized substrate motifs for identifying ubiquitination sites. The proposed method presents a practical means of preliminary analysis and greatly diminishes the total number of potential targets required for further experimental confirmation. This method may help unravel their mechanisms and roles in E3 recognition and ubiquitin-mediated protein degradation.

Arabidopsis ATG4 cysteine proteases specificity toward ATG8 substrates

PubMed Central

Park, Eunsook; Woo, Jongchan; Dinesh-Kumar, SP

2014-01-01

Macroautophagy (hereafter autophagy) is a regulated intracellular process during which cytoplasmic cargo engulfed by double-membrane autophagosomes is delivered to the vacuole or lysosome for degradation and recycling. Atg8 that is conjugated to phosphatidylethanolamine (PE) during autophagy plays an important role not only in autophagosome biogenesis but also in cargo recruitment. Conjugation of PE to Atg8 requires processing of the C-terminal conserved glycine residue in Atg8 by the Atg4 cysteine protease. The Arabidopsis plant genome contains 9 Atg8 (AtATG8a to AtATG8i) and 2 Atg4 (AtATG4a and AtATG4b) family members. To understand AtATG4’s specificity toward different AtATG8 substrates, we generated a unique synthetic substrate C-AtATG8-ShR (citrine-AtATG8-Renilla luciferase SuperhRLUC). In vitro analyses indicated that AtATG4a is catalytically more active and has broad AtATG8 substrate specificity compared with AtATG4b. Arabidopsis transgenic plants expressing the synthetic substrate C-AtAtg8a-ShR is efficiently processed by endogenous AtATG4s and targeted to the vacuole during nitrogen starvation. These results indicate that the synthetic substrate mimics endogenous AtATG8, and its processing can be monitored in vivo by a bioluminescence resonance energy transfer (BRET) assay. The synthetic Atg8 substrates provide an easy and versatile method to study plant autophagy during different biological processes. PMID:24658121

Cloning and characterization of a new broadspecific β-glucosidase from Lactococcus sp. FSJ4.

PubMed

Fang, Shujun; Chang, Jie; Lee, Yong Seok; Guo, Weiliang; Choi, Yong Lark; Zhou, Yongcan

2014-01-01

A β-glucosidase gene bglX was cloned from Lactococcus sp. FSJ4 by the method of shotgun. The bglX open reading frame consisted of 1,437 bp, encoding 478 amino acids. SDS-PAGE showed a recombinant bglX monomer of 54 kDa. Substrate specificity study revealed that the enzyme exhibited multifunctional catalysis activity against pNPG, pNPX and pNPGal. This enzyme shows higher activity against aryl glycosides of xylose than those of glucose or galactose. The enzyme exhibited the maximal activity at 40 °C, and the optimal pH was 6.0 with pNPG and 6.5 with pNPX as the substrates. Molecular modeling and substrate docking showed that there should be one active center responsible for the mutifuntional activity in this enzyme, since the active site pocket was substantially wide to allow the entry of pNPG, pNPX and pNPGal, which elucidated the structure-function relationship in substrate specificities. Substrate docking results indicated that Glu180 and Glu377 were the essential catalytic residues of the enzyme. The CDOCKER_ENERGY values obtained by substrate docking indicated that the enzyme has higher activity against pNPX than those of pNPG and pNPGal. These observations are in conformity with the results obtained from experimental investigation. Therefore, such substrate specificity makes this β-glucosidase of great interest for further study on physiological and catalytic reaction processes.

Use of Activity-Based Probes to Develop High Throughput Screening Assays That Can Be Performed in Complex Cell Extracts

PubMed Central

Deu, Edgar; Yang, Zhimou; Wang, Flora; Klemba, Michael; Bogyo, Matthew

2010-01-01

Background High throughput screening (HTS) is one of the primary tools used to identify novel enzyme inhibitors. However, its applicability is generally restricted to targets that can either be expressed recombinantly or purified in large quantities. Methodology and Principal Findings Here, we described a method to use activity-based probes (ABPs) to identify substrates that are sufficiently selective to allow HTS in complex biological samples. Because ABPs label their target enzymes through the formation of a permanent covalent bond, we can correlate labeling of target enzymes in a complex mixture with inhibition of turnover of a substrate in that same mixture. Thus, substrate specificity can be determined and substrates with sufficiently high selectivity for HTS can be identified. In this study, we demonstrate this method by using an ABP for dipeptidyl aminopeptidases to identify (Pro-Arg)2-Rhodamine as a specific substrate for DPAP1 in Plasmodium falciparum lysates and Cathepsin C in rat liver extracts. We then used this substrate to develop highly sensitive HTS assays (Z’>0.8) that are suitable for use in screening large collections of small molecules (i.e >300,000) for inhibitors of these proteases. Finally, we demonstrate that it is possible to use broad-spectrum ABPs to identify target-specific substrates. Conclusions We believe that this approach will have value for many enzymatic systems where access to large amounts of active enzyme is problematic. PMID:20700487

Molecular mechanisms of substrate recognition and specificity of botulinum neurotoxin serotype F.

PubMed

Chen, Sheng; Wan, Hoi Ying

2011-01-15

BoNTs (botulinum neurotoxins) are both deadly neurotoxins and natural toxins that are widely used in protein therapies to treat numerous neurological disorders of dystonia and spinal spasticity. Understanding the mechanism of action and substrate specificity of BoNTs is a prerequisite to develop antitoxin and novel BoNT-derived protein therapy. To date, there is a lack of detailed information with regard to how BoNTs recognize and hydrolyse the substrate VAMP-2 (vesicle-associated membrane protein 2), even though it is known to be cleaved by four of the seven BoNT serotypes, B, D, F, G and TeNT (tetanus neurotoxin). In the present study we dissected the molecular mechanisms of VAMP-2 recognition by BoNT serotype F for the first time. The initial substrate recognition was mediated through sequential binding of VAMP-2 to the B1, B2 and B3 pockets in LC/F (light chain of BoNT serotype F), which directed VAMP-2 to the active site of LC/F and stabilized the active site substrate recognition, where the P2, P1' and P2' sites of VAMP-2 were specifically recognized by the S2, S1' and S2' pockets of LC/F to promote substrate hydrolysis. The understanding of the molecular mechanisms of LC/F substrate recognition provides insights into the development of antitoxins and engineering novel BoNTs to optimize current therapy and extend therapeutic interventions.

Mismatch repair factor MSH2-MSH3 binds and alters the conformation of branched DNA structures predicted to form during genetic recombination.

PubMed

Surtees, Jennifer A; Alani, Eric

2006-07-14

Genetic studies in Saccharomyces cerevisiae predict that the mismatch repair (MMR) factor MSH2-MSH3 binds and stabilizes branched recombination intermediates that form during single strand annealing and gene conversion. To test this model, we constructed a series of DNA substrates that are predicted to form during these recombination events. We show in an electrophoretic mobility shift assay that S. cerevisiae MSH2-MSH3 specifically binds branched DNA substrates containing 3' single-stranded DNA and that ATP stimulates its release from these substrates. Chemical footprinting analyses indicate that MSH2-MSH3 specifically binds at the double-strand/single-strand junction of branched substrates, alters its conformation and opens up the junction. Therefore, MSH2-MSH3 binding to its substrates creates a unique nucleoprotein structure that may signal downstream steps in repair that include interactions with MMR and nucleotide excision repair factors.

Aerobic biological treatment of leachates from municipal solid waste landfill.

PubMed

Andrés, P; Gutierrez, F; Arrabal, C; Cortijo, M

2004-01-01

The main objective of the study was to improve chemical oxygen demand (COD) elimination by secondary biological treatment from leachate of municipal solid waste landfill. This effluent was a supernatant liquid obtained after physicochemical processes and coagulating with Al3+ followed by ammoniacal stripping. First, respirometric assays were carried out to determine the substrate biodegradability. Specific sludge respiration rate (R(s)) vs. concentration of substrate (S), showed an increasing specific rate of assimilation of substrate (Rs), which reached the highest value, when the substrate concentration (COD) was between 75 and 200 mg O2 L(-1). Second, continuous experiments were made in an aerobic digester to test the previous respirometric data and the results showed removal efficiency of COD between 83 and 69%, and a substrate assimilation rate between 1.3 and 3.1 g COD g(-1) volatile suspended solids d(-1).

"Choice" and destiny: the substrate composition and mechanical stability of settlement structures can mediate coral recruit fate in post-bleached reefs

NASA Astrophysics Data System (ADS)

Yadav, Shreya; Rathod, Pooja; Alcoverro, Teresa; Arthur, Rohan

2016-03-01

Increasingly frequent and intense ocean warming events seriously test the buffer and recovery capacities of tropical coral reefs. Post-disturbance, available settlement structures on a reef (often dead coral skeletons) vary considerably in their mechanical stability and substrate composition, critically influencing coral recruit settlement choice and fate. In the wake of a coral mass mortality in the Lakshadweep archipelago, we examine (1) the relative availability of recruit settlement structures (from stable to unstable: reef platform, dead massive coral, consolidated rubble, dead corymbose coral, dead tabular coral, and unconsolidated rubble) in 12 recovering reefs across three atolls in the archipelago, (2) the substrate composition [crustose coralline algae (CCA), mixed turf, macroalgae] of these structural forms, and (3) whether the choice and fate of young coral are mediated by the substrate and stability of different structural forms. For this, we measured the abundance and distribution of recruit (<1 cm), juvenile (1-5 cm), and young adult (5-10) corals of 24 common coral genera. Four years after the mass mortality, reefs differed considerably in composition of settlement structures. The structures themselves varied significantly in substrate cover with dead tables largely covered in CCA [60 ± 6.05 % (SE)] and dead corymbose coral dominated by mixed turf (61.83 ± 3.8 %). The youngest visible recruits (<1 cm) clearly preferred CCA-rich structures such as dead massives and tables. However, older size classes were rarely found on unstable structures (strongly "avoiding" tables, Ivlev's electivity index, E = -0.5). Our results indicate that while substrate cover might mediate coral choice, the mechanical stability of settlement structures is critical in determining post-settlement coral survival. The composition and availability of settlement structures on a reef may serve as a characteristic signature of its recovery potential, aiding in assessments of reef resilience.

Sputtering characteristics, crystal structures, and transparent conductive properties of TiOxNy films deposited on α-Al2O3(0 0 0 1) and glass substrates

NASA Astrophysics Data System (ADS)

Akazawa, Housei

2012-12-01

Adding N2 gas during reactive sputtering of a Ti target prevented the target surface from being severely poisoned by oxygen atoms and sustained a high deposition rate for titanium oxynitride films under metal-mode-like sputtering conditions. With progress in the degree of oxidization, films deposited onto a glass substrate varied from TiO1-xNx having a face-centered cubic (fcc) structure to TiO2-xNx having an anatase structure. Titanium oxynitride films deposited on an Al2O3(0 0 0 1) substrate were epitaxial with major orientations toward the (1 1 1) and (2 0 0) directions for fcc-TiO1-xNx and (1 1 2) for anatase-TiO2-xNx. Intermediately oxidized films between TiO1-xNx and TiO2-xNx were amorphous on the glass substrate but crystallized into a Magneli phase, TinO(N)2n-1, on the Al2O3(0 0 0 1) substrate. Partially substituting oxygen in TiO2 with nitrogen as well as continuously irradiating the growing film surface with a Xe plasma stream preferentially formed anatase rather than rutile. However, the occupation of anion sites with enough oxygen rather than nitrogen was the required condition for anatase crystals to form. The transparent conductive properties of epitaxial TiO2-xNx films on Al2O3(0 0 0 1) were superior to those of microcrystalline films on the glass substrate. Since resistivity and optical transmittance of TiOxNy films vary continuously with changing N2 flow rate, their transparent conductive properties can be controlled more easily than TiOx. Nb5+ ions could be doped as donors in TiO2-xNx anatase crystals.

Effect of tacticity on the structure and glass transition temperature of polystyrene adsorbed onto solid surfaces

NASA Astrophysics Data System (ADS)

Negash, Solomon; Tatek, Yergou B.; Tsige, Mesfin

2018-04-01

We have carried out atomistic (all-atom) molecular dynamics simulations to investigate the effect of tacticity on the structure and glass transition temperature (Tg) of polystyrene (PS) thin films adsorbed on two distinct types of solid substrates. The systems consist of thin films made of atactic, isotactic, and syndiotactic PS chains supported by graphite or hydroxylated α-quartz substrates, which are known to be atomically flat but chemically and structurally different. We have observed a marked dependence of the film structure on substrate type as well as on tacticity. For instance, rings' orientation near substrate surfaces depends on substrate type for atactic PS and isotactic PS films, while no such dependence is observed for syndiotactic PS films whose interfacial structure seems to result from their propensity to adopt the trans conformation rather than their specific interaction with the substrates. Moreover, our results indicate that glass transition temperatures of substrate supported polystyrene films are higher compared to those of the corresponding free-standing films. More specifically, PS films on graphite exhibit larger Tg values than those on α-quartz, and we have noticed that syndiotactic PS has the largest Tg irrespective of the substrate type. Furthermore, the local Tg in the region of the film in contact with the substrates shows a strong tacticity and substrate dependence, whereas no dependencies were found for the local Tg in the middle of the film. Substrate-film interaction energy and chains' dynamics near substrate-film interfaces were subsequently investigated in order to substantiate the obtained Tgs, and it was found that films with higher Tgs are strongly adsorbed on the substrates and/or exhibit smaller interfacial chains' dynamics essentially due to steric hindrance.

Comparison of different substrates for laser-induced electron transfer desorption/ionization of metal complexes

NASA Astrophysics Data System (ADS)

Grechnikov, A. A.; Georgieva, V. B.; Donkov, N.; Borodkov, A. S.; Pento, A. V.; Raicheva, Z. G.; Yordanov, Tc A.

2016-03-01

Four different substrates, namely, graphite, tungsten, amorphous silicon (α-Si) and titanium dioxide (TiO2) films, were compared in view of the laser-induced electron transfer desorption/ionization (LETDI) of metal coordination complexes. A rhenium complex with 8-mercaptoquinoline, a copper complex with diphenylthiocarbazone and chlorophyll A were studied as the test analytes. The dependencies of the ion yield and the surface temperature on the incident radiation fluence were investigated experimentally and theoretically. The temperature was estimated using the numerical solution of a one-dimensional heat conduction problem with a heat source distributed in time and space. It was found that at the same temperature, the ion yield from the different substrates varies in the range of three orders of magnitude. The direct comparison of all studied substrates revealed that LETDI from the TiO2 and α-Si films offer a better choice for producing molecular ions of metal coordination complexes.

Morphology Controlled Fabrication of InN Nanowires on Brass Substrates

PubMed Central

Li, Huijie; Zhao, Guijuan; Wang, Lianshan; Chen, Zhen; Yang, Shaoyan

2016-01-01

Growth of semiconductor nanowires on cheap metal substrates could pave the way to the large-scale manufacture of low-cost nanowire-based devices. In this work, we demonstrated that high density InN nanowires can be directly grown on brass substrates by metal-organic chemical vapor deposition. It was found that Zn from the brass substrates is the key factor in the formation of nanowires by restricting the lateral growth of InN. The nanowire morphology is highly dependent on the growth temperature. While at a lower growth temperature, the nanowires and the In droplets have large diameters. At the elevated growth temperature, the lateral sizes of the nanowires and the In droplets are much smaller. Moreover, the nanowire diameter can be controlled in situ by varying the temperature in the growth process. This method is very instructive to the diameter-controlled growth of nanowires of other materials. PMID:28335323

Detection of anions by normal Raman spectroscopy and surface-enhanced Raman spectroscopy of cationic-coated substrates.

PubMed

Mosier-Boss, P A; Lieberman, S H

2003-09-01

The use of normal Raman spectroscopy and surface-enhanced Raman spectroscopy (SERS) of cationic-coated silver and gold substrates to detect polyatomic anions in aqueous environments is examined. For normal Raman spectroscopy, using near-infrared excitation, linear concentration responses were observed. Detection limits varied from 84 ppm for perchlorate to 2600 ppm for phosphate. In general, detection limits in the ppb to ppm concentration range for the polyatomic anions were achieved using cationic-coated SERS substrates. Adsorption of the polyatomic anions on the cationic-coated SERS substrates was described by a Frumkin isotherm. The SERS technique could not be used to detect dichromate, as this anion reacted with the coatings to form thiol esters. A competitive complexation method was used to evaluate the interaction of chloride ion with the cationic coatings. Hydrogen bonding and pi-pi interactions play significant roles in the selectivity of the cationic coatings.

Defect-induced magnetic order in pure ZnO films

NASA Astrophysics Data System (ADS)

Khalid, M.; Ziese, M.; Setzer, A.; Esquinazi, P.; Lorenz, M.; Hochmuth, H.; Grundmann, M.; Spemann, D.; Butz, T.; Brauer, G.; Anwand, W.; Fischer, G.; Adeagbo, W. A.; Hergert, W.; Ernst, A.

2009-07-01

We have investigated the magnetic properties of pure ZnO thin films grown under N2 pressure on a -, c -, and r -plane Al2O3 substrates by pulsed-laser deposition. The substrate temperature and the N2 pressure were varied from room temperature to 570°C and from 0.007 to 1.0 mbar, respectively. The magnetic properties of bare substrates and ZnO films were investigated by SQUID magnetometry. ZnO films grown on c - and a -plane Al2O3 substrates did not show significant ferromagnetism. However, ZnO films grown on r -plane Al2O3 showed reproducible ferromagnetism at 300 K when grown at 300-400°C and 0.1-1.0 mbar N2 pressure. Positron annihilation spectroscopy measurements as well as density-functional theory calculations suggest that the ferromagnetism in ZnO films is related to Zn vacancies.

Monte Carlo study of the hetero-polytypical growth of cubic on hexagonal silicon carbide polytypes

NASA Astrophysics Data System (ADS)

Camarda, Massimo

2012-08-01

In this article we use three dimensional kinetic Monte Carlo simulations on super-lattices to study the hetero-polytypical growth of cubic silicon carbide polytype (3C-SiC) on misoriented hexagonal (4H and 6H) substrates. We analyze the quality of the 3C-SiC film varying the polytype, the miscut angle and the initial surface morphology of the substrate. We find that the use of 6H misoriented (4°-10° off) substrates, with step bunched surfaces, can strongly improve the quality of the cubic epitaxial film whereas the 3C/4H growth is affected by the generation of dislocations, due to the incommensurable periodicity of the 3C (3) and the 4H (4) polytypes. For these reasons, a proper pre-growth treatment of 6H misoriented substrates can be the key for the growth of high quality, twin free, 3C-SiC films.

Growth of epitaxial Pb(Zr,Ti)O3 films by pulsed laser deposition

NASA Astrophysics Data System (ADS)

Lee, J.; Safari, A.; Pfeffer, R. L.

1992-10-01

Lead zirconate titanate (PZT) thin films with a composition near the morphotropic phase boundary have been grown on MgO (100) and Y1Ba2Cu3Ox (YBCO) coated MgO substrates. Substrate temperature and oxygen pressure were varied to achieve ferroelectric films with a perovskite structure. Films grown on MgO had the perovskite structure with an epitaxial relationship with the MgO substrate. On the other hand, films grown on the YBCO/MgO substrate had an oriented structure to the surface normal with a misorientation in the plane parallel to the surface. The measured dielectric constant and loss tangent at 1 kHz were 670 and 0.05, respectively. The remnant polarization and coercive field were 42 μC/cm2 and 53 kV/cm. A large internal bias field (12 kV/cm) was observed in the as-deposited state of the undoped PZT films.

Composite Pillars with a Tunable Interface for Adhesion to Rough Substrates

PubMed Central

2016-01-01

The benefits of synthetic fibrillar dry adhesives for temporary and reversible attachment to hard objects with smooth surfaces have been successfully demonstrated in previous studies. However, surface roughness induces a dramatic reduction in pull-off stresses and necessarily requires revised design concepts. Toward this aim, we introduce cylindrical two-phase single pillars, which are composed of a mechanically stiff stalk and a soft tip layer. Adhesion to smooth and rough substrates is shown to exceed that of conventional pillar structures. The adhesion characteristics can be tuned by varying the thickness of the soft tip layer, the ratio of the Young’s moduli and the curvature of the interface between the two phases. For rough substrates, adhesion values similar to those obtained on smooth substrates were achieved. Our concept of composite pillars overcomes current practical limitations caused by surface roughness and opens up fields of application where roughness is omnipresent. PMID:27997118

Effect of film thickness on localized surface plasmon enhanced chemical sensor

NASA Astrophysics Data System (ADS)

Kassu, Aschalew; Farley, Carlton; Sharma, Anup; Kim, Wonkyu; Guo, Junpeng

2014-05-01

A highly-sensitive, reliable, simple and inexpensive chemical detection and identification platform is demonstrated. The sensing technique is based on localized surface plasmon enhanced Raman scattering measurements from gold-coated highly-ordered symmetric nanoporous ceramic membranes fabricated from anodic aluminum oxide. To investigate the effects of the thickness of the sputter-coated gold films on the sensitivity of sensor, and optimize the performance of the substrates, the geometry of the nanopores and the film thicknesses are varied in the range of 30 nm to 120 nm. To characterize the sensing technique and the detection limits, surface enhanced Raman scatterings of low concentrations of a standard chemical adsorbed on the gold coated substrates are collected and analyzed. The morphology of the proposed substrates is characterized by atomic force microscopy and the optical properties including transmittance, reflectance and absorbance of each substrate are also investigated.

System and method for floating-substrate passive voltage contrast

DOEpatents

Jenkins, Mark W [Albuquerque, NM; Cole, Jr., Edward I.; Tangyunyong, Paiboon [Albuquerque, NM; Soden, Jerry M [Placitas, NM; Walraven, Jeremy A [Albuquerque, NM; Pimentel, Alejandro A [Albuquerque, NM

2009-04-28

A passive voltage contrast (PVC) system and method are disclosed for analyzing ICs to locate defects and failure mechanisms. During analysis a device side of a semiconductor die containing the IC is maintained in an electrically-floating condition without any ground electrical connection while a charged particle beam is scanned over the device side. Secondary particle emission from the device side of the IC is detected to form an image of device features, including electrical vias connected to transistor gates or to other structures in the IC. A difference in image contrast allows the defects or failure mechanisms be pinpointed. Varying the scan rate can, in some instances, produce an image reversal to facilitate precisely locating the defects or failure mechanisms in the IC. The system and method are useful for failure analysis of ICs formed on substrates (e.g. bulk semiconductor substrates and SOI substrates) and other types of structures.

Graphene plasmonic nanogratings for biomolecular sensing in liquid

NASA Astrophysics Data System (ADS)

Chorsi, Meysam T.; Chorsi, Hamid T.

2017-12-01

We design a surface plasmon resonance (SPR) molecular sensor based on graphene and biomolecule adsorption at graphene-liquid interfaces. The sensor configuration consists of two opposing arrays of graphene nanograting mounted on a substrate, with a liquid-phase sensing medium confined between them. We characterize the design in simulation on a variety of substrates by altering the refractive index of the sensing medium and varying the absorbance-transmittance characteristics. The influence of various parameters on the biosensor's performance, including the Fermi level of graphene, the dielectric constant of the substrate, and the incident angle for plasmon excitation, is investigated. Numerical simulations demonstrate the sensitivity higher than 3000 nm/RIU (refractive index unit). The device supports a wide range of substrates in which graphene can be epitaxially grown. The proposed biosensor works independent of the incident angle and can be tuned to cover a broadband wavelength range.