Sample records for restricted substrate specificity
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Host Specificity of Salmonella typhimurium Deoxyribonucleic Acid Restriction and Modification
Slocum, Harvey; Boyer, Herbert W.
1973-01-01
The restriction and modification genes of Salmonella typhimurium which lie near the thr locus were transferred to a restrictionless mutant of Escherichia coli. These genes were found to be allelic to the E. coli K, B, and A restriction and modification genes. E. coli recombinants with the restriction and modification host specificity of S. typhimurium restricted phage λ that had been modified by each of the seven known host specificities of E. coli at efficiency of plating levels of about 10−2. Phage λ modified with the S. typhimurium host specificity was restricted by six of the seven E. coli host specificities but not by the RII (fi− R-factor controlled) host specificity. It is proposed that the restriction and modification enzymes of this S. typhimurium host specificity have two substrates, one of which is a substrate for the RII host specificity enzymes. PMID:4570605
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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
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The active site of O-GlcNAc transferase imposes constraints on substrate sequence
Rafie, Karim; Blair, David E.; Borodkin, Vladimir S.; Albarbarawi, Osama; van Aalten, Daan M. F.
2016-01-01
O-GlcNAc transferase (OGT) glycosylates a diverse range of intracellular proteins with O-linked N-acetylglucosamine (O-GlcNAc), an essential and dynamic post-translational modification in metazoa. Although this enzyme modifies hundreds of proteins with O-GlcNAc, it is not understood how OGT achieves substrate specificity. In this study, we describe the application of a high-throughput OGT assay on a library of peptides. The sites of O-GlcNAc modification were mapped by ETD-mass spectrometry, and found to correlate with previously detected O-GlcNAc sites. Crystal structures of four acceptor peptides in complex with human OGT suggest that a combination of size and conformational restriction defines sequence specificity in the −3 to +2 subsites. This work reveals that while the N-terminal TPR repeats of hOGT may play a role in substrate recognition, the sequence restriction imposed by the peptide-binding site makes a significant contribution to O-GlcNAc site specificity. PMID:26237509
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A new restriction endonuclease from Citrobacter freundii
Janulaitis, A.A.; Stakenas, P.S.; Lebedenko, E.N.; Berlin, Yu.A.
1982-01-01
CfrI, a new restriction endonuclease of unique substrate specificity, has been isolated from a Citrobacter freundii strain. The enzyme recognizes a degenerated sequence PyGGCCPu in double-strand DNA and cleaves it between Py and G residues to yield 5′ -protruding tetranucleotide ends GGCC. Images PMID:6294607
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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
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Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease.
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.
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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
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CNG site-specific and methyl-sensitive endonuclease WEN1 from wheat seedlings.
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.
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Stable isotope tracers and exercise physiology: past, present and future.
Wilkinson, Daniel J; Brook, Matthew S; Smith, Kenneth; Atherton, Philip J
2017-05-01
Stable isotope tracers have been invaluable assets in physiological research for over 80 years. The application of substrate-specific stable isotope tracers has permitted exquisite insight into amino acid, fatty-acid and carbohydrate metabolic regulation (i.e. incorporation, flux, and oxidation, in a tissue-specific and whole-body fashion) in health, disease and response to acute and chronic exercise. Yet, despite many breakthroughs, there are limitations to 'substrate-specific' stable isotope tracers, which limit physiological insight, e.g. the need for intravenous infusions and restriction to short-term studies (hours) in controlled laboratory settings. In recent years significant interest has developed in alternative stable isotope tracer techniques that overcome these limitations, in particular deuterium oxide (D 2 O or heavy water). The unique properties of this tracer mean that through oral administration, the turnover and flux through a number of different substrates (muscle proteins, lipids, glucose, DNA (satellite cells)) can be monitored simultaneously and flexibly (hours/weeks/months) without the need for restrictive experimental control. This makes it uniquely suited for the study of 'real world' human exercise physiology (amongst many other applications). Moreover, using D 2 O permits evaluation of turnover of plasma and muscle proteins (e.g. dynamic proteomics) in addition to metabolomics (e.g. fluxomics) to seek molecular underpinnings, e.g. of exercise adaptation. Here, we provide insight into the role of stable isotope tracers, from substrate-specific to novel D 2 O approaches, in facilitating our understanding of metabolism. Further novel potential applications of stable isotope tracers are also discussed in the context of integration with the snowballing field of 'omic' technologies. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.
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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.
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mTOR kinase structure, mechanism and regulation by the rapamycin-binding domain
Yang, Haijuan; Rudge, Derek G.; Koos, Joseph D.; Vaidialingam, Bhamini; Yang, Hyo J.; Pavletich, Nikola P.
2015-01-01
The mammalian target of rapamycin (mTOR), a phosphoinositide 3-kinase related protein kinase, controls cell growth in response to nutrients and growth factors and is frequently deregulated in cancer. Here we report co-crystal structures of a truncated mTOR-mLST8 complex with an ATP transition state mimic and with ATP-site inhibitors. The structures reveal an intrinsically active kinase conformation, with catalytic residues and mechanism remarkably similar to canonical protein kinases. The active site is highly recessed due to the FKBP12-Rapamycin binding (FRB) domain and an inhibitory helix protruding from the catalytic cleft. mTOR activating mutations map to the structural framework that holds these elements in place, indicating the kinase is controlled by restricted access. In vitro biochemistry indicates that the FRB domain acts as a gatekeeper, with its rapamycin-binding site interacting with substrates to grant them access to the restricted active site. FKBP12-rapamycin inhibits by directly blocking substrate recruitment and by further restricting active site access. The structures also reveal active site residues and conformational changes that underlie inhibitor potency and specificity. PMID:23636326
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Helbling, Damian E; Hammes, Frederik; Egli, Thomas; Kohler, Hans-Peter E
2014-02-01
The fundamentals of growth-linked biodegradation occurring at low substrate concentrations are poorly understood. Substrate utilization kinetics and microbial growth yields are two critically important process parameters that can be influenced by low substrate concentrations. Standard biodegradation tests aimed at measuring these parameters generally ignore the ubiquitous occurrence of assimilable organic carbon (AOC) in experimental systems which can be present at concentrations exceeding the concentration of the target substrate. The occurrence of AOC effectively makes biodegradation assays conducted at low substrate concentrations mixed-substrate assays, which can have profound effects on observed substrate utilization kinetics and microbial growth yields. In this work, we introduce a novel methodology for investigating biodegradation at low concentrations by restricting AOC in our experiments. We modified an existing method designed to measure trace concentrations of AOC in water samples and applied it to systems in which pure bacterial strains were growing on pesticide substrates between 0.01 and 50 mg liter(-1). We simultaneously measured substrate concentrations by means of high-performance liquid chromatography with UV detection (HPLC-UV) or mass spectrometry (MS) and cell densities by means of flow cytometry. Our data demonstrate that substrate utilization kinetic parameters estimated from high-concentration experiments can be used to predict substrate utilization at low concentrations under AOC-restricted conditions. Further, restricting AOC in our experiments enabled accurate and direct measurement of microbial growth yields at environmentally relevant concentrations for the first time. These are critical measurements for evaluating the degradation potential of natural or engineered remediation systems. Our work provides novel insights into the kinetics of biodegradation processes and growth yields at low substrate concentrations.
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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
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Inorganic phosphate blocks binding of pre-miRNA to Dicer-2 via its PAZ domain
Fukunaga, Ryuya; Colpan, Cansu; Han, Bo W; Zamore, Phillip D
2014-01-01
In Drosophila, Dicer-1 produces microRNAs (miRNAs) from pre-miRNAs, whereas Dicer-2 generates small interfering RNAs from long double-stranded RNA (dsRNA), a process that requires ATP hydrolysis. We previously showed that inorganic phosphate inhibits Dicer-2 cleavage of pre-miRNAs, but not long dsRNAs. Here, we report that phosphate-dependent substrate discrimination by Dicer-2 reflects dsRNA substrate length. Efficient processing by Dicer-2 of short dsRNA requires a 5′ terminal phosphate and a two-nucleotide, 3′ overhang, but does not require ATP. Phosphate inhibits cleavage of such short substrates. In contrast, cleavage of longer dsRNA requires ATP but no specific end structure: phosphate does not inhibit cleavage of these substrates. Mutation of a pair of conserved arginine residues in the Dicer-2 PAZ domain blocked cleavage of short, but not long, dsRNA. We propose that inorganic phosphate occupies a PAZ domain pocket required to bind the 5′ terminal phosphate of short substrates, blocking their use and restricting pre-miRNA processing in flies to Dicer-1. Our study helps explain how a small molecule can alter the substrate specificity of a nucleic acid processing enzyme. PMID:24488111
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Tahara, Erich B; Cunha, Fernanda M; Basso, Thiago O; Della Bianca, Bianca E; Gombert, Andreas K; Kowaltowski, Alicia J
2013-01-01
Calorie restriction (CR) is an intervention known to extend the lifespan of a wide variety of organisms. In S. cerevisiae, chronological lifespan is prolonged by decreasing glucose availability in the culture media, a model for CR. The mechanism has been proposed to involve an increase in the oxidative (versus fermentative) metabolism of glucose. Here, we measured wild-type and respiratory incompetent (ρ(0)) S. cerevisiae biomass formation, pH, oxygen and glucose consumption, and the evolution of ethanol, glycerol, acetate, pyruvate and succinate levels during the course of 28 days of chronological aging, aiming to identify metabolic changes responsible for the effects of CR. The concomitant and quantitative measurements allowed for calculations of conversion factors between different pairs of substrates and products, maximum specific substrate consumption and product formation rates and maximum specific growth rates. Interestingly, we found that the limitation of glucose availability in CR S. cerevisiae cultures hysteretically increases oxygen consumption rates many hours after the complete exhaustion of glucose from the media. Surprisingly, glucose-to-ethanol conversion and cellular growth supported by glucose were not quantitatively altered by CR. Instead, we found that CR primed the cells for earlier, faster and more efficient metabolism of respiratory substrates, especially ethanol. Since lifespan-enhancing effects of CR are absent in respiratory incompetent ρ(0) cells, we propose that the hysteretic effect of glucose limitation on oxidative metabolism is central toward chronological lifespan extension by CR in this yeast.
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Delta ribozyme has the ability to cleave in transan mRNA.
Roy, G; Ananvoranich, S; Perreault, J P
1999-01-01
We report here the first demonstration of the cleavage of an mRNA in trans by delta ribozyme derived from the antigenomic version of the human hepatitis delta virus (HDV). We characterized potential delta ribozyme cleavage sites within HDV mRNA sequence (i.e. C/UGN6), using oligonucleotide binding shift assays and ribonuclease H hydrolysis. Ribozymes were synthesized based on the structural data and then tested for their ability to cleave the mRNA. Of the nine ribozymes examined, three specifically cleaved a derivative HDV mRNA. All three active ribozymes gave consistent indications that they cleaved single-stranded regions. Kinetic characterization of the ability of ribozymes to cleave both the full-length mRNA and either wild-type or mutant small model substrate suggests: (i) delta ribozyme has turnovers, that is to say, several mRNA molecules can be successively cleaved by one ribozyme molecule; and (ii) the substrate specificity of delta ribozyme cleavage is not restricted to C/UGN6. Specifically, substrates with a higher guanosine residue content upstream of the cleavage site (i.e. positions -4 to -2) were always cleaved more efficiently than wild-type substrate. This work shows that delta ribozyme constitutes a potential catalytic RNA for further gene-inactivation therapy. PMID:9927724
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Tang, Baishan; Zhou, Weijun; Du, Jingwen; He, Yanjie; Li, Yuhua
2015-08-01
T-cell-mediated immunotherapy of hematological malignancies requires selection of targeted tumor-associated antigens and T-cell epitopes contained in these tumor proteins. Epidermal growth factor receptor pathway substrate 8 (EPS8), whose function is pivotal for tumor proliferation, progression and metastasis, has been found to be overexpressed in most human tumor types, while its expression in normal tissue is low. The aim of the present study was to identify human leukemia antigen (HLA)-A*0201-restricted epitopes of EPS8 by using a reverse immunology approach. To achieve this, computer algorithms were used to predict HLA-A*0201 molecular binding, proteasome cleavage patterns as well as translocation of transporters associated with antigen processing. Candidate peptides were experimentally validated by T2 binding affinity assay and brefeldin-A decay assay. The functional avidity of peptide-specific cytotoxic T lymphocytes (CTLs) induced from peripheral blood mononuclear cells of healthy volunteers were evaluated by using an enzyme-linked immunosorbent spot assay and a cytotoxicity assay. Four peptides, designated as P455, P92, P276 and P360, had high affinity and stability of binding towards the HLA-A*0201 molecule, and specific CTLs induced by them significantly responded to the corresponding peptides and secreted IFN-γ. At the same time, the CTLs were able to specifically lyse EPS8-expressing cell lines in an HLA-A*0201-restricted manner. The present study demonstrated that P455, P92, P276 and P360 were CTL epitopes of EPS8, and were able to be used for epitope-defined adoptive T-cell transfer and multi-epitope-based vaccine design.
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Rathore, Anurag; Carpenter, Michael A; Demir, Özlem; Ikeda, Terumasa; Li, Ming; Shaban, Nadine; Law, Emily K.; Anokhin, Dmitry; Brown, William L.; Amaro, Rommie E.; Harris, Reuben S.
2013-01-01
APOBEC3A and APOBEC3G are DNA cytosine deaminases with biological functions in foreign DNA and retrovirus restriction, respectively. APOBEC3A has an intrinsic preference for cytosine preceded by thymine (5′-TC) in single-stranded DNA substrates, whereas APOBEC3G prefers the target cytosine to be preceded by another cytosine (5′-CC). To determine the amino acids responsible for these strong dinucleotide preferences, we analyzed a series of chimeras in which putative DNA binding loop regions of APOBEC3G were replaced with the corresponding regions from APOBEC3A. Loop 3 replacement enhanced APOBEC3G catalytic activity but did not alter its intrinsic 5′-CC dinucleotide substrate preference. Loop 7 replacement caused APOBEC3G to become APOBEC3A-like and strongly prefer 5′-TC substrates. Simultaneous loop 3/7 replacement resulted in a hyperactive APOBEC3G variant that also preferred 5′-TC dinucleotides. Single amino acid exchanges revealed D317 as a critical determinant of dinucleotide substrate specificity. Multi-copy explicitly solvated all-atom molecular dynamics simulations suggested a model in which D317 acts as a helix-capping residue by constraining the mobility of loop 7, forming a novel binding pocket that favorably accommodates cytosine. All catalytically active APOBEC3G variants, regardless of dinucleotide preference, retained HIV-1 restriction activity. These data support a model in which the loop 7 region governs the selection of local dinucleotide substrates for deamination but is unlikely to be part of the higher level targeting mechanisms that direct these enzymes to biological substrates such as HIV-1 cDNA. PMID:23938202
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Das, Sreetama; Pawale, Vijaykumar S.; Dadireddy, Venkatareddy; Singh, Avinash Kumar; Ramakumar, Suryanarayanarao; Roy, Rajendra P.
2017-01-01
Surface proteins in Gram-positive bacteria are incorporated into the cell wall through a peptide ligation reaction catalyzed by transpeptidase sortase. Six main classes (A–F) of sortase have been identified of which class A sortase is meant for housekeeping functions. The prototypic housekeeping sortase A (SaSrtA) from Staphylococcus aureus cleaves LPXTG-containing proteins at the scissile T–G peptide bond and ligates protein-LPXT to the terminal Gly residue of the nascent cross-bridge of peptidoglycan lipid II precursor. Sortase-mediated ligation (“sortagging”) of LPXTG-containing substrates and Gly-terminated nucleophiles occurs in vitro as well as in cellulo in the presence of Ca2+ and has been applied extensively for protein conjugations. Although the majority of applications emanate from SaSrtA, low catalytic efficiency, LPXTG specificity restriction, and Ca2+ requirement (particularly for in cellulo applications) remain a drawback. Given that Gram-positive bacteria genomes encode a variety of sortases, natural sortase mining can be a viable complementary approach akin to engineering of wild-type SaSrtA. Here, we describe the structure and specificity of a new class E sortase (SavSrtE) annotated to perform housekeeping roles in Streptomyces avermitilis. Biochemical experiments define the attributes of an optimum peptide substrate, demonstrate Ca2+-independent activity, and provide insights about contrasting functional characteristics of SavSrtE and SaSrtA. Crystal structure, substrate docking, and mutagenesis experiments have identified a critical residue that dictates the preference for a non-canonical LAXTG recognition motif over LPXTG. These results have implications for rational tailoring of substrate tolerance in sortases. Besides, Ca2+-independent orthogonal specificity of SavSrtE is likely to expand the sortagging toolkit. PMID:28270507
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Jin, Zhinan; Johnson, Kenneth A.
2011-01-01
A nucleotide-induced change in DNA polymerase structure governs the kinetics of polymerization by high fidelity DNA polymerases. Mutation of a GAG hinge (G542A/G544A) in T7 DNA polymerase resulted in a 1000-fold slower rate of conformational change, which then limited the rate of correct nucleotide incorporation. Rates of misincorporation were comparable to that seen for wild-type enzyme so that the net effect of the mutation was a large decrease in fidelity. We demonstrate that a presumably modest change from glycine to alanine 20 Å from the active site can severely restrict the flexibility of the enzyme structure needed to recognize and incorporate correct substrates with high specificity. These results emphasize the importance of the substrate-induced conformational change in governing nucleotide selectivity by accelerating the incorporation of correct base pairs but not mismatches. PMID:20978284
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Microbenthic distribution of Proterozoic tidal flats: environmental and taphonomic considerations
NASA Technical Reports Server (NTRS)
Kah, L. C.; Knoll, A. H.
1996-01-01
Silicified carbonates of the late Mesoproterozoic to early Neoproterozoic Society Cliffs Formation, Baffin Island, contain distinctive microfabrics and microbenthic assemblages whose paleo-environmental distribution within the formation parallels the distribution of these elements through Proterozoic time. In the Society Cliffs Formation, restricted carbonates--including microdigitate stromatolites, laminated tufa, and tufted microbial mats--consist predominantly of synsedimentary cements; these facies and the cyanobacterial fossils they contain are common in Paleoproterozoic successions but rare in Neoproterozoic and younger rocks. Less restricted tidal-flat facies in the formation are composed of laminated microbialites dominated by micritic carbonate lithified early, yet demonstrably after compaction; these strata contain cyanobacteria that are characteristic in Neoproterozoic rocks. Within the formation, the facies-dependent distribution of microbial populations reflects both the style and timing of carbonate deposition because of the strong substrate specificity of benthic cyanobacteria. A reasonable conclusion is that secular changes in microbenthic assemblages through Proterozoic time reflect a decrease in the overall representation of rapidly lithified carbonate substrates in younger peritidal environments, as well as concomitant changes in the taphonomic window of silicification through which early life is observed.
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Structural Basis of Substrate Recognition by Hematopoietic Tyrosine Phosphatase (HePTP)
DOE Office of Scientific and Technical Information (OSTI.GOV)
Critton, D.; Tortajada, A; Stetson, G
2008-01-01
Hematopoietic tyrosine phosphatase (HePTP) is one of three members of the kinase interaction motif (KIM) phosphatase family which also includes STEP and PCPTP1. The KIM-PTPs are characterized by a 15 residue sequence, the KIM, which confers specific high-affinity binding to their only known substrates, the MAP kinases Erk and p38, an interaction which is critical for their ability to regulate processes such as T cell differentiation (HePTP) and neuronal signaling (STEP). The KIM-PTPs are also characterized by a unique set of residues in their PTP substrate binding loops, where 4 of the 13 residues are differentially conserved among the KIM-PTPsmore » as compared to more than 30 other class I PTPs. One of these residues, T106 in HePTP, is either an aspartate or asparagine in nearly every other PTP. Using multiple techniques, we investigate the role of these KIM-PTP specific residues in order to elucidate the molecular basis of substrate recognition by HePTP. First, we used NMR spectroscopy to show that Erk2-derived peptides interact specifically with HePTP at the active site. Next, to reveal the molecular details of this interaction, we solved the high-resolution three-dimensional structures of two distinct HePTP-Erk2 peptide complexes. Strikingly, we were only able to obtain crystals of these transient complexes using a KIM-PTP specific substrate-trapping mutant, in which the KIM-PTP specific residue T106 was mutated to an aspartic acid (T106D). The introduced aspartate side chain facilitates the coordination of the bound peptides, thereby stabilizing the active dephosphorylation complex. These structures establish the essential role of HePTP T106 in restricting HePTP specificity to only those substrates which are able to interact with KIM-PTPs via the KIM (e.g., Erk2, p38). Finally, we describe how this interaction of the KIM is sufficient for overcoming the otherwise weak interaction at the active site of KIM-PTPs.« less
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Mahor, Durga; Priyanka, Anu; Prasad, Gandham S; Thakur, Krishan Gopal
2016-01-01
Consumption of foods and beverages with high purine content increases the risk of hyperuricemia, which causes gout and can lead to cardiovascular, renal, and other metabolic disorders. As patients often find dietary restrictions challenging, enzymatically lowering purine content in popular foods and beverages offers a safe and attractive strategy to control hyperuricemia. Here, we report structurally and functionally characterized purine nucleoside phosphorylase (PNP) from Kluyveromyces lactis (KlacPNP), a key enzyme involved in the purine degradation pathway. We report a 1.97 Å resolution crystal structure of homotrimeric KlacPNP with an intrinsically bound hypoxanthine in the active site. KlacPNP belongs to the nucleoside phosphorylase-I (NP-I) family, and it specifically utilizes 6-oxopurine substrates in the following order: inosine > guanosine > xanthosine, but is inactive towards adenosine. To engineer enzymes with broad substrate specificity, we created two point variants, KlacPNPN256D and KlacPNPN256E, by replacing the catalytically active Asn256 with Asp and Glu, respectively, based on structural and comparative sequence analysis. KlacPNPN256D not only displayed broad substrate specificity by utilizing both 6-oxopurines and 6-aminopurines in the order adenosine > inosine > xanthosine > guanosine, but also displayed reversal of substrate specificity. In contrast, KlacPNPN256E was highly specific to inosine and could not utilize other tested substrates. Beer consumption is associated with increased risk of developing gout, owing to its high purine content. Here, we demonstrate that KlacPNP and KlacPNPN256D could be used to catalyze a key reaction involved in lowering beer purine content. Biochemical properties of these enzymes such as activity across a wide pH range, optimum activity at about 25°C, and stability for months at about 8°C, make them suitable candidates for food and beverage industries. Since KlacPNPN256D has broad substrate specificity, a combination of engineered KlacPNP and other enzymes involved in purine degradation could effectively lower the purine content in foods and beverages. PMID:27768715
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Deacylation transition states of a bacterial DD-peptidase.
Adediran, S A; Kumar, I; Pratt, R F
2006-10-31
Beta-lactam antibiotics restrict bacterial growth by inhibiting DD-peptidases. These enzymes catalyze the final transpeptidation step in bacterial cell wall biosynthesis. Although much structural information is now available for these enzymes, the mechanism of the actual transpeptidation reaction has not been studied in detail. The reaction is known to involve a double-displacement mechanism with an acyl-enzyme intermediate, which can be attacked by water, specific amino acids, peptides, and other acyl acceptors. We describe in this paper an investigation of acyl acceptor specificity and assess the need for general base catalysis in the deacylation transition state of the Streptomyces R61 DD-peptidase. We show, by the criterion of solvent deuterium kinetic isotope effect measurements and proton inventories, that the transition states of specific and nonspecific substrates are very similar, at least with respect to proton motion. The transition states for attack (tetrahedral intermediate formation) by d-amino acids and Gly-l-Xaa dipeptides do not include a general base catalyst, while such catalysis is essential for reaction with water and d-alpha-hydroxy acids. D-Alpha-hydroxy acids act as acyl acceptors for glycyl substrates but not for more specific d-alanyl substrates; hydroxy acids actually behave, more generally, as mixed inhibitors of the DD-peptidase. The structural and mechanistic bases of these observations are discussed; they should inform transition state analogue design.
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Huang, Hua; Yury, Patskovsky; Toro, Rafael; Farelli, Jeremiah D.; Pandya, Chetanya; Almo, Steven C.; Allen, Karen N.; Dunaway-Mariano, Debra
2012-01-01
The explosion of protein sequence information requires that current strategies for function assignment must evolve to complement experimental approaches with computationally-based function prediction. This necessitates the development of strategies based on the identification of sequence markers in the form of specificity determinants and a more informed definition of orthologues. Herein, we have undertaken the function assignment of the unknown Haloalkanoate Dehalogenase superfamily member BT2127 (Uniprot accession # Q8A5V9) from Bacteroides thetaiotaomicron using an integrated bioinformatics/structure/mechanism approach. The substrate specificity profile and steady-state rate constants of BT2127 (with kcat/Km value for pyrophosphate of ∼1 × 105 M−1 s−1), together with the gene context, supports the assigned in vivo function as an inorganic pyrophosphatase. The X-ray structural analysis of the wild-type BT2127 and several variants generated by site-directed mutagenesis shows that substrate discrimination is based, in part, on active site space restrictions imposed by the cap domain (specifically by residues Tyr76 and Glu47). Structure guided site directed mutagenesis coupled with kinetic analysis of the mutant enzymes identified the residues required for catalysis, substrate binding, and domain-domain association. Based on this structure-function analysis, the catalytic residues Asp11, Asp13, Thr113, and Lys147 as well the metal binding residues Asp171, Asn172 and Glu47 were used as markers to confirm BT2127 orthologues identified via sequence searches. This bioinformatic analysis demonstrated that the biological range of BT2127 orthologue is restricted to the phylum Bacteroidetes/Chlorobi. The key structural determinants in the divergence of BT2127 and its closest homologue β-phosphoglucomutase control the leaving group size (phosphate vs. glucose-phosphate) and the position of the Asp acid/base in the open vs. closed conformations. HADSF pyrophosphatases represent a third mechanistic and fold type for bacterial pyrophosphatases. PMID:21894910
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Graphene-Mesoporous Si Nanocomposite as a Compliant Substrate for Heteroepitaxy.
Boucherif, Abderrahim Rahim; Boucherif, Abderraouf; Kolhatkar, Gitanjali; Ruediger, Andreas; Arès, Richard
2017-05-01
The ultimate performance of a solid state device is limited by the restricted number of crystalline substrates that are available for epitaxial growth. As a result, only a small fraction of semiconductors are usable. This study describes a novel concept for a tunable compliant substrate for epitaxy, based on a graphene-porous silicon nanocomposite, which extends the range of available lattice constants for epitaxial semiconductor alloys. The presence of graphene and its effect on the strain of the porous layer lattice parameter are discussed in detail and new remarkable properties are demonstrated. These include thermal stability up to 900 °C, lattice tuning up to 0.9 % mismatch, and compliance under stress for virtual substrate thicknesses of several micrometers. A theoretical model is proposed to define the compliant substrate design rules. These advances lay the foundation for the fabrication of a compliant substrate that could unlock the lattice constant restrictions for defect-free new epitaxial semiconductor alloys and devices. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Cheng, Ai-Xia; Han, Xiao-Juan; Wu, Yi-Feng; Lou, Hong-Xiang
2014-01-01
Flavonoids are secondary metabolites derived from phenylalanine and acetate metabolism. They fulfil a variety of functions in plants and have health benefits for humans. During the synthesis of the tricyclic flavonoid natural products in plants, oxidative modifications to the central C ring are catalyzed by four of FeII and 2-oxoglutarate dependent (2-ODD) oxygenases, namely flavone synthase I (FNS I), flavonol synthase (FLS), anthocyanidin synthase (ANS) and flavanone 3β-hydroxylase (FHT). FNS I, FLS and ANS are involved in desaturation of C2–C3 of flavonoids and FHT in hydroxylation of C3. FNS I, which is restricted to the Apiaceae species and in rice, is predicted to have evolved from FHT by duplication. Due to their sequence similarity and substrate specificity, FLS and ANS, which interact with the α surface of the substrate, belong to a group of dioxygenases having a broad substrate specificity, while FNS I and FHT are more selective, and interact with the naringenin β surface. Here, we summarize recent findings regarding the function of the four 2-ODD oxygenases and the relationship between their catalytic activity, their polypeptide sequence and their tertiary structure. PMID:24434621
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Kise, Hiroki; Fujii, Takuma; Masucci, Giovanni Diego; Biondi, Piera; Reimer, James Davis
2017-01-01
In this study, three new species of macrocnemic zoantharians (Hexacorallia, Zoantharia) are described from localities in the Indo-Pacific Ocean including the Red Sea, the Maldives, Palau, and southern Japan: Antipathozoanthus obscurus sp. n. , A. remengesaui sp. n. , and A. cavernus sp. n. Although the genus Antipathozoanthus is currently restricted to species living on antipatharians, A. obscurus sp. n. is not associated with any living substrate and instead is found on coral reef carbonate substrate within narrow caves or cracks. The two new species that have association with antipatharians, A. remengesaui sp. n. and A. cavernus sp. n. , can be distinguished by their relative coenenchyme development and the antipatharian species that each uses as substrate. Additionally, all new species described in this study have unique nuclear internal transcribed spacer region of ribosomal DNA (ITS-rDNA) sequences. Our results indicate that more phylogenetic studies focusing on increasing the numbers of species examined within each of the genera of Parazoanthidae are required in order to better understand the evolutionary history of substrate specificity within the family Parazoanthidae.
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Kise, Hiroki; Fujii, Takuma; Masucci, Giovanni Diego; Biondi, Piera; Reimer, James Davis
2017-01-01
Abstract In this study, three new species of macrocnemic zoantharians (Hexacorallia, Zoantharia) are described from localities in the Indo-Pacific Ocean including the Red Sea, the Maldives, Palau, and southern Japan: Antipathozoanthus obscurus sp. n., A. remengesaui sp. n., and A. cavernus sp. n. Although the genus Antipathozoanthus is currently restricted to species living on antipatharians, A. obscurus sp. n. is not associated with any living substrate and instead is found on coral reef carbonate substrate within narrow caves or cracks. The two new species that have association with antipatharians, A. remengesaui sp. n. and A. cavernus sp. n., can be distinguished by their relative coenenchyme development and the antipatharian species that each uses as substrate. Additionally, all new species described in this study have unique nuclear internal transcribed spacer region of ribosomal DNA (ITS-rDNA) sequences. Our results indicate that more phylogenetic studies focusing on increasing the numbers of species examined within each of the genera of Parazoanthidae are required in order to better understand the evolutionary history of substrate specificity within the family Parazoanthidae. PMID:29362542
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Scent evolution in Chinese roses
Scalliet, Gabriel; Piola, Florence; Douady, Christophe J.; Réty, Stéphane; Raymond, Olivier; Baudino, Sylvie; Bordji, Karim; Bendahmane, Mohammed; Dumas, Christian; Cock, J. Mark; Hugueney, Philippe
2008-01-01
The phenolic methyl ether 3,5-dimethoxytoluene (DMT) is a major scent compound of many modern rose varieties, and its fragrance participates in the characteristic “tea scent” that gave their name to Tea and Hybrid Tea roses. Among wild roses, phenolic methyl ether (PME) biosynthesis is restricted to Chinese rose species, but the progenitors of modern roses included both European and Chinese species (e.g., Rosa chinensis cv Old Blush), so this trait was transmitted to their hybrid progeny. The last steps of the biosynthetic pathways leading to DMT involve two methylation reactions catalyzed by the highly similar orcinol O-methyltransferases (OOMT) 1 and 2. OOMT1 and OOMT2 enzymes exhibit different substrate specificities that are consistent with their operating sequentially in DMT biosynthesis. Here, we show that these different substrate specificities are mostly due to a single amino acid polymorphism in the phenolic substrate binding site of OOMTs. An analysis of the OOMT gene family in 18 species representing the diversity of the genus Rosa indicated that only Chinese roses possess both the OOMT2 and the OOMT1 genes. In addition, we provide evidence that the Chinese-rose-specific OOMT1 genes most probably evolved from an OOMT2-like gene that has homologues in the genomes of all extant roses. We propose that the emergence of the OOMT1 gene may have been a critical step in the evolution of scent production in Chinese roses. PMID:18413608
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Structure and substrate recruitment of the human spindle checkpoint kinase Bub1.
Kang, Jungseog; Yang, Maojun; Li, Bing; Qi, Wei; Zhang, Chao; Shokat, Kevan M; Tomchick, Diana R; Machius, Mischa; Yu, Hongtao
2008-11-07
In mitosis, the spindle checkpoint detects a single unattached kinetochore, inhibits the anaphase-promoting complex or cyclosome (APC/C), and prevents premature sister chromatid separation. The checkpoint kinase Bub1 contributes to checkpoint sensitivity through phosphorylating the APC/C activator, Cdc20, and inhibiting APC/C catalytically. We report here the crystal structure of the kinase domain of Bub1, revealing the requirement of an N-terminal extension for its kinase activity. Though the activation segment of Bub1 is ordered and has structural features indicative of active kinases, the C-terminal portion of this segment sterically restricts substrate access to the active site. Bub1 uses docking motifs, so-called KEN boxes, outside its kinase domain to recruit Cdc20, one of two known KEN box receptors. The KEN boxes of Bub1 are required for the spindle checkpoint in human cells. Therefore, its unusual active-site conformation and mode of substrate recruitment suggest that Bub1 has an exquisitely tuned specificity for Cdc20.
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Robinson, Clifford R.; Sligar, Stephen G.
1998-01-01
Restriction endonucleases such as EcoRI bind and cleave DNA with great specificity and represent a paradigm for protein–DNA interactions and molecular recognition. Using osmotic pressure to induce water release, we demonstrate the participation of bound waters in the sequence discrimination of substrate DNA by EcoRI. Changes in solvation can play a critical role in directing sequence-specific DNA binding by EcoRI and are also crucial in assisting site discrimination during catalysis. By measuring the volume change for complex formation, we show that at the cognate sequence (GAATTC) EcoRI binding releases about 70 fewer water molecules than binding at an alternate DNA sequence (TAATTC), which differs by a single base pair. EcoRI complexation with nonspecific DNA releases substantially less water than either of these specific complexes. In cognate substrates (GAATTC) kcat decreases as osmotic pressure is increased, indicating the binding of about 30 water molecules accompanies the cleavage reaction. For the alternate substrate (TAATTC), release of about 40 water molecules accompanies the reaction, indicated by a dramatic acceleration of the rate when osmotic pressure is raised. These large differences in solvation effects demonstrate that water molecules can be key players in the molecular recognition process during both association and catalytic phases of the EcoRI reaction, acting to change the specificity of the enzyme. For both the protein–DNA complex and the transition state, there may be substantial conformational differences between cognate and alternate sites, accompanied by significant alterations in hydration and solvent accessibility. PMID:9482860
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Mobility restrictions and glass transition behaviour of an epoxy resin under confinement.
Djemour, A; Sanctuary, R; Baller, J
2015-04-07
Confinement can have a big influence on the dynamics of glass formers in the vicinity of the glass transition. Already 40 to 50 K above the glass transition temperature, thermal equilibration of glass formers can be strongly influenced by the confining substrate. We investigate the linear thermal expansion and the specific heat capacity cp of an epoxy resin (diglycidyl ether of bisphenol A, DGEBA) in a temperature interval of 120 K around the glass transition temperature. The epoxy resin is filled into controlled pore glasses with pore diameters between 4 and 111 nm. Since DGEBA can form H-bonds with silica surfaces, we also investigate the influence of surface silanization of the porous substrates. In untreated substrates a core/shell structure of the epoxy resin can be identified. The glass transition behaviours of the bulk phase and that of the shell phase are different. In silanized substrates, the shell phase disappears. At a temperature well above the glass transition, a second transition is found for the bulk phase - both in the linear expansion data as well as in the specific heat capacity. The cp data do not allow excluding the glass transition of a third phase as being the cause for this transition, whereas the linear expansion data do so. The additional transition temperature is interpreted as a separation between two regimes: above this temperature, macroscopic flow of the bulk phase inside the porous structure is possible to balance the mismatch of thermal expansion coefficients between DGEBA and the substrate. Below the transition temperature, this degree of freedom is hindered by geometrical constraints of the porous substrates. Moreover, this second transition could also be found in the linear expansion data of the shell phase.
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Purification of Restriction Endonuclease EcoRII and its Co-Crystallization
NASA Technical Reports Server (NTRS)
Karpova, E. A.; Chen, L.; Meehan, E.; Pusey, M.; Rose, M. Franklin (Technical Monitor)
2000-01-01
Restriction endonuclease EcoRII (EcoRII) is a homodimeric DNA-binding protein. It belongs to the type II family of restriction-modification enzymes (subclass IIe). EcoRII recognizes the nucleotide sequence 5'-CCWGG (W=A or T) and cleaves the phosphodiester bond preceding the first cytosine. Methylation at C5 of the second cytosine inhibits cleavage. The enzyme has a unique ability to search for the presence of two substrate sites before cleavage. To the best of our knowledge no other subclass IIe restriction endonuclease has been crystallized yet, without or with a DNA-substrate. We have recently grown and characterized the crystals of this enzyme (1) Here we report on the result of co-crystallization experiments of EcoRII with an 11 b.p. oligonucleotide substrate. The dissociation constant (Kd) EcoRII: 11 b.p. was determined earlier (unpublished results). The needle-like crystals of oligonucleotide-EcoRII protein complex were obtained with this substrate by the technique of vapor diffusion hanging drops. The crystals obtained were washed and dissolved in an aliquot of 10 mM Tris-HCl buffer, pH=7.5. Running a portion of this solution on the SDS-get indicated the presence of endonuclease in the solution. A UV-spectrophotometric test of a second portion confirmed the presence of DNA. We are now working on improvement of the DNA-EcoRII protein crystals. Results obtained from these and ongoing efforts will be reported.
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Chen, Yuanyuan; Farquhar, Erik R.; Chance, Mark R.; Palczewski, Krzysztof; Kiser, Philip D.
2012-01-01
Aminopeptidases are key enzymes involved in the regulation of signaling peptide activity. Here, we present a detailed biochemical and structural analysis of an evolutionary highly conserved aspartyl aminopeptidase called DNPEP. We show that this peptidase can cleave multiple physiologically relevant substrates, including angiotensins, and thus may play a key role in regulating neuron function. Using a combination of x-ray crystallography, x-ray absorption spectroscopy, and single particle electron microscopy analysis, we provide the first detailed structural analysis of DNPEP. We show that this enzyme possesses a binuclear zinc-active site in which one of the zinc ions is readily exchangeable with other divalent cations such as manganese, which strongly stimulates the enzymatic activity of the protein. The plasticity of this metal-binding site suggests a mechanism for regulation of DNPEP activity. We also demonstrate that DNPEP assembles into a functionally relevant tetrahedral complex that restricts access of peptide substrates to the active site. These structural data allow rationalization of the enzyme's preference for short peptide substrates with N-terminal acidic residues. This study provides a structural basis for understanding the physiology and bioinorganic chemistry of DNPEP and other M18 family aminopeptidases. PMID:22356908
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How Does the Brain Implement Adaptive Decision Making to Eat?
Walsh, B. Timothy; Kaye, Walter; Geliebter, Allan
2015-01-01
Adaptive decision making to eat is crucial for survival, but in anorexia nervosa, the brain persistently supports reduced food intake despite a growing need for energy. How the brain persists in reducing food intake, sometimes even to the point of death and despite the evolution of multiple mechanisms to ensure survival by governing adaptive eating behaviors, remains mysterious. Neural substrates belong to the reward-habit system, which could differ among the eating disorders. The present review provides an overview of neural circuitry of restrictive food choice, binge eating, and the contribution of specific serotonin receptors. One possibility is that restrictive food intake critically engages goal-directed (decision making) systems and “habit,” supporting the view that persistent caloric restriction mimics some aspects of addiction to drugs of abuse. SIGNIFICANCE STATEMENT An improved understanding of the neural basis of eating disorders is a timely challenge because these disorders can be deadly. Up to 70 million of people in the world suffer from eating disorders. Anorexia nervosa affects 1–4% of women in United States and is the first cause of death among adolescents in Europe. Studies relying on animal models suggest that decision making to eat (or not) can prevail over actual energy requirements due to emotional disturbances resulting in abnormal habitual behavior, mimicking dependence. These recent studies provide a foundation for developing more specific and effective interventions for these disorders. PMID:26468187
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Evitt, Andrew S; Cox, Russell J
2011-05-01
Inhibitors of the enzyme aspartate semialdehyde dehydrogenase, a key biological target for the generation of a new class of antibiotic compounds, have been developed. To investigate improvements to binding within an inhibitor series, the lowering of the entropic barrier to binding through conformational restriction was investigated. A library of linear and cyclic substrate analogues was generated and computational docking used to aid in structure selection. The cyclic phosphonate inhibitor 18 was thus identified as complimentary to the enzyme active-site. Synthesis and in vitro inhibition assay revealed a K(i) of 3.8 mM against natural substrate, where the linear analogue of 18, compound 15, had previously shown no inhibitory activity. Two further inhibitors, phosphate analogue diastereoisomers 17a and 17b, were synthesised and also found to have low millimolar K(i) values. As a result of the computational docking investigations, a novel substrate binding interaction was discovered: hydrogen bonding between the substrate (phosphate hydroxy-group as the hydrogen bond donor) and the NADPH cofactor (2'-oxygen as the hydrogen bond acceptor).
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Butterer, Annika; Pernstich, Christian; Smith, Rachel M.; Sobott, Frank; Szczelkun, Mark D.; Tóth, Júlia
2014-01-01
Fundamental aspects of the biochemistry of Type III restriction endonucleases remain unresolved despite being characterized by numerous research groups in the past decades. One such feature is the subunit stoichiometry of these hetero-oligomeric enzyme complexes, which has important implications for the reaction mechanism. In this study, we present a series of results obtained by native mass spectrometry and size exclusion chromatography with multi-angle light scattering consistent with a 1:2 ratio of Res to Mod subunits in the EcoP15I, EcoPI and PstII complexes as the main holoenzyme species and a 1:1 stoichiometry of specific DNA (sDNA) binding by EcoP15I and EcoPI. Our data are also consistent with a model where ATP hydrolysis activated by recognition site binding leads to release of the enzyme from the site, dissociation from the substrate via a free DNA end and cleavage of the DNA. These results are discussed critically in the light of the published literature, aiming to resolve controversies and discuss consequences in terms of the reaction mechanism. PMID:24510100
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Superhydrophobic SERS substrates based on silicon hierarchical nanostructures
NASA Astrophysics Data System (ADS)
Chen, Xuexian; Wen, Jinxiu; Zhou, Jianhua; Zheng, Zebo; An, Di; Wang, Hao; Xie, Weiguang; Zhan, Runze; Xu, Ningsheng; Chen, Jun; She, Juncong; Chen, Huanjun; Deng, Shaozhi
2018-02-01
Silicon nanostructures have been cultivated as promising surface enhanced Raman scattering (SERS) substrates in terms of their low-loss optical resonance modes, facile functionalization, and compatibility with today’s state-of-the-art CMOS techniques. However, unlike their plasmonic counterparts, the electromagnetic field enhancements induced by silicon nanostructures are relatively small, which restrict their SERS sensing limit to around 10-7 M. To tackle this problem, we propose here a strategy for improving the SERS performance of silicon nanostructures by constructing silicon hierarchical nanostructures with a superhydrophobic surface. The hierarchical nanostructures are binary structures consisted of silicon nanowires (NWs) grown on micropyramids (MPs). After being modified with perfluorooctyltriethoxysilane (PFOT), the nanostructure surface shows a stable superhydrophobicity with a high contact angle of ˜160°. The substrate can allow for concentrating diluted analyte solutions into a specific area during the evaporation of the liquid droplet, whereby the analytes are aggregated into a small volume and can be easily detected by the silicon nanostructure SERS substrate. The analyte molecules (methylene blue: MB) enriched from an aqueous solution lower than 10-8 M can be readily detected. Such a detection limit is ˜100-fold lower than the conventional SERS substrates made of silicon nanostructures. Additionally, the detection limit can be further improved by functionalizing gold nanoparticles onto silicon hierarchical nanostructures, whereby the superhydrophobic characteristics and plasmonic field enhancements can be combined synergistically to give a detection limit down to ˜10-11 M. A gold nanoparticle-functionalized superhydrophobic substrate was employed to detect the spiked melamine in liquid milk. The results showed that the detection limit can be as low as 10-5 M, highlighting the potential of the proposed superhydrophobic SERS substrate in practical food safety inspection applications.
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Taweesak, Viyachai; Lee Abdullah, Thohirah; Hassan, Siti Aishah; Kamarulzaman, Nitty Hirawaty; Wan Yusoff, Wan Abdullah
2014-01-01
Influences of irrigation frequency on the growth and flowering of chrysanthemum grown under restricted root volume were tested. Chrysanthemum cuttings (Chrysanthemum morifolium “Reagan White”) were grown in seedling tray which contained coconut peat in volumes of 73 and 140 cm3. Plants were irrigated with drip irrigation at irrigation frequencies of 4 (266 mL), 6 (400 mL), and 8 (533 mL) times/day to observe their growth and flowering performances. There was interaction between irrigation frequency and substrate volume on plant height of chrysanthemum. Plants grown in 140 cm3 substrates and irrigated 6 times/day produced the tallest plant of 109.25 cm. Plants irrigated 6 and 8 times/day had significantly higher level of phosphorus content in their leaves than those plants irrigated 4 times/day. The total leaf area, number of internodes, leaf length, and leaf width of chrysanthemums grown in 140 cm3 substrate were significantly higher than those grown in 73 cm3 substrate. The numbers of flowers were affected by both irrigation frequencies and substrate volumes. Chrysanthemums irrigated 8 times/day had an average of 19.56 flowers while those irrigated 4 times/day had an average of 16.63 flowers. Increasing irrigation frequency can improve the growth and flowering of chrysanthemums in small substrate volumes. PMID:25478586
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Brewer, Shannon K.; Dyer, Joseph J.
2016-01-01
Human activities can alter the environment to the point that it is unsuitable to the native species resulting in a loss of biodiversity. Ecologists understand the importance of biodiversity and the conservation of vulnerable species. Species that are narrowly endemic are considered to be particularly vulnerable because they often use specific habitats that are highly susceptible to human disturbance. The basic components of species conservation are 1) delineation of the spatial distribution of the species, 2) understanding how the species interacts with its environment, and 3) employing management strategies based on the ecology of the species. In this study, we investigated several crayfish species endemic to the Ouachita Mountains in Oklahoma and Arkansas. We established the spatial distributions (i.e., range) of the crayfish using Maximum Entropy species distribution modeling. We then investigated crayfish habitat use with quantitative sampling and a paired movement study. Finally, we evaluated the ability of crayfish to burrow under different environmental conditions in a controlled laboratory setting. Crayfish distribution at the landscape scale was largely driven by climate, geology and elevation. In general, the endemic crayfish in this study occurred above 300-m elevation where the geology was dominated by sandstone and shale, and rainfall totals were the highest compared to the rest of the study region. Our quantitative data indicated crayfish did not select for specific habitat types at the reach scale; however, crayfish appeared to continue to use shallow and dry habitat even as the streams dried. Movement by passive integrated transponder (PIT) tagged crayfish was highly variable but crayfish tended to burrow in response to drought rather than migrate to wet habitat. Controlled laboratory experiments revealed smaller substrate size (pebble) restricted crayfish burrowing more than larger substrates (cobble). We also found excess fine sediment restricted crayfish burrowing regardless of dominant substrate size. Our results suggest climate change and sedimentation resulting from land-use practices, combined with increased water withdrawals have the potential to alter crayfish distributions and affect persistence of some crayfish populations.
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Chemical biology-based approaches on fluorescent labeling of proteins in live cells.
Jung, Deokho; Min, Kyoungmi; Jung, Juyeon; Jang, Wonhee; Kwon, Youngeun
2013-05-01
Recently, significant advances have been made in live cell imaging owing to the rapid development of selective labeling of proteins in vivo. Green fluorescent protein (GFP) was the first example of fluorescent reporters genetically introduced to protein of interest (POI). While GFP and various types of engineered fluorescent proteins (FPs) have been actively used for live cell imaging for many years, the size and the limited windows of fluorescent spectra of GFP and its variants set limits on possible applications. In order to complement FP-based labeling methods, alternative approaches that allow incorporation of synthetic fluorescent probes to target POIs were developed. Synthetic fluorescent probes are smaller than fluorescent proteins, often have improved photochemical properties, and offer a larger variety of colors. These synthetic probes can be introduced to POIs selectively by numerous approaches that can be largely categorized into chemical recognition-based labeling, which utilizes metal-chelating peptide tags and fluorophore-carrying metal complexes, and biological recognition-based labeling, such as (1) specific non-covalent binding between an enzyme tag and its fluorophore-carrying substrate, (2) self-modification of protein tags using substrate variants conjugated to fluorophores, (3) enzymatic reaction to generate a covalent binding between a small molecule substrate and a peptide tag, and (4) split-intein-based C-terminal labeling of target proteins. The chemical recognition-based labeling reaction often suffers from compromised selectivity of metal-ligand interaction in the cytosolic environment, consequently producing high background signals. Use of protein-substrate interactions or enzyme-mediated reactions generally shows improved specificity but each method has its limitations. Some examples are the presence of large linker protein, restriction on the choice of introducible probes due to the substrate specificity of enzymes, and competitive reaction mediated by an endogenous analogue of the introduced protein tag. These limitations have been addressed, in part, by the split-intein-based labeling approach, which introduces fluorescent probes with a minimal size (~4 amino acids) peptide tag. In this review, the advantages and the limitations of each labeling method are discussed.
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2005-01-01
We have identified and characterized a novel NADP+-dependent D-arabitol dehydrogenase and the corresponding gene from the rust fungus Uromyces fabae, a biotrophic plant pathogen on broad bean (Vicia faba). The new enzyme was termed ARD1p (D-arabitol dehydrogenase 1). It recognizes D-arabitol and mannitol as substrates in the forward reaction, and D-xylulose, D-ribulose and D-fructose as substrates in the reverse reaction. Co-factor specificity was restricted to NADP(H). Kinetic data for the major substrates and co-factors are presented. A detailed analysis of the organization and expression pattern of the ARD1 gene are also given. Immunocytological data indicate a localization of the gene product predominantly in haustoria, the feeding structures of these fungi. Analyses of metabolite levels during pathogenesis indicate that the D-arabitol concentration rises dramatically as infection progresses, and D-arabitol was shown in an in vitro system to be capable of quenching reactive oxygen species involved in host plant defence reactions. ARD1p may therefore play an important role in carbohydrate metabolism and in establishing and/or maintaining the biotrophic interaction in U. fabae. PMID:15796718
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NASA Astrophysics Data System (ADS)
Wang, Xiu; Zhou, Zhiming; Liang, Zhiyu; Zhuang, Zanyong; Yu, Yan
2017-11-01
The Fe0/C3N4/MoS2 heterostructure was fabricated through photochemical synthesis that was free of NaBH4. Specifically, the g-C3N4/MoS2 (GCNM) composite was used as the substrate. Visible light excited the electrons from the valence band of the GCNM in the substrate, and the excited electrons reduced the Fe2+ ions in the solution nearby GCNM to Fe0 and then created the Fe0/C3N4/MoS2 heterostructure. Small Fe0 (<9 nm) dots well dispersed on the GCNM surface were obtained, because the diffusion of the Fe ions in the solution and the diffusion of the electrons on the GCNM substrate restricted the growth of Fe0 nanoparticles. The smaller size of Fe0 provided a larger number of active metal centers and improved the carrier separation efficiency. As a result, the Fe0/C3N4/MoS2 heterostructure exhibited superior catalytic properties in the redox reactions of rhodamine B, Cr(VI), Pb(II), and Cd(II). It could also be readily recycled without severe loss of catalytic performance.
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Mitochondrial oxidative metabolism during respiratory infection in riboflavin deficient mice.
Brijlal, S; Lakshmi, A V; Bamji, M S
1999-12-01
Studies in children and mice have shown that respiratory infection alters riboflavin metabolism, resulting in increased urinary loss of this vitamin. This could be due to mobilization of riboflavin from the liver to blood because liver Flavin adenine dinucleotide (FAD) levels were lowered in the mice during infection. To understand the functional implications of lowered hepatic FAD levels during respiratory infection, flavoprotein functions such as oxidative phosphorylation and beta-oxidation of the liver mitochondria were examined during infection in mice. Weanling mice were fed either riboflavin-restricted or control diet for 18 days and then injected with a sublethal dose of Klebsiella pneumoniae. During infection, the state 3 respiratory rate with palmitoyl-L-carnitine and glutamate were significantly lowered (27-29%) in the riboflavin-restricted group, whereas in the control group 10% reduction was observed with palmitoyl-L-carnitine as substrate. A 22% reduction in the respiratory control ratio with palmitoyl-L-carnitine as substrate was observed during infection in the riboflavin-restricted group. The beta-oxidation of palmitoyl-L-carnitine was significantly lowered (29%) in the riboflavin-restricted infected group. The results of the study suggest that the effects of infection on vital physiologic functions were more pronounced in the riboflavin-restricted mice than in the control mice. (c) Elsevier Science Inc. 1999.
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Method for applying a diffusion barrier interlayer for high temperature components
Wei, Ronghua; Cheruvu, Narayana S.
2016-03-08
A coated substrate and a method of forming a diffusion barrier coating system between a substrate and a MCrAl coating, including a diffusion barrier coating deposited onto at least a portion of a substrate surface, wherein the diffusion barrier coating comprises a nitride, oxide or carbide of one or more transition metals and/or metalloids and a MCrAl coating, wherein M includes a transition metal or a metalloid, deposited on at least a portion of the diffusion barrier coating, wherein the diffusion barrier coating restricts the inward diffusion of aluminum of the MCrAl coating into the substrate.
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Vascular flora and geoecology of Mont de la Table, Gaspesie, Quebec
Scott W. Bailey; Joann Hoy; Charles V. Cogbill
2015-01-01
The influence of substrate lithology on the distribution of many vascular and nonvascular plants has long been recognized, especially in alpine, subalpine, and other rocky habitats. In particular, plants have been classified as dependent on high-calcium substrates (i.e., calcicoles) based on common restriction to habitats developed in calcareous rocks, such as...
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High density electronic circuit and process for making
Morgan, William P.
1999-01-01
High density circuits with posts that protrude beyond one surface of a substrate to provide easy mounting of devices such as integrated circuits. The posts also provide stress relief to accommodate differential thermal expansion. The process allows high interconnect density with fewer alignment restrictions and less wasted circuit area than previous processes. The resulting substrates can be test platforms for die testing and for multi-chip module substrate testing. The test platform can contain active components and emulate realistic operational conditions, replacing shorts/opens net testing.
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Tröndle, Julia; Albermann, Christoph; Weiner, Michael; Sprenger, Georg A; Weuster-Botz, Dirk
2018-05-01
Usually perturbation of the metabolism of cells by addition of substrates is applied for metabolic analysis of production organisms, but perturbation studies are restricted to the endogenous substrates of the cells under study. The goal of this study is to overcome this limitation by making phosphoenolpyruvate (PEP) available for perturbation studies with Escherichia coli producing L-phenylalanine. A production strain overexpressing a PEP-transporter variant (UhpT-D388C) is applied in a standardized fed-batch production-process on a 42 L-scale. Four parallel short-term perturbation experiments of 20 min are performed with glucose and glycerol as fed-batch carbon sources after rapid media transition of cells from the production-process. PEP is added after 9 min and is immediately consumed by the cells with up to 1.5 mmol g CDW -1 h -1 . L-phenylalanine production rates increased by up to 200% after addition of PEP. This clearly indicates an intracellular PEP-limitation in the L-phenylalanine production strain under study. Thus, it is shown that overexpressing specific transporters for analytical reasons makes exogenous substrates available as perturbation substrates for metabolic analyses of cells sampled from production-processes and thereby allows a very targeted perturbation of whole-cell metabolism. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Schwarz, Friedrich W.; van Aelst, Kara; Tóth, Júlia; Seidel, Ralf; Szczelkun, Mark D.
2011-01-01
DNA cleavage by the Type III Restriction–Modification enzymes requires communication in 1D between two distant indirectly-repeated recognitions sites, yet results in non-specific dsDNA cleavage close to only one of the two sites. To test a recently proposed ATP-triggered DNA sliding model, we addressed why one site is selected over another during cleavage. We examined the relative cleavage of a pair of identical sites on DNA substrates with different distances to a free or protein blocked end, and on a DNA substrate using different relative concentrations of protein. Under these conditions a bias can be induced in the cleavage of one site over the other. Monte-Carlo simulations based on the sliding model reproduce the experimentally observed behaviour. This suggests that cleavage site selection simply reflects the dynamics of the preceding stochastic enzyme events that are consistent with bidirectional motion in 1D and DNA cleavage following head-on protein collision. PMID:21724613
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Akrami, Hassan; Soheili, Zahra-Soheila; Sadeghizadeh, Majid; Khalooghi, Keynoush; Ahmadieh, Hamid; Kanavi, Mojgan Rezaie; Samiei, Shahram; Pakravesh, Jalil
2011-06-01
The retinal pigment epithelium (RPE) plays a key role in the maintenance of the normal functions of the retina. Tissue engineering using amniotic membrane as a substrate to culture RPE cells may provide a promising new strategy to replace damaged RPE. We established a method of culturing RPE cells over the amniotic membrane as a support for their growth and transplantation. The transcription of specific genes involved in cellular function of native RPE, including RPE65, CRALBP, VEGF, CD68, and tyrosinase, were then measured using quantitative real-time PCR. Data showed a considerable increase in transcription of RPE65, CD68, and VEGF in RPE cells cultured on amniotic membrane. The amounts of CRALBP and tyrosinase transcripts were not affected. This may simply indicate that amniotic membrane restricted dedifferentiation of RPE cells in culture. The results suggest that amniotic membrane may be considered as an elective biological substrate for RPE cell culture.
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Fessner, Wolf-Dieter
2015-12-25
Systems Biocatalysis is an emerging concept of organizing enzymes in vitro to construct complex reaction cascades for an efficient, sustainable synthesis of valuable chemical products. The strategy merges the synthetic focus of chemistry with the modular design of biological systems, which is similar to metabolic engineering of cellular production systems but can be realized at a far lower level of complexity from a true reductionist approach. Such operations are free from material erosion by competing metabolic pathways, from kinetic restrictions by physical barriers and regulating circuits, and from toxicity problems with reactive foreign substrates, which are notorious problems in whole-cell systems. A particular advantage of cell-free concepts arises from the inherent opportunity to construct novel biocatalytic reaction systems for the efficient synthesis of non-natural products ("artificial metabolisms") by using enzymes specifically chosen or engineered for non-natural substrate promiscuity. Examples illustrating the technology from our laboratory are discussed. Copyright © 2014 Elsevier B.V. All rights reserved.
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2012-01-01
Background The influence of feed restriction and different diet's caloric value on the aerobic and anaerobic capacity is unclear in the literature. Thus, the objectives of this study were to determine the possible influences of two diets with different caloric values and the influence of feed restriction on the aerobic (anaerobic threshold: AT) and anaerobic (time to exhaustion: Tlim) variables measured by a lactate minimum test (LM) in rats. Methods We used 40 adult Wistar rats. The animals were divided into four groups: ad libitum commercial Purina® diet (3028.0 Kcal/kg) (ALP), restricted commercial Purina® diet (RAP), ad libitum semi-purified AIN-93 diet (3802.7 Kcal/kg) (ALD) and restricted semi-purified AIN-93 diet (RAD). The animals performed LM at the end of the experiment, 48 h before euthanasia. Comparisons between groups were performed by analysis of variance (p < 0,05). Results At the end of the experiment, the weights of the rats in the groups with the restricted diets were significantly lower than those in the groups with ad libitum diet intakes. In addition, the ALD group had higher amounts of adipose tissue. With respect to energetic substrates, the groups subjected to diet restriction had significantly higher levels of liver and muscle glycogen. There were no differences between the groups with respect to AT; however, the ALD group had lower lactatemia at the AT intensity and higher Tlim than the other groups. Conclusions We conclude that dietary restriction induces changes in energetic substrates and that ad libitum intake of a semi-purified AIN-93 diet results in an increase in adipose tissue, likely reducing the density of the animals in water and favouring their performance during the swimming exercises. PMID:22448911
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PhosphoregDB: The tissue and sub-cellular distribution of mammalian protein kinases and phosphatases
Forrest, Alistair RR; Taylor, Darrin F; Fink, J Lynn; Gongora, M Milena; Flegg, Cameron; Teasdale, Rohan D; Suzuki, Harukazu; Kanamori, Mutsumi; Kai, Chikatoshi; Hayashizaki, Yoshihide; Grimmond, Sean M
2006-01-01
Background Protein kinases and protein phosphatases are the fundamental components of phosphorylation dependent protein regulatory systems. We have created a database for the protein kinase-like and phosphatase-like loci of mouse that integrates protein sequence, interaction, classification and pathway information with the results of a systematic screen of their sub-cellular localization and tissue specific expression data mined from the GNF tissue atlas of mouse. Results The database lets users query where a specific kinase or phosphatase is expressed at both the tissue and sub-cellular levels. Similarly the interface allows the user to query by tissue, pathway or sub-cellular localization, to reveal which components are co-expressed or co-localized. A review of their expression reveals 30% of these components are detected in all tissues tested while 70% show some level of tissue restriction. Hierarchical clustering of the expression data reveals that expression of these genes can be used to separate the samples into tissues of related lineage, including 3 larger clusters of nervous tissue, developing embryo and cells of the immune system. By overlaying the expression, sub-cellular localization and classification data we examine correlations between class, specificity and tissue restriction and show that tyrosine kinases are more generally expressed in fewer tissues than serine/threonine kinases. Conclusion Together these data demonstrate that cell type specific systems exist to regulate protein phosphorylation and that for accurate modelling and for determination of enzyme substrate relationships the co-location of components needs to be considered. PMID:16504016
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Kwong, Gabriel A; Radu, Caius G; Hwang, Kiwook; Shu, Chengyi J; Ma, Chao; Koya, Richard C; Comin-Anduix, Begonya; Hadrup, Sine Reker; Bailey, Ryan C; Witte, Owen N; Schumacher, Ton N; Ribas, Antoni; Heath, James R
2009-07-22
The human immune system consists of a large number of T cells capable of recognizing and responding to antigens derived from various sources. The development of peptide-major histocompatibility (p/MHC) tetrameric complexes has enabled the direct detection of these antigen-specific T cells. With the goal of increasing throughput and multiplexing of T cell detection, protein microarrays spotted with defined p/MHC complexes have been reported, but studies have been limited due to the inherent instability and reproducibility of arrays produced via conventional spotted methods. Herein, we report on a platform for the detection of antigen-specific T cells on glass substrates that offers significant advantages over existing surface-bound schemes. In this approach, called "Nucleic Acid Cell Sorting (NACS)", single-stranded DNA oligomers conjugated site-specifically to p/MHC tetramers are employed to immobilize p/MHC tetramers via hybridization to a complementary-printed substrate. Fully assembled p/MHC arrays are used to detect and enumerate T cells captured from cellular suspensions, including primary human T cells collected from cancer patients. NACS arrays outperform conventional spotted arrays assessed in key criteria such as repeatability and homogeneity. The versatility of employing DNA sequences for cell sorting is exploited to enable the programmed, selective release of target populations of immobilized T cells with restriction endonucleases for downstream analysis. Because of the performance, facile and modular assembly of p/MHC tetramer arrays, NACS holds promise as a versatile platform for multiplexed T cell detection.
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Optimisation of substrate blends in anaerobic co-digestion using adaptive linear programming.
García-Gen, Santiago; Rodríguez, Jorge; Lema, Juan M
2014-12-01
Anaerobic co-digestion of multiple substrates has the potential to enhance biogas productivity by making use of the complementary characteristics of different substrates. A blending strategy based on a linear programming optimisation method is proposed aiming at maximising COD conversion into methane, but simultaneously maintaining a digestate and biogas quality. The method incorporates experimental and heuristic information to define the objective function and the linear restrictions. The active constraints are continuously adapted (by relaxing the restriction boundaries) such that further optimisations in terms of methane productivity can be achieved. The feasibility of the blends calculated with this methodology was previously tested and accurately predicted with an ADM1-based co-digestion model. This was validated in a continuously operated pilot plant, treating for several months different mixtures of glycerine, gelatine and pig manure at organic loading rates from 1.50 to 4.93 gCOD/Ld and hydraulic retention times between 32 and 40 days at mesophilic conditions. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Solomonia, Revaz O; Apkhazava, David; Nozadze, Maia; Jackson, Antony P; McCabe, Brian J; Horn, Gabriel
2008-06-01
There is strong evidence that a restricted part of the chick forebrain, the IMM (formerly IMHV), stores information acquired through the learning process of visual imprinting. Twenty-four hours after imprinting training, a learning-specific increase in amount of myristoylated, alanine-rich C-kinase substrate (MARCKS) protein is known to occur in the homogenate fraction of IMM. We investigated the two components of this fraction, membrane-bound and cytoplasmic-phosphorylated MARCKS. In IMM, amount of membrane-bound MARCKS, but not of cytoplasmic-phosphorylated MARCKS, increased as chicks learned. No changes were observed for either form of MARCKS in PPN, a control forebrain region. The results indicate that there is a learning-specific increase in membrane-bound, non-phosphorylated MARCKS 24 h after training. This increase might contribute to stabilization of synaptic morphology.
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Placental Adaptations in Growth Restriction
Zhang, Song; Regnault, Timothy R.H.; Barker, Paige L.; Botting, Kimberley J.; McMillen, Isabella C.; McMillan, Christine M.; Roberts, Claire T.; Morrison, Janna L.
2015-01-01
The placenta is the primary interface between the fetus and mother and plays an important role in maintaining fetal development and growth by facilitating the transfer of substrates and participating in modulating the maternal immune response to prevent immunological rejection of the conceptus. The major substrates required for fetal growth include oxygen, glucose, amino acids and fatty acids, and their transport processes depend on morphological characteristics of the placenta, such as placental size, morphology, blood flow and vascularity. Other factors including insulin-like growth factors, apoptosis, autophagy and glucocorticoid exposure also affect placental growth and substrate transport capacity. Intrauterine growth restriction (IUGR) is often a consequence of insufficiency, and is associated with a high incidence of perinatal morbidity and mortality, as well as increased risk of cardiovascular and metabolic diseases in later life. Several different experimental methods have been used to induce placental insufficiency and IUGR in animal models and a range of factors that regulate placental growth and substrate transport capacity have been demonstrated. While no model system completely recapitulates human IUGR, these animal models allow us to carefully dissect cellular and molecular mechanisms to improve our understanding and facilitate development of therapeutic interventions. PMID:25580812
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Whigham, L D; Butz, D E; Johnson, L K; Schoeller, D A; Abbott, D H; Porter, W P; Cook, M E
2014-09-01
Rapid detection of shifts in substrate utilization and energy balance would provide a compelling biofeedback tool for individuals attempting weight loss. As a proof of concept, we tested whether the natural abundance of exhaled carbon stable isotope ratios (breath δ(13)C) reflects shifts between negative and positive energy balance. Volunteers (n=5) consumed a 40% energy-restricted diet for 6 days followed by 50% excess on day 7. Breath was sampled immediately before and 1 h and 2 h after breakfast, lunch and dinner. Exhaled breath δ(13)C values were measured by cavity ring-down spectroscopy. Using repeated measures analysis of variance (ANOVA) followed by Dunnett's contrasts, pre-breakfast breath values on days 2-6 were compared with day 1, and postprandial day 7 time points were compared with pre-breakfast day 7. Energy restriction diminished pre-breakfast breath δ(13)C by day 3 (P<0.05). On day 7, increased energy intake was first detected immediately before dinner (-23.8±0.6 vs -21.9±0.7‰, P=0.002 (means±s.d.)), and breath δ(13)C remained elevated at least 2 h post dinner. In conclusion, when shifting between negative and positive energy balance, breath δ(13)C showed anticipated isotopic changes. Although additional research is needed to determine specificity and repeatability, this method may provide a biomarker for marked increases in caloric intake.
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Hardin, Shane C; Tang, Guo-Qing; Scholz, Anke; Holtgraewe, Daniela; Winter, Heike; Huber, Steven C
2003-09-01
Sequence analysis identified serine 170 (S170) of the maize (Zea mays L.) SUS1 sucrose synthase (SUS) protein as a possible, second phosphorylation site. Maize leaves contained two calcium-dependent protein kinase activities and a calcium-independent kinase activity with characteristics of an sucrose non-fermenting 1 (SNF1)-related protein kinase. Phosphorylation of the novel S170 and the known serine 15 (S15) site by these protein kinases was determined in peptide substrates and detected in SUS1 protein substrates utilizing sequence- and phosphorylation-specific antibodies. We demonstrate phosphorylation of S170 in vitro and in vivo. The calcium-dependent protein kinases phosphorylated both S170 and S15, whereas SNF1-related protein kinase activity was restricted to S15. Calcium-dependent protein-kinase-mediated S170 and S15 phosphorylation kinetics were determined in wild-type and mutant SUS1 substrates. These analyses revealed that kinase specificity for S170 was threefold lower than that for S15, and that phosphorylation of S170 was stimulated by prior phosphorylation at the S15 site. The SUS-binding peptides encoded by early nodulin 40 (ENOD40) specifically antagonized S170 phosphorylation in vitro. A model wherein S170 phosphorylation functions as part of a mechanism targeting SUS for proteasome-mediated degradation is supported by the observations that SUS proteolytic fragments: (i) were detected and possessed relatively high phosphorylated-S170 (pS170) stoichiometry; (ii) were spatially coincident with proteasome activity within developing leaves; and (iii) co-sedimented with proteasome activity. In addition, full-length pS170-SUS protein was less stable than S170-SUS in cultured leaf segments and was stabilized by proteasome inhibition. Post-translational control of SUS protein level through pS170-promoted proteolysis may explain the specific and significant decrease in SUS abundance that accompanies the sink-to-source transition in developing maize leaves.
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High density electronic circuit and process for making
Morgan, W.P.
1999-06-29
High density circuits with posts that protrude beyond one surface of a substrate to provide easy mounting of devices such as integrated circuits are disclosed. The posts also provide stress relief to accommodate differential thermal expansion. The process allows high interconnect density with fewer alignment restrictions and less wasted circuit area than previous processes. The resulting substrates can be test platforms for die testing and for multi-chip module substrate testing. The test platform can contain active components and emulate realistic operational conditions, replacing shorts/opens net testing. 8 figs.
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Dikshit, Pritam Kumar; Moholkar, Vijayanand S
2016-09-01
The present study has investigated kinetic features of bioconversion of biodiesel-derived crude glycerol to dihydroxyacetone with immobilized Gluconobacter oxydans cells using modified Haldane substrate-inhibition model. The results have been compared against free cells and pure glycerol. Relative variations in the kinetic parameters KS, KI, Vmax, n and X reveal that immobilized G. oxydans cells (on PU foam substrate) with crude glycerol as substrate give higher order of inhibition (n) and lower maximum reaction velocities (Vmax). These results are essentially implications of substrate transport restrictions across immobilization matrix, which causes retention of substrate in the matrix and reduction in fractional available substrate (X) for the cells. This causes reduction in both KS (substrate concentration at Vmax/2) and KI (inhibition constant) as compared to free cells. For immobilized cells, substrate concentration (Smax) corresponding to Vmax is practically same for both pure and crude glycerol as substrate. Copyright © 2016 Elsevier Ltd. All rights reserved.
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ERIC Educational Resources Information Center
Ngo, Duc Minh
2009-01-01
Current methodologies used for the inference of thin film stresses through curvatures are strictly restricted to stress and curvature states which are assumed to remain uniform over the entire film/substrate system. In this dissertation, we extend these methodologies to non-uniform stress and curvature states for the single layer of thin film or…
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Saneja, Ankit; Dubey, Ravindra Dhar; Alam, Noor; Khare, Vaibhav; Gupta, Prem N
2014-01-01
Scientific community is striving to understand the role of P-glycoprotein (P-gp) in drug discovery programs due to its impact on pharmacokinetic and multi-drug resistance (MDR) of anticancer drugs. A number of efforts to resolve the crystal structure and understanding the mechanism of P-gp mediated efflux have been made. Several generations of Pgp inhibitors have been developed to tackle this multi-specific efflux protein. Unfortunately, these inhibitors lack selectivity, exhibit poor solubility and severe pharmacokinetic interactions restricting their clinical use. The nanocarrier drug delivery systems (NDDS) are receiving increasing attention for P-gp modulating activity of pharmaceutical excipients which are used in their fabrication. In addition, NDDS can enhance the solubility and exhibited ability to bypass P-gp mediated efflux. The co-formulation of P-gp inhibitors and substrate anticancer drugs in single drug delivery system offers the advantage of bypassing P-gp mediated drug efflux as well as inhibiting the P-gp. Moreover, severe pharmacokinetic interactions between P-gp inhibitor and substrate anticancer drugs could be avoided by using this strategy. In this article we describe the co-formulation strategies using nanocarriers for modulation of pharmacokinetics as well as multi-drug resistance of anticancer drugs along with the challenges in this area.
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NASA Astrophysics Data System (ADS)
Marchena, Miriam; Wagner, Frederic; Arliguie, Therese; Zhu, Bin; Johnson, Benedict; Fernández, Manuel; Lai Chen, Tong; Chang, Theresa; Lee, Robert; Pruneri, Valerio; Mazumder, Prantik
2018-07-01
We demonstrate the direct transfer of graphene from Cu foil to rigid and flexible substrates, such as glass and PET, using as an intermediate layer a thin film of polyimide (PI) mixed with an aminosilane (3-aminopropyltrimethoxysilane) or only PI, respectively. While the dry removal of graphene by an adhesive has been previously demonstrated—being removed from graphite by scotch tape or from a Cu foil by thick epoxy (~20 µm) on Si—our work is the first step towards making a substrate ready for device fabrication using the polymer-free technique. Our approach leads to an article that is transparent, thermally stable—up to 350 °C—and free of polymer residues on the device side of the graphene, which is contrary to the case of the standard wet-transfer process using PMMA. Also, in addition to previous novelty, our technique is fast and easier by using current industrial technology—a hot press and a laminator—with Cu recycling by its mechanical peel-off; it provides high interfacial stability in aqueous media and it is not restricted to a specific material—polyimide and polyamic acids can be used. All the previous reasons demonstrate a feasible process that enables device fabrication.
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Structure and function of APH(4)-Ia, a hygromycin B resistance enzyme.
Stogios, Peter J; Shakya, Tushar; Evdokimova, Elena; Savchenko, Alexei; Wright, Gerard D
2011-01-21
The aminoglycoside phosphotransferase (APH) APH(4)-Ia is one of two enzymes responsible for bacterial resistance to the atypical aminoglycoside antibiotic hygromycin B (hygB). The crystal structure of APH(4)-Ia enzyme was solved in complex with hygB at 1.95 Å resolution. The APH(4)-Ia structure adapts a general two-lobe architecture shared by other APH enzymes and eukaryotic kinases, with the active site located at the interdomain cavity. The enzyme forms an extended hydrogen bond network with hygB primarily through polar and acidic side chain groups. Individual alanine substitutions of seven residues involved in hygB binding did not have significant effect on APH(4)-Ia enzymatic activity, indicating that the binding affinity is spread across a distributed network. hygB appeared as the only substrate recognized by APH(4)-Ia among the panel of 14 aminoglycoside compounds. Analysis of the active site architecture and the interaction with the hygB molecule demonstrated several unique features supporting such restricted substrate specificity. Primarily the APH(4)-Ia substrate-binding site contains a cluster of hydrophobic residues that provides a complementary surface to the twisted structure of the substrate. Similar to APH(2″) enzymes, the APH(4)-Ia is able to utilize either ATP or GTP for phosphoryl transfer. The defined structural features of APH(4)-Ia interactions with hygB and the promiscuity in regard to ATP or GTP binding could be exploited for the design of novel aminoglycoside antibiotics or inhibitors of this enzyme.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Stogios, Peter J.; Shakya, Tushar; Evdokimova, Elena
The aminoglycoside phosphotransferase (APH) APH(4)-Ia is one of two enzymes responsible for bacterial resistance to the atypical aminoglycoside antibiotic hygromycin B (hygB). The crystal structure of APH(4)-Ia enzyme was solved in complex with hygB at 1.95 {angstrom} resolution. The APH(4)-Ia structure adapts a general two-lobe architecture shared by other APH enzymes and eukaryotic kinases, with the active site located at the interdomain cavity. The enzyme forms an extended hydrogen bond network with hygB primarily through polar and acidic side chain groups. Individual alanine substitutions of seven residues involved in hygB binding did not have significant effect on APH(4)-Ia enzymatic activity,more » indicating that the binding affinity is spread across a distributed network. hygB appeared as the only substrate recognized by APH(4)-Ia among the panel of 14 aminoglycoside compounds. Analysis of the active site architecture and the interaction with the hygB molecule demonstrated several unique features supporting such restricted substrate specificity. Primarily the APH(4)-Ia substrate-binding site contains a cluster of hydrophobic residues that provides a complementary surface to the twisted structure of the substrate. Similar to APH(2{double_prime}) enzymes, the APH(4)-Ia is able to utilize either ATP or GTP for phosphoryl transfer. The defined structural features of APH(4)-Ia interactions with hygB and the promiscuity in regard to ATP or GTP binding could be exploited for the design of novel aminoglycoside antibiotics or inhibitors of this enzyme.« less
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Depth and type of substrate influence the ability of Nasonia vitripennis to locate a host
Frederickx, Christine; Dekeirsschieter, Jessica; Verheggen, François J.; Haubruge, Eric
2014-01-01
Abstract The foraging behaviour of a parasitoid insect species includes the host’s habitat and subsequent location of the host. Habitats substrate, substrate moisture, and light levels can affect the host searching of different species of parasitoids. However, the depth at which parasitoids concentrate their search effort is another important ecological characteristic and plays an important role in locating a host. Here, we investigated the ability of a pupal parasitoid, Nasonia vitripennis Walker (Hymenoptera: Pteromalidae), to penetrate and kill fly pupae located at different depths of the substrate. Three different types of substrate were tested: loam soil, compost, and vermiculite substrate. In both loam soil and compost, all of the parasitism activity was restricted to pupae placed directly on the surface. Parasitism activity in vermiculite showed that the average number of pupae parasitized decreased with depth of substrate. These results suggest that fly pupae situated deeper in the substrate are less subjected to parasitism by N. vitripennis . PMID:25373205
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Exploiting CRISPR/Cas systems for biotechnology
Sampson, Timothy R.; Weiss, David S.
2015-01-01
The Cas9 endonuclease is the central component of the Type II CRISPR/Cas system, a prokaryotic adaptive restriction system against invading nucleic acids, such as those originating from bacteriophages and plasmids. Recently, this RNA-directed DNA endonuclease has been harnessed to target DNA sequences of interest. Here, we review the development of Cas9 as an important tool to not only edit the genomes of a number of different prokaryotic and eukaryotic species, but also as an efficient system for site-specific transcriptional repression or activation. Additionally, a specific Cas9 protein has been observed to target an RNA substrate, suggesting that Cas9 may have the ability to be programmed to target RNA as well. Cas proteins from other CRISPR/Cas subtypes may also be exploited in this regard. Thus, CRISPR/Cas systems represent an effective and versatile biotechnological tool, which will have significant impact on future advancements in genome engineering. PMID:24323919
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Exploiting CRISPR/Cas systems for biotechnology.
Sampson, Timothy R; Weiss, David S
2014-01-01
The Cas9 endonuclease is the central component of the Type II CRISPR/Cas system, a prokaryotic adaptive restriction system against invading nucleic acids, such as those originating from bacteriophages and plasmids. Recently, this RNA-directed DNA endonuclease has been harnessed to target DNA sequences of interest. Here, we review the development of Cas9 as an important tool to not only edit the genomes of a number of different prokaryotic and eukaryotic species, but also as an efficient system for site-specific transcriptional repression or activation. Additionally, a specific Cas9 protein has been observed to target an RNA substrate, suggesting that Cas9 may have the ability to be programmed to target RNA as well. Cas proteins from other CRISPR/Cas subtypes may also be exploited in this regard. Thus, CRISPR/Cas systems represent an effective and versatile biotechnological tool, which will have significant impact on future advancements in genome engineering. © 2014 WILEY Periodicals, Inc.
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Møller, Jan Kloppenborg; Bergmann, Kirsten Riber; Christiansen, Lasse Engbo; Madsen, Henrik
2012-07-21
In the present study, bacterial growth in a rich media is analysed in a Stochastic Differential Equation (SDE) framework. It is demonstrated that the SDE formulation and smoothened state estimates provide a systematic framework for data driven model improvements, using random walk hidden states. Bacterial growth is limited by the available substrate and the inclusion of diffusion must obey this natural restriction. By inclusion of a modified logistic diffusion term it is possible to introduce a diffusion term flexible enough to capture both the growth phase and the stationary phase, while concentration is restricted to the natural state space (substrate and bacteria non-negative). The case considered is the growth of Salmonella and Enterococcus in a rich media. It is found that a hidden state is necessary to capture the lag phase of growth, and that a flexible logistic diffusion term is needed to capture the random behaviour of the growth model. Further, it is concluded that the Monod effect is not needed to capture the dynamics of bacterial growth in the data presented. Copyright © 2012 Elsevier Ltd. All rights reserved.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Cummings, J.; Fedorov, A; Xu, C
The catalytic activities of three members of the amidohydrolase superfamily were discovered using amino acid substrate libraries. Bb3285 from Bordetella bronchiseptica, Gox1177 from Gluconobacter oxidans, and Sco4986 from Streptomyces coelicolor are currently annotated as d-aminoacylases or N-acetyl-d-glutamate deacetylases. These three enzymes are 22-34% identical to one another in amino acid sequence. Substrate libraries containing nearly all combinations of N-formyl-d-Xaa, N-acetyl-d-Xaa, N-succinyl-d-Xaa, and l-Xaa-d-Xaa were used to establish the substrate profiles for these enzymes. It was demonstrated that Bb3285 is restricted to the hydrolysis of N-acyl-substituted derivatives of d-glutamate. The best substrates for this enzyme are N-formyl-d-glutamate (k{sub cat}/K{sub m} =more » 5.8 x 10{sup 6} M{sup -1} s{sup -1}), N-acetyl-d-glutamate (k{sub cat}/K{sub m} = 5.2 x 10{sup 6} M{sup -1} s{sup -1}), and l-methionine-d-glutamate (k{sub cat}/K{sub m} = 3.4 x 10{sup 5} M{sup -1} s{sup -1}). Gox1177 and Sco4986 preferentially hydrolyze N-acyl-substituted derivatives of hydrophobic d-amino acids. The best substrates for Gox1177 are N-acetyl-d-leucine (k{sub cat}/K{sub m} = 3.2 x 104 M{sup -1} s-1), N-acetyl-d-tryptophan (kcat/Km = 4.1 x 104 M-1 s-1), and l-tyrosine-d-leucine (kcat/Km = 1.5 x 104 M-1 s-1). A fourth protein, Bb2785 from B. bronchiseptica, did not have d-aminoacylase activity. The best substrates for Sco4986 are N-acetyl-d-phenylalanine and N-acetyl-d-tryptophan. The three-dimensional structures of Bb3285 in the presence of the product acetate or a potent mimic of the tetrahedral intermediate were determined by X-ray diffraction methods. The side chain of the d-glutamate moiety of the inhibitor is ion-paired to Arg-295, while the {alpha}-carboxylate is ion-paired with Lys-250 and Arg-376. These results have revealed the chemical and structural determinants for substrate specificity in this protein. Bioinformatic analyses of an additional {approx}250 sequences identified as members of this group suggest that there are no simple motifs that allow prediction of substrate specificity for most of these unknowns, highlighting the challenges for computational annotation of some groups of homologous proteins.« less
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R Factor-Controlled Restriction and Modification of Deoxyribonucleic Acid: Restriction Mutants
Yoshimori, Robert; Roulland-Dussoix, Daisy; Boyer, Herbert W.
1972-01-01
Restriction mutants of two different R factor-controlled host specificities (RI and RII) were isolated. All of the restriction mutants examined had a normal modification phenotype. No complementation was observed between the RI and RII host specificities. It is concluded that for each host specificity no protein subunit is shared by the restriction endonuclease and modification methylase. PMID:4565538
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The GM1 and GM2 Gangliosidoses: Natural History and Progress toward Therapy.
Regier, Debra S; Proia, Richard L; D'Azzo, Alessandra; Tifft, Cynthia J
2016-06-01
The gangliosidoses are lysosomal storage disorders caused by accumulation of GM1 or GM2 gangliosides. GM1 gangliosidosis has both central nervous system and systemic findings; while, GM2 gangliosidosis is restricted primarily to the central nervous system. Both disorders have autosomal recessive modes of inheritance and a continuum of clinical presentations from a severe infantile form to a milder, chronic adult form. Both are devastating diseases without cure or specific treatment however, with the use of supportive aggressive medical management, the lifespan and quality of life has been extended for both diseases. Naturally occurring and engineered animal models that mimic the human diseases have enhanced our understanding of the pathogenesis of disease progression. Some models have shown significant improvement in symptoms and lifespan with enzyme replacement, substrate reduction, and anti-inflammatory treatments alone or in combination. More recently gene therapy has shown impressive results in large and small animal models. Treatment with FDA-approved glucose analogs to reduce the amount of ganglioside substrate is used as off-label treatments for some patients. Therapies also under clinical development include small molecule chaperones and gene therapy.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Artavanis-Tsakonas, Katerina; Weihofen, Wilhelm A.; Antos, John M.
Like their human hosts, Plasmodium falciparum parasites rely on the ubiquitin-proteasome system for survival. We previously identified PfUCHL3, a deubiquitinating enzyme, and here we characterize its activity and changes in active site architecture upon binding to ubiquitin. We find strong evidence that PfUCHL3 is essential to parasite survival. The crystal structures of both PfUCHL3 alone and in complex with the ubiquitin-based suicide substrate UbVME suggest a rather rigid active site crossover loop that likely plays a role in restricting the size of ubiquitin adduct substrates. Molecular dynamics simulations of the structures and a model of the PfUCHL3-PfNedd8 complex allowed themore » identification of shared key interactions of ubiquitin and PfNedd8 with PfUCHL3, explaining the dual specificity of this enzyme. Distinct differences observed in ubiquitin binding between PfUCHL3 and its human counterpart make it likely that the parasitic DUB can be selectively targeted while leaving the human enzyme unaffected.« less
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Dual-Responsive SPMA-Modified Polymer Photonic Crystals and Their Dynamic Display Patterns.
Gao, Zewen; Gao, Dongsheng; Huang, Chao; Zhang, Hanbing; Guo, Jinbao; Wei, Jie
2018-05-28
Light and electrothermal responsive polymer photonic crystals (PCs) modified with 1'-acryloyl chloride-3',3'-dimethyl-6-nitro-spiro(2H-1-benzopyran-2,2'-indoline) (SPMA) are proposed, and their dynamic display patterns are achieved through the combination of the SPMA-modified PCs and a patterned graphite layer. These PCs exhibit fluorescence under UV light irradiation because of the isomerization of the SPMA, which is restricted in the shell of the polymer colloidal spheres. After a voltage is applied to the patterned graphite layer, the fluorescence of PCs in the specific area disappears, and dynamic display patterns are obtained. Under UV light irradiation, the PCs change from the "partial-fluorescence" state to the initial "fluorescence" state, and the patterns disappear. Using this technique, the PC pattern "M L N" on the glass substrate and PC patterns from "0" to "9" on the paper substrate are fabricated. Thus, these dual-responsive PCs have potential applications in information recording, anticounterfeiting, dynamic display, and photoelectric devices. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Key advances in the chemical modification of nanocelluloses.
Habibi, Youssef
2014-03-07
Nanocelluloses, including nanocrystalline cellulose, nanofibrillated cellulose and bacterial cellulose nanofibers, have become fascinating building blocks for the design of new biomaterials. Derived from the must abundant and renewable biopolymer, they are drawing a tremendous level of attention, which certainly will continue to grow in the future driven by the sustainability trend. This growing interest is related to their unsurpassed quintessential physical and chemical properties. Yet, owing to their hydrophilic nature, their utilization is restricted to applications involving hydrophilic or polar media, which limits their exploitation. With the presence of a large number of chemical functionalities within their structure, these building blocks provide a unique platform for significant surface modification through various chemistries. These chemical modifications are prerequisite, sometimes unavoidable, to adapt the interfacial properties of nanocellulose substrates or adjust their hydrophilic-hydrophobic balance. Therefore, various chemistries have been developed aiming to surface-modify these nano-sized substrates in order to confer to them specific properties, extending therefore their use to highly sophisticated applications. This review collocates current knowledge in the research and development of nanocelluloses and emphasizes more particularly on the chemical modification routes developed so far for their functionalization.
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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
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Kaminishi, Tatsuya; Schedlbauer, Andreas; Fabbretti, Attilio; Brandi, Letizia; Ochoa-Lizarralde, Borja; He, Cheng-Guang; Milón, Pohl; Connell, Sean R.; Gualerzi, Claudio O.; Fucini, Paola
2015-01-01
Hygromycin A (HygA) binds to the large ribosomal subunit and inhibits its peptidyl transferase (PT) activity. The presented structural and biochemical data indicate that HygA does not interfere with the initial binding of aminoacyl-tRNA to the A site, but prevents its subsequent adjustment such that it fails to act as a substrate in the PT reaction. Structurally we demonstrate that HygA binds within the peptidyl transferase center (PTC) and induces a unique conformation. Specifically in its ribosomal binding site HygA would overlap and clash with aminoacyl-A76 ribose moiety and, therefore, its primary mode of action involves sterically restricting access of the incoming aminoacyl-tRNA to the PTC. PMID:26464437
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Rohrer, Karin M; Haug, Markus; Schwörer, Daniela; Kalbacher, Hubert; Holzer, Ursula
2014-01-01
Heat-shock protein 70 (Hsp70)–peptide complexes are involved in MHC class I-and II-restricted antigen presentation, enabling enhanced activation of T cells. As shown previously, mammalian cytosolic Hsp70 (Hsc70) molecules interact specifically with HLA-DR molecules. This interaction might be of significance as Hsp70 molecules could transfer bound antigenic peptides in a ternary complex into the binding groove of HLA-DR molecules. The present study provides new insights into the distinct interaction of Hsp70 with HLA-DR molecules. Using a quantitative binding assay, it could be demonstrated that a point mutation of amino acids alanine 406 and valine 438 in the substrate binding pocket led to reduced peptide binding compared with the wild-type Hsp70 whereas HLA-DR binding remains unaffected. The removal of the C-terminal lid neither altered the substrate binding capacity nor the Hsp70 binding characteristics to HLA-DR. A truncated variant lacking the nucleotide binding domain showed no binding interactions with HLA-DR. Furthermore, the truncated ATPase subunit of constitutively expressed Hsc70 revealed similar binding affinities to HLA-DR compared with the complete Hsc70. Hence, it can be assumed that the Hsp70–HLA-DR interaction takes place outside the peptide binding groove and is attributed to the ATPase domain of HSP70 molecules. The Hsp70-chaperoned peptides might thereby be directly transferred into the binding groove of HLA-DR, so enabling enhanced presentation of the peptide on antigen-presenting cells and leading to an improved proliferation of responding T cells as shown previously. PMID:24428437
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Datta, Kamal; Weinfeld, Michael; Neumann, Ronald D; Winters, Thomas A
2007-02-01
End groups contribute to the structural complexity of radiation-induced DNA double-strand breaks (DSBs). As such, end-group structures may affect a cell's ability to repair DSBs. The 3'-end groups of strand breaks caused by gamma radiation, or oxidative processes, under oxygenated aqueous conditions have been shown to be distributed primarily between 3'-phosphoglycolate and 3'-phosphate, with 5'-phosphate ends in both cases. In this study, end groups of the high-LET-like DSBs caused by 125I decay were investigated. Site-specific DNA double-strand breaks were produced in plasmid pTC27 in the presence or absence of 2 M DMSO by 125I-labeled triplex-forming oligonucleotide targeting. End-group structure was assessed enzymatically as a function of the DSB end to serve as a substrate for ligation and various forms of end labeling. Using this approach, we have demonstrated 3'-hydroxyl (3'-OH) and 3'-phosphate (3'-P) end groups and 5'-ends (> or = 42%) terminated by phosphate. A 32P postlabeling assay failed to detect 3'-phosphoglycolate in a restriction fragment terminated by the 125I-induced DNA double-strand break, and this is likely due to restricted oxygen diffusion during irradiation as a frozen aqueous solution. Even so, end-group structure and relative distribution varied as a function of the free radical scavenging capacity of the irradiation buffer.
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Santos-López, Gerardo; Borraz-Argüello, María T; Márquez-Domínguez, Luis; Flores-Alonso, Juan Carlos; Ramírez-Mendoza, Humberto; Priem, Bernard; Fort, Sébastien; Vallejo-Ruiz, Verónica; Reyes-Leyva, Julio; Herrera-Camacho, Irma
2017-10-01
Porcine rubulavirus (PorPV), also known as La Piedad Michoacan Virus (LPMV) causes encephalitis and reproductive failure in newborn and adult pigs, respectively. The hemagglutinin-neuraminidase (HN) glycoprotein is the most exposed and antigenic of the virus proteins. HN plays central roles in PorPV infection; i.e., it recognizes sialic acid-containing cell receptors that mediate virus attachment and penetration; in addition, its neuraminidase (sialic acid releasing) activity has been proposed as a virulence factor. This work describes the purification and characterization of PorPV HN protein (isolate PAC1). The specificity of neuraminidase is restricted to sialyl(α2,3)lactose (3SL). HN showed typical Michaelis-Menten kinetics with fetuin as substrate (km=0.029μM, Vmax=522.8nmolmin -1 mg -1 ). When 3SL was used as substrate, typical cooperative kinetics were found (S 50 =0.15μM, Vmax=154.3nmolmin -1 mg -1 ). The influenza inhibitor zanamivir inhibited the PorPV neuraminidase with IC 50 of 0.24μM. PorPV neuraminidase was activated by Ca 2+ and inhibited by nucleoside triphosphates with the level of inhibition depending on phosphorylation level. The present results open possibilities to study the role of neuraminidase in the pathogenicity of PorPV infection and its potential inhibitors. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Prasad, Bhim Bali; Madhuri, Rashmi; Tiwari, Mahavir Prasad; Sharma, Piyush Sindhu
2010-05-15
Molecularly imprinted polymers (MIPs) are often electrically insulating materials. Due to the presence of diffusion barrier(s) in between such MIP coating and electrode surface and the absence of a direct path for the conduction of electrons from the binding sites to the electrode, the development of electrochemical sensor is significantly restricted. The direct use of MIPs those possess intrinsic electron-transport properties, is highly limited. These problems are resolved by the design of an original, substrate-selective MIP-fiber sensor that combines conventional insulating MIP and conducting carbon powder in consolidated phase. A layer of conducting carbon particles, arranged orderly as 'carbon strip', is inducted in the polymer for direct electronic conduction. MIP-carbon composite (monolithic fiber) in this work is prepared via in situ free radical polymerization of a new monomer (2,4,6-trisacrylamido-1,3,5-triazine, TAT) and subsequent cross-linkage with ethylene glycol dimethacrylate, in the presence of carbon powder and template (folic acid), at 55 degrees C in a glass capillary. The detection of folic acid with the MIP-fiber sensor was found to be specific and quantitative (detection limit 0.20 ng mL(-1), RSD=1.3%, S/N=3), in aqueous, blood serum and pharmaceutical samples, without any problem of non-specific false-positive contribution and cross-reactivity. 2010 Elsevier B.V. All rights reserved.
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Arroyo-Caro, José María; Chileh, Tarik; Kazachkov, Michael; Zou, Jitao; Alonso, Diego López; García-Maroto, Federico
2013-02-01
The multigene family encoding proteins related to lysophosphatidyl-acyltransferases (LPATs) has been analyzed in the castor plant Ricinus communis. Among them, two genes designated RcLPAT2 and RcLPATB, encoding proteins with LPAT activity and expressed in the developing seed, have been cloned and characterized in some detail. RcLPAT2 groups with well characterized members of the so-called A-class LPATs and it shows a generalized expression pattern in the plant and along seed development. Enzymatic assays of RcLPAT2 indicate a preference for ricinoleoyl-CoA over other fatty acid thioesters when ricinoleoyl-LPA is used as the acyl acceptor, while oleoyl-CoA is the preferred substrate when oleoyl-LPA is employed. RcLPATB groups with B-class LPAT enzymes described as seed specific and selective for unusual fatty acids. However, RcLPATB exhibit a broad specificity on the acyl-CoAs, with saturated fatty acids (12:0-16:0) being the preferred substrates. RcLPATB is upregulated coinciding with seed triacylglycerol accumulation, but its expression is not restricted to the seed. These results are discussed in the light of a possible role for LPAT isoenzymes in the channelling of ricinoleic acid into castor bean triacylglycerol. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.
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Seafloor mapping and benthic habitat GIS for southern California, volume III
Cochrane, Guy R.; Golden, Nadine E.; Dartnell, Pete; Schroeder, Donna M.; Finlayson, David P.
2007-01-01
From August 8-27, 2005, more than 75 km of the continental shelf (Fig. 1) in water depths of 20-70m southeast of Santa Barbara, were surveyed during the USGS cruise S-1-05-SC (http://walrus.wr.usgs.gov/infobank/s/s105sc/html/s-1-05-sc.meta.html). Both Interferometric sonar and 14 hours of both vertical and oblique georeferenced submarine digital video were collected to (1) obtain geophysical data (bathymetry and acoustic reflectance), (2) examine and record geologic characteristics of the sea floor, and (3) construct maps of seafloor geomorphology and habitat distribution. Substrate distribution is predicted using a modified version of Cochrane and Lafferty (2002) video-supervised statistical classification of sonar data that includes derivatives of bathymetry data. Specific details of the methods can be found in the meatadata of the bathymetry data file. Substrates observed are predominantly sand with some rock. Rocky substrates were restricted primarily to an east-west trending bathymetric high 2,000 m north of oil platforms. This is an updated report (version 2.0) from the earlier 2007-1271 (version 1.0) open-file report. This updated report re-releases the data files in UTM, zone 11, WGS84 coordinates. Also, the bathymetry data has been corrected for a vertical offset discovered in the earlier 2007-1271 (version 1.0) report.
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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
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NASA Astrophysics Data System (ADS)
To, Anthony; Downs, Corey; Fu, Elain
2017-05-01
Wax printing has become a common method of fabricating channels in cellulose-based microfluidic devices. However, a limitation of wax printing is that it is restricted to relatively thin, smooth substrates that are compatible with processing by a commercial wax printer. In the current report, we describe a simple patterning method that extends the utility of wax printers for creating hydrophobic barriers on non-standard porous substrates via a process called wax transfer printing. We demonstrate the use of multiple wax transfer cycles to create well-defined, robust, and reproducible barriers in a thick cellulose substrate that is not compatible with feeding through a wax printer. We characterize the method for (i) wax spreading within the substrate as a function of heating time, (ii) the ability to create functional barriers in a substrate, and (iii) reproducibility in line width.
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NASA Astrophysics Data System (ADS)
Azzam, Rasheed M. A.; Angel, Wade W.
1992-12-01
A reflective division-of-wavefront polarizing beam splitter is described that uses a dual- thickness transparent thin-film coating on a metal substrate. A previous design that used a partially clad substrate at the principal angle of the metal [Azzam, JOSA A 5, 1576 (1988)] is replaced by a more general one in which the substrate is coated throughout and the film thickness alternates between two non-zero levels. The incident linear polarization azimuth is chosen near, but not restricted to, 45 degree(s) (measured from the plane of incidence), and the angle of incidence may be selected over a range of values. The design procedure, which uses the two-dimensional Newton-Raphson method, is applied to the SiO2-Au film- substrate system at 633 nm wavelength, as an example, and the characteristics of the various possible coatings are presented.
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Antenatal glucocorticoid treatment of the growth-restricted fetus: benefit or cost?
Morrison, Janna L; Orgeig, Sandra
2009-06-01
Women at risk of preterm labor are commonly treated with antenatal glucocorticoids to reduce neonatal complications, including respiratory distress syndrome. Despite the benefits of antenatal glucocorticoid for neonatal lung function, they are associated with negative cardiovascular outcomes. Among this population, there is a group of intrauterine growth-restricted fetuses in which substrate supply is reduced and these fetuses must undergo a range of cardiovascular adaptations to survive. Interestingly, the cardiovascular changes caused by antenatal glucocorticoid in normally grown fetuses are contrary to the cardiovascular adaptations that the intrauterine growth-restricted fetus must make to survive. Hence, the possibility exists that antenatal glucocorticoid in intrauterine growth-restricted infants may compromise cardiovascular development. This review first provides an overview of general antenatal glucocorticoid effects, before outlining the effects on cardiorespiratory development in normally grown fetuses, the cardiovascular adaptations that occur in the intrauterine growth-restricted fetus and finally integrating this with the very limited evidence for the effect of antenatal glucocorticoid in intrauterine growth-restricted infants.
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10 CFR 810.9 - Restrictions on general and specific authorization.
Code of Federal Regulations, 2013 CFR
2013-01-01
... 10 Energy 4 2013-01-01 2013-01-01 false Restrictions on general and specific authorization. 810.9 Section 810.9 Energy DEPARTMENT OF ENERGY ASSISTANCE TO FOREIGN ATOMIC ENERGY ACTIVITIES § 810.9 Restrictions on general and specific authorization. A general or specific authorization granted by the...
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10 CFR 810.9 - Restrictions on general and specific authorization.
Code of Federal Regulations, 2012 CFR
2012-01-01
... 10 Energy 4 2012-01-01 2012-01-01 false Restrictions on general and specific authorization. 810.9 Section 810.9 Energy DEPARTMENT OF ENERGY ASSISTANCE TO FOREIGN ATOMIC ENERGY ACTIVITIES § 810.9 Restrictions on general and specific authorization. A general or specific authorization granted by the...
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10 CFR 810.9 - Restrictions on general and specific authorization.
Code of Federal Regulations, 2011 CFR
2011-01-01
... 10 Energy 4 2011-01-01 2011-01-01 false Restrictions on general and specific authorization. 810.9 Section 810.9 Energy DEPARTMENT OF ENERGY ASSISTANCE TO FOREIGN ATOMIC ENERGY ACTIVITIES § 810.9 Restrictions on general and specific authorization. A general or specific authorization granted by the...
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10 CFR 810.9 - Restrictions on general and specific authorization.
Code of Federal Regulations, 2014 CFR
2014-01-01
... 10 Energy 4 2014-01-01 2014-01-01 false Restrictions on general and specific authorization. 810.9 Section 810.9 Energy DEPARTMENT OF ENERGY ASSISTANCE TO FOREIGN ATOMIC ENERGY ACTIVITIES § 810.9 Restrictions on general and specific authorization. A general or specific authorization granted by the...
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10 CFR 810.9 - Restrictions on general and specific authorization.
Code of Federal Regulations, 2010 CFR
2010-01-01
... 10 Energy 4 2010-01-01 2010-01-01 false Restrictions on general and specific authorization. 810.9 Section 810.9 Energy DEPARTMENT OF ENERGY ASSISTANCE TO FOREIGN ATOMIC ENERGY ACTIVITIES § 810.9 Restrictions on general and specific authorization. A general or specific authorization granted by the...
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NASA Astrophysics Data System (ADS)
Abe, Masanori; Nakagawa, Hidenobu; Gomi, Manabu; Nomura, Shoichiro
1982-01-01
The film thickness allowance and the waveguide length in a 3-layer (substrate/film/air) magneto-optical unidirectional TE-TM mode converter which utilizes the intrinsic birefringence in an anisotropic material are calculated at λ0{=}1.55 μm. The film material should be gyrotropic in order to make the waveguide length short, and the film thickness allowance is relaxed by reducing the ratio of the dielectric constant of the film to that of the substrate. When the waveguide is made of an isotropic gyrotropic film of YIG deposited on an anisotropic substrate (which may be gyrotropic or not), the restriction on the film thickness can in practice be removed, but this requires precise control of the dielectric constant of the film and the substrate instead.
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Gentili, Sheridan; Morrison, Janna L; McMillen, I Caroline
2009-06-01
Fetal adaptations to periods of substrate deprivation can result in the programming of glucose intolerance, insulin resistance, and metabolic dysfunction in later life. Placental insufficiency can be associated with either sparing or sacrifice of fetal liver growth, and these different responses may have different metabolic consequences. It is unclear what intrahepatic mechanisms determine the differential responses of the fetal liver to substrate restriction. We investigated the effects of placental restriction (PR) on liver growth and the hepatic expression of SLC2A1, IGF1, IGF2, IGF1R, IGF2R, PPARGC1A, PPARA, PRKAA1, PRKAA2, PCK2, and HSDL1 mRNA in fetal sheep at 140-145 days of gestation. A mean gestational arterial partial pressure of oxygen less than 17 mmHg was defined as hypoxic, and a relative liver of weight more than 2 SD below the mean liver weight of controls was defined as reduced liver growth. Fetuses therefore were defined as control-normoxic (C-N; n = 9), PR-normoxic (PR-N; n = 7), PR-hypoxic (PR-H; n = 8), or PR-hypoxic reduced liver growth (PR-H RLG; n = 4). Hepatic SLC2A1 mRNA expression was highest (P < 0.05) in the PR-H fetuses, in which liver growth was maintained. Expression of IGF1 mRNA was decreased (P < 0.05) only in the PR-H RLG group. Hepatic expression of HSDL1, PPARGC1A, and PCK2 mRNA also were increased (P < 0.05) in the PR-H RLG fetuses. The present study highlights that intrahepatic responses to fetal substrate restriction may exist that protect the liver from decreased growth and, potentially, from a decreased responsiveness to the actions of insulin in postnatal life.
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2014-01-01
Background Bacterial species coexist commonly in mixed communities, for instance those occurring in microbial infections of humans. Interspecies effects contribute to alterations in composition of communities with respect to species and thus, to the course and severity of infection. Therefore, knowledge concerning growth and viability of single species in medically-relevant mixed communities is of high interest to resolve complexity of interspecies dynamics and to support development of treatment strategies. In this study, a flow cytometric method was established to assess the species-specific viability in defined three-species mixed cultures. The method enables the characterization of viability of Pseudomonas aeruginosa, Burkholderia cepacia and Staphylococcus aureus, which are relevant to lung infections of Cystic Fibrosis (CF) patients. The method combines fluorescence detection by antibody and lectin labeling with viability fluorescence staining using SYBR®Green I and propidium iodide. In addition, species-specific cell enumeration analysis using quantitative terminal restriction fragment length polymorphisms (qT-RFLP) was used to monitor the growth dynamics. Finally, to investigate the impact of substrate availability on growth and viability, concentrations of main substrates and metabolites released were determined. Results For each species, the time course of growth and viability during mixed culture cultivations was obtained by using qT-RFLP analysis in combination with flow cytometry. Comparison between mixed and pure cultures revealed for every species differences in growth properties, e.g. enhanced growth of P. aeruginosa in mixed culture. Differences were also observed for B. cepacia and S. aureus in the time course of viability, e.g. an early and drastic reduction of viability of S. aureus in mixed culture. Overall, P. aeruginosa clearly dominated the mixed culture with regard to obtained cell concentrations. Conclusions In combination with qT-RFLP analysis, the methods enabled monitoring of species-specific cell concentrations and viability during co-cultivation of theses strains. Experimental findings suggest that the predominance of P. aeruginosa over B. cepacia and S. aureus in mixed culture under the chosen cultivation conditions is promoted by more efficient substrate consumption of P. aeruginosa, and antagonistic interspecies effects induced by P. aeruginosa. PMID:24606608
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Steroid hormones specifically modify the activity of organic anion transporting polypeptides.
Koenen, Anna; Köck, Kathleen; Keiser, Markus; Siegmund, Werner; Kroemer, Heyo K; Grube, Markus
2012-11-20
Previously, the steroid hormone progesterone has been demonstrated to stimulate OATP2B1-mediated transport of estrone-3-sulphate (E(1)S), dehydroepiandrosterone sulphate (DHEAS) and pregnenolone sulphate (PS), which may influence the uptake of precursor molecules for steroid hormone synthesis. However, it is unclear whether OATP2B1 drug substrates like atorvastatin or glibenclamide are also affected by this phenomenon. In addition, it has not been studied so far if this stimulatory effect is specific for OATP2B1. To address these questions, we examined the influence of progesterone on OATP2B1-mediated atorvastatin and glibenclamide uptake and studied the impact of steroid hormones on the transport activity of OATP1A2, OATP1B1 and OATP1B3. Comparison of the substrate spectrum of the investigated OATPs revealed that DHEAS and atorvastatin are substrates of all transporters, while E(1)S was only significantly transported by OATP1A2, OATP2B1 and OATP1B1. Glibenclamide uptake was limited to OATP1A2, OATP1B1 and OATP2'B1. Subsequent interaction studies indicated that progesterone only increases OATP2B1-mediated E(1)S and DHEAS transport, whereas uptake of BSP, atorvastatin and glibenclamide was either inhibited or not affected. Moreover, the steroid hormone effect was specific for OATP2B1; neither OATP1B1, OATP1B3 nor OATP1A2 function was stimulated in the presence of progesterone. Similar to progesterone, the glucocorticoide dexamethasone stimulated OATP2B1-mediated transport of E(1)S and DHEAS (EC(50) for E(1)S: 10.2 ± 5.6 μM and 17.9 ± 15.4 μM for DHEAS). In conclusion, our data demonstrate that among the tested compounds the stimulatory effect of progesterone is specific for OATP2B1 and restricted to sulphated steroids like E(1)S and DHEAS while the OATP-mediated drug transport is not enhanced. Copyright © 2012 Elsevier B.V. All rights reserved.
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21 CFR 809.30 - Restrictions on the sale, distribution and use of analyte specific reagents.
Code of Federal Regulations, 2013 CFR
2013-04-01
... analyte specific reagents. 809.30 Section 809.30 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT... Requirements for Manufacturers and Producers § 809.30 Restrictions on the sale, distribution and use of analyte specific reagents. (a) Analyte specific reagents (ASR's) (§ 864.4020 of this chapter) are restricted...
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21 CFR 809.30 - Restrictions on the sale, distribution and use of analyte specific reagents.
Code of Federal Regulations, 2012 CFR
2012-04-01
... analyte specific reagents. 809.30 Section 809.30 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT... Requirements for Manufacturers and Producers § 809.30 Restrictions on the sale, distribution and use of analyte specific reagents. (a) Analyte specific reagents (ASR's) (§ 864.4020 of this chapter) are restricted...
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21 CFR 809.30 - Restrictions on the sale, distribution and use of analyte specific reagents.
Code of Federal Regulations, 2014 CFR
2014-04-01
... analyte specific reagents. 809.30 Section 809.30 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT... Requirements for Manufacturers and Producers § 809.30 Restrictions on the sale, distribution and use of analyte specific reagents. (a) Analyte specific reagents (ASR's) (§ 864.4020 of this chapter) are restricted...
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Substrate channel in nitrogenase revealed by a molecular dynamics approach.
Smith, Dayle; Danyal, Karamatullah; Raugei, Simone; Seefeldt, Lance C
2014-04-15
Mo-dependent nitrogenase catalyzes the biological reduction of N2 to two NH3 molecules at FeMo-cofactor buried deep inside the MoFe protein. Access of substrates, such as N2, to the active site is likely restricted by the surrounding protein, requiring substrate channels that lead from the surface to the active site. Earlier studies on crystallographic structures of the MoFe protein have suggested three putative substrate channels. Here, we have utilized submicrosecond atomistic molecular dynamics simulations to allow the nitrogenase MoFe protein to explore its conformational space in an aqueous solution at physiological ionic strength, revealing a putative substrate channel. The viability of this observed channel was tested by examining the free energy of passage of N2 from the surface through the channel to FeMo-cofactor, resulting in the discovery of a very low energy barrier. These studies point to a viable substrate channel in nitrogenase that appears during thermal motions of the protein in an aqueous environment and that approaches a face of FeMo-cofactor earlier implicated in substrate binding.
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An AAA Motor-Driven Mechanical Switch in Rpn11 Controls Deubiquitination at the 26S Proteasome.
Worden, Evan J; Dong, Ken C; Martin, Andreas
2017-09-07
Poly-ubiquitin chains direct protein substrates to the 26S proteasome, where they are removed by the deubiquitinase Rpn11 during ATP-dependent substrate degradation. Rapid deubiquitination is required for efficient degradation but must be restricted to committed substrates that are engaged with the ATPase motor to prevent premature ubiquitin chain removal and substrate escape. Here we reveal the ubiquitin-bound structure of Rpn11 from S. cerevisiae and the mechanisms for mechanochemical coupling of substrate degradation and deubiquitination. Ubiquitin binding induces a conformational switch of Rpn11's Insert-1 loop from an inactive closed state to an active β hairpin. This switch is rate-limiting for deubiquitination and strongly accelerated by mechanical substrate translocation into the AAA+ motor. Deubiquitination by Rpn11 and ubiquitin unfolding by the ATPases are in direct competition. The AAA+ motor-driven acceleration of Rpn11 is therefore important to ensure that poly-ubiquitin chains are removed only from committed substrates and fast enough to prevent their co-degradation. Copyright © 2017 Elsevier Inc. All rights reserved.
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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.
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Control of Growth Rate by Initial Substrate Concentration at Values Below Maximum Rate
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
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High-throughput kinase assays with protein substrates using fluorescent polymer superquenching.
Rininsland, Frauke; Stankewicz, Casey; Weatherford, Wendy; McBranch, Duncan
2005-05-31
High-throughput screening is used by the pharmaceutical industry for identifying lead compounds that interact with targets of pharmacological interest. Because of the key role that aberrant regulation of protein phosphorylation plays in diseases such as cancer, diabetes and hypertension, kinases have become one of the main drug targets. With the exception of antibody-based assays, methods to screen for specific kinase activity are generally restricted to the use of small synthetic peptides as substrates. However, the use of natural protein substrates has the advantage that potential inhibitors can be detected that affect enzyme activity by binding to a site other than the catalytic site. We have previously reported a non-radioactive and non-antibody-based fluorescence quench assay for detection of phosphorylation or dephosphorylation using synthetic peptide substrates. The aim of this work is to develop an assay for detection of phosphorylation of chemically unmodified proteins based on this polymer superquenching platform. Using a modified QTL Lightspeed assay, phosphorylation of native protein was quantified by the interaction of the phosphorylated proteins with metal-ion coordinating groups co-located with fluorescent polymer deposited onto microspheres. The binding of phospho-protein inhibits a dye-labeled "tracer" peptide from associating to the phosphate-binding sites present on the fluorescent microspheres. The resulting inhibition of quench generates a "turn on" assay, in which the signal correlates with the phosphorylation of the substrate. The assay was tested on three different proteins: Myelin Basic Protein (MBP), Histone H1 and Phosphorylated heat- and acid-stable protein (PHAS-1). Phosphorylation of the proteins was detected by Protein Kinase Calpha (PKCalpha) and by the Interleukin -1 Receptor-associated Kinase 4 (IRAK4). Enzyme inhibition yielded IC50 values that were comparable to those obtained using peptide substrates. Statistical parameters that are used in the high-throughput community to determine assay robustness (Z'-value) demonstrate the suitability of this format for high-throughput screening applications for detection of inhibitors of enzyme activity. The QTL Lightspeed protein detection system provides a simple mix and measure "turn on" assay for the detection of kinase activity using natural protein substrates. The platform is robust and allows for identification of inhibitors of kinase activity.
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Janowski, Karen M.; Ledbetter, Stephanie; Mayo, Matthew S.; Hockett, Richard D.
1997-01-01
Control of the rearrangement and expression of the T cell receptor α and δ chains is critical for determining T cell type. The process of δ deletion is a candidate mechanism for maintaining separation of the α and δ loci. Mice harboring a transgenic reporter δ deletion construct show α/β T cell lineage–specific use of the transgenic elements. A 48-basepair segment of DNA, termed HPS1A, when deleted from this reporter construct, loses tight lineage-specific rearrangement control of transgenic elements, with abundant rearrangements of transgenic δ-deleting elements now in γ/δ T cells. Furthermore, HPS1A augments recombination frequency of extrachromosomal substrates in an in vitro recombination assay. DNA binding proteins recognizing HPS1A have been identified and are restricted to early B and T cells, during the time of active rearrangement of endogenous TCR and immunoglobulin loci. These data are consistent with δ deletion playing an important role in maintaining separate TCR α and δ loci. PMID:9207011
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NASA Astrophysics Data System (ADS)
Kang, Dongseok; Lee, Sung-Min; Kwong, Anthony; Yoon, Jongseung
2015-03-01
Despite many unique advantages, vertical cavity surface emitting lasers (VCSELs) have been available mostly on rigid, planar wafers over restricted areas, thereby limiting their usage for applications that can benefit from large-scale, programmable assemblies, hybrid integration with dissimilar materials and devices, or mechanically flexible constructions. Here, materials design and fabrication strategies that address these limitations of conventional VCSELs are presented. Specialized design of epitaxial materials and etching processes, together with printing-based deterministic assemblies and substrate thermal engineering, enabled defect-free release of microscale VCSELs and their device- and circuit-level implementation on non-native, flexible substrates with performance comparable to devices on the growth substrate.
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Trim25 Is an RNA-Specific Activator of Lin28a/TuT4-Mediated Uridylation.
Choudhury, Nila Roy; Nowak, Jakub S; Zuo, Juan; Rappsilber, Juri; Spoel, Steven H; Michlewski, Gracjan
2014-11-20
RNA binding proteins have thousands of cellular RNA targets and often exhibit opposite or passive molecular functions. Lin28a is a conserved RNA binding protein involved in pluripotency and tumorigenesis that was previously shown to trigger TuT4-mediated pre-let-7 uridylation, inhibiting its processing and targeting it for degradation. Surprisingly, despite binding to other pre-microRNAs (pre-miRNAs), only pre-let-7 is efficiently uridylated by TuT4. Thus, we hypothesized the existence of substrate-specific cofactors that stimulate Lin28a-mediated pre-let-7 uridylation or restrict its functionality on non-let-7 pre-miRNAs. Through RNA pull-downs coupled with quantitative mass spectrometry, we identified the E3 ligase Trim25 as an RNA-specific cofactor for Lin28a/TuT4-mediated uridylation. We show that Trim25 binds to the conserved terminal loop (CTL) of pre-let-7 and activates TuT4, allowing for more efficient Lin28a-mediated uridylation. These findings reveal that protein-modifying enzymes, only recently shown to bind RNA, can guide the function of canonical ribonucleoprotein (RNP) complexes in cis, thereby providing an additional level of specificity.
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[Placental epigenetic programming in intrauterine growth restriction (IUGR)].
Casanello, Paola; Castro-Rodríguez, José A; Uauy, Ricardo; Krause, Bernardo J
2016-01-01
Intrauterine growth restriction (IUGR) is a perinatal condition affecting foetal growth, with under the 10th percentile of the weight curve expected for gestational age. This condition has been associated with higher cardiovascular and metabolic risk and post-natal obesity. There are also major changes in placental function, and particularly in a key molecule in this regulation, nitric oxide. The synthesis of nitric oxide has numerous control mechanisms and competition with arginase for their common substrate, the amino acid L-arginine. This competition is reflected in various vascular diseases and particularly in the endothelium of the umbilical vessels of babies with IUGR. Along with this, there is regulation at the epigenetic level, where methylation in specific regions of some gene promoters, such as the nitric oxide synthase, regulating their expression. It is currently of great interest to understand the mechanisms by which diseases such as IUGR may be conditioned, particularly by maternal nutritional and metabolic conditions, and epigenetic mechanisms that could eventually be modifiable, and thus a focus of interest for health interventions. Copyright © 2016 Sociedad Chilena de Pediatría. Publicado por Elsevier España, S.L.U. All rights reserved.
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Diversity of metabolic capacities among strains degrading polycyclic aromatic hydrocarbons
DOE Office of Scientific and Technical Information (OSTI.GOV)
Bouchez, M.; Besnaienou, B.; Blanchet, D.
1995-12-31
Strains of Pseudomonas and Rhodococcus genera were isolated for their capacity to use, as a sole carbon and energy source, one of the following polycyclic aromatic hydrocarbons (PAHs): naphthalene (NAP), fluorene (FLU), phenanthrene (PHE), anthracene (ANT), fluoranthene (FLT), and pyrene (PYR). The range of PAHs supporting growth of these pure strains was usually restricted, but several other hydrocarbons were used by Rhodococcus sp. All strains could grow on simple organic acids. Maximal specific growth rates ({mu}{sub max}) of all strains on their PAH growth substrates were determined by respirometry. No clear relationships between {mu}{sub max} values and the molecular weightmore » or water solubility of PAHs were apparent, but Pseudomonas sp. exhibited the highest {mu}{sub max} values. Carbon balances for PAH biodegradation were established. Differences between strains were observed, but high mineralization rates and low production of soluble metabolites were obtained for all PAHs. Bacterial biomass represented 16% to 35% of the carbon consumed. Strain diversity was also apparent in the interactions observed in the degradation of a mixture of two PAHs by individual strains, which often involved inhibition of PAH substrate degradation, with or without cometabolization of the second PAH.« less
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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
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Cushing's syndrome mutant PKA L205R exhibits altered substrate specificity
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
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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.
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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…
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McClelland, M; Nelson, M; Raschke, E
1994-01-01
Restriction endonucleases have site-specific interactions with DNA that can often be inhibited by site-specific DNA methylation and other site-specific DNA modifications. However, such inhibition cannot generally be predicted. The empirically acquired data on these effects are tabulated for over 320 restriction endonucleases. In addition, a table of known site-specific DNA modification methyltransferases and their specificities is presented along with EMBL database accession numbers for cloned genes. PMID:7937074
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Hepatic autophagy contributes to the metabolic response to dietary protein restriction.
Henagan, Tara M; Laeger, Thomas; Navard, Alexandra M; Albarado, Diana; Noland, Robert C; Stadler, Krisztian; Elks, Carrie M; Burk, David; Morrison, Christopher D
2016-06-01
Autophagy is an essential cellular response which acts to release stored cellular substrates during nutrient restriction, and particularly plays a key role in the cellular response to amino acid restriction. However, there has been limited work testing whether the induction of autophagy is required for adaptive metabolic responses to dietary protein restriction in the whole animal. Here, we found that moderate dietary protein restriction led to a series of metabolic changes in rats, including increases in food intake and energy expenditure, the downregulation of hepatic fatty acid synthesis gene expression and reduced markers of hepatic mitochondrial number. Importantly, these effects were also associated with an induction of hepatic autophagy. To determine if the induction of autophagy contributes to these metabolic effects, we tested the metabolic response to dietary protein restriction in BCL2-AAA mice, which bear a genetic mutation that impairs autophagy induction. Interestingly, BCL2-AAA mice exhibit exaggerated responses in terms of both food intake and energy expenditure, whereas the effects of protein restriction on hepatic metabolism were significantly blunted. These data demonstrate that restriction of dietary protein is sufficient to trigger hepatic autophagy, and that disruption of autophagy significantly alters both hepatic and whole animal metabolic response to dietary protein restriction. Copyright © 2016 Elsevier Inc. All rights reserved.
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Skov, Peter V.; Lund, Ivar; Pargana, Alexandre M.
2015-01-01
Sustained swimming at moderate speeds is considered beneficial in terms of the productive performance of salmonids, but the causative mechanisms have yet to be unequivocally established. In the present study, the effects of moderate exercise on the bioenergetics of rainbow trout were assessed during a 15 week growth experiment, in which fish were reared at three different current speeds: 1 BL s−1, 0.5 BL s−1 and still water (≈ 0 BL s−1). Randomly selected groups of 100 fish were distributed among twelve 600 L tanks and maintained on a restricted diet regime. Specific growth rate (SGR) and feed conversion ratio (FCR) were calculated from weight and length measurements every 3 weeks. Routine metabolic rate (RMR) was measured every hour as rate of oxygen consumption in the tanks, and was positively correlated with swimming speed. Total ammonia nitrogen (TAN) excretion rates showed a tendency to decrease with increasing swimming speeds, yet neither they nor the resulting nitrogen quotients (NQ) indicated that swimming significantly reduced the fraction of dietary protein used to fuel metabolism. Energetic budgets revealed a positive correlation between energy expenditure and the current speed at which fish were reared, fish that were forced to swim and were fed restrictively consequentially had poorer growth and feed utilization. The results show that for rainbow trout, water current can negatively affect growth despite promoting minor positive changes in substrate utilization. We hypothesize that this may be the result of either a limited dietary energy supply from diet restriction being insufficient for both covering the extra costs of swimming and supporting enhanced growth. PMID:25705195
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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.
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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.
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Gondim, A I; Dias, T L P; Christoffersen, M L
2013-05-01
The genus Diadema presently consists of seven species, two of which are known from the Brazilian coast: D. antillarum and D. ascensionis. The first is usually known for shallow coastal areas, while the second was apparently restricted to oceanic islands. In February 2011, a dense population of D. ascensionis was observed on the coastal reefs of Praia do Francês (Alagoas State, northeastern Brazil). Five specimens were collected and transported to the laboratory where morphological studies of the test and pedicellariae were conducted. Subsequently, visits were made to scientific collections in order to compare and confirm species identifications. Our observations confirm the presence of tridentate pedicellariae with narrow and strongly curved valves. The axial cavity in the tips of the spines is filled with dense nonreticular tissue. This taxonomic data confirms the occurrence of D. ascensionis in coastal areas. On the coastal reefs of Praia do Francês, animals were observed from the beach to the reef formations about 200 m offshore in areas with a sandy substrate and in reef cavities, usually in clear and well illuminated waters. Solitary individuals or groups of up to 15 individuals formed dense populations in the area. We stress the importance of pedicellariae for the specific identification of the Diadematidae, considering that they are quite constant and reliable at this taxonomic level. Our results demonstrate that D. ascensionis is not restricted to insular environments and that this species may be common in shallow coastal habitats.
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Sun, Liou; Sadighi Akha, Amir A.; Miller, Richard A.
2009-01-01
Life span can be extended in rodents by restricting food availability (caloric restriction [CR]) or by providing food low in methionine (Meth-R). Here, we show that a period of food restriction limited to the first 20 days of life, via a 50% enlargement of litter size, shows extended median and maximal life span relative to mice from normal sized litters and that a Meth-R diet initiated at 12 months of age also significantly increases longevity. Furthermore, mice exposed to a CR diet show changes in liver messenger RNA patterns, in phosphorylation of Erk, Jnk2, and p38 kinases, and in phosphorylation of mammalian target of rapamycin and its substrate 4EBP1, HE-binding protein 1 that are not observed in liver from age-matched Meth-R mice. These results introduce new protocols that can increase maximal life span and suggest that the spectrum of metabolic changes induced by low-calorie and low-methionine diets may differ in instructive ways. PMID:19414512
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Vuong, Thu V; Foumani, Maryam; MacCormick, Benjamin; Kwan, Rachel; Master, Emma R
2016-11-21
Glucose oxidase (GO) activity is generally restricted to glucose and is susceptible to inactivation by H 2 O 2 . By comparison, the Y300A variant of gluco-oligosaccharide oxidase (GOOX) from Sarocladium strictum showed broader substrate range and higher H 2 O 2 stability. Specifically, Y300A exhibited up to 40 times higher activity on all tested sugars except glucose, compared to GO. Moreover, fusion of the Y300A variant to a family 22 carbohydrate binding module from Clostridium thermocellum (CtCBM22A) nearly doubled its catalytic efficiency on glucose, while retaining significant activity on oligosaccharides. In the presence of 200 mM of H 2 O 2 , the recombinant CtCBM22A_Y300A retained 80% of activity on glucose and 100% of activity on cellobiose, the preferred substrate for this enzyme. By contrast, a commercial glucose oxidase reported to contain ≤0.1 units catalase/ mg protein, retained 60% activity on glucose under the same conditions. GOOX variants appear to undergo a different mechanism of inactivation, as a loss of histidine instead of methionine was observed after H 2 O 2 incubation. The addition of CtCBM22A also promoted functional binding of the fusion enzyme to xylan, facilitating its simultaneous purification and immobilization using edible oat spelt xylan, which might benefit the usage of this enzyme preparation in food and baking applications.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Bashiri, G.; Squire, C.J.; Moreland, N.J.
2009-05-11
The modified flavin coenzyme F{sub 420} is found in a restricted number of microorganisms. It is widely distributed in mycobacteria, however, where it is important in energy metabolism, and in Mycobacterium tuberculosis (Mtb) is implicated in redox processes related to non-replicating persistence. In Mtb, the F{sub 420}-dependent glucose-6-phosphate dehydrogenase FGD1 provides reduced F{sub 420} for the in vivo activation of the nitroimidazopyran prodrug PA-824, currently being developed for anti-tuberculosis therapy against both replicating and persistent bacteria. The structure of M. tuberculosis FGD1 has been determined by x-ray crystallography both in its apo state and in complex with F{sub 420} andmore » citrate at resolutions of 1.90 and 1.95{angstrom}, respectively. The structure reveals a highly specific F{sub 420} binding mode, which is shared with several other F{sub 420}-dependent enzymes. Citrate occupies the substrate binding pocket adjacent to F{sub 420} and is shown to be a competitive inhibitor (IC{sub 50} 43 {micro}m). Modeling of the binding of the glucose 6-phosphate (G6P) substrate identifies a positively charged phosphate binding pocket and shows that G6P, like citrate, packs against the isoalloxazine moiety of F{sub 420} and helps promote a butterfly bend conformation that facilitates F{sub 420} reduction and catalysis.« less
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Crystal Structures of Human SIRT[subscript 3] Displaying Substrate-induced Conformational Changes
DOE Office of Scientific and Technical Information (OSTI.GOV)
Jin, Lei; Wei, Wentao; Jiang, Yaobin
2009-11-04
SIRT3 is a major mitochondrial NAD{sup +}-dependent protein deacetylase playing important roles in regulating mitochondrial metabolism and energy production and has been linked to the beneficial effects of exercise and caloric restriction. SIRT3 is emerging as a potential therapeutic target to treat metabolic and neurological diseases. We report the first sets of crystal structures of human SIRT3, an apo-structure with no substrate, a structure with a peptide containing acetyl lysine of its natural substrate acetyl-CoA synthetase 2, a reaction intermediate structure trapped by a thioacetyl peptide, and a structure with the dethioacetylated peptide bound. These structures provide insights into themore » conformational changes induced by the two substrates required for the reaction, the acetylated substrate peptide and NAD+. In addition, the binding study by isothermal titration calorimetry suggests that the acetylated peptide is the first substrate to bind to SIRT3, before NAD{sup +}. These structures and biophysical studies provide key insight into the structural and functional relationship of the SIRT3 deacetylation activity.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Smith, Dayle; Danyal, Karamatullah; Raugei, Simone
Mo-dependent nitrogenase catalyzes the biological reduction of N 2 to 2NH 3 at the FeMo-cofactor buried deep inside the MoFe protein. Access of substrates, such as N 2, to the active site is likely restricted by the surrounding protein, requiring substrate channels that lead from the surface to the active site. Earlier studies on crystallographic structures of the MoFe protein have suggested three putative substrate channels. Here, we have utilized sub-microsecond atomistic molecular dynamics simulations to allow the nitrogenase MoFe protein to explore its conformational space in an aqueous solution at physiological ionic strength, revealing a putative substrate channel notmore » previously reported. The viability of the proposed channel was tested by examining the free energy of passage of N 2 from the surface through the channel to FeMo-cofactor, with discovery of a very low energy barrier. These studies point to a viable substrate channel in nitrogenase that appears during thermal motions of the protein in an aqueous environment that approaches a face of FeMo-cofactor earlier implicated in substrate binding.« less
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Performance and Reliability of Bonded Interfaces for High-Temperature Packaging. Annual Report
DOE Office of Scientific and Technical Information (OSTI.GOV)
DeVoto, Douglas
2016-04-01
Current generation automotive power electronics packages utilize silicon devices and lead-free solder alloys. To meet stringent technical targets for 2020 and beyond (for cost, power density, specific power, efficiency and reliability), wide-bandgap devices are being considered since they offer advantages such as operation at higher frequencies, voltages, and temperatures. Traditional power electronics packages must be redesigned to utilize the full potential of wide-bandgap devices, and the die- and substrate-attach layers are key areas where new material development and validation is required. Present solder alloys do not meet the performance requirements for these new package designs while also meeting cost andmore » hazardous substance restrictions. Sintered silver (Ag) promises to meet the needs for die- and substrate-attach interfaces but synthesis optimization and reliability evaluation must be completed. Sintered Ag material was proposed as an alternative solution in power electronics packages almost 20 years back. However, synthesis pressure requirements up 40 MPa caused a higher complexity in the production process and more stringent flatness specifications for the substrates. Recently, several manufacturers have developed sintered Ag materials that require lower (3-5 MPa) or even no bonding pressures. Degradation mechanisms for these sintered Ag materials are not well known and need to be addressed. We are addressing these aspects to some extent in this project. We are developing generalized (i.e., independent of geometry) stress intensity factor versus cycles-to-failure relations for sintered Ag. Because sintered Ag is a relatively new material for automotive power electronics, the industry currently does not have a good understanding of recommended synthesis parameters or expected reliability under prescribed conditions. It is an important deliverable of this project to transfer findings to industry to eliminate barriers to using sintered Ag as a viable and commercialized die- and substrate-attach material. Only a few manufacturers produce sintered Ag pastes and may consider some processing conditions as proprietary. It is the goal of this project to openly explore and define best practices in order to impact the maximum number of power electronics module manufacturers and suppliers.« less
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Reductive trapping of substrate to bovine plasma amine oxidase
DOE Office of Scientific and Technical Information (OSTI.GOV)
Hartmann, C.; Klinman, J.P.
1987-01-25
Plasma amine oxidases catalyze the oxidative deamination of amines to aldehydes, followed by a 2e- reduction of O/sub 2/ to H/sub 2/O/sub 2/. Pyrroloquinoline quinone (PQQ), previously believed to be restricted to prokaryotes, has recently been proposed to be the cofactor undergoing reduction in the first half-reaction of bovine plasma amine oxidase (Ameyama, M., Hayashi, U., Matsushita, K., Shinagawa, E., and Adachi, O. (1984) Agric. Biol. Chem. 48, 561-565; Lobenstein-Verbeek, C. L., Jongejan, J. A., Frank, J., and Duine, J. A. (1984) FEBS Lett. 170, 305-309). This result is unexpected, since model studies with PQQ implicate Schiff's base formation betweenmore » a reactive carbonyl and substrates, whereas experiments with bovine plasma amine oxidase have failed to provide evidence for a carbonyl cofactor. We have, therefore, re-examined putative adducts between substrate and enzyme-bound cofactor, employing a combination of (/sup 14/C)benzylamine and (/sup 3/H)NaCNBH/sub 3/. The use of the relatively weak reductant, NaCNBH/sub 3/, affords Schiff's base specificity and permits the study of enzyme below pH 7.0. As we show, enzyme can only be inactivated by NaCNBH/sub 3/ in the presence of substrate, leading to the incorporation of 1 mol of (/sup 14/C)benzylamine/mol of enzyme subunit at complete inactivation. By contrast, we are unable to detect any labeling with (/sup 3/H)NaCNBH/sub 3/, analogous to an earlier study with (/sup 3/H)NaCNBH/sub 4/ (Suva, R. H., and Abeles, R. H. (1978) Biochemistry 17, 3538-3545). We conclude, first, that our inability to obtain adducts containing both carbon 14 and tritium rules out the reductive trapping either of amine substrate with pyridoxal phosphate or of aldehyde product with a lysyl side chain and, second, that the observed pattern of labeling is fully consistent with the presence of PQQ at the active site of bovine plasma amine oxidase.« less
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Substrate specificity of the ubiquitin and Ubl proteases
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
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Altered Substrate Specificity of Drug-Resistant Human Immunodeficiency Virus Type 1 Protease
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
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Using N-Limiting Growth Conditions to Remove Atrazine from Groundwater: Laboratory Studies.
USDA-ARS?s Scientific Manuscript database
Typically, respiratory redox reactions are the driving mechanism behind in situ bioremediations that use a carbon substrate. This is because electron (e-) donor availability generally restricts subsurface microbial activity. Thus, microbial growth and respiration can be greatly stimulated by the a...
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NASA Technical Reports Server (NTRS)
Karpova, E. A.; Kubareva, E. A.; Shabarova, Z. A.
1999-01-01
To elucidate the mechanism of interaction of restriction endonuclease EcoRII with DNA, we studied by native gel electrophoresis the binding of this endonuclease to a set of synthetic DNA-duplexes containing the modified or canonical recognition sequence 5'-d(CCA/TGG)-3'. All binding substrate or substrate analogues tested could be divided into two major groups: (i) duplexes that, at the interaction with endonuclease EcoRII, form two types of stable complexes on native gel in the absence of Mg2+ cofactor; (ii) duplexes that form only one type of complex, observed both in the presence and absence of Mg2+. Unlike the latter, duplexes under the first group can be hydrolyzed by endonuclease. Data obtained suggest that the active complex is most likely formed by one protein subunit and one DNA recognition sequence. A model of EcoRII endonuclease action is presented.
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Cleavage Entropy as Quantitative Measure of Protease Specificity
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
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Morphology Controlled Fabrication of InN Nanowires on Brass Substrates
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
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Substrate specificity of sheep liver sorbitol dehydrogenase.
Lindstad, R I; Köll, P; McKinley-McKee, J S
1998-01-01
The substrate specificity of sheep liver sorbitol dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of sorbitol dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo> D-ribo > L-xylo > D-lyxo approximately L-arabino > D-arabino > L-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH > -CH2NH2 > -CH2OCH3 approximately -CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of sorbitol dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of stereospecificity at C2 in some polyols. PMID:9461546
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Design and characterization of hirulogs: A novel class of bivalent peptide inhibitors of thrombin
DOE Office of Scientific and Technical Information (OSTI.GOV)
Maraganore, J.M.; Bourdon, P.; Jablonski, J.
1990-07-31
A novel class of synthetic peptides has been designed that inhibit the thrombin catalytic site and exhibit specificity for the anion-binding exosite (ABE) of {alpha}-thrombin. These peptides, called hirulogs, consist of (i) an active-site specificity sequence with a restricted Arg-Pro scissile bond, (ii) a polymeric linker of glycyl residues from 6 to 18 {angstrom} in length, and (iii) an ABE recognition sequence such as that in the hirudin C-terminus. Hirulog-1 ((D-Phe)-Pro-Arg-Pro-(Gly){sub 4}-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Tyr-Leu) inhibits the thrombin-catalyzed hydrolysis of a tripeptide p-nitroanilide substrate with K{sub i} = 2.3 nM. In contrast, the synthetic C-terminal hirudin peptide S-Hir{sub 53-64}, which binds to themore » thrombin ABE, blocked the fibrinogen clotting activity of the enzyme with K{sub i} = 144 nM but failed to inhibit the hydrolysis of p-nitroanilide substrates at concentrations as high as 1 mM. Hirulog-1, but not S-Hir{sub 53-64}, was found to inhibit the incorporation of ({sup 14}C)diisopropyl fluorophosphate in thrombin. Hirulog-1 appears specific for thrombin as it lacks inhibitory activities toward human factor Xa, human plasmin, and bovine trypsin at inhibitor:enzyme concentrations 3 orders of magnitude higher than those required to inhibit thrombin. The optimal inhibitory activity of hirulog-1 depends upon all three components of its structure. Comparison of anticoagulant activities of hirulog-1, hirudin, and S-Hir{sub 53-64} showed that the synthetic hirulog-1 is 2-fold more potent than hirudin and 100-fold more active than S-Hir{sub 53-64} in increasing the activated partial thromboplastin time of normal human plasma.« less
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Diversity Arrays Technology (DArT) for Pan-Genomic Evolutionary Studies of Non-Model Organisms
James, Karen E.; Schneider, Harald; Ansell, Stephen W.; Evers, Margaret; Robba, Lavinia; Uszynski, Grzegorz; Pedersen, Niklas; Newton, Angela E.; Russell, Stephen J.; Vogel, Johannes C.; Kilian, Andrzej
2008-01-01
Background High-throughput tools for pan-genomic study, especially the DNA microarray platform, have sparked a remarkable increase in data production and enabled a shift in the scale at which biological investigation is possible. The use of microarrays to examine evolutionary relationships and processes, however, is predominantly restricted to model or near-model organisms. Methodology/Principal Findings This study explores the utility of Diversity Arrays Technology (DArT) in evolutionary studies of non-model organisms. DArT is a hybridization-based genotyping method that uses microarray technology to identify and type DNA polymorphism. Theoretically applicable to any organism (even one for which no prior genetic data are available), DArT has not yet been explored in exclusively wild sample sets, nor extensively examined in a phylogenetic framework. DArT recovered 1349 markers of largely low copy-number loci in two lineages of seed-free land plants: the diploid fern Asplenium viride and the haploid moss Garovaglia elegans. Direct sequencing of 148 of these DArT markers identified 30 putative loci including four routinely sequenced for evolutionary studies in plants. Phylogenetic analyses of DArT genotypes reveal phylogeographic and substrate specificity patterns in A. viride, a lack of phylogeographic pattern in Australian G. elegans, and additive variation in hybrid or mixed samples. Conclusions/Significance These results enable methodological recommendations including procedures for detecting and analysing DArT markers tailored specifically to evolutionary investigations and practical factors informing the decision to use DArT, and raise evolutionary hypotheses concerning substrate specificity and biogeographic patterns. Thus DArT is a demonstrably valuable addition to the set of existing molecular approaches used to infer biological phenomena such as adaptive radiations, population dynamics, hybridization, introgression, ecological differentiation and phylogeography. PMID:18301759
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Kinetics of reactions of the Actinomadura R39 DD-peptidase with specific substrates.
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.
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E3Net: a system for exploring E3-mediated regulatory networks of cellular functions.
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.
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Glycan microarray screening assay for glycosyltransferase specificities.
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.
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Characterizing Protease Specificity: How Many Substrates Do We Need?
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
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Aquatic wood -- an insect perspective
Peter S. Cranston; Brendan McKie
2006-01-01
Immersed wood provides refugia and substrate for a diverse array of macroinvertebrates, and food for a more restricted genuinely xylophagous fauna. Worldwide, xylophages are found across aquatic insect orders, including Coleoptera, Diptera, Trichoptera and Plecoptera. Xylophages often are specialised, feeding on the wood surface or mining deep within. Many feed...
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Using Nitrogen Limiting Growth Conditions to Remove Atrazine from Groundwater: Laboratory Studies
USDA-ARS?s Scientific Manuscript database
In the past microbial redox reactions have been the driving mechanism behind in situ bioremediations that use a carbon substrate. This is because subsurface microbial activity is generally restricted by electron (e-) donor availability and microbial activity, growth and respiration, can be stimulat...
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1986-01-01
We have examined requirements for antigen presentation to a panel of MHC class I-and class II-restricted, influenza virus-specific CTL clones by controlling the form of virus presented on the target cell surface. Both H-2K/D- and I region-restricted CTL recognize target cells exposed to infectious virus, but only the I region-restricted clones efficiently lysed histocompatible target cells pulsed with inactivated virus preparations. The isolated influenza hemagglutinin (HA) polypeptide also could sensitize target cells for recognition by class II-restricted, HA-specific CTL, but not by class I-restricted, HA- specific CTL. Inhibition of nascent viral protein synthesis abrogated the ability of target cells to present viral antigen relevant for class I-restricted CTL recognition. Significantly, presentation for class II- restricted recognition was unaffected in target cells exposed to preparations of either inactivated or infectious virus. This differential sensitivity suggested that these H-2I region-restricted CTL recognized viral polypeptides derived from the exogenously introduced virions, rather than viral polypeptides newly synthesized in the infected cell. In support of this contention, treatment of the target cells with the lysosomotropic agent chloroquine abolished recognition of infected target cells by class II-restricted CTL without diminishing class I-restricted recognition of infected target cells. Furthermore, when the influenza HA gene was introduced into target cells without exogenous HA polypeptide, the target cells that expressed the newly synthesized protein product of the HA gene were recognized only by H-2K/D-restricted CTL. These observations suggest that important differences may exist in requirements for antigen presentation between H-2K/D and H-2I region-restricted CTL. These differences may reflect the nature of the antigenic epitopes recognized by these two CTL subsets. PMID:3485173
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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.
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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
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USDA-ARS?s Scientific Manuscript database
Nitrogen-limited soil microbial activity has important implications for soil carbon storage and nutrient availability, but previous methods for assessing resource limitation have been restricted, due to enrichment criteria (i.e., long incubation periods, high substrate amendments) and/or logistical ...
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Hay, William W; Brown, Laura D; Rozance, Paul J; Wesolowski, Stephanie R; Limesand, Sean W
2016-08-01
Previous attempts to improve growth and development of the intrauterine growth-restricted (IUGR) foetus during pregnancy have not worked or caused harm. Our research identifies tissue-specific mechanisms underlying foetal growth restriction and then tests strategies to improve growth and ameliorate many of the metabolic problems before the infant is born. The goal of our studies is to reduce the impact of foetal growth restriction at critical stages of development on the lifelong complications of IUGR offspring. Defining specific mechanisms that cause growth restriction in the foetus might identify specific nutrients and hormones that could be given to the mother to improve foetal growth and reduce metabolic complications, using strategies first tested in our IUGR animal model. ©2016 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.
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Trim25 Is an RNA-Specific Activator of Lin28a/TuT4-Mediated Uridylation
Choudhury, Nila Roy; Nowak, Jakub S.; Zuo, Juan; Rappsilber, Juri; Spoel, Steven H.; Michlewski, Gracjan
2014-01-01
Summary RNA binding proteins have thousands of cellular RNA targets and often exhibit opposite or passive molecular functions. Lin28a is a conserved RNA binding protein involved in pluripotency and tumorigenesis that was previously shown to trigger TuT4-mediated pre-let-7 uridylation, inhibiting its processing and targeting it for degradation. Surprisingly, despite binding to other pre-microRNAs (pre-miRNAs), only pre-let-7 is efficiently uridylated by TuT4. Thus, we hypothesized the existence of substrate-specific cofactors that stimulate Lin28a-mediated pre-let-7 uridylation or restrict its functionality on non-let-7 pre-miRNAs. Through RNA pull-downs coupled with quantitative mass spectrometry, we identified the E3 ligase Trim25 as an RNA-specific cofactor for Lin28a/TuT4-mediated uridylation. We show that Trim25 binds to the conserved terminal loop (CTL) of pre-let-7 and activates TuT4, allowing for more efficient Lin28a-mediated uridylation. These findings reveal that protein-modifying enzymes, only recently shown to bind RNA, can guide the function of canonical ribonucleoprotein (RNP) complexes in cis, thereby providing an additional level of specificity. PMID:25457611
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Diallinas, George
2014-01-01
Transporters are ubiquitous proteins mediating the translocation of solutes across cell membranes, a biological process involved in nutrition, signaling, neurotransmission, cell communication and drug uptake or efflux. Similarly to enzymes, most transporters have a single substrate binding-site and thus their activity follows Michaelis-Menten kinetics. Substrate binding elicits a series of structural changes, which produce a transporter conformer open toward the side opposite to the one from where the substrate was originally bound. This mechanism, involving alternate outward- and inward-facing transporter conformers, has gained significant support from structural, genetic, biochemical and biophysical approaches. Most transporters are specific for a given substrate or a group of substrates with similar chemical structure, but substrate specificity and/or affinity can vary dramatically, even among members of a transporter family that show high overall amino acid sequence and structural similarity. The current view is that transporter substrate affinity or specificity is determined by a small number of interactions a given solute can make within a specific binding site. However, genetic, biochemical and in silico modeling studies with the purine transporter UapA of the filamentous ascomycete Aspergillus nidulans have challenged this dogma. This review highlights results leading to a novel concept, stating that substrate specificity, but also transport kinetics and transporter turnover, are determined by subtle intramolecular interactions between a major substrate binding site and independent outward- or cytoplasmically-facing gating domains, analogous to those present in channels. This concept is supported by recent structural evidence from several, phylogenetically and functionally distinct transporter families. The significance of this concept is discussed in relationship to the role and potential exploitation of transporters in drug action. PMID:25309439
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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.
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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
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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
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Substrate- and isoform-specific proteome stability in normal and stressed cardiac mitochondria.
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.
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NASA Technical Reports Server (NTRS)
Dingell, Charles W. (Inventor); Quintana, Clemente E. (Inventor); Le, Suy (Inventor); Clark, Michael R. (Inventor); Cloutier, Robert E. (Inventor); Hafermalz, David Scott (Inventor)
2009-01-01
A sublimator includes a sublimation plate having a thermal element disposed adjacent to a feed water channel and a control point disposed between at least a portion of the thermal element and a large pore substrate. The control point includes a sintered metal material. A method of dissipating heat using a sublimator includes a sublimation plate having a thermal element and a control point. The thermal element is disposed adjacent to a feed water channel and the control point is disposed between at least a portion of the thermal element and a large pore substrate. The method includes controlling a flow rate of feed water to the large pore substrate at the control point and supplying heated coolant to the thermal element. Sublimation occurs in the large pore substrate and the controlling of the flow rate of feed water is independent of time. A sublimator includes a sublimation plate having a thermal element disposed adjacent to a feed water channel and a control point disposed between at least a portion of the thermal element and a large pore substrate. The control point restricts a flow rate of feed water from the feed water channel to the large pore substrate independent of time.
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Hibi, Masanobu; Kubota, Chie; Mizuno, Tomohito; Aritake, Sayaka; Mitsui, Yuki; Katashima, Mitsuhiro; Uchida, Sunao
2017-01-10
The effects of sleep restriction on energy metabolism and appetite remain controversial. We examined the effects of shortened sleep duration on energy metabolism, core body temperature (CBT), and appetite profiles. Nine healthy men were evaluated in a randomised crossover study under two conditions: a 3.5-h sleep duration and a 7-h sleep duration for three consecutive nights followed by one 7-h recovery sleep night. The subjects' energy expenditure (EE), substrate utilisation, and CBT were continually measured for 48 h using a whole-room calorimeter. The subjects completed an appetite questionnaire every hour while in the calorimeter. Sleep restriction did not affect total EE or substrate utilisation. The 48-h mean CBT decreased significantly during the 3.5-h sleep condition compared with the 7-h sleep condition (7-h sleep, 36.75 ± 0.11 °C; 3.5-h sleep, 36.68 ± 0.14 °C; p = 0.016). After three consecutive nights of sleep restriction, fasting peptide YY levels and fullness were significantly decreased (p = 0.011), whereas hunger and prospective food consumption were significantly increased, compared to those under the 7-h sleep condition. Shortened sleep increased appetite by decreasing gastric hormone levels, but did not affect EE, suggesting that greater caloric intake during a shortened sleep cycle increases the risk of weight gain.
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40 CFR 279.61 - Restrictions on burning.
Code of Federal Regulations, 2013 CFR
2013-07-01
... 40 Protection of Environment 28 2013-07-01 2013-07-01 false Restrictions on burning. 279.61... (CONTINUED) STANDARDS FOR THE MANAGEMENT OF USED OIL Standards for Used Oil Burners Who Burn Off-Specification Used Oil for Energy Recovery § 279.61 Restrictions on burning. (a) Off-specification used oil fuel...
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40 CFR 279.61 - Restrictions on burning.
Code of Federal Regulations, 2012 CFR
2012-07-01
... 40 Protection of Environment 28 2012-07-01 2012-07-01 false Restrictions on burning. 279.61... (CONTINUED) STANDARDS FOR THE MANAGEMENT OF USED OIL Standards for Used Oil Burners Who Burn Off-Specification Used Oil for Energy Recovery § 279.61 Restrictions on burning. (a) Off-specification used oil fuel...
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40 CFR 279.61 - Restrictions on burning.
Code of Federal Regulations, 2011 CFR
2011-07-01
... 40 Protection of Environment 27 2011-07-01 2011-07-01 false Restrictions on burning. 279.61... (CONTINUED) STANDARDS FOR THE MANAGEMENT OF USED OIL Standards for Used Oil Burners Who Burn Off-Specification Used Oil for Energy Recovery § 279.61 Restrictions on burning. (a) Off-specification used oil fuel...
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40 CFR 279.61 - Restrictions on burning.
Code of Federal Regulations, 2014 CFR
2014-07-01
... 40 Protection of Environment 27 2014-07-01 2014-07-01 false Restrictions on burning. 279.61... (CONTINUED) STANDARDS FOR THE MANAGEMENT OF USED OIL Standards for Used Oil Burners Who Burn Off-Specification Used Oil for Energy Recovery § 279.61 Restrictions on burning. (a) Off-specification used oil fuel...
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Maternal–Fetal Nutrient Transport in Pregnancy Pathologies: The Role of the Placenta
Brett, Kendra Elizabeth; Ferraro, Zachary Michael; Yockell-Lelievre, Julien; Gruslin, Andrée; Adamo, Kristi Bree
2014-01-01
Appropriate in utero growth is essential for offspring development and is a critical contributor to long-term health. Fetal growth is largely dictated by the availability of nutrients in maternal circulation and the ability of these nutrients to be transported into fetal circulation via the placenta. Substrate flux across placental gradients is dependent on the accessibility and activity of nutrient-specific transporters. Changes in the expression and activity of these transporters is implicated in cases of restricted and excessive fetal growth, and may represent a control mechanism by which fetal growth rate attempts to match availability of nutrients in maternal circulation. This review provides an overview of placenta nutrient transport with an emphasis on macro-nutrient transporters. It highlights the changes in expression and activity of these transporters associated with common pregnancy pathologies, including intrauterine growth restriction, macrosomia, diabetes and obesity, as well as the potential impact of maternal diet. Molecular signaling pathways linking maternal nutrient availability and placenta nutrient transport are discussed. How sexual dimorphism affects fetal growth strategies and the placenta’s response to an altered intrauterine environment is considered. Further knowledge in this area may be the first step in the development of targeted interventions to help optimize fetal growth. PMID:25222554
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Occurrence of fungi in combs of fungus-growing termites (Isoptera: Termitidae, Macrotermitinae).
Guedegbe, Herbert J; Miambi, Edouard; Pando, Anne; Roman, Jocelyne; Houngnandan, Pascal; Rouland-Lefevre, Corinne
2009-10-01
Fungus-growing termites cultivate their mutualistic basidiomycete Termitomyces species on a substrate called a fungal comb. Here, the Suicide Polymerase Endonuclease Restriction (SuPER) method was adapted for the first time to a fungal study to determine the entire fungal community of fungal combs and to test whether fungi other than the symbiotic cultivar interact with termite hosts. Our molecular analyses show that although active combs are dominated by Termitomyces fungi isolated with direct Polymerase Endonuclease Restriction - Denaturing Gradient Gel Electrophoresis (PCR-DGGE), they can also harbor some filamentous fungi and yeasts only revealed by SuPER PCR-DGGE. This is the first molecular evidence of the presence of non-Termitomyces species in active combs. However, because there is no evidence for a species-specific relationship between these fungi and termites, they are mere transient guests with no specialization in the symbiosis. It is however surprising to notice that termite-associated Xylaria strains were not isolated from active combs even though they are frequently retrieved when nests are abandoned by termites. This finding highlights the implication of fungus-growing termites in the regulation of fungi occurring within the combs and also suggests that they might not have any particular evolutionary-based association with Xylaria species.
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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
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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
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Liu, Xiaomei; Qi, Ying; Gao, Hong; Jiao, Yisheng; Gu, Hui; Miao, Jianing; Yuan, Zhengwei
2013-01-01
It is well recognized that intrauterine growth restriction leads to the development of insulin resistance and type 2 diabetes mellitus in adulthood. To investigate the mechanisms behind this "metabolic imprinting" phenomenon, we examined the impact of maternal undernutrition on insulin signaling pathway and the ATP sensitive potassium channel expression in the hypothalamus of intrauterine growth restriction fetus. Intrauterine growth restriction rat model was developed through maternal low protein diet. The expression and activated levels of insulin signaling molecules and K(ATP) protein in the hypothalami which were dissected at 20 days of gestation, were analyzed by western blot and real time PCR. The tyrosine phosphorylation levels of the insulin receptor substrate 2 and phosphatidylinositol 3'-kinase p85α in the hypothalami of intrauterine growth restriction fetus were markedly reduced. There was also a downregulation of the hypothalamic ATP sensitive potassium channel subunit, sulfonylurea receptor 1, which conveys the insulin signaling. Moreover, the abundances of gluconeogenesis enzymes were increased in the intrauterine growth restriction livers, though no correlation was observed between sulfonylurea receptor 1 and gluconeogenesis enzymes. Our data suggested that aberrant intrauterine milieu impaired insulin signaling in the hypothalamus, and these alterations early in life might contribute to the predisposition of the intrauterine growth restriction fetus toward the adult metabolic disorders.
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Tanaka, Kohei; Zelenitsky, Darla K; Therrien, François; Kobayashi, Yoshitsugu
2018-03-15
Dinosaurs thrived and reproduced in various regions worldwide, including the Arctic. In order to understand their nesting in diverse or extreme environments, the relationships between nests, nesting environments, and incubation methods in extant archosaurs were investigated. Statistical analyses reveal that species of extant covered nesters (i.e., crocodylians and megapodes) preferentially select specific sediments/substrates as a function of their nesting style and incubation heat sources. Relationships between dinosaur eggs and the sediments in which they occur reveal that hadrosaurs and some sauropods (i.e., megaloolithid eggs) built organic-rich mound nests that relied on microbial decay for incubation, whereas other sauropods (i.e., faveoloolithid eggs) built sandy in-filled hole nests that relied on solar or potentially geothermal heat for incubation. Paleogeographic distribution of mound nests and sandy in-filled hole nests in dinosaurs reveals these nest types produced sufficient incubation heat to be successful up to mid latitudes (≤47°), 10° higher than covered nesters today. However, only mound nesting and likely brooding could have produced sufficient incubation heat for nesting above the polar circle (>66°). As a result, differences in nesting styles may have placed restrictions on the reproduction of dinosaurs and their dispersal at high latitudes.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Hodder, Anthony N.; Malby, Robyn L.; Clarke, Oliver B.
The sera genes of the malaria-causing parasite Plasmodium encode a family of unique proteins that are maximally expressed at the time of egress of parasites from infected red blood cells. These multi-domain proteins are unique, containing a central papain-like cysteine-protease fragment enclosed between the disulfide-linked N- and C-terminal domains. However, the central fragment of several members of this family, including serine repeat antigen 5 (SERA5), contains a serine (S596) in place of the active-site cysteine. Here we report the crystal structure of the central protease-like domain of Plasmodium falciparum SERA5, revealing a number of anomalies in addition to the putativemore » nucleophilic serine: (1) the structure of the putative active site is not conducive to binding substrate in the canonical cysteine-protease manner; (2) the side chain of D594 restricts access of substrate to the putative active site; and (3) the S{sub 2} specificity pocket is occupied by the side chain of Y735, reducing this site to a small depression on the protein surface. Attempts to determine the structure in complex with known inhibitors were not successful. Thus, despite having revealed its structure, the function of the catalytic domain of SERA5 remains an enigma.« less
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Alternative function for the mitochondrial SAM complex in biogenesis of alpha-helical TOM proteins.
Stojanovski, Diana; Guiard, Bernard; Kozjak-Pavlovic, Vera; Pfanner, Nikolaus; Meisinger, Chris
2007-12-03
The mitochondrial outer membrane contains two preprotein translocases: the general translocase of outer membrane (TOM) and the beta-barrel-specific sorting and assembly machinery (SAM). TOM functions as the central entry gate for nuclear-encoded proteins. The channel-forming Tom40 is a beta-barrel protein, whereas all Tom receptors and small Tom proteins are membrane anchored by a transmembrane alpha-helical segment in their N- or C-terminal portion. Synthesis of Tom precursors takes place in the cytosol, and their import occurs via preexisting TOM complexes. The precursor of Tom40 is then transferred to SAM for membrane insertion and assembly. Unexpectedly, we find that the biogenesis of alpha-helical Tom proteins with a membrane anchor in the C-terminal portion is SAM dependent. Each SAM protein is necessary for efficient membrane integration of the receptor Tom22, whereas assembly of the small Tom proteins depends on Sam37. Thus, the substrate specificity of SAM is not restricted to beta-barrel proteins but also includes the majority of alpha-helical Tom proteins.
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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
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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
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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
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Cáceres, Tamar B; Thakur, Abhishek; Price, Owen M; Ippolito, Nicole; Li, Jun; Qu, Jun; Acevedo, Orlando; Hevel, Joan M
2018-02-27
Protein arginine methyltransferase 7 (PRMT7) is unique within the PRMT family as it is the only isoform known to exclusively make monomethylarginine (MMA). Given its role in epigenetics, the mechanistic basis for the strict monomethylation activity is under investigation. It is thought that PRMT7 enzymes are unable to add a second methyl group because of steric hindrance in the active site that restricts them to monomethylation. To test this, we probed the active site of trypanosomal PRMT7 (TbPRMT7) using accelerated molecular dynamics, site-directed mutagenesis, kinetic, binding, and product analyses. Both the dynamics simulations and experimental results show that the mutation of Phe71 to Ile converts the enzyme from a type III methyltransferase into a mixed type I/II, that is, an enzyme that can now perform dimethylation. In contrast, the serine and alanine mutants of Phe71 preserve the type III behavior of the native enzyme. These results are inconsistent with a sterics-only model to explain product specificity. Instead, molecular dynamics simulations of these variants bound to peptides show hydrogen bonding between would-be substrates and Glu172 of TbPRMT7. Only in the case of the Phe71 to Ile mutation is this interaction between MMA and the enzyme maintained, and the geometry for optimal S N 2 methyl transfer is obtained. The results of these studies highlight the benefit of combined computational and experimental methods in providing a better understanding for how product specificity is dictated by PRMTs.
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Substrate-specific regulation of ubiquitination by the anaphase-promoting complex
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
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Exploring the specific features of interfacial enzymology based on lipase studies.
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.
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Everson, Carol A.; Szabo, Aniko
2011-01-01
Chronic sleep disruption in laboratory rats leads to increased energy expenditure, connective tissue abnormalities, and increased weights of major organs relative to body weight. Here we report on expanded findings and the extent to which abnormalities become long-lasting, potentially permanent changes to health status after apparent recuperation from chronic sleep disruption. Rats were exposed 6 times to long periods of disrupted sleep or control conditions during 10 weeks to produce adaptations and then were permitted nearly 4 months of undisturbed sleep. Measurements were made in tissues from these groups and in preserved tissue from the experimental and control groups of an antecedent study that lacked a lengthy recuperation period. Cycles of sleep restriction resulted in energy deficiency marked by a progressive course of hyperphagia and major (15%) weight loss. Analyses of tissue composition in chronically sleep-restricted rats indicated that protein and lipid amounts in internal organs were largely spared, while adipose tissue depots appeared depleted. This suggests high metabolic demands may have preserved the size of the vital organs relative to expectations of severe energy deficiency alone. Low plasma corticosterone and leptin concentrations appear to reflect low substrate availability and diminished adiposity. After nearly 4 months of recuperation, sleep-restricted rats were consuming 20% more food and 35% more water than did comparison control rats, despite normalized weight, normalized adipocytes, and elevated plasma leptin concentrations. Plasma cholesterol levels in recuperated sleep-restricted rats were diminished relative to those of controls. The chronically increased intake of nutriments and water, along with altered negative feedback regulation and substrate use, indicate that internal processes are modified long after a severe period of prolonged and insufficient sleep has ended. PMID:21853062
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Murase, Hirotaka; Noguchi, Tomoharu; Sasaki, Shigeki
2018-06-01
Chromomycin A3 (CMA3) is an aureolic acid-type antitumor antibiotic. CMA3 forms dimeric complexes with divalent cations, such as Mg 2+ , which strongly binds to the GC rich sequence of DNA to inhibit DNA replication and transcription. In this study, the binding property of CMA3 to the DNA sequence containing multiple GC-rich binding sites was investigated by measuring the protection from hydrolysis by the restriction enzymes, AccII and Fnu4HI, for the center of the CGCG site and the 5'-GC↓GGC site, respectively. In contrast to the standard DNase I footprinting method, the DNA substrates are fully hydrolyzed by the restriction enzymes, therefore, the full protection of DNA at all the cleavable sites indicates that CMA3 simultaneously binds to all the binding sites. The restriction enzyme assay has suggested that CMA3 has a high tendency to bind the successive CGCG sites and the CGG repeat. Copyright © 2018 Elsevier Ltd. All rights reserved.
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ERIC Educational Resources Information Center
Freund, Barbara; Petrakos, Davithoula
2008-01-01
We developed driving restrictions that are linked to specific driving errors, allowing cognitively impaired individuals to continue to independently meet mobility needs while minimizing risk to themselves and others. The purpose of this project was to evaluate the efficacy and duration expectancy of these restrictions in promoting safe continued…
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Wu, Qian; Zhu, Liying; Xu, Qing; Huang, He; Jiang, Ling; Yang, Shang-Tian
2017-10-11
Fermentations employing anaerobes always suffer from the restriction of stringent anaerobic conditions during the production of bulk and fine chemicals. This work aims to improve the oxidative stress tolerance of C. tyrobutyricum CCTCC W428, an ideal butyric-acid-producing anaerobe, via the introduction of trehalose biosynthesis capability. Compared with the wild type, the engineered strain showed a wider substrate spectrum, an improved metabolic profile, and a significantly increased specific growth rate upon aeration and acid challenge. Molecular simulation experiments indicated that CoA transferase maintained its native folded state when protected by the trehalose system. Furthermore, qRT-PCR was combined assays for acid-related enzyme activities under various conditions to verify the effects of trehalose. These results demonstrate that introducing a trehalose biosynthetic pathway, which is redundant for the metabolism of C. tyrobutyricum, can increase the robustness of the host to achieve a better oxidative resistance.
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Laser-induced breakdown spectroscopy for detection of heavy metals in environmental samples
NASA Astrophysics Data System (ADS)
Wisbrun, Richard W.; Schechter, Israel; Niessner, Reinhard; Schroeder, Hartmut
1993-03-01
The application of LIBS technology as a sensor for heavy metals in solid environmental samples has been studied. This specific application introduces some new problems in the LIBS analysis. Some of them are related to the particular distribution of contaminants in the grained samples. Other problems are related to mechanical properties of the samples and to general matrix effects, like the water and organic fibers content of the sample. An attempt has been made to optimize the experimental set-up for the various involved parameters. The understanding of these factors has enabled the adjustment of the technique to the substrates of interest. The special importance of the grain size and of the laser-induced aerosol production is pointed out. Calibration plots for the analysis of heavy metals in diverse sand and soil samples have been carried out. The detection limits are shown to be usually below the recent regulation restricted concentrations.
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Charting organellar importomes by quantitative mass spectrometry
Peikert, Christian D.; Mani, Jan; Morgenstern, Marcel; Käser, Sandro; Knapp, Bettina; Wenger, Christoph; Harsman, Anke; Oeljeklaus, Silke; Schneider, André; Warscheid, Bettina
2017-01-01
Protein import into organelles is essential for all eukaryotes and facilitated by multi-protein translocation machineries. Analysing whether a protein is transported into an organelle is largely restricted to single constituents. This renders knowledge about imported proteins incomplete, limiting our understanding of organellar biogenesis and function. Here we introduce a method that enables charting an organelle's importome. The approach relies on inducible RNAi-mediated knockdown of an essential subunit of a translocase to impair import and quantitative mass spectrometry. To highlight its potential, we established the mitochondrial importome of Trypanosoma brucei, comprising 1,120 proteins including 331 new candidates. Furthermore, the method allows for the identification of proteins with dual or multiple locations and the substrates of distinct protein import pathways. We demonstrate the specificity and versatility of this ImportOmics method by targeting import factors in mitochondria and glycosomes, which demonstrates its potential for globally studying protein import and inventories of organelles. PMID:28485388
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Deciphering kinase-substrate relationships by analysis of domain-specific phosphorylation network.
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.
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Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease*
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
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Column experiments on organic micropollutants - applications and limitations
NASA Astrophysics Data System (ADS)
Banzhaf, Stefan; Hebig, Klaus
2016-04-01
As organic micropollutants become more and more ubiquitous in the aquatic environment, a sound understanding of their fate and transport behaviour is needed. This is to assure both safe and clean drinking water supply for mankind in the future and to protect the aquatic environment from pollution and negative consequences caused by manmade contamination. Apart from countless field studies, column experiments were and are frequently used to study transport of organic micropollutants. As the transport of (organic) solutes in groundwater is controlled by the chemical and physical properties of the compounds, the solvent (the groundwater including all solutes), and the substrate (the aquifer material), the adjustment and control of these boundary conditions allow to study a multitude of different experimental setups and to address specific research questions. The main purpose, however, remains to study the transport of a specific compound and its sorption and degradation behaviour in a specific sediment or substrate. Apart from the effective control of the individual boundary conditions, the main advantage of columns studies compared to other experimental setups (such as field studies, batch/microcosm studies), is that conservative and reactive solute breakthrough curves are obtained, which represent the sum of the transport processes. The analysis of these curves is well-developed and established. Additionally, limitations of this experimental method are presented here: the effects observed in column studies are often a result of dynamic, non-equilibrium processes. Time (or flow velocity) plays a major role in contrast to batch experiments, in which all processes will be observed until equilibrium is reached in the substrate-solution-system. Slightly modifying boundary conditions in different experiments have a strong influence on transport and degradation behaviour of organic micropollutants. This is a significant severe issue when it comes to general findings on the transport behaviour of a specific organic compound that are transferable to any given hydrogeochemical environment. Unfortunately, results of most column experiments therefore remain restricted to their specific setup. Column experiments can provide good estimates of all relevant transport parameters. However, the obtained results will almost always be limited to the scale they were obtained from. This means that direct application to field scale studies is infeasible as too many parameters are exclusive for the laboratory column setup. The remaining future challenge is to develop standard column experiments on organic micropollutants that overcome this issue. Here, we present a review of column experiments on organic micropollutants. We present different setups and discuss weaknesses, problems and advantages and provide ideas how to obtain more comparable results on the transport of organic micropollutants in the future.
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Substrate Sorting by a Supercharged Nanoreactor
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
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Allosteric regulation of rhomboid intramembrane proteolysis.
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.
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Allosteric regulation of rhomboid intramembrane proteolysis
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
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Buttet, Géraldine F.; Holliger, Christof
2013-01-01
Reductive dehalogenases are the key enzymes involved in the anaerobic respiration of organohalides such as the widespread groundwater pollutant tetrachloroethene. The increasing number of available bacterial genomes and metagenomes gives access to hundreds of new putative reductive dehalogenase genes that display a high level of sequence diversity and for which substrate prediction remains very challenging. In this study, we present the development of a functional genotyping method targeting the diverse reductive dehalogenases present in Sulfurospirillum spp., which allowed us to unambiguously identify a new reductive dehalogenase from our tetrachloroethene-dechlorinating SL2 bacterial consortia. The new enzyme, named PceATCE, shows 92% sequence identity with the well-characterized PceA enzyme of Sulfurospirillum multivorans, but in contrast to the latter, it is restricted to tetrachloroethene as a substrate. Its apparent higher dechlorinating activity with tetrachloroethene likely allowed its selection and maintenance in the bacterial consortia among other enzymes showing broader substrate ranges. The sequence-substrate relationships within tetrachloroethene reductive dehalogenases are also discussed. PMID:23995945
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Evolutionary dynamics of enzymes.
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.
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Browning, Jeffrey D.; Weis, Brian; Davis, Jeannie; Satapati, Santhosh; Merritt, Matthew; Malloy, Craig R.; Burgess, Shawn C.
2009-01-01
Carbohydrate-restriction is a common weight-loss approach that modifies hepatic metabolism by increasing gluconeogenesis and ketosis. Because little is known regarding the effect of carbohydrate-restriction on the origin of gluconeogenic precursors (gluconeogenesis from glycerol (GNGglycerol) and lactate/amino acids (GNGPEP)) or its consequence to hepatic energy homeostasis, we studied these parameters in a group of overweight/obese subjects undergoing weight-loss via dietary restriction. We used 2H and 13C tracers and nuclear magnetic resonance spectroscopy to measure the sources of hepatic glucose and TCA cycle flux in weight-stable subjects(n=7) and subjects following carbohydrate-(n=7) or calorie-restriction(n=7). The majority of hepatic glucose production in carbohydrate-restricted subjects came from GNGPEP. The contribution of glycerol to gluconeogenesis was similar in all groups despite evidence of increased fat oxidation in carbohydrate-restricted subjects. A strong correlation between TCA cycle flux and GNGPEP was found, though the reliance on TCA cycle energy production for gluconeogenesis was attenuated in subjects undergoing carbohydrate restriction. Together, these data imply that the TCA cycle is the energetic patron of gluconeogenesis. However, the relationship between these two pathways is modified by carbohydrate restriction, suggesting an increased reliance of the hepatocyte on energy generated outside of the TCA cycle when GNGPEP is maximal. In conclusion, carbohydrate-restriction modifies hepatic gluconeogenesis by increasing reliance on substrates like lactate or amino acids but not glycerol. This modification is associated with a reorganization of hepatic energy metabolism suggestive of enhanced hepatic β-oxidation. PMID:18925642
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Engineering the Substrate Specificity of the DhbE Adenylation Domain by Yeast Cell Surface Display
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
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Ahn, Soyon; Phillips, Anthony G
2012-06-01
The relationship between food restriction and subsequent dysregulation of food intake is complex, variable and long-lasting. The present study investigated in rats whether repeated cycles of food restriction and binge feeding opportunities may alter regulation of food intake by employing a test for sensory-specific satiety. Rats that experienced repeated food restriction-binge cycles maintained heavier body weights compared to rats that remained on continuous food restriction. In contrast to the control subjects, rats that alternated between food restriction and binge feeding failed to display sensory-specific satiety. During the first meal, there was a gradual decrease in the amount of food intake over a 40 min period. When presented with a second meal of the same food, these rats responded to the familiar food in a manner similar as to a novel food (i.e., comparable quantities of both types of food were consumed). Food restriction-binge feeding cycles may be considered as a form of stress, which in turn is associated with cross-sensitization to numerous behavioral responses. Therefore, we propose that stress-induced disruption of sensory-specific satiety reflects a sensitized response to food, in which the interaction between sensory and satiety factors are no longer the key regulators of food choice and meal cessation. Furthermore, a role for sensory-specific satiety in terminating food intake appeared to decline with the progression of the cycles, thereby contributing to a steady increase in body weight of rats that experienced restriction with bouts of binge feeding opportunities. Copyright © 2012 Elsevier B.V. All rights reserved.
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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.
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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
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Qureshi, N.; Maddox, I.S.; Friedl, A.
1992-09-01
The technique of continuous substrate feeding has been applied to the batch fermentation process using freely suspended cells, for ABE (acetone-butanol-ethanol) production. To avoid the product inhibition which normally restricts ABE production to less than 20 g/L and sugar utilization to 60 g/L, a product removal technique has been integrated into the fermentation process. The techniques investigated were liquid-liquid extraction, perstraction, gas-stripping, and pervaporation. By using a substrate of whey permeate, the reactor productivity has been improved over that observed in a traditional batch fermentation, while equivalent lactose utilization and ABE production values of 180 g and 69 g, respectively,more » have been achieved in a 1-L culture volume. 17 refs., 14 figs., 5 tabs.« less
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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.
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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.
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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.
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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.
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21 CFR 809.30 - Restrictions on the sale, distribution and use of analyte specific reagents.
Code of Federal Regulations, 2011 CFR
2011-04-01
... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Restrictions on the sale, distribution and use of... Requirements for Manufacturers and Producers § 809.30 Restrictions on the sale, distribution and use of analyte... include the statement for class I exempt ASR's: “Analyte Specific Reagent. Analytical and performance...
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Substrate Specificities and Conformational Flexibility of 3-Ketosteroid 9α-Hydroxylases*
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
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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
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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.
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NASA Astrophysics Data System (ADS)
Huang, Qi-Zhang; Fang, Yue-Yun; Liu, Peng-Yi; Zhu, Yan-Qing; Shi, Ji-Fu; Xu, Gang
2018-01-01
The practical application of superhydrophobic coatings on glass is usually restricted by their poor wear resistance due to the insufficient adhesion. A double-silica-layered structure was proposed to reinforce the coating adhesion on glass substrate. The wettability, surface morphologies, and chemical composition were investigated by water contact angle measurement, scanning electron microscopy (SEM), and fourier transformed infrared (FTIR) spectroscopy. The prepared superhydrophobic coating displays a good wear-resistance by emery paper and sand abrasion, which also has excellent thermal stability and UV resistance. This strategy shows a bright future for durable superhydrophobic coating on glass.
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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.
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The hepatic transcriptome of young suckling and aging intrauterine growth restricted male rats
Freije, William A.; Thamotharan, Shanthie; Lee, Regina; Shin, Bo-Chul; Devaskar, Sherin U.
2015-01-01
Intrauterine growth restriction leads to the development of adult onset obesity/metabolic syndrome, diabetes mellitus, cardiovascular disease, hypertension, stroke, dyslipidemia, and non-alcoholic fatty liver disease/steatohepatitis. Continued postnatal growth restriction has been shown to ameliorate many of these sequelae. To further our understanding of the mechanism of how intrauterine and early postnatal growth affects adult health we have employed Affymetrix microarray-based expression profiling to characterize hepatic gene expression of male offspring in a rat model of maternal nutrient restriction in early and late life. At day 21 of life (p21) combined intrauterine and postnatal calorie restriction treatment led to expression changes in circadian, metabolic, and insulin-like growth factor genes as part of a larger transcriptional response that encompasses 144 genes. Independent and controlled experiments at p21 confirm the early life circadian, metabolic, and growth factor perturbations. In contrast to the p21 transcriptional response, at day 450 of life (d450) only seven genes, largely uncharacterized, were differentially expressed. This lack of a transcriptional response identifies non-transcriptional mechanisms mediating the adult sequelae of intrauterine growth restriction. Independent experiments at d450 identify a circadian defect as well as validate expression changes to four of the genes identified by the microarray screen which have a novel association with growth restriction. Emerging from this rich dataset is a portrait of how the liver responds to growth restriction through circadian dysregulation, energy/substrate management, and growth factor modulation. PMID:25371150
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The hepatic transcriptome of young suckling and aging intrauterine growth restricted male rats.
Freije, William A; Thamotharan, Shanthie; Lee, Regina; Shin, Bo-Chul; Devaskar, Sherin U
2015-04-01
Intrauterine growth restriction leads to the development of adult onset obesity/metabolic syndrome, diabetes mellitus, cardiovascular disease, hypertension, stroke, dyslipidemia, and non-alcoholic fatty liver disease/steatohepatitis. Continued postnatal growth restriction has been shown to ameliorate many of these sequelae. To further our understanding of the mechanism of how intrauterine and early postnatal growth affects adult health we have employed Affymetrix microarray-based expression profiling to characterize hepatic gene expression of male offspring in a rat model of maternal nutrient restriction in early and late life. At day 21 of life (p21) combined intrauterine and postnatal calorie restriction treatment led to expression changes in circadian, metabolic, and insulin-like growth factor genes as part of a larger transcriptional response that encompasses 144 genes. Independent and controlled experiments at p21 confirm the early life circadian, metabolic, and growth factor perturbations. In contrast to the p21 transcriptional response, at day 450 of life (d450) only seven genes, largely uncharacterized, were differentially expressed. This lack of a transcriptional response identifies non-transcriptional mechanisms mediating the adult sequelae of intrauterine growth restriction. Independent experiments at d450 identify a circadian defect as well as validate expression changes to four of the genes identified by the microarray screen which have a novel association with growth restriction. Emerging from this rich dataset is a portrait of how the liver responds to growth restriction through circadian dysregulation, energy/substrate management, and growth factor modulation. © 2014 Wiley Periodicals, Inc.
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Udatha, D B R K Gupta; Kouskoumvekaki, Irene; Olsson, Lisbeth; Panagiotou, Gianni
2011-01-01
One of the most intriguing groups of enzymes, the feruloyl esterases (FAEs), is ubiquitous in both simple and complex organisms. FAEs have gained importance in biofuel, medicine and food industries due to their capability of acting on a large range of substrates for cleaving ester bonds and synthesizing high-added value molecules through esterification and transesterification reactions. During the past two decades extensive studies have been carried out on the production and partial characterization of FAEs from fungi, while much less is known about FAEs of bacterial or plant origin. Initial classification studies on FAEs were restricted on sequence similarity and substrate specificity on just four model substrates and considered only a handful of FAEs belonging to the fungal kingdom. This study centers on the descriptor-based classification and structural analysis of experimentally verified and putative FAEs; nevertheless, the framework presented here is applicable to every poorly characterized enzyme family. 365 FAE-related sequences of fungal, bacterial and plantae origin were collected and they were clustered using Self Organizing Maps followed by k-means clustering into distinct groups based on amino acid composition and physico-chemical composition descriptors derived from the respective amino acid sequence. A Support Vector Machine model was subsequently constructed for the classification of new FAEs into the pre-assigned clusters. The model successfully recognized 98.2% of the training sequences and all the sequences of the blind test. The underlying functionality of the 12 proposed FAE families was validated against a combination of prediction tools and published experimental data. Another important aspect of the present work involves the development of pharmacophore models for the new FAE families, for which sufficient information on known substrates existed. Knowing the pharmacophoric features of a small molecule that are essential for binding to the members of a certain family opens a window of opportunities for tailored applications of FAEs. Copyright © 2010 Elsevier Inc. All rights reserved.
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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.
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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.
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Substrate Specificity and Possible Heterologous Targets of Phytaspase, a Plant Cell Death Protease.
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.
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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
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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.
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Precise micropatterning of silver nanoparticles on plastic substrates
NASA Astrophysics Data System (ADS)
Ammosova, Lena; Jiang, Yu; Suvanto, Mika; Pakkanen, Tapani A.
2017-04-01
Conventional fabrication methods to obtain metal patterns on polymer substrates are restricted by high operating temperature and complex preparation steps. The present study demonstrates a simple yet versatile method for preparation of silver nanoparticle micropatterns on polymer substrates with various surface geometry. With the microworking robot technique, we were able not only to directly structure the surface, but also precisely deposit silver nanoparticle ink on the desired surface location with the minimum usage of ink material. The prepared silver nanoparticle ink, containing silver cations and polyethylene glycol (PEG) as a reducing agent, yields silver nanoparticle micropatterns on plastic substrates at low sintering temperature without any contamination. The influence of the ink behaviour was studied, such as substrate wettability, ink volume, and sintering temperature. The ultraviolet visible (UV-vis), scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS) measurements revealed the formation of micropatterns with uniformly distributed silver nanoparticles. The prepared patterns are expected to have a broad range of applications in optics, medicine, and sensor devices owing to the unique properties of silver. Furthermore, the deposition of a chemical compound, which is different from the substrate material, not only adds a fourth dimension to the prestructured three-dimensional (3D) surfaces, but also opens new application areas to the conventional surface structures.
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Pupal colour plasticity in a tropical butterfly, Mycalesis mineus (Nymphalidae: Satyrinae).
Mayekar, Harshad Vijay; Kodandaramaiah, Ullasa
2017-01-01
Lepidopteran insects have provided excellent study systems for understanding adaptive phenotypic plasticity. Although there are a few well-studied examples of adult plasticity among tropical butterflies, our understanding of plasticity of larval and pupal stages is largely restricted to temperate butterflies. The environmental parameters inducing phenotypic plasticity and the selective pressures acting on phenotypes are likely to differ across tropical and temperate climate regimes. We tested the influence of relative humidity (RH), a prominent yet under-appreciated tropical climatic component, along with pupation substrate, larval development time, pupal sex and weight in determining pupal colour in the tropical satyrine butterfly Mycalesis mineus. Pupae of this butterfly are either brown or green or very rarely intermediate. Larvae were reared at high (85%) and low (60%) RH at a constant temperature. Proportions of green and brown pupae were expected to vary across low and high RH and pupation substrates in order to enhance crypsis. Brown pupae were more common at low RH than at high RH, as predicted, and developed faster than green pupae. Pupal colour was correlated with pupation substrate. Choice of pupation substrate differed across RH treatments. It is unclear whether pupal colour influences substrate selection or whether substrate influences pupal colour. Our study underscores the need for further work to understand the basis of pupal plasticity in tropical butterflies.
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Patterning N-type and S-type neuroblastoma cells with Pluronic F108 and ECM proteins.
Corey, Joseph M; Gertz, Caitlyn C; Sutton, Thomas J; Chen, Qiaoran; Mycek, Katherine B; Wang, Bor-Shuen; Martin, Abbey A; Johnson, Sara L; Feldman, Eva L
2010-05-01
Influencing cell shape using micropatterned substrates affects cell behaviors, such as proliferation and apoptosis. Cell shape may also affect these behaviors in human neuroblastoma (NBL) cancer, but to date, no substrate design has effectively patterned multiple clinically important human NBL lines. In this study, we investigated whether Pluronic F108 was an effective antiadhesive coating for human NBL cells and whether it would localize three NBL lines to adhesive regions of tissue culture plastic or collagen I on substrate patterns. The adhesion and patterning of an S-type line, SH-EP, and two N-type lines, SH-SY5Y and IMR-32, were tested. In adhesion assays, F108 deterred NBL adhesion equally as well as two antiadhesive organofunctional silanes and far better than bovine serum albumin. Patterned stripes of F108 restricted all three human NBL lines to adhesive stripes of tissue culture plastic. We then investigated four schemes of applying collagen and F108 to different regions of a substrate. Contact with collagen obliterates the ability of F108 to deter NBL adhesion, limiting how both materials can be applied to substrates to produce high fidelity NBL patterning. This patterned substrate design should facilitate investigations of the role of cell shape in NBL cell behavior. Copyright 2009 Wiley Periodicals, Inc.
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Patterning N-type and S-type Neuroblastoma Cells with Pluronic F108 and ECM Proteins
Corey, Joseph M.; Gertz, Caitlyn C.; Sutton, Thomas J.; Chen, Qiaoran; Mycek, Katherine B.; Wang, Bor-Shuen; Martin, Abbey A.; Johnson, Sara L.; Feldman, Eva L.
2009-01-01
Influencing cell shape using micropatterned substrates affects cell behaviors, such as proliferation and apoptosis. Cell shape may also affect these behaviors in human neuroblastoma (NBL) cancer, but to date, no substrate design has effectively patterned multiple clinically important human NBL lines. In this study, we investigated whether Pluronic F108 was an effective anti-adhesive coating for human NBL cells and whether it would localize three NBL lines to adhesive regions of tissue culture plastic or collagen I on substrate patterns. The adhesion and patterning of an S-type line, SH-EP, and two N-type lines, SH-SY5Y and IMR-32, were tested. In adhesion assays, F108 deterred NBL adhesion equally as well as two anti-adhesive organofunctional silanes and far better than bovine serum albumin. Patterned stripes of F108 restricted all three human NBL lines to adhesive stripes of tissue culture plastic. We then investigated four schemes of applying collagen and F108 to different regions of a substrate. Contact with collagen obliterates the ability of F108 to deter NBL adhesion, limiting how both materials can be applied to substrates to produce high fidelity NBL patterning. This patterned substrate design should facilitate investigations of the role of cell shape in NBL cell behavior. PMID:19609877
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Langerhans Cells: the 'Yin and Yang' of HIV Restriction and Transmission.
Mayr, Luzia; Su, Bin; Moog, Christiane
2017-03-01
Langerhans cells are specialized sentinels present in the epidermis expressing Langerin, a specific C-type lectin receptor involved in HIV capture and destruction. Recently, the specific mechanism leading to this HIV restriction was discovered. Nevertheless, Langerhans cells can be infected and the way HIV escapes this restriction needs to be unraveled. Copyright © 2017. Published by Elsevier Ltd.
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CgII cleaves DNA using a mechanism distinct from other ATP-dependent restriction endonucleases
Toliusis, Paulius; Silanskas, Arunas; Szczelkun, Mark D.
2017-01-01
Abstract The restriction endonuclease CglI from Corynebacterium glutamicum recognizes an asymmetric 5′-GCCGC-3′ site and cleaves the DNA 7 and 6/7 nucleotides downstream on the top and bottom DNA strands, respectively, in an NTP-hydrolysis dependent reaction. CglI is composed of two different proteins: an endonuclease (R.CglI) and a DEAD-family helicase-like ATPase (H.CglI). These subunits form a heterotetrameric complex with R2H2 stoichiometry. However, the R2H2·CglI complex has only one nuclease active site sufficient to cut one DNA strand suggesting that two complexes are required to introduce a double strand break. Here, we report studies to evaluate the DNA cleavage mechanism of CglI. Using one- and two-site circular DNA substrates we show that CglI does not require two sites on the same DNA for optimal catalytic activity. However, one-site linear DNA is a poor substrate, supporting a mechanism where CglI complexes must communicate along the one-dimensional DNA contour before cleavage is activated. Based on experimental data, we propose that adenosine triphosphate (ATP) hydrolysis by CglI produces translocation on DNA preferentially in a downstream direction from the target, although upstream translocation is also possible. Our results are consistent with a mechanism of CglI action that is distinct from that of other ATP-dependent restriction-modification enzymes. PMID:28854738
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Hibi, Masanobu; Kubota, Chie; Mizuno, Tomohito; Aritake, Sayaka; Mitsui, Yuki; Katashima, Mitsuhiro; Uchida, Sunao
2017-01-01
The effects of sleep restriction on energy metabolism and appetite remain controversial. We examined the effects of shortened sleep duration on energy metabolism, core body temperature (CBT), and appetite profiles. Nine healthy men were evaluated in a randomised crossover study under two conditions: a 3.5-h sleep duration and a 7-h sleep duration for three consecutive nights followed by one 7-h recovery sleep night. The subjects’ energy expenditure (EE), substrate utilisation, and CBT were continually measured for 48 h using a whole-room calorimeter. The subjects completed an appetite questionnaire every hour while in the calorimeter. Sleep restriction did not affect total EE or substrate utilisation. The 48-h mean CBT decreased significantly during the 3.5-h sleep condition compared with the 7-h sleep condition (7-h sleep, 36.75 ± 0.11 °C; 3.5-h sleep, 36.68 ± 0.14 °C; p = 0.016). After three consecutive nights of sleep restriction, fasting peptide YY levels and fullness were significantly decreased (p = 0.011), whereas hunger and prospective food consumption were significantly increased, compared to those under the 7-h sleep condition. Shortened sleep increased appetite by decreasing gastric hormone levels, but did not affect EE, suggesting that greater caloric intake during a shortened sleep cycle increases the risk of weight gain. PMID:28071649
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38 CFR 17.33 - Patients' rights.
Code of Federal Regulations, 2013 CFR
2013-07-01
... refuse visitors. (iii) If a patient's right to receive unopened mail is restricted pursuant to paragraph... believes that the full exercise of the specific right would— (i) Adversely affect the patient's physical or... the specific right should be restricted. In determining whether a patient's specific right should be...
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38 CFR 17.33 - Patients' rights.
Code of Federal Regulations, 2011 CFR
2011-07-01
... refuse visitors. (iii) If a patient's right to receive unopened mail is restricted pursuant to paragraph... believes that the full exercise of the specific right would— (i) Adversely affect the patient's physical or... the specific right should be restricted. In determining whether a patient's specific right should be...
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38 CFR 17.33 - Patients' rights.
Code of Federal Regulations, 2012 CFR
2012-07-01
... refuse visitors. (iii) If a patient's right to receive unopened mail is restricted pursuant to paragraph... believes that the full exercise of the specific right would— (i) Adversely affect the patient's physical or... the specific right should be restricted. In determining whether a patient's specific right should be...
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38 CFR 17.33 - Patients' rights.
Code of Federal Regulations, 2014 CFR
2014-07-01
... refuse visitors. (iii) If a patient's right to receive unopened mail is restricted pursuant to paragraph... believes that the full exercise of the specific right would— (i) Adversely affect the patient's physical or... the specific right should be restricted. In determining whether a patient's specific right should be...
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Substrate specificity and pH dependence of homogeneous wheat germ acid phosphatase.
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.
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Federal Register 2010, 2011, 2012, 2013, 2014
2011-05-24
... Restrictions: Site-Specific Treatment Variance for Hazardous Selenium-Bearing Waste Treated by U.S. Ecology.... Ecology Nevada in Beatty, Nevada and withdrew an existing site- specific treatment variance issued to... 268.44(o)) by granting a site-specific treatment variance to U.S. Ecology Nevada in Beatty, Nevada and...
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Temperate bioerosion: ichnodiversity and biodiversity from intertidal to bathyal depths (Azores).
Wisshak, M; Tribollet, A; Golubic, S; Jakobsen, J; Freiwald, A
2011-11-01
In the temperate Azores carbonate factory, a substantial fraction of the calcareous skeletal components is recycled by a remarkable biodiversity of biota producing bioerosion traces (incipient trace fossils). To study this biodiversity, experimental carbonate substrates were exposed to colonisation by epilithic and endolithic organisms along a bathymetrical gradient from 0 to 500 m depth, during 1 and 2 years of exposure. The overall bioerosion ichnodiversity is very high and comprises 56 ichnotaxa and ichnoforms attributed to cyanobacteria, chlorophytes, fungi, other micro-chemotrophs, macroborers, grazers and epilithic attachment scars. In the intertidal, hydrodynamic force, partial emersion and strong temperature fluctuations lead to the lowest ichnospecies richness. This contrasts with the highest ichnodiversity found at 15 m under the most favourable environmental conditions. Towards aphotic depths, a gradual depletion in ichnodiversity is observed, most probably because of the restricted light availability and a slowdown in ichnocoenosis development. Analysis of similarity (ANOSIM), in combination with non-metrical multidimensional scaling (NMDS), was used to highlight variability in the relative abundance of traces among depths, substrate orientations and exposure times. Ichnodiversity and abundance of traces decrease significantly with depth and are higher on up-facing versus down-facing substrates, whereas differences between years were not as pronounced. This study demonstrates that statistical methods of biodiversity analysis are not per se restricted to biotaxa but may well be applied also to ichnotaxa. In the analysis of trace fossil assemblages, this approach supports the recognition of diversity patterns and their relation to environmental gradients. © 2011 Blackwell Publishing Ltd.
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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.
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Bioerosion and encrustation: Evidences from the Middle ‒ Upper Jurassic of central Saudi Arabia
NASA Astrophysics Data System (ADS)
El-Hedeny, Magdy; El-Sabbagh, Ahmed; Al Farraj, Saleh
2017-10-01
The Middle ‒ Upper Jurassic hard substrates of central Saudi Arabia displayed considerable signs of bioerosion and encrustations. They include organic (oysters, other bivalves, gastropods, corals and brachiopods) and an inorganic carbonate hardground that marks the boundary between the Middle Jurassic Tuwaiq Mountain Limestone and the Upper Jurassic Hanifa Formation. Traces of bioerosion in organic substrates include seven ichnotaxa produced by bivalves (Gastrochaenolites Leymerie, 1842), polychaete annelids (Trypanites Mägdefrau, 1932; MaeandropolydoraVoigt, 1965 and CaulostrepsisClarke, 1908), sponges (Entobia Bronn, 1837), acrothoracican cirripedes (Rogerella Saint-Seine, 1951), gastropods (Oichnus Bromley, 1981) and probable ?Centrichnus cf. eccentricus. The encrusting epifauna on these substrates consist of several organisms, including oysters, serpulid worms, corals and foraminifera. In contrast, the carbonate hardground was only bioeroded by Gastrochaenolite, Trypanites and Entobia. Epibionts on this hardground include ;Liostrea Douvillé, 1904-type; oysters, Nanogyra nana Sowerby, 1822 and serpulids. In general, bioerosion and encrustation are less diversified in hardground than in organic substrates, indicating a long time of exposition of organic substrates with slow to moderate rate of deposition in a restricted marine environment. Both organic and inorganic commuinities are correlated with those of other equatorial, subtropical and temperate equivalents.
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Structural properties of atactic polystyrene adsorbed onto solid surfaces.
Tatek, Yergou B; Tsige, Mesfin
2011-11-07
In the present work, we are studying the local conformation of chains in a thin film of polystyrene adsorbed on a solid substrate by using atomistically detailed simulations. The simulations are carried out by using the readily available and massively parallel molecular dynamics code known as LAMMPS. In particular, a special emphasis is given to the density and orientation of side chains (which consist of phenyl groups and methylene units) at solid/polymer and polymer/vacuum interfaces. Three types of substrates were used in our study: α-quartz, graphite, and amorphous silica. Our investigation was restricted to atactic polystyrene. Our results show that the density and structural properties of side chains depend on the type of surface. An excess of phenyl rings is observed near the α-quartz substrate while the film adsorbed on graphite is depleted in C(6)H(5). Moreover, the orientation of the rings and methylene units on the substrate/film interface show a strong dependence on the type of the substrate, while the rings at the film/vacuum interface show a marked tendency to point outward, away from the film. The results we obtained are in a large part in good agreement with previous experimental and simulation results.
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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.
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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.
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Resveratrol serves as a protein-substrate interaction stabilizer in human SIRT1 activation.
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.
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Engineering the substrate specificity of the DhbE adenylation domain by yeast cell surface display.
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.
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Neural Substrates Underlying Effort, Time, and Risk-Based Decision Making in Motivated Behavior
Bailey, Matthew R.; Simpson, Eleanor H.; Balsam, Peter D
2016-01-01
All mobile organisms rely on adaptive motivated behavior to overcome the challenges of living in an environment in which essential resources may be limited. A variety of influences ranging from an organism’s environment, experiential history, and physiological state all influence a cost-benefit analysis which allows motivation to energize behavior and direct it toward specific goals. Here we review the substantial amount of research aimed at discovering the interconnected neural circuits which allow organisms to carry-out the cost-benefit computations which allow them to behave in adaptive ways. We specifically focus on how the brain deals with different types of costs, including effort requirements, delays to reward and payoff riskiness. An examination of this broad literature highlights the importance of the extended neural circuits which enable organisms to make decisions about these different types of costs. This involves Cortical Structures, including the Anterior Cingulate Cortex (ACC), the Orbital Frontal Cortex (OFC), the Infralimbic Cortex (IL), and prelimbic Cortex (PL), as well as the Baso-Lateral Amygdala (BLA), the Nucleus Accumbens (Nacc), the Ventral Pallidal (VP), the Sub Thalamic Nucleus (STN) among others. Some regions are involved in multiple aspects of cost-benefit computations while the involvement of other regions is restricted to information relating to specific types of costs. PMID:27427327
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Neural substrates underlying effort, time, and risk-based decision making in motivated behavior.
Bailey, Matthew R; Simpson, Eleanor H; Balsam, Peter D
2016-09-01
All mobile organisms rely on adaptive motivated behavior to overcome the challenges of living in an environment in which essential resources may be limited. A variety of influences ranging from an organism's environment, experiential history, and physiological state all influence a cost-benefit analysis which allows motivation to energize behavior and direct it toward specific goals. Here we review the substantial amount of research aimed at discovering the interconnected neural circuits which allow organisms to carry-out the cost-benefit computations which allow them to behave in adaptive ways. We specifically focus on how the brain deals with different types of costs, including effort requirements, delays to reward and payoff riskiness. An examination of this broad literature highlights the importance of the extended neural circuits which enable organisms to make decisions about these different types of costs. This involves Cortical Structures, including the Anterior Cingulate Cortex (ACC), the Orbital Frontal Cortex (OFC), the Infralimbic Cortex (IL), and prelimbic Cortex (PL), as well as the Baso-Lateral Amygdala (BLA), the Nucleus Accumbens (NAcc), the Ventral Pallidal (VP), the Sub Thalamic Nucleus (STN) among others. Some regions are involved in multiple aspects of cost-benefit computations while the involvement of other regions is restricted to information relating to specific types of costs. Copyright © 2016 Elsevier Inc. All rights reserved.
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Substrate-Driven Mapping of the Degradome by Comparison of Sequence Logos
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
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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.
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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.
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Somyoonsap, Peechapack; Kitpreechavanich, Vichein
2013-01-01
A sequence-specific nicking endonuclease from Streptomyces designated as DC13 was purified to near homogeneity. Starting with 30 grams of wet cells, the enzyme was purified by ammonium sulfate fractionation, DEAE cellulose, and phenyl-Sepharose chromatography. The purified protein had a specific activity 1000 units/mg and migrated on SDS-PAGE gel with an estimated molecular weight of 71 kDa. Determination of subunit composition by gel filtration chromatography indicated that the native enzyme is a monomer. When incubated with different DNA substrates including pBluescript II KS, pUC118, pET-15b, and pET-26b, the enzyme converted these supercoiled plasmids to a mixture of open circular and linear DNA products, with the open circular DNA as the major cleavage product. Analysis of the kinetic of DNA cleavage showed that the enzyme appeared to cleave super-coiled plasmid in two distinct steps: a rapid cleavage of super-coiled plasmid to an open circular DNA followed a much slower step to linear DNA. The DNA cleavage reaction of the enzyme required Mg2+ as a cofactor. Based on the monomeric nature of the enzyme, the kinetics of DNA cleavage exhibited by the enzyme, and cofactor requirement, it is suggested here that the purified enzyme is a sequence-specific nicking endonuclease that is similar to type IIS restriction endonuclease. PMID:25937959
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Freund, Barbara; Petrakos, Davithoula
2008-01-01
We developed driving restrictions that are linked to specific driving errors, allowing cognitively impaired individuals to continue to independently meet mobility needs while minimizing risk to themselves and others. The purpose of this project was to evaluate the efficacy and duration expectancy of these restrictions in promoting safe continued driving. We followed 47 drivers age 60 years and older for 18 months, evaluating driving performance at 6-month intervals. Results demonstrated restricted drivers had safety profiles similar to safe drivers and gained additional driving time to transition to nondrivers.
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Divakaruni, Ajit S; Rogers, George W; Murphy, Anne N
2014-05-27
Measurements of mitochondrial respiration in intact cells can help define metabolism and its dysregulation in fields such as cancer, metabolic disease, immunology, and neurodegeneration. Although cells can be offered various substrates in the assay medium, many cell types can oxidize stored pools of energy substrates. A general bioenergetic profile can therefore be obtained using intact cells, but the inability to control substrate provision to the mitochondria can restrict an in-depth, mechanistic understanding. Mitochondria can be isolated from intact cells, but the yield and quality of the end product is often poor and prone to subselection during isolation. Plasma membrane permeabilization of cells provides a solution to this challenge, allowing experimental control of the medium surrounding the mitochondria. This unit describes techniques to measure respiration in permeabilized adherent cells using a Seahorse XF Analyzer or permeabilized suspended cells in a Hansatech Oxygraph. Copyright © 2014 John Wiley & Sons, Inc.
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Kang, Dongseok; Kim, Won-Jun; Lim, Jung Ah; Song, Yong-Won
2012-07-25
Using only a simple tube furnace, we demonstrate the synthesis of patterned graphene directly on a designed substrate without the need for an external carbon source. Carbon atoms are absorbed onto Ni evaporator sources as impurities, and incorporated into catalyst layers during the deposition. Heat treatment conditions were optimized so that the atoms diffused out along the grain boundaries to form nanocrystals at the catalyst-substrate interfaces. Graphene patterns were obtained under patterned catalysts, which restricted graphene formation to within patterned areas. The resultant multilayer graphene was characterized by Raman spectroscopy and transmission electron microscopy to verify the high crystallinity and two-dimensional nanomorphology. Finally, a metal-semiconductor diode with a catalyst-graphene contact structure were fabricated and characterized to assess the semiconducting properties of the graphene sheets with respect to the display of asymmetric current-voltage behavior.
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Hamson, Elizabeth J; Keane, Fiona M; Tholen, Stefan; Schilling, Oliver; Gorrell, Mark D
2014-06-01
Fibroblast activation protein (FAP) is best known for its heightened expression in tumour stroma. This atypical serine protease has both dipeptidyl peptidase and endopeptidase activities, cleaving substrates at a post-proline bond. FAP expression is difficult to detect in non-diseased adult organs, but is greatly upregulated in sites of tissue remodelling, which include liver fibrosis, lung fibrosis, atherosclerosis, arthritis, tumours and embryonic tissues. Due to its restricted expression pattern and dual enzymatic activities, FAP is emerging as a unique therapeutic target. However, methods to exploit and target this protease are advancing more rapidly than knowledge of the fundamental biology of FAP. This review highlights this imbalance, emphasising the need to better define the substrate repertoire and expression patterns of FAP to elucidate its role in biological and pathological processes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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Kaye, Nicholas M; Christian, Eric L; Harris, Michael E
2002-04-09
The tRNA processing endonuclease ribonuclease P contains an essential and highly conserved RNA molecule (RNase P RNA) that is the catalytic subunit of the enzyme. To identify and characterize functional groups involved in RNase P RNA catalysis, we applied self-cleaving ribozyme-substrate conjugates, on the basis of the RNase P RNA from Escherichia coli, in nucleotide analogue interference mapping (NAIM) and site-specific modification experiments. At high monovalent ion concentrations (3 M) that facilitate protein-independent substrate binding, we find that the ribozyme is largely insensitive to analogue substitution and that concentrations of Mg2+ (1.25 mM) well below that necessary for optimal catalytic rate (>100 mM) are required to produce interference effects because of modification of nucleotide bases. An examination of the pH dependence of the reaction rate at 1.25 mM Mg2+ indicates that the increased sensitivity to analogue interference is not due to a change in the rate-limiting step. The nucleotide positions detected by NAIM under these conditions are located exclusively in the catalytic domain, consistent with the proposed global structure of the ribozyme, and predominantly occur within the highly conserved P1-P4 multihelix junction. Several sensitive positions in J3/4 and J2/4 are proximal to a previously identified site of divalent metal ion binding in the P1-P4 element. Kinetic analysis of ribozymes with site-specific N7-deazaadenosine and deazaguanosine modifications in J3/4 was, in general, consistent with the interference results and also permitted the analysis of sites not accessible by NAIM. These results show that, in this region only, modification of the N7 positions of A62, A65, and A66 resulted in measurable effects on reaction rate and modification at each position displayed distinct sensitivities to Mg2+ concentration. These results reveal a restricted subset of individual functional groups within the catalytic domain that are particularly important for substrate cleavage and demonstrate a close association between catalytic function and metal ion-dependent structure in the highly conserved P1-P4 multihelix junction.
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Shrestha, Swastina; Dave, Amish J; Losina, Elena; Katz, Jeffrey N
2016-07-07
Administrative health care data are frequently used to study disease burden and treatment outcomes in many conditions including osteoarthritis (OA). OA is a chronic condition with significant disease burden affecting over 27 million adults in the US. There are few studies examining the performance of administrative data algorithms to diagnose OA. The purpose of this study is to perform a systematic review of administrative data algorithms for OA diagnosis; and, to evaluate the diagnostic characteristics of algorithms based on restrictiveness and reference standards. Two reviewers independently screened English-language articles published in Medline, Embase, PubMed, and Cochrane databases that used administrative data to identify OA cases. Each algorithm was classified as restrictive or less restrictive based on number and type of administrative codes required to satisfy the case definition. We recorded sensitivity and specificity of algorithms and calculated positive likelihood ratio (LR+) and positive predictive value (PPV) based on assumed OA prevalence of 0.1, 0.25, and 0.50. The search identified 7 studies that used 13 algorithms. Of these 13 algorithms, 5 were classified as restrictive and 8 as less restrictive. Restrictive algorithms had lower median sensitivity and higher median specificity compared to less restrictive algorithms when reference standards were self-report and American college of Rheumatology (ACR) criteria. The algorithms compared to reference standard of physician diagnosis had higher sensitivity and specificity than those compared to self-reported diagnosis or ACR criteria. Restrictive algorithms are more specific for OA diagnosis and can be used to identify cases when false positives have higher costs e.g. interventional studies. Less restrictive algorithms are more sensitive and suited for studies that attempt to identify all cases e.g. screening programs.
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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.
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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
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Mondal, D.; RoyChaudhuri, C., E-mail: chirosreepram@yahoo.com; Pal, D.
2015-07-28
Oxidized porous silicon (PS) is a common topographical biocompatible substrate that potentially provides a distinct in vitro environment for better understanding of in vivo behavior. But in the reported studies on oxidized PS, cell-cell and cell-substrate interactions have been detected only by fluorescent labeling. This paper is the first attempt to investigate real-time sensing of these interactions on HaCaT cells by label-free impedance spectroscopy on oxidized PS of two pore diameters (50 and 500 nm). One of the major requirements for successful impedance spectroscopy measurement is to restrict the channeling of electric field lines through the pores. To satisfy this criterion,more » we have designed the pore depths after analyzing the penetration of the medium by using computational fluid dynamics simulation. A distributed electrical model was also developed for estimating the various cellular attributes by considering a pseudorandom distribution of pores. It is observed from the impedance measurements and from the model that the proliferation rate increases for 50 nm pores but decreases for 500 nm pores compared to that for planar substrates. The rate of decrease in cell substrate separation (h) in the initial stage is more than the rate of increase in cell-cell junction resistance (R{sub b}) corresponding to the initial adhesion phase of cells. It is observed that R{sub b} and h are higher for 50 nm pores than those for planar substrates, corresponding to the fact that substrates more conducive toward cell adhesion encourage cell-cell interactions than direct cell-substrate interactions. Thus, the impedance spectroscopy coupled with the proposed theoretical framework for PS substrates can sense and quantify the cellular interactions.« less
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Bitterlich, Michael; Franken, Philipp; Graefe, Jan
2018-01-01
Arbuscular mycorrhizal fungi (AMF) proliferate in soils and are known to affect soil structure. Although their contribution to structure is extensively investigated, the consequences of those processes for soil water extractability and transport has, so far, gained surprisingly little attention. Therefore we asked, whether AMF can affect water retention and unsaturated hydraulic conductivity under exclusion of root ingrowth, in order to minimize plant driven effects. We carried out experiments with tomato inoculated with Rhizoglomus irregulare in a soil substrate with sand and vermiculite that created variation in colonization by mixed pots with wild type (WT) plants and mycorrhiza resistant (RMC) mutants. Sampling cores were introduced and used to assess substrate moisture retention dynamics and modeling of substrate water retention and hydraulic conductivity. AMF reduced the saturated water content and total porosity, but maintained air filled porosity in soil spheres that excluded root ingrowth. The water content between field capacity and the permanent wilting point (6-1500 kPa) was only reduced in mycorrhizal substrates that contained at least one RMC mutant. Plant available water contents correlated positively with soil protein contents. Soil protein contents were highest in pots that possessed the strongest hyphal colonization, but not significantly affected. Substrate conductivity increased up to 50% in colonized substrates in the physiologically important water potential range between 6 and 10 kPa. The improvements in hydraulic conductivity are restricted to substrates where at least one WT plant was available for the fungus, indicating a necessity of a functional symbiosis for this effect. We conclude that functional mycorrhiza alleviates the resistance to water movement through the substrate in substrate areas outside of the root zone.
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Importance of benthic prey for fishes in coral reef-associated sediments
DeFelice, R.C.; Parrish, J.D.
2003-01-01
The importance of open, sandy substrate adjacent to coral reefs as habitat and a food source for fishes has been little studied in most shallow tropical waters in the Pacific, including Hawai'i. In this study, in Hanalei Bay, Hiwai'i, we identified and quantified the major invertebrate fauna (larger than 0.5 mm) in the well-characterized sands adjoining the shallow fringing reefs. Concurrently, we identified the fish species that seemed to make substantial use of these sand habitats, estimated their density there, sampled their gut contents to examine trophic links with the sand habitat, and made other observations and collections to determine the times, locations, and types of activity there. A variety of (mostly small) polychaeres were dominant in the sediments at most sampling stations, along with many small crustaceans (e.g., amphipods, isopods, ostracods, and small shrimps) and fair numbers of mollusks (especially bivalves) and small echinoids. Fish guts examined contained ???77% of the total number of benthic taxa collected, including nearly all those just listed. However, fish consumption was selective, and the larger shrimps, crabs, and small cryptic fishes were dominant in the diets of most of the numerous predator taxa. Diets of benthic-feeding fishes showed relatively low specific overlap. The fish fauna in this area included substrate-indifferent pelagics, species with various degrees of reef relatedness, reef-restricted species, and (at the other extreme) permanent cryptic sand dwellers. Data on occurrence and movements of fishes indicated that a band of sandy substrate several tens of meters wide next to the reef was an active area for fishes, and activity was considerably different at different times of day and for fish of different ages. These results imply an important trophic role for the benthos in these near-reef habitats in support of reef-associated fishes.
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Micali, E; Chehade, K A; Isaacs, R J; Andres, D A; Spielmann, H P
2001-10-16
Farnesylation is a posttranslational lipid modification in which a 15-carbon farnesyl isoprenoid is linked via a thioether bond to specific cysteine residues of proteins in a reaction catalyzed by protein farnesyltransferase (FTase). We synthesized the benzyloxyisoprenyl pyrophosphate (BnPP) series of transferable farnesyl pyrophosphate (FPP) analogues (1a-e) to test the length dependence of the isoprenoid substrate on the FTase-catalyzed transfer of lipid to protein substrate. Kinetic analyses show that pyrophosphates 1a-e and geranyl pyrophosphate (GPP) transfer with a lower efficiency than FPP whereas geranylgeranyl pyrophosphate (GGPP) does not transfer at all. While a correlation was found between K(m) and analogue hydrophobicity and length, there was no correlation between k(cat) and these properties. Potential binding geometries of FPP, GPP, GGPP, and analogues 1a-e were examined by modeling the molecules into the active site of the FTase crystal structure. We found that analogue 1d displaces approximately the same volume of the active site as does FPP, whereas GPP and analogues 1a-c occupy lesser volumes and 1e occupies a slightly larger volume. Modeling also indicated that GGPP adopts a different conformation than the farnesyl chain of FPP, partially occluding the space occupied by the Ca(1)a(2)X peptide in the ternary X-ray crystal structure. Within the confines of the FTase pocket, the double bonds and branched methyl groups of the geranylgeranyl chain significantly restrict the number of possible conformations relative to the more flexible lipid chain of analogues 1a-e. The modeling results also provide a molecular explanation for the observation that an aromatic ring is a good isostere for the terminal isoprene of FPP.
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NASA Astrophysics Data System (ADS)
Chu, Elza
Phase separation in soft matter has been the crucial element in generating hybrid materials, such as polymer blends and mixed polymer brushes. This dissertation discusses two methods of developing self-assembled nanoscale templates via microphase separation induced by polymer brush synthesis. This work introduces a novel soft substrate approach with renewable grafting sites where polyacrylamide is "grafted through" chitosan soft substrates. The mechanism of grafting leads to ordered arrays of filament-like nanostructures spanning the chitosan-air interface. Additionally, the chemical composition of the filaments allows for post-chemical modification to change the physical properties of the filaments, and subsequently tailor surfaces for specific application. Unlike traditional materials, multi-functional or "smart" materials, such as binary polymer brushes (BPB) are capable of spontaneously changing the spatial distribution of functional groups and morphology at the surface upon external stimuli. Although promising in principle, the limited range of available complementary polymers with common non-selective solvents confines the diversity of usable materials and restricts any further advancement in the field. This dissertation also covers the fabrication and characterization of responsive nanoscale polystyrene templates or "mosaic" brushes that are capable of changing interfacial composition upon exposure to varying solvent qualities. Using a "mosaic" brush template is a unique approach that allows the fabrication of strongly immiscible polymer BPB without the need for a common solvent. The synthesis of such BPB is exemplified by two strongly immiscible polymers, i.e. polystyrene (polar) and polyacrylamide (non-polar), where polyacrylamide brush is "graft through" a Si-substrate modified with the polystyrene collapsed "mosaic" brush. The surface exhibits solvent-triggered responses, as well as application potential for anti-biofouling.
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Padilla, Nelly; Junqué, Carme; Figueras, Francesc; Sanz-Cortes, Magdalena; Bargalló, Núria; Arranz, Angela; Donaire, Antonio; Figueras, Josep; Gratacos, Eduard
2014-01-30
Intrauterine growth restriction (IUGR) is associated with a high risk of abnormal neurodevelopment. Underlying neuroanatomical substrates are partially documented. We hypothesized that at 12 months preterm infants would evidence specific white-matter microstructure alterations and gray-matter differences induced by severe IUGR. Twenty preterm infants with IUGR (26-34 weeks of gestation) were compared with 20 term-born infants and 20 appropriate for gestational age preterm infants of similar gestational age. Preterm groups showed no evidence of brain abnormalities. At 12 months, infants were scanned sleeping naturally. Gray-matter volumes were studied with voxel-based morphometry. White-matter microstructure was examined using tract-based spatial statistics. The relationship between diffusivity indices in white matter, gray matter volumes, and perinatal data was also investigated. Gray-matter decrements attributable to IUGR comprised amygdala, basal ganglia, thalamus and insula bilaterally, left occipital and parietal lobes, and right perirolandic area. Gray-matter volumes positively correlated with birth weight exclusively. Preterm infants had reduced FA in the corpus callosum, and increased FA in the anterior corona radiata. Additionally, IUGR infants had increased FA in the forceps minor, internal and external capsules, uncinate and fronto-occipital white matter tracts. Increased axial diffusivity was observed in several white matter tracts. Fractional anisotropy positively correlated with birth weight and gestational age at birth. These data suggest that IUGR differentially affects gray and white matter development preferentially affecting gray matter. At 12 months IUGR is associated with a specific set of structural gray-matter decrements. White matter follows an unusual developmental pattern, and is apparently affected by IUGR and prematurity combined. Copyright © 2013 Elsevier B.V. All rights reserved.
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Zhou, Ying; Carpenter, Zachary W.; Brennan, Gregory
2009-01-01
Drosophila Morgue is a unique ubiquitination protein that facilitates programmed cell death and associates with DIAP1, a critical cell death inhibitor with E3 ubiquitin ligase activity. Morgue possesses a unique combination of functional domains typically associated with distinct types of ubiquitination enzymes. This includes an F box characteristic of the substrate-binding subunit in Skp, Cullin, and F box (SCF)-type ubiquitin E3 ligase complexes and a variant ubiquitin E2 conjugase domain where the active site cysteine is replaced by a glycine. Morgue also contains a single C4-type zinc finger motif. This architecture suggests potentially novel ubiquitination activities for Morgue. In this study, we address the evolutionary origins of this distinctive protein utilizing a combination of bioinformatics and molecular biology approaches. We find that Morgue exhibits widespread but restricted phylogenetic distribution among metazoans. Morgue proteins were identified in a wide range of Protostome phyla, including Arthropoda, Annelida, Mollusca, Nematoda, and Platyhelminthes. However, with one potential exception, Morgue was not detected in Deuterostomes, including Chordates, Hemichordates, or Echinoderms. Morgue was also not found in Ctenophora, Cnidaria, Placozoa, or Porifera. Characterization of Morgue sequences within specific animal lineages suggests that gene deletion or acquisition has occurred during divergence of nematodes and that at least one arachnid expresses an atypical form of Morgue consisting only of the variant E2 conjugase domain. Analysis of the organization of several morgue genes suggests that exon-shuffling events have contributed to the evolution of the Morgue protein. These results suggest that Morgue mediates conserved and distinctive ubiquitination functions in specific cell death pathways. PMID:19602541
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Bitterlich, Michael; Sandmann, Martin; Graefe, Jan
2018-01-01
Arbuscular mycorrhizal fungi (AMF) proliferate in soil pores, on the surface of soil particles and affect soil structure. Although modifications in substrate moisture retention depend on structure and could influence plant water extraction, mycorrhizal impacts on water retention and hydraulic conductivity were rarely quantified. Hence, we asked whether inoculation with AMF affects substrate water retention, water transport properties and at which drought intensity those factors become limiting for plant transpiration. Solanum lycopersicum plants were set up in the glasshouse, inoculated or not with Funneliformis mosseae , and grown for 35 days under ample water supply. After mycorrhizal establishment, we harvested three sets of plants, one before (36 days after inoculation) and the second (day 42) and third (day 47) within a sequential drying episode. Sampling cores were introduced into pots before planting. After harvest, moisture retention and substrate conductivity properties were assessed and water retention and hydraulic conductivity models were fitted. A root water uptake model was adopted in order to identify the critical substrate moisture that induces soil derived transpiration limitation. Neither substrate porosity nor saturated water contents were affected by inoculation, but both declined after substrates dried. Drying also caused a decline in pot water capacity and hydraulic conductivity. Plant available water contents under wet (pF 1.8-4.2) and dry (pF 2.5-4.2) conditions increased in mycorrhizal substrates and were conserved after drying. Substrate hydraulic conductivity was higher in mycorrhizal pots before and during drought exposure. After withholding water from pots, higher substrate drying rates and lower substrate water potentials were found in mycorrhizal substrates. Mycorrhiza neither affected leaf area nor root weight or length. Consistently with higher substrate drying rates, AMF restored the plant hydraulic status, and increased plant transpiration when soil moisture declined. The water potential at the root surface and the resistance to water flow in the rhizosphere were restored in mycorrhizal pots although the bulk substrate dried more. Finally, substrates colonized by AMF can be more desiccated before substrate water flux quantitatively limits transpiration. This is most pronounced under high transpiration demands and complies with a difference of over 1,000 hPa in substrate water potential.
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NASA Technical Reports Server (NTRS)
Sergeev, V. N.; Knoll, A. H.
1997-01-01
Silicified carbonates of the latest Mesoproterozoic Sukhaya Tunguska Formation, northwestern Siberia, contain abundant and diverse permineralized microfossils. Peritidal environments are dominated by microbial mats built by filamentous cyanobacteria comparable to modern species of Lyngbya and Phormidium. In subtidal to lower intertidal settings, mat-dwelling microbenthos and possible coastal microplankton are abundant. In contrast, densely woven mat populations with few associated taxa characterize more restricted parts of tidal flats; the preservation of vertically oriented sheath bundles and primary fenestrae indicates that in these mats carbonate cementation was commonly penecontemporaneous with mat growth. Eoentophysalis mats are limited to restricted environments where microlaminated carbonate precipitates formed on or just beneath the sediment surface. Most microbenthic populations are cyanobacterial, although eukaryotic microfossils may occur among the simple spheroidal cells interpreted as coastal plankton. Protists are more securely represented by large (up to 320 micrometers in diameter) but poorly preserved acritarchs in basinal facies. The Sukhaya Tunguska assemblage contains 27 species in 18 genera. By virtue of their stratigraphic longevity and their close and predictable association with specific paleoenvironmental conditions, including substrates, Proterozoic cyanobacteria support a model of bacterial evolution in which populations adapt rapidly to novel environments and, thereafter, resist competitive replacement. The resulting evolutionary pattern is one of accumulation and stasis rather than the turnover and replacement characteristic of Phanerozoic plants and animals.
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Sergeev, V N; Knoll, A H; Petrov PYu
1997-12-01
Silicified carbonates of the latest Mesoproterozoic Sukhaya Tunguska Formation, northwestern Siberia, contain abundant and diverse permineralized microfossils. Peritidal environments are dominated by microbial mats built by filamentous cyanobacteria comparable to modern species of Lyngbya and Phormidium. In subtidal to lower intertidal settings, mat-dwelling microbenthos and possible coastal microplankton are abundant. In contrast, densely woven mat populations with few associated taxa characterize more restricted parts of tidal flats; the preservation of vertically oriented sheath bundles and primary fenestrae indicates that in these mats carbonate cementation was commonly penecontemporaneous with mat growth. Eoentophysalis mats are limited to restricted environments where microlaminated carbonate precipitates formed on or just beneath the sediment surface. Most microbenthic populations are cyanobacterial, although eukaryotic microfossils may occur among the simple spheroidal cells interpreted as coastal plankton. Protists are more securely represented by large (up to 320 micrometers in diameter) but poorly preserved acritarchs in basinal facies. The Sukhaya Tunguska assemblage contains 27 species in 18 genera. By virtue of their stratigraphic longevity and their close and predictable association with specific paleoenvironmental conditions, including substrates, Proterozoic cyanobacteria support a model of bacterial evolution in which populations adapt rapidly to novel environments and, thereafter, resist competitive replacement. The resulting evolutionary pattern is one of accumulation and stasis rather than the turnover and replacement characteristic of Phanerozoic plants and animals.
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Wherry, E John; Golovina, Tatiana N; Morrison, Susan E; Sinnathamby, Gomathinayagam; McElhaugh, Michael J; Shockey, David C; Eisenlohr, Laurence C
2006-02-15
The proteasome is primarily responsible for the generation of MHC class I-restricted CTL epitopes. However, some epitopes, such as NP(147-155) of the influenza nucleoprotein (NP), are presented efficiently in the presence of proteasome inhibitors. The pathways used to generate such apparently "proteasome-independent" epitopes remain poorly defined. We have examined the generation of NP(147-155) and a second proteasome-dependent NP epitope, NP(50-57), using cells adapted to growth in the presence of proteasome inhibitors and also through protease overexpression. We observed that: 1) Ag processing and presentation proceeds in proteasome-inhibitor adapted cells but may become more dependent, at least in part, on nonproteasomal protease(s), 2) tripeptidyl peptidase II does not substitute for the proteasome in the generation of NP(147-155), 3) overexpression of leucine aminopeptidase, thymet oligopeptidase, puromycin-sensitive aminopeptidase, and bleomycin hydrolase, has little impact on the processing and presentation of NP(50-57) or NP(147-155), and 4) proteasome-inhibitor treatment altered the specificity of substrate cleavage by the proteasome using cell-free digests favoring NP(147-155) epitope preservation. Based on these results, we propose a central role for the proteasome in epitope generation even in the presence of proteasome inhibitors, although such inhibitors will likely alter cleavage patterns and may increase the dependence of the processing pathway on postproteasomal enzymes.
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Specificity of hammerhead ribozyme cleavage.
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
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Fang, Xian; Wang, Xueting; Li, Guiling; Zeng, Jun; Li, Jian; Liu, Jingwen
2018-05-01
PEGylation is one of the most promising and extensively studied strategies for improving the properties of proteins as well as enzymic physical and thermal stability. Phospholipase C, hydrolyzing the phospholipids offers tremendous applications in diverse fields. However, the poor thermal stability and higher cost of production have restricted its industrial application. This study focused on improving the stabilization of recombinant PLC by chemical modification with methoxypolyethylene glycol-Succinimidyl Succinate (SS-mPEG, MW 5000). PLC gene from isolate Bacillus cereus HSL3 was fused with SUMO, a novel small ubiquitin-related modifier expression vector and over expressed in Escherichia coli. The soluble fraction of SUMO-PLC reached 80% of the total recombinant protein. The enzyme exhibited maximum catalytic activity at 80 °C and was relatively thermostable at 40-70 °C. It showed extensive substrate specificity pattern and marked activity toward phosphatidylcholine, which made it a typical non-specific PLC for industrial purpose. SS-mPEG-PLC complex exhibited an enhanced thermal stability at 70-80 °C and the catalytic efficiency (K cat /K m ) had increased by 3.03 folds compared with free PLC. CD spectrum of SS-mPEG-PLC indicated a possible enzyme aggregation after chemical modification, which contributed to the higher thermostability of SS-mPEG-PLC. The increase of antiparallel β sheets in secondary structure also made it more stable than parallel β sheets. The presence of SS-mPEG chains on the enzyme molecule surface somewhat changed the binding rate of the substrates, leading to a significant improvement in catalytic efficiency. This study provided an insight into the addition of SS-mPEG for enhancing the industrial applications of phospholipase C at higher temperature. Copyright © 2018 Elsevier B.V. All rights reserved.
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A Proteasome Cap Subunit Required for Spindle Pole Body Duplication in Yeast
McDonald, Heather B.; Byers, Breck
1997-01-01
Proteasome-mediated protein degradation is a key regulatory mechanism in a diversity of complex processes, including the control of cell cycle progression. The selection of substrates for degradation clearly depends on the specificity of ubiquitination mechanisms, but further regulation may occur within the proteasomal 19S cap complexes, which attach to the ends of the 20S proteolytic core and are thought to control entry of substrates into the core. We have characterized a gene from Saccharomyces cerevisiae that displays extensive sequence similarity to members of a family of ATPases that are components of the 19S complex, including human subunit p42 and S. cerevisiae SUG1/ CIM3 and CIM5 products. This gene, termed PCS1 (for proteasomal cap subunit), is identical to the recently described SUG2 gene (Russell, S.J., U.G. Sathyanarayana, and S.A. Johnston. 1996. J. Biol. Chem. 271:32810– 32817). We have shown that PCS1 function is essential for viability. A temperature-sensitive pcs1 strain arrests principally in the second cycle after transfer to the restrictive temperature, blocking as large-budded cells with a G2 content of unsegregated DNA. EM reveals that each arrested pcs1 cell has failed to duplicate its spindle pole body (SPB), which becomes enlarged as in other monopolar mutants. Additionally, we have shown localization of a functional Pcs1–green fluorescent protein fusion to the nucleus throughout the cell cycle. We hypothesize that Pcs1p plays a role in the degradation of certain potentially nuclear component(s) in a manner that specifically is required for SPB duplication. PMID:9151663
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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
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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
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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
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Dissecting substrate specificities of the mitochondrial AFG3L2 protease.
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.
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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
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Computational Study on Substrate Specificity of a Novel Cysteine Protease 1 Precursor from Zea mays
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
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A multiwell format assay for heparanase.
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.
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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.
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Interfacial characterization of flexible hybrid electronics
NASA Astrophysics Data System (ADS)
Najafian, Sara; Amirkhizi, Alireza V.; Stapleton, Scott
2018-03-01
Flexible Hybrid Electronics (FHEs) are the new generation of electronics combining flexible plastic film substrates with electronic devices. Besides the electrical features, design improvements of FHEs depend on the prediction of their mechanical and failure behavior. Debonding of electronic components from the flexible substrate is one of the most common and critical failures of these devices, therefore, the experimental determination of material and interface properties is of great importance in the prediction of failure mechanisms. Traditional interface characterization involves isolated shear and normal mode tests such as the double cantilever beam (DCB) and end notch flexure (ENF) tests. However, due to the thin, flexible nature of the materials and manufacturing restrictions, tests mirroring traditional interface characterization experiments may not always be possible. The ideal goal of this research is to design experiments such that each mode of fracture is isolated. However, due to the complex nonlinear nature of the response and small geometries of FHEs, design of the proper tests to characterize the interface properties can be significantly time and cost consuming. Hence numerical modeling has been implemented to design these novel characterization experiments. This research involves loading case and specimen geometry parametric studies using numerical modeling to design future experiments where either shear or normal fracture modes are dominant. These virtual experiments will provide a foundation for designing similar tests for many different types of flexible electronics and predicting the failure mechanism independent of the specific FHE materials.
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NASA Astrophysics Data System (ADS)
He, Y.; Farokhzadeh, K.; Edrisy, A.
2017-04-01
Perfluoroalkoxy (PFA) is a potential polymer coating material for low-temperature waste heat recovery in heat exchangers. Nonetheless, poor thermal conductivity, low strength and susceptibility to surface degradation by erosion/wear pose restrictions in its application. In this study, four types of fillers, namely graphite, silicon carbide, alumina and boron nitride, were introduced to enhance the thermal, mechanical and tribological properties in PFA coatings. The thermal diffusivity and specific heat capacity of the composites (reinforced with 20 wt.% filler) were also measured using laser flash and differential scanning calorimetry techniques, respectively. The results indicated that the addition of graphite or boron nitride increased the thermal conductivity of PFA by at least 2.8 orders of magnitude, while the composites with the same weight fraction of alumina or silicon carbide showed 20-80% rise in thermal conductivity. The micromechanical deformation and tribological behavior of composite coatings, electrostatically sprayed on steel substrates, were investigated by means of instrumented indentation and scratch tests. The deformation response and friction characteristics were investigated, and the failure mechanisms were identified. Surface hardness, roughness and structure of fillers influenced the sliding performance of the composite coatings. PFA coatings filled with Al2O3 or SiC particles showed high load-bearing capacity under sliding conditions. Conversely, BN- and graphite-filled PFA coatings exhibited lower interfacial adhesion to steel substrate and were prone to failure at relatively lower applied loads.
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Visual cues for woodpeckers: light reflectance of decayed wood varies by decay fungus
O'Daniels, Sean T.; Kesler, Dylan C.; Mihail, Jeanne D.; Webb, Elisabeth B.; Werner, Scott J.
2018-01-01
The appearance of wood substrates is likely relevant to bird species with life histories that require regular interactions with wood for food and shelter. Woodpeckers detect decayed wood for cavity placement or foraging, and some species may be capable of detecting trees decayed by specific fungi; however, a mechanism allowing for such specificity remains unidentified. We hypothesized that decay fungi associated with woodpecker cavity sites alter the substrate reflectance in a species-specific manner that is visually discriminable by woodpeckers. We grew 10 species of wood decay fungi from pure cultures on sterile wood substrates of 3 tree species. We then measured the relative reflectance spectra of decayed and control wood wafers and compared them using the receptor noise-limited (RNL) color discrimination model. The RNL model has been used in studies of feather coloration, egg shells, flowers, and fruit to model how the colors of objects appear to birds. Our analyses indicated 6 of 10 decayed substrate/control comparisons were above the threshold of discrimination (i.e., indicating differences discriminable by avian viewers), and 12 of 13 decayed substrate comparisons were also above threshold for a hypothetical woodpecker. We conclude that woodpeckers should be capable of visually detecting decayed wood on trees where bark is absent, and they should also be able to detect visually species-specific differences in wood substrates decayed by fungi used in this study. Our results provide evidence for a visual mechanism by which woodpeckers could identify and select substrates decayed by specific fungi, which has implications for understanding ecologically important woodpecker–fungus interactions.
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Lauter, F R; Ninnemann, O; Bucher, M; Riesmeier, J W; Frommer, W B
1996-01-01
Root hairs as specialized epidermal cells represent part of the outermost interface between a plant and its soil environment. They make up to 70% of the root surface and, therefore, are likely to contribute significantly to nutrient uptake. To study uptake systems for mineral nitrogen, three genes homologous to Arabidopsis nitrate and ammonium transporters (AtNrt1 and AtAmt1) were isolated from a root hair-specific tomato cDNA library. Accumulation of LeNrt1-1, LeNrt1-2, and LeAmt1 transcripts was root-specific, with no detectable transcripts in stems or leaves. Expression was root cell type-specific and regulated by nitrogen availability. LeNrt1-2 mRNA accumulation was restricted to root hairs that had been exposed to nitrate. In contrast, LeNrt1-1 transcripts were detected in root hairs as well as other root tissues under all nitrogen treatments applied. Analogous to LeNrt1-1, the gene LeAmt1 was expressed under all nitrogen conditions tested, and root hair-specific mRNA accumulation was highest following exposure to ammonium. Expression of LeAMT1 in an ammonium uptake-deficient yeast strain restored growth on low ammonium medium, confirming its involvement in ammonium transport. Root hair specificity and characteristics of substrate regulation suggest an important role of the three genes in uptake of mineral nitrogen. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8755617
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Monitoring of protease catalyzed reactions by quantitative MALDI MS using metal labeling.
Gregorius, Barbara; Jakoby, Thomas; Schaumlöffel, Dirk; Tholey, Andreas
2013-05-21
Quantitative mass spectrometry is a powerful tool for the determination of enzyme activities as it does not require labeled substrates and simultaneously allows for the identification of reaction products. However, major restrictions are the limited number of samples which can be measured in parallel due to the need for isotope labeled internal standards. Here we describe the use of metal labeling of peptides for the setup of multiplexed enzyme activity assays. After proteolytic reaction, using the protease trypsin, remaining substrates and peptide products formed in the reaction were labeled with metal chelators complexing rare earth metal ions. Labeled peptides were quantified with high accuracy and over a wide dynamic range (at least 2 orders of magnitude) using MALDI MS in case of simple peptide mixtures or by LC-MALDI MS for complex substrate mixtures and used for the monitoring of time-dependent product formation and substrate consumption. Due to multiplexing capabilities and accuracy, the presented approach will be useful for the determination of enzyme activities with a wide range of biochemical and biotechnological applications.
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Carol E. Johnston; Wendell R. Haag
1996-01-01
The Yazoo darter, Etheostoma raneyi, is restricted to tributaries of the Little Tallahatchie and Yocona Rivers (Yazoo River drainage) in north-central Mississippi. The species inhabits small, clear streams, many of which are spring-fed, and have a variety of substrate types including silt, clay, sand and gravel. In Morris Creek, a second-order stream...
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Substrate specificity effects of lipoxygenase products and inhibitors on soybean lipoxygenase-1.
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.
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Identification of ribozymes within a ribozyme library that efficiently cleave a long substrate RNA.
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
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Enhanced Optical Transmission Mediated by Localized Plasmons in Anisotropic, 3D Nanohole Arrays
Yang, Jiun-Chan; Gao, Hanwei; Suh, Jae Yong; Zhou, Wei; Lee, Min Hyung; Odom, Teri W.
2010-01-01
This paper describes 3D nanohole arrays whose high optical transmission is mediated more by localized surface plasmon (LSP) excitations than by surface plasmon polaritons (SPPs). First, LSPs on 3D hole arrays lead to optical transmission an order of magnitude higher than 2D planar hole arrays. Second, LSP-mediated transmission is broadband and more tunable than SPP-enhanced transmission which is restricted by Bragg coupling. Third, for the first time, two types of surface plasmons can be selectively excited and manipulated on the same plasmonic substrate. This new plasmonic substrate fabricated by high-throughput nanolithography techniques paves the way for cutting-edge optoelectronic and biomedical applications. PMID:20698633
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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.
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NASA Astrophysics Data System (ADS)
Böcking, Dominique; Wiltschka, Oliver; Niinimäki, Jenni; Shokry, Hussein; Brenner, Rolf; Lindén, Mika; Sahlgren, Cecilia
2014-01-01
Biochemical cues are critical to control stem cell function and can be utilized to develop smart biomaterials for stem cell engineering. The challenge is to deliver these cues in a restricted manner with spatial and temporal control. Here we have developed bilayer films of mesoporous silica nanoparticles for delayed cellular delivery of Notch modulators to promote muscle stem cell differentiation. We demonstrate that drug-loaded particles are internalized from the particle-covered surface, which allows for direct delivery of the drug into the cell and a delayed and confined drug release. Substrates of particles loaded with γ-secretase-inhibitors, which block the Notch signalling pathway, promoted efficient differentiation of myoblasts. The particle substrates were fully biocompatible and did not interfere with the inherent differentiation process. We further demonstrate that impregnating commercially available, biocompatible polymer scaffolds with MSNs allows for a free standing substrate for cell directed drug delivery.Biochemical cues are critical to control stem cell function and can be utilized to develop smart biomaterials for stem cell engineering. The challenge is to deliver these cues in a restricted manner with spatial and temporal control. Here we have developed bilayer films of mesoporous silica nanoparticles for delayed cellular delivery of Notch modulators to promote muscle stem cell differentiation. We demonstrate that drug-loaded particles are internalized from the particle-covered surface, which allows for direct delivery of the drug into the cell and a delayed and confined drug release. Substrates of particles loaded with γ-secretase-inhibitors, which block the Notch signalling pathway, promoted efficient differentiation of myoblasts. The particle substrates were fully biocompatible and did not interfere with the inherent differentiation process. We further demonstrate that impregnating commercially available, biocompatible polymer scaffolds with MSNs allows for a free standing substrate for cell directed drug delivery. Electronic supplementary information (ESI) available: (1) Particle characterization. (2) Immunohistochemistry and SEM analyses of C2C12 cells grown on films for 3, 6, 24 and 72 h. Light microscopy and WST1 analyses of cells grown on cover slips and films for 6, 24 and 72 h (3) Quantification of protein levels of C2C12 cells differentiating on cover slips versus MSN films. (4) Stability of MSN films in biological solution and the influence on cell viability. (5) Cell internalization of particles from MSN films and intracellular drug release at 12 and 24 h (6) Cell internalization and intracellular DiI release of MSNs from (3Dtro®) fiber scaffolds impregnated with MSNs. See DOI: 10.1039/c3nr04022d
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Cabezas, Alicia; Ribeiro, João Meireles; Rodrigues, Joaquim Rui; López-Villamizar, Iralis; Fernández, Ascensión; Canales, José; Pinto, Rosa María; Costas, María Jesús; Cameselle, José Carlos
2015-01-01
Among metallo-dependent phosphatases, ADP-ribose/CDP-alcohol diphosphatases form a protein family (ADPRibase-Mn-like) mainly restricted, in eukaryotes, to vertebrates and plants, with preferential expression, at least in rodents, in immune cells. Rat and zebrafish ADPRibase-Mn, the only biochemically studied, are phosphohydrolases of ADP-ribose and, somewhat less efficiently, of CDP-alcohols and 2´,3´-cAMP. Furthermore, the rat but not the zebrafish enzyme displays a unique phosphohydrolytic activity on cyclic ADP-ribose. The molecular basis of such specificity is unknown. Human ADPRibase-Mn showed similar activities, including cyclic ADP-ribose phosphohydrolase, which seems thus common to mammalian ADPRibase-Mn. Substrate docking on a homology model of human ADPRibase-Mn suggested possible interactions of ADP-ribose with seven residues located, with one exception (Cys253), either within the metallo-dependent phosphatases signature (Gln27, Asn110, His111), or in unique structural regions of the ADPRibase-Mn family: s2s3 (Phe37 and Arg43) and h7h8 (Phe210), around the active site entrance. Mutants were constructed, and kinetic parameters for ADP-ribose, CDP-choline, 2´,3´-cAMP and cyclic ADP-ribose were determined. Phe37 was needed for ADP-ribose preference without catalytic effect, as indicated by the increased ADP-ribose K m and unchanged k cat of F37A-ADPRibase-Mn, while the K m values for the other substrates were little affected. Arg43 was essential for catalysis as indicated by the drastic efficiency loss shown by R43A-ADPRibase-Mn. Unexpectedly, Cys253 was hindering for cADPR phosphohydrolase, as indicated by the specific tenfold gain of efficiency of C253A-ADPRibase-Mn with cyclic ADP-ribose. This allowed the design of a triple mutant (F37A+L196F+C253A) for which cyclic ADP-ribose was the best substrate, with a catalytic efficiency of 3.5´104 M-1s-1 versus 4´103 M-1s-1 of the wild type. PMID:25692488
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Structural Features of Antiviral APOBEC3 Proteins are Linked to Their Functional Activities
Kitamura, Shingo; Ode, Hirotaka; Iwatani, Yasumasa
2011-01-01
Human APOBEC3 (A3) proteins are cellular cytidine deaminases that potently restrict the replication of retroviruses by hypermutating viral cDNA and/or inhibiting reverse transcription. There are seven members of this family including A3A, B, C, DE, F, G, and H, all encoded in a tandem array on human chromosome 22. A3F and A3G are the most potent inhibitors of HIV-1, but only in the absence of the virus-encoded protein, Vif. HIV-1 utilizes Vif to abrogate A3 functions in the producer cells. More specifically, Vif, serving as a substrate receptor, facilitates ubiquitination of A3 proteins by forming a Cullin5 (Cul5)-based E3 ubiquitin ligase complex, which targets A3 proteins for rapid proteasomal degradation. The specificity of A3 degradation is determined by the ability of Vif to bind to the target. Several lines of evidence have suggested that three distinct regions of A3 proteins are involved in the interaction with Vif. Here, we review the biological functions of A3 family members with special focus on A3G and base our analysis on the available structural information. PMID:22203821
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Serine proteases in rodent hippocampus.
Davies, B J; Pickard, B S; Steel, M; Morris, R G; Lathe, R
1998-09-04
Brain serine proteases are implicated in developmental processes, synaptic plasticity, and in disorders including Alzheimer's disease. The spectrum of the major enzymes expressed in brain has not been established previously. We now present a systematic study of the serine proteases expressed in adult rat and mouse hippocampus. Using a combination of techniques including polymerase chain reaction amplification and Northern blotting we show that tissue-type plasminogen activator (t-PA) is the major species represented. Unexpectedly, the next most abundant species were RNK-Met-1, a lymphocyte protease not reported previously in brain, and two new family members, BSP1 (brain serine protease 1) and BSP2. We report full-length sequences of the two new proteases; homologies indicate that these are of tryptic specificity. Although BSP2 is expressed in several brain regions, BSP1 expression is strikingly restricted to hippocampus. Other enzymes represented, but at lower levels, included elastase IV, proteinase 3, complement C2, chymotrypsin B, chymotrypsin-like protein, and Hageman factor. Although thrombin and urokinase-type plasminogen activator were not detected in the primary screen, low level expression was confirmed using specific polymerase chain reaction primers. In contrast, and despite robust expression of t-PA, the usual t-PA substrate plasminogen was not expressed at detectable levels.
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Epitaxy versus oriented heterogeneous nucleation of organic crystals on ionic substrates
NASA Astrophysics Data System (ADS)
Sarma, K. R.; Shlichta, P. J.; Wilcox, W. R.; Lefever, R. A.
1997-04-01
It is plausible to assume that epitaxy is a special case of heterogeneous nucleation in which a restrictive crystallographic relationship exists between substrate and deposit orientations. This would mean that epitaxial substrates should always induce a perceptible reduction in the critical supercooling for nucleation of the deposit. To test this hypothesis, the critical supercoolings of six organic compounds were measured on glass and 11 single-crystal cleaved substrates including (0001) graphite, (001) mica, (111) BaF 2, SrF 2, and CaF 2, and (100) KCl, KBr, KI, NaCl, NaF, and LiF. Reductions in supercooling (with reference to glass substrates) were checked many times for repeatability and reproducibility and shown in almost all cases to have a standard deviation of 1 C or less. Acetanilide, benzoic acid, and p-bromochlorobenzene showed a wide range of supercooling reductions and were oriented on all crystalline substrates. Naphthalene and p-dibromobenzene showed only slight supercooling reductions but were oriented on all substrates, including glass. Benzil showed strong supercooling reductions only for mica and KI but was oriented not only in these cases but also with KI, BaF 2, CaF 2, and graphite. There was little correlation between degree of lattice match and either supercooling reduction or degree of preferred orientation. These results suggest that, for the systems and geometry studied, forces such as molecular dipole binding and growth anisotropy had a stronger effect than lattice match.
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Problem-Solving Test: Restriction Endonuclease Mapping
ERIC Educational Resources Information Center
Szeberenyi, Jozsef
2011-01-01
The term "restriction endonuclease mapping" covers a number of related techniques used to identify specific restriction enzyme recognition sites on small DNA molecules. A method for restriction endonuclease mapping of a 1,000-basepair (bp)-long DNA molecule is described in the fictitious experiment of this test. The most important fact needed to…
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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.
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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
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A Bottom-Up Proteomic Approach to Identify Substrate Specificity of Outer-Membrane Protease OmpT.
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.
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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.
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[Substrate specificities of bile salt hydrolase 1 and its mutants from Lactobacillus salivarius].
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.
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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.
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Sotiropoulos, C; Coloe, P J; Smith, S C
1994-01-01
Chromosomal DNA restriction enzyme analysis and Southern blot hybridization were used to characterize Serpulina hyodysenteriae strains. When chromosomal DNAs from selected strains (reference serotypes) of S. hyodysenteriae were digested with the restriction endonuclease Sau3A and hybridized with a 1.1-kb S. hyodysenteriae-specific DNA probe, a common 3-kb band was always detected in S. hyodysenteriae strains but was absent from Serpulina innocens strains. When the chromosomal DNA was digested with the restriction endonuclease Asp 700 and hybridized with two S. hyodysenteriae-specific DNA probes (0.75 and 1.1 kb of DNA), distinct hybridization patterns for each S. hyodysenteriae reference strain and the Australian isolate S. hyodysenteriae 5380 were detected. Neither the 1.1-kb nor the 0.75-kb DNA probe hybridized with Asp 700- or Sau3A-digested S. innocens chromosomal DNA. The presence of the 3-kb Sau3A DNA fragment in S. hyodysenteriae reference strains from diverse geographical locations shows that this fragment is conserved among S. hyodysenteriae strains and can be used as a species-specific marker. Restriction endonuclease analysis and Southern blot hybridization with these well-defined DNA probes are reliable and accurate methods for species-specific and strain-specific identification of S. hyodysenteriae. Images PMID:7914209
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Karas, Panagiotis A; Makri, Sotirina; Papadopoulou, Evangelia S; Ehaliotis, Constantinos; Menkissoglu-Spiroudi, Urania; Karpouzas, Dimitrios G
2016-02-01
Citrus fruit-packaging plants (FPP) produce large wastewater volumes with high loads of fungicides like ortho-phenylphenol (OPP) and imazalil (IMZ). No methods are in place for the treatment of those effluents and biobeds appear as a viable alternative. We employed a column study to investigate the potential of spent mushroom substrate (SMS) of Pleurotus ostreatus, either alone or in mixture with straw and soil plus a mixture of straw /soil to retain and dissipate IMZ and OPP. The role of P. ostreatus on fungicides dissipation was also investigated by studying in parallel the performance of fresh mushroom substrate of P. ostreatus (FMS) and measuring lignolytic enzymatic activity in the leachates. All substrates effectively reduced the leaching of OPP and IMZ which corresponded to 0.014-1.1% and 0.120-0.420% of their initial amounts respectively. Mass balance analysis revealed that FMS and SMS/Straw/Soil (50/25/25 by vol) offered the most efficient removal of OPP and IMZ from wastewaters respectively. Regardless of the substrate, OPP was restricted in the top 0-20cm of the columns and was bioavailable (extractable with water), compared to IMZ which was less bioavailable (extractable with acetonitrile) but diffused at deeper layers (20-50, 50-80cm) in the SMS- and Straw/Soil-columns. PLFAs showed that fungal abundance was significantly lower in the top layer of all substrates from where the highest pesticide amounts were recovered suggesting an inhibitory effect of fungicides on total fungi in the substrates tested. Our data suggest that biobeds packed with SMS-rich substrates could ensure the efficient removal of IMZ and OPP from wastewaters of citrus FPP. Copyright © 2015 Elsevier Inc. All rights reserved.
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2011-01-01
Background Ethnobotanical research was carried out with speakers of Iquito, a critically endangered Amazonian language of the Zaparoan family. The study focused on the concept of "dieting" (siyan++ni in Iquito), a practice involving prohibitions considered necessary to the healing process. These restrictions include: 1) foods and activities that can exacerbate illness, 2) environmental influences that conflict with some methods of healing (e.g. steam baths or enemas) and 3) foods and activities forbidden by the spirits of certain powerful medicinal plants. The study tested the following hypotheses: H1 - Each restriction will correlate with specific elements in illness explanatory models and H2 - Illnesses whose explanatory models have personalistic elements will show a greater number and variety of restrictions than those based on naturalistic reasoning. Methods The work was carried out in 2009 and 2010 in the Alto Nanay region of Peru. In structured interviews, informants gave explanatory models for illness categories, including etiologies, pathophysiologies, treatments and dietary restrictions necessary for 49 illnesses. Seventeen botanical vouchers for species said to have powerful spirits that require diets were also collected. Results All restrictions found correspond to some aspect of illness explanatory models. Thirty-five percent match up with specific illness etiologies, 53% correspond to particular pathophysiologies, 18% correspond with overall seriousness of the illness and 18% are only found with particular forms of treatment. Diets based on personalistic reasoning have a significantly higher average number of restrictions than those based on naturalistic reasoning. Conclusions Dieting plays a central role in healing among Iquito speakers. Specific prohibitions can be explained in terms of specific aspects of illness etiologies, pathophysiologies and treatments. Although the Amazonian literature contains few studies focusing on dietary proscriptions over a wide range of illnesses, some specific restrictions reported here do correspond with trends seen in other Amazonian societies, particularly those related to sympathetic reasoning and for magical and spiritual uses of plants. PMID:21745400
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Jernigan, Kevin A
2011-07-11
Ethno botanical research was carried out with speakers of Iquitos, a critically endangered Amazonian language of the Zaparoan family. The study focused on the concept of "dieting" (siyan++ni in Iquitos), a practice involving prohibitions considered necessary to the healing process. These restrictions include: 1) foods and activities that can exacerbate illness, 2) environmental influences that conflict with some methods of healing (e.g. steam baths or enemas) and 3) foods and activities forbidden by the spirits of certain powerful medicinal plants. The study tested the following hypotheses: H1--Each restriction will correlate with specific elements in illness explanatory models and H2--Illnesses whose explanatory models have personality elements will show a greater number and variety of restrictions than those based on naturalistic reasoning. The work was carried out in 2009 and 2010 in the Alto Nanay region of Peru. In structured interviews, informants gave explanatory models for illness categories, including etiologies, pathophysiologies, treatments and dietary restrictions necessary for 49 illnesses. Seventeen botanical vouchers for species said to have powerful spirits that require diets were also collected. All restrictions found correspond to some aspect of illness explanatory models. Thirty-five percent match up with specific illness etiologies, 53% correspond to particular pathophysiologies, 18% correspond with overall seriousness of the illness and 18% are only found with particular forms of treatment. Diets based on personalistic reasoning have a significantly higher average number of restrictions than those based on naturalistic reasoning. Dieting plays a central role in healing among Iquitos speakers. Specific prohibitions can be explained in terms of specific aspects of illness etiologies, pathophysiologies and treatments. Although the Amazonian literature contains few studies focusing on dietary proscriptions over a wide range of illnesses, some specific restrictions reported here do correspond with trends seen in other Amazonian societies, particularly those related to sympathetic reasoning and for magical and spiritual uses of plants.
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25 CFR 213.41 - Leases executed but not approved before restrictions removed from land.
Code of Federal Regulations, 2010 CFR
2010-04-01
.... Leases executed before the removal of restrictions against alienation on land from all of which restrictions against alienation shall be removed after such execution, if such leases contain specific...
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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
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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
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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.
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Nocker, Andreas; Lepo, Joe Eugene; Martin, Linda Lin; Snyder, Richard Allan
2007-10-01
The information content and responsiveness of microbial biofilm community structure, as an integrative indicator of water quality, was assessed against short-term changes in oxygen and nutrient loading in an open-water estuarine setting. Biofilms were grown for 7-day periods on artificial substrates in the Pensacola Bay estuary, Florida, in the vicinity of a wastewater treatment plant (WWTP) outfall and a nearby reference site. Substrates were deployed floating at the surface and near the benthos in 5.4 m of water. Three sampling events covered a 1-month period coincident with declining seasonal WWTP flow and increasing dissolved oxygen (DO) levels in the bottom waters. Biomass accumulation in benthic biofilms appeared to be controlled by oxygen rather than nutrients. The overriding effect of DO was also seen in DNA fingerprints of community structure by terminal restriction fragment length polymorphism (T-RFLP) of amplified 16S rRNA genes. Ribotype diversity in benthic biofilms at both sites dramatically increased during the transition from hypoxic to normoxic. Terminal restriction fragment length polymorphism patterns showed pronounced differences between benthic and surface biofilm communities from the same site in terms of signal type, strength, and diversity, but minor differences between sites. Sequencing of 16S rRNA gene clone libraries from benthic biofilms at the WWTP site suggested that low DO levels favored sulfate-reducing prokaryotes (SRP), which decreased with rising oxygen levels and increasing overall diversity. A 91-bp ribotype in the CfoI-restricted 16S rRNA gene T-RFLP profiles, indicative of SRP, tracked the decrease in relative SRP abundance over time.
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Graham, Simon P.; Pellé, Roger; Yamage, Mat; Mwangi, Duncan M.; Honda, Yoshikazu; Mwakubambanya, Ramadhan S.; de Villiers, Etienne P.; Abuya, Evelyne; Awino, Elias; Gachanja, James; Mbwika, Ferdinand; Muthiani, Anthony M.; Muriuki, Cecelia; Nyanjui, John K.; Onono, Fredrick O.; Osaso, Julius; Riitho, Victor; Saya, Rosemary M.; Ellis, Shirley A.; McKeever, Declan J.; MacHugh, Niall D.; Gilbert, Sarah C.; Audonnet, Jean-Christophe; Morrison, W. Ivan; van der Bruggen, Pierre; Taracha, Evans L. N.
2008-01-01
Immunity against the bovine intracellular protozoan parasite Theileria parva has been shown to be mediated by CD8 T cells. Six antigens targeted by CD8 T cells from T. parva-immune cattle of different major histocompatibility complex (MHC) genotypes have been identified, raising the prospect of developing a subunit vaccine. To facilitate further dissection of the specificity of protective CD8 T-cell responses and to assist in the assessment of responses to vaccination, we set out to identify the epitopes recognized in these T. parva antigens and their MHC restriction elements. Nine epitopes in six T. parva antigens, together with their respective MHC restriction elements, were successfully identified. Five of the cytotoxic-T-lymphocyte epitopes were found to be restricted by products of previously described alleles, and four were restricted by four novel restriction elements. Analyses of CD8 T-cell responses to five of the epitopes in groups of cattle carrying the defined restriction elements and immunized with live parasites demonstrated that, with one exception, the epitopes were consistently recognized by animals of the respective genotypes. The analysis of responses was extended to animals immunized with multiple antigens delivered in separate vaccine constructs. Specific CD8 T-cell responses were detected in 19 of 24 immunized cattle. All responder cattle mounted responses specific for antigens for which they carried an identified restriction element. By contrast, only 8 of 19 responder cattle displayed a response to antigens for which they did not carry an identified restriction element. These data demonstrate that the identified antigens are inherently dominant in animals with the corresponding MHC genotypes. PMID:18070892
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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
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NASA Astrophysics Data System (ADS)
Mangano, M. G.; Buatois, L. A.
The paleoecologic and paleoenvironmental significance of trace fossils related to discontinuity surfaces in the Lower Cretaceous marine deposits of the Aconcagua area are analysed here. Carbonate-evaporite shoaling-upward cycles, developed by high organic production in a shallow hypersaline restricted environment, make up the section. Two types of cycles are defined, being mainly distinguished by their subtidal unit. Cycle I begins with a highly dolomitized lower subtidal unit (Facies A), followed upward by an intensely bioturbated upper subtidal unit (Facies B). The nodular packstone facies (B 1) is capped by a discontinuity surface (firmground or hardground) and occasionally overlain by an oystreid bed (Facies C). Cycle II is characterized by a pelletoidal subtidal unit (Facies B 2) with an abnormal salinity impoverished fauna. Both cycles end with intertidal to supratidal evaporite deposits (Facies D and E, respectively). Attention is particularly focused on cycle I due to its ichologic content. The mode of preservation and the distribution of trace fossils in nodular packstone facies are controlled by original substrate consolidation. Thalassinoides paradoxicus (pre-omission suite) represents colonization in a soft bottom, while Thalassinoides suevicus (omission suite pre-lithification) is apparently restricted to firm substrates. When consolidation processes are interrupted early, only an embryonic hard-ground that represents a minor halt in sedimentation was developed. Sometimes, consolidation processes continued leading to an intraformational hardground. Colonization by Trypanites solitarius (omission suite post-lithification) and Exogyra-like oystreids possibly characterizes hard substrate stage. When two discontinuity surfaces follow closely, a post-omission suite may be defined in relation to the lower cemented surface. As trace fossils are so closely related to changes in the degree of bottom lithification, they prove to be very useful as indicators of substrate evolution. The presence of discontinuity surfaces, evidenced by trace fossil association, suggests changes of sedimentary rate and environmental conditions that should be taken into account in future studies seeking to erect depositional models for these Cretaceous deposits.
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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…
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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.
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Keane, Fiona M; Yao, Tsun-Wen; Seelk, Stefanie; Gall, Margaret G; Chowdhury, Sumaiya; Poplawski, Sarah E; Lai, Jack H; Li, Youhua; Wu, Wengen; Farrell, Penny; Vieira de Ribeiro, Ana Julia; Osborne, Brenna; Yu, Denise M T; Seth, Devanshi; Rahman, Khairunnessa; Haber, Paul; Topaloglu, A Kemal; Wang, Chuanmin; Thomson, Sally; Hennessy, Annemarie; Prins, John; Twigg, Stephen M; McLennan, Susan V; McCaughan, Geoffrey W; Bachovchin, William W; Gorrell, Mark D
2013-01-01
The protease fibroblast activation protein (FAP) is a specific marker of activated mesenchymal cells in tumour stroma and fibrotic liver. A specific, reliable FAP enzyme assay has been lacking. FAP's unique and restricted cleavage of the post proline bond was exploited to generate a new specific substrate to quantify FAP enzyme activity. This sensitive assay detected no FAP activity in any tissue or fluid of FAP gene knockout mice, thus confirming assay specificity. Circulating FAP activity was ∼20- and 1.3-fold less in baboon than in mouse and human plasma, respectively. Serum and plasma contained comparable FAP activity. In mice, the highest levels of FAP activity were in uterus, pancreas, submaxillary gland and skin, whereas the lowest levels were in brain, prostate, leukocytes and testis. Baboon organs high in FAP activity included skin, epididymis, bladder, colon, adipose tissue, nerve and tongue. FAP activity was greatly elevated in tumours and associated lymph nodes and in fungal-infected skin of unhealthy baboons. FAP activity was 14- to 18-fold greater in cirrhotic than in non-diseased human liver, and circulating FAP activity was almost doubled in alcoholic cirrhosis. Parallel DPP4 measurements concorded with the literature, except for the novel finding of high DPP4 activity in bile. The new FAP enzyme assay is the first to be thoroughly characterised and shows that FAP activity is measurable in most organs and at high levels in some. This new assay is a robust tool for specific quantitation of FAP enzyme activity in both preclinical and clinical samples, particularly liver fibrosis.
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Keane, Fiona M.; Yao, Tsun-Wen; Seelk, Stefanie; Gall, Margaret G.; Chowdhury, Sumaiya; Poplawski, Sarah E.; Lai, Jack H.; Li, Youhua; Wu, Wengen; Farrell, Penny; Vieira de Ribeiro, Ana Julia; Osborne, Brenna; Yu, Denise M.T.; Seth, Devanshi; Rahman, Khairunnessa; Haber, Paul; Topaloglu, A. Kemal; Wang, Chuanmin; Thomson, Sally; Hennessy, Annemarie; Prins, John; Twigg, Stephen M.; McLennan, Susan V.; McCaughan, Geoffrey W.; Bachovchin, William W.; Gorrell, Mark D.
2013-01-01
The protease fibroblast activation protein (FAP) is a specific marker of activated mesenchymal cells in tumour stroma and fibrotic liver. A specific, reliable FAP enzyme assay has been lacking. FAP's unique and restricted cleavage of the post proline bond was exploited to generate a new specific substrate to quantify FAP enzyme activity. This sensitive assay detected no FAP activity in any tissue or fluid of FAP gene knockout mice, thus confirming assay specificity. Circulating FAP activity was ∼20- and 1.3-fold less in baboon than in mouse and human plasma, respectively. Serum and plasma contained comparable FAP activity. In mice, the highest levels of FAP activity were in uterus, pancreas, submaxillary gland and skin, whereas the lowest levels were in brain, prostate, leukocytes and testis. Baboon organs high in FAP activity included skin, epididymis, bladder, colon, adipose tissue, nerve and tongue. FAP activity was greatly elevated in tumours and associated lymph nodes and in fungal-infected skin of unhealthy baboons. FAP activity was 14- to 18-fold greater in cirrhotic than in non-diseased human liver, and circulating FAP activity was almost doubled in alcoholic cirrhosis. Parallel DPP4 measurements concorded with the literature, except for the novel finding of high DPP4 activity in bile. The new FAP enzyme assay is the first to be thoroughly characterised and shows that FAP activity is measurable in most organs and at high levels in some. This new assay is a robust tool for specific quantitation of FAP enzyme activity in both preclinical and clinical samples, particularly liver fibrosis. PMID:24371721
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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.
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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.
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Song, Sung E; Choi, Gwan H; Yi, Gi-Ra; Yoo, Pil J
2017-11-01
Polymeric thin films coated on non-wettable substrates undergo film-instabilities, which are usually manifested as surface deformation in the form of dewetting or wrinkling. The former takes place in fluidic films, whereas the latter occurs in solid films. Therefore, there have rarely been reports of systems involving simultaneous deformations of dewetting and wrinkling. In this study, we propose polymeric thin films of liquid crystalline (LC) mesogens prepared on a non-wettable Si substrate and apply a treatment of plasma irradiation to form a thin polymerized layer at the surface. The resulting compressive stress generated in the surface region drives the formation of wrinkles, while at the same time, dipolar attraction between LC molecules induces competitive cohesive dewetting. Intriguing surface structures were obtained whereby dewetting-like hole arrays are nested inside the randomly propagated wrinkles. The structural features are readily controlled by the degree of surface cross-linking, hydrophilicity of the substrates, and the LC film thickness. In particular, dewetting of LC mesogens is observed to be restricted to occur at the trough regions of wrinkles, exhibiting the typical behavior of geometrically confined dewetting. Finally, wrinkling-dewetting mixed structures are separated from the substrate in the form of free standing films to demonstrate the potential applicability as membranes.
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NASA Astrophysics Data System (ADS)
Mannsfeld, S. C.; Fritz, T.
2004-02-01
The physical structure of organic-inorganic heteroepitaxial thin films is usually governed by a fine balance between weak molecule-molecule interactions and a weakly laterally varying molecule-substrate interaction potential. Therefore, in order to investigate the energetics of such a layer system one has to consider large molecular domains. So far, layer potential calculations for large domains of organic thin films on crystalline substrates were difficult to perform concerning the computational effort which stems from the vast number of atoms which have to be included. Here, we present a technique which enables the calculation of the molecule-substrate interaction potential for large molecular domains by utilizing potential energy grid files. This technique allows the investigation of the substrate influence in systems prepared by organic molecular beam epitaxy (OMBE), like 3,4,9,10-perylenetetracarboxylicdianhydride on highly oriented pyrolytic graphite. For this system the so-called point-on-line coincidence was proposed, a growth mode which has been controversially discussed in literature. Furthermore, we are able to provide evidence for a general energetic advantage of such point-on-line coincident domain orientations over arbitrarily oriented domains which substantiates that energetically favorable lattice structures in OMBE systems are not restricted to commensurate unit cells or coincident super cells.
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Utilizing a Dynamical Description of IspH to Aid in the Development of Novel Antimicrobial Drugs
Blachly, Patrick G.; de Oliveira, César A. F.; Williams, Sarah L.; McCammon, J. Andrew
2013-01-01
The nonmevalonate pathway is responsible for isoprenoid production in microbes, including H. pylori, M. tuberculosis and P. falciparum, but is nonexistent in humans, thus providing a desirable route for antibacterial and antimalarial drug discovery. We coordinate a structural study of IspH, a [4Fe-4S] protein responsible for converting HMBPP to IPP and DMAPP in the ultimate step in the nonmevalonate pathway. By performing accelerated molecular dynamics simulations on both substrate-free and HMBPP-bound [Fe4S4]2+ IspH, we elucidate how substrate binding alters the dynamics of the protein. Using principal component analysis, we note that while substrate-free IspH samples various open and closed conformations, the closed conformation observed experimentally for HMBPP-bound IspH is inaccessible in the absence of HMBPP. In contrast, simulations with HMBPP bound are restricted from accessing the open states sampled by the substrate-free simulations. Further investigation of the substrate-free simulations reveals large fluctuations in the HMBPP binding pocket, as well as allosteric pocket openings – both of which are achieved through the hinge motions of the individual domains in IspH. Coupling these findings with solvent mapping and various structural analyses reveals alternative druggable sites that may be exploited in future drug design efforts. PMID:24367248
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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.
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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.
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USDA-ARS?s Scientific Manuscript database
Feed costs account for over 70% of the annual expenditures in cow/calf production. The majority of the nutrients are used to support the cow’s maintenance requirements and substrate cycling has been identified as one of the major contributors toward this type of energy expenditure. The objective of ...
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Cell-Imprinted Substrates Modulate Differentiation, Redifferentiation, and Transdifferentiation.
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.
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Insights into the Specificity of Lysine Acetyltransferases
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
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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.
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Low energy electron catalyst: the electronic origin of catalytic strategies.
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.
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Resveratrol derivatives as a pharmacological tool.
Biasutto, Lucia; Mattarei, Andrea; Azzolini, Michele; La Spina, Martina; Sassi, Nicola; Romio, Matteo; Paradisi, Cristina; Zoratti, Mario
2017-09-01
Prodrugs of resveratrol are under development. Among the long-term goals, still largely elusive, are (1) modulating physical properties (e.g., water-soluble derivatives bearing polyethylene glycol chains), (2) changing distribution in the body (e.g., galactosyl derivatives restricted to the intestinal lumen), (3) increasing absorption from the gastrointestinal tract (e.g., derivatives imitating the natural substrates of endogenous transporters), and (4) hindering phase II metabolism (e.g., temporarily blocking the hydroxyls), all contributing to (5) increasing bioavailability. The chemical bonds that have been tested for functionalization include carboxyester, acetal, and carbamate groups. A second approach, which can be combined with the first, seeks to reinforce or modify the biochemical activities of resveratrol by concentrating the compound at specific subcellular sites. An example is provided by mitochondria-targeted derivatives. These proved to be pro-oxidant and cytotoxic in vitro, selectively killing fast-growing and tumor cells when supplied in the low micromolar range. This suggests the possibility of anticancer applications. © 2017 New York Academy of Sciences.
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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.
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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.
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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
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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
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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.
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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
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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
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Biochemistry students' ideas about how an enzyme interacts with a substrate.
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.
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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.
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Radial restricted solid-on-solid and etching interface-growth models
NASA Astrophysics Data System (ADS)
Alves, Sidiney G.
2018-03-01
An approach to generate radial interfaces is presented. A radial network recursively obtained is used to implement discrete model rules designed originally for the investigation in flat substrates. I used the restricted solid-on-solid and etching models as to test the proposed scheme. The results indicate the Kardar, Parisi, and Zhang conjecture is completely verified leading to a good agreement between the interface radius fluctuation distribution and the Gaussian unitary ensemble. The evolution of the radius agrees well with the generalized conjecture, and the two-point correlation function exhibits also a good agreement with the covariance of the Airy2 process. The approach can be used to investigate radial interfaces evolution for many other classes of universality.
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Radial restricted solid-on-solid and etching interface-growth models.
Alves, Sidiney G
2018-03-01
An approach to generate radial interfaces is presented. A radial network recursively obtained is used to implement discrete model rules designed originally for the investigation in flat substrates. I used the restricted solid-on-solid and etching models as to test the proposed scheme. The results indicate the Kardar, Parisi, and Zhang conjecture is completely verified leading to a good agreement between the interface radius fluctuation distribution and the Gaussian unitary ensemble. The evolution of the radius agrees well with the generalized conjecture, and the two-point correlation function exhibits also a good agreement with the covariance of the Airy_{2} process. The approach can be used to investigate radial interfaces evolution for many other classes of universality.
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Bitterlich, Michael; Sandmann, Martin; Graefe, Jan
2018-01-01
Arbuscular mycorrhizal fungi (AMF) proliferate in soil pores, on the surface of soil particles and affect soil structure. Although modifications in substrate moisture retention depend on structure and could influence plant water extraction, mycorrhizal impacts on water retention and hydraulic conductivity were rarely quantified. Hence, we asked whether inoculation with AMF affects substrate water retention, water transport properties and at which drought intensity those factors become limiting for plant transpiration. Solanum lycopersicum plants were set up in the glasshouse, inoculated or not with Funneliformis mosseae, and grown for 35 days under ample water supply. After mycorrhizal establishment, we harvested three sets of plants, one before (36 days after inoculation) and the second (day 42) and third (day 47) within a sequential drying episode. Sampling cores were introduced into pots before planting. After harvest, moisture retention and substrate conductivity properties were assessed and water retention and hydraulic conductivity models were fitted. A root water uptake model was adopted in order to identify the critical substrate moisture that induces soil derived transpiration limitation. Neither substrate porosity nor saturated water contents were affected by inoculation, but both declined after substrates dried. Drying also caused a decline in pot water capacity and hydraulic conductivity. Plant available water contents under wet (pF 1.8–4.2) and dry (pF 2.5–4.2) conditions increased in mycorrhizal substrates and were conserved after drying. Substrate hydraulic conductivity was higher in mycorrhizal pots before and during drought exposure. After withholding water from pots, higher substrate drying rates and lower substrate water potentials were found in mycorrhizal substrates. Mycorrhiza neither affected leaf area nor root weight or length. Consistently with higher substrate drying rates, AMF restored the plant hydraulic status, and increased plant transpiration when soil moisture declined. The water potential at the root surface and the resistance to water flow in the rhizosphere were restored in mycorrhizal pots although the bulk substrate dried more. Finally, substrates colonized by AMF can be more desiccated before substrate water flux quantitatively limits transpiration. This is most pronounced under high transpiration demands and complies with a difference of over 1,000 hPa in substrate water potential. PMID:29503655
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75 FR 17561 - Amendment to Restricted Area R-2510A; El Centro, CA
Federal Register 2010, 2011, 2012, 2013, 2014
2010-04-07
...-0346; Airspace Docket No. 10-AWP-3] RIN 2120-AA66 Amendment to Restricted Area R-2510A; El Centro, CA... Restricted Area R-2510A, El Centro, CA. Specifically, the action inadvertently omitted the reference to.... 92-AWP-15 entitled ``Alteration and Subdivision of Restricted Area R-2510A; El Centro, CA''. That...
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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.
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The Structure of Lombricine Kinase
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
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Biochemical profiling in silico--predicting substrate specificities of large enzyme families.
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.
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NASA Astrophysics Data System (ADS)
Sukhishvili, Svetlana A.; Granick, Steve
1999-05-01
We contrast the adsorption of human serum albumin (HSA) onto two solid substrates previously primed with the same polyelectrolyte of net opposite charge to form one of two alternative structures: randomly adsorbed polymer and the "brush" configuration. These structures were formed either by the adsorption of quaternized poly-4-vinylpyridine (QPVP) or by end-grafting QPVP chains of the same chemical makeup and the same molecular weight to surfaces onto which QPVP segments did not adsorb. The adsorption of HSA was quantified by using Fourier transform infrared spectroscopy in attenuated total reflection (FTIR-ATR). The two substrates showed striking differences with regard to HSA adsorption. First, the brush substrate induced lesser perturbations in the secondary structure of the adsorbed HSA, reflecting easier conformational adjustment for longer free segments of polyelectrolyte upon binding with the protein. Second, the penetration of HSA into the brush substrate was kinetically retarded relative to the randomly adsorbed polymer, probably due to both pore size restriction and electrostatic sticking between charged groups of HSA and QPVP molecules. Third, release of HSA from the adsorbed layer, as the ionic strength was increased from a low level up to the high level of 1 M NaCl, was largely inhibited for the brush substrate, but occurred easily and rapidly for the substrate with statistically adsorbed QPVP chains. Finally, even after addition of a strong polymeric adsorption competitor (sodium polystyrene sulfonate), HSA remained trapped within a brush substrate though it desorbed slowly from the preadsorbed QPVP layer. This method to produce irreversible trapping of the protein within a brush substrate without major conformational change may find application in biosensor design.
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2011-01-06
identified viral restriction factors that inhibit infection mediated by the influenza A virus ( IAV ) hemagglutinin (HA) protein. Here we show that IFITM...observations, interferon-b specifically restricted filovirus and IAV entry processes. IFITM proteins also inhibited replication of infectious MARV and EBOV...We observed distinct patterns of IFITM-mediated restriction: compared with IAV , the entry processes of MARV and EBOV were less restricted by IFITM3
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Ji, Seok Young; Choi, Wonsuk; Jeon, Jin-Woo; Chang, Won Seok
2018-01-01
The development of printing technologies has enabled the realization of electric circuit fabrication on a flexible substrate. However, the current technique remains restricted to single-layer patterning. In this paper, we demonstrate a fully solution-processable patterning approach for multi-layer circuits using a combined method of laser sintering and ablation. Selective laser sintering of silver (Ag) nanoparticle-based ink is applied to make conductive patterns on a heat-sensitive substrate and insulating layer. The laser beam path and irradiation fluence are controlled to create circuit patterns for flexible electronics. Microvia drilling using femtosecond laser through the polyvinylphenol-film insulating layer by laser ablation, as well as sequential coating of Ag ink and laser sintering, achieves an interlayer interconnection between multi-layer circuits. The dimension of microvia is determined by a sophisticated adjustment of the laser focal position and intensity. Based on these methods, a flexible electronic circuit with chip-size-package light-emitting diodes was successfully fabricated and demonstrated to have functional operations. PMID:29425144
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Methyl Transfer by Substrate Signaling from a Knotted Protein Fold
Christian, Thomas; Sakaguchi, Reiko; Perlinska, Agata P.; Lahoud, Georges; Ito, Takuhiro; Taylor, Erika A.; Yokoyama, Shigeyuki; Sulkowska, Joanna I.; Hou, Ya-Ming
2017-01-01
Proteins with knotted configurations are restricted in conformational space relative to unknotted proteins. Little is known if knotted proteins have sufficient dynamics to communicate between spatially separated substrate-binding sites. In bacteria, TrmD is a methyl transferase that uses a knotted protein fold to catalyze methyl transfer from S-adenosyl methionine (AdoMet) to G37-tRNA. The product m1G37-tRNA is essential for life as a determinant to maintain protein synthesis reading-frame. Using an integrated approach of structure, kinetic, and computational analysis, we show here that the structurally constrained TrmD knot is required for its catalytic activity. Unexpectedly, the TrmD knot has complex internal movements that respond to AdoMet binding and signaling. Most of the signaling propagates the free energy of AdoMet binding to stabilize tRNA binding and to assemble the active site. This work demonstrates new principles of knots as an organized structure that captures the free energies of substrate binding to facilitate catalysis. PMID:27571175
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Elucidating the impact of micro-scale heterogeneous bacterial distribution on biodegradation
NASA Astrophysics Data System (ADS)
Schmidt, Susanne I.; Kreft, Jan-Ulrich; Mackay, Rae; Picioreanu, Cristian; Thullner, Martin
2018-06-01
Groundwater microorganisms hardly ever cover the solid matrix uniformly-instead they form micro-scale colonies. To which extent such colony formation limits the bioavailability and biodegradation of a substrate is poorly understood. We used a high-resolution numerical model of a single pore channel inhabited by bacterial colonies to simulate the transport and biodegradation of organic substrates. These high-resolution 2D simulation results were compared to 1D simulations that were based on effective rate laws for bioavailability-limited biodegradation. We (i) quantified the observed bioavailability limitations and (ii) evaluated the applicability of previously established effective rate concepts if microorganisms are heterogeneously distributed. Effective bioavailability reductions of up to more than one order of magnitude were observed, showing that the micro-scale aggregation of bacterial cells into colonies can severely restrict the bioavailability of a substrate and reduce in situ degradation rates. Effective rate laws proved applicable for upscaling when using the introduced effective colony sizes.
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Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates
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
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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
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Hypoxia promotes Mycobacterium tuberculosis-specific up-regulation of granulysin in human T cells.
Zenk, Sebastian F; Vollmer, Michael; Schercher, Esra; Kallert, Stephanie; Kubis, Jan; Stenger, Steffen
2016-06-01
Oxygen tension affects local immune responses in inflammation and infection. In tuberculosis mycobacteria avoid hypoxic areas and preferentially persist and reactivate in the oxygen-rich apex of the lung. Oxygen restriction activates antimicrobial effector mechanisms in macrophages and restricts growth of intracellular Mycobacterium tuberculosis (M.Tb). The effect of oxygen restriction on T cell-mediated antimicrobial effector mechanisms is unknown. Therefore we determined the influence of hypoxia on the expression of granulysin, an antimicrobial peptide of lymphocytes. Hypoxia increased the antigen-specific up-regulation of granulysin mRNA and protein in human CD4(+) and CD8(+) T lymphocytes. This observation was functionally relevant, because oxygen restriction supported the growth-limiting effect of antigen-specific T cells against virulent M.Tb residing in primary human macrophages. Our results provide evidence that oxygen restriction promotes the expression of granulysin and suggest that this effect-in conjunction with additional T cell-mediated immune responses-supports protection against mycobacteria. The therapeutic modulation of oxygen availability may offer a new strategy for the host-directed therapy of infectious diseases with intracellular pathogens.
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Conformal Robotic Stereolithography
Stevens, Adam G.; Oliver, C. Ryan; Kirchmeyer, Matthieu; Wu, Jieyuan; Chin, Lillian; Polsen, Erik S.; Archer, Chad; Boyle, Casey; Garber, Jenna
2016-01-01
Abstract Additive manufacturing by layerwise photopolymerization, commonly called stereolithography (SLA), is attractive due to its high resolution and diversity of materials chemistry. However, traditional SLA methods are restricted to planar substrates and planar layers that are perpendicular to a single-axis build direction. Here, we present a robotic system that is capable of maskless layerwise photopolymerization on curved surfaces, enabling production of large-area conformal patterns and the construction of conformal freeform objects. The system comprises an industrial six-axis robot and a custom-built maskless projector end effector. Use of the system involves creating a mesh representation of the freeform substrate, generation of a triangulated toolpath with curved layers that represents the target object to be printed, precision mounting of the substrate in the robot workspace, and robotic photopatterning of the target object by coordinated motion of the robot and substrate. We demonstrate printing of conformal photopatterns on spheres of various sizes, and construction of miniature three-dimensional objects on spheres without requiring support features. Improvement of the motion accuracy and development of freeform toolpaths would enable construction of polymer objects that surpass the size and support structure constraints imparted by traditional SLA systems. PMID:29577062
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More than just orphans: are taxonomically-restricted genes important in evolution?
Khalturin, Konstantin; Hemmrich, Georg; Fraune, Sebastian; Augustin, René; Bosch, Thomas C G
2009-09-01
Comparative genome analyses indicate that every taxonomic group so far studied contains 10-20% of genes that lack recognizable homologs in other species. Do such 'orphan' or 'taxonomically-restricted' genes comprise spurious, non-functional ORFs, or does their presence reflect important evolutionary processes? Recent studies in basal metazoans such as Nematostella, Acropora and Hydra have shed light on the function of these genes, and now indicate that they are involved in important species-specific adaptive processes. Here we focus on evidence from Hydra suggesting that taxonomically-restricted genes play a role in the creation of phylum-specific novelties such as cnidocytes, in the generation of morphological diversity, and in the innate defence system. We propose that taxon-specific genes drive morphological specification, enabling organisms to adapt to changing conditions.
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Kayombo, S; Mbwette, T S A; Katima, J H Y; Jorgensen, S E
2003-07-01
This paper presents the effect of substrate concentration on the growth of a mixed culture of algae and heterotrophic bacteria in secondary facultative ponds (SFPs) utilizing settled domestic sewage as a sole source of organic carbon. The growth of the mixed culture was studied at the concentrations ranging between 200 and 800 mg COD/l in a series of batch chemostat reactors. From the laboratory data, the specific growth rate (micro) was determined using the modified Gompertz model. The maximum specific growth rate ( micro(max)) and half saturation coefficients (K(s)) were calculated using the Monod kinetic equation. The maximum observed growth rate ( micro(max)) for heterotrophic bacteria was 3.8 day(-1) with K(s) of 200 mg COD/l. The micro(max) for algal biomass based on suspended volatile solids was 2.7 day(-1) with K(s) of 110 mg COD/l. The micro(max) of algae based on the chlorophyll-a was 3.5 day(-1) at K(s) of 50mg COD/l. The observed specific substrate removal by heterotrophic bacteria varied between the concentrations of substrate used and the average value was 0.82 (mg COD/mg biomass). The specific substrate utilization rate in the bioreactors was direct proportional to the specific growth rate. Hence, the determined Monod kinetic parameters are useful for the definition of the operation of SFPs.
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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.
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UFD4 lacking the proteasome-binding region catalyses ubiquitination but is impaired in proteolysis.
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.
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Substrate Curvature Restricts Spreading and Induces Differentiation of Human Mesenchymal Stem Cells.
Lee, Sang Joo; Yang, Shengyuan
2017-09-01
While cells attach, spread, migrate, proliferate, and differentiate in three-dimensional (3D) micromechanical environments, the mechanical factors of these environments influence the shapes, sizes, and adhesion forces of the cells. Here, the authors culture human mesenchymal stem cells (hMSCs) on a unique class of curvature-defined substrates, micro glass ball embedded polyacrylamide gels, prepared with an improved protocol, and investigate the spreading responses of the hMSCs on the glass balls to study the effects of substrate curvature on the spreading of hMSCs. The authors find that, among the used diameters of glass balls, the minimum diameter of a glass ball on which an hMSC can attach and spread is 500 μm. In contrast to the well-spread morphologies with randomly-multiple lamellipodia for the hMSCs growing on the flat glass plates, the morphologies of the hMSCs growing on the glass balls are almost uniformly spindle-shaped with two lamellipodia. The sensitivities of the attachment and spreading morphology of an hMSC to substrate curvature are very different from those of a fibroblast. The RT-PCR analysis reveals that the substrate curvature alone can induce adipogenesis of the hMSCs. These findings imply that substrate curvature has profound effects on stem cell behaviors, and detailed and in-depth studies on these effects and their underlying biophysical mechanisms are necessary. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
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ERIC Educational Resources Information Center
Fragman, Cathy; Goodluck, Helen; Heggie, Lindsay
2007-01-01
We report four act-out experiments testing the sensitivity of adults and three- to five-year-old children to the distinction between restrictive and non-restrictive relative clauses in English. Specifically, we test knowledge of the fact that restrictive relative clauses cannot modify a proper name head, and of the fact that relatives introduced…
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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.
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NASA Astrophysics Data System (ADS)
Schlichting, Johannes; Winkler, Kerstin; Koerner, Lienhard; Schletterer, Thomas; Burghardt, Berthold; Kahlert, Hans-Juergen
2000-10-01
The productive and accurate ablation of microstructures demands the precise imaging of a mask pattern onto the substrate under work. The job can be done with high performance wide field lenses as a key component of ablation equipment. The image field has dimensions of 20 to 30 mm. Typical dimensions and accuracy of the microstructures are in the order of some microns. On the other hand, the working depth of focus (DOF) has to be in the order of some 10 microns to be successful on drilling through 20 to 50 μm substrates. All these features have to be reached under the conditions of high power laser UV light. Some design principles for such systems are applied, such as optimum number of elements, minimum tolerance sensitivity, material restrictions for the lens elements as well as mechanical parts (mounting), restrictions of possible power densities on lens surfaces (including ghosts), matched quality for the manufactures system. The special applications require appropriate performance criteria for theoretical calculation and measurements, which allow to conclude the performance of the application. The base is wave front calculation and measurement (using Shack- Hartmann sensor) in UV. Derived criteria are calculated and compared with application results.
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Investigating the causes of low birth weight in contrasting ovine paradigms
Wallace, JM; Regnault, TRH; Limesand, SW; Hay, WW; Anthony, RV
2005-01-01
Intrauterine growth restriction (IUGR) still accounts for a large incidence of infant mortality and morbity worldwide. Many of the circulatory and transport properties of the sheep placenta are similar to those of the human placenta and as such, the pregnant sheep offers an excellent model in which to study the development of IUGR. Two natural models of ovine IUGR are those of hyperthermic exposure during pregnancy, and adolescent overfeeding, also during pregnancy. Both models yield significantly reduced placental weights and an asymmetrically growth-restricted fetus, and display altered maternal hormone concentrations, indicative of an impaired trophoblast capacity. Additionally, impaired placental angiogenesis and uteroplacental blood flow appears to be an early defect in both the hyperthermic and adolescent paradigms. The effects of these alterations in placental functional development appear to be irreversible. IUGR fetuses are both hypoxic and hypoglycaemic, and have reduced insulin and insulin-like growth factor-1 (IGF-1), and elevated concentrations of lactate. However, fetal utilization of oxygen and glucose, on a weight basis, remain constant compared with control pregnancies. Maintained utilization of these substrates, in a substrate-deficient environment, suggests increased sensitivities to metabolic signals, which may play a role in the development of metabolic diseases in later adult life. PMID:15774527
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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.
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Towards an Understanding of Energy Impairment in Huntington’s Disease Brain
Dubinsky, Janet M.
2017-01-01
This review systematically examines the evidence for shifts in flux through energy generating biochemical pathways in Huntington’s disease (HD) brains from humans and model systems. Compromise of the electron transport chain (ETC) appears not to be the primary or earliest metabolic change in HD pathogenesis. Rather, compromise of glucose uptake facilitates glucose flux through glycolysis and may possibly decrease flux through the pentose phosphate pathway (PPP), limiting subsequent NADPH and GSH production needed for antioxidant protection. As a result, oxidative damage to key glycolytic and tricarboxylic acid (TCA) cycle enzymes further restricts energy production so that while basal needs may be met through oxidative phosphorylation, those of excessive stimulation cannot. Energy production may also be compromised by deficits in mitochondrial biogenesis, dynamics or trafficking. Restrictions on energy production may be compensated for by glutamate oxidation and/or stimulation of fatty acid oxidation. Transcriptional dysregulation generated by mutant huntingtin also contributes to energetic disruption at specific enzymatic steps. Many of the alterations in metabolic substrates and enzymes may derive from normal regulatory feedback mechanisms and appear oscillatory. Fine temporal sequencing of the shifts in metabolic flux and transcriptional and expression changes associated with mutant huntingtin expression remain largely unexplored and may be model dependent. Differences in disease progression among HD model systems at the time of experimentation and their varying states of metabolic compensation may explain conflicting reports in the literature. Progressive shifts in metabolic flux represent homeostatic compensatory mechanisms that maintain the model organism through presymptomatic and symptomatic stages. PMID:29125492
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Th-POK regulates mammary gland lactation through mTOR-SREBP pathway.
Zhang, Rui; Ma, Huimin; Gao, Yuan; Wu, Yanjun; Qiao, Yuemei; Geng, Ajun; Cai, Cheguo; Han, Yingying; Zeng, Yi Arial; Liu, Xiaolong; Ge, Gaoxiang
2018-02-01
The Th-inducing POK (Th-POK, also known as ZBTB7B or cKrox) transcription factor is a key regulator of lineage commitment of immature T cell precursors. It is yet unclear the physiological functions of Th-POK besides helper T cell differentiation. Here we show that Th-POK is restrictedly expressed in the luminal epithelial cells in the mammary glands that is upregulated at late pregnancy and lactation. Lineage restrictedly expressed Th-POK exerts distinct biological functions in the mammary epithelial cells and T cells in a tissue-specific manner. Th-POK is not required for mammary epithelial cell fate determination. Mammary gland morphogenesis in puberty and alveologenesis in pregnancy are phenotypically normal in the Th-POK-deficient mice. However, Th-POK-deficient mice are defective in triggering the onset of lactation upon parturition with large cellular lipid droplets retained within alveolar epithelial cells. As a result, Th-POK knockout mice are unable to efficiently secret milk lipid and to nurse the offspring. Such defect is mainly attributed to the malfunctioned mammary epithelial cells, but not the tissue microenvironment in the Th-POK deficient mice. Th-POK directly regulates expression of insulin receptor substrate-1 (IRS-1) and insulin-induced Akt-mTOR-SREBP signaling. Th-POK deficiency compromises IRS-1 expression and Akt-mTOR-SREBP signaling in the lactating mammary glands. Conversely, insulin induces Th-POK expression. Thus, Th-POK functions as an important feed-forward regulator of insulin signaling in mammary gland lactation.
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Th-POK regulates mammary gland lactation through mTOR-SREBP pathway
Wu, Yanjun; Qiao, Yuemei; Geng, Ajun; Cai, Cheguo; Han, Yingying; Zeng, Yi Arial
2018-01-01
The Th-inducing POK (Th-POK, also known as ZBTB7B or cKrox) transcription factor is a key regulator of lineage commitment of immature T cell precursors. It is yet unclear the physiological functions of Th-POK besides helper T cell differentiation. Here we show that Th-POK is restrictedly expressed in the luminal epithelial cells in the mammary glands that is upregulated at late pregnancy and lactation. Lineage restrictedly expressed Th-POK exerts distinct biological functions in the mammary epithelial cells and T cells in a tissue-specific manner. Th-POK is not required for mammary epithelial cell fate determination. Mammary gland morphogenesis in puberty and alveologenesis in pregnancy are phenotypically normal in the Th-POK-deficient mice. However, Th-POK-deficient mice are defective in triggering the onset of lactation upon parturition with large cellular lipid droplets retained within alveolar epithelial cells. As a result, Th-POK knockout mice are unable to efficiently secret milk lipid and to nurse the offspring. Such defect is mainly attributed to the malfunctioned mammary epithelial cells, but not the tissue microenvironment in the Th-POK deficient mice. Th-POK directly regulates expression of insulin receptor substrate-1 (IRS-1) and insulin-induced Akt-mTOR-SREBP signaling. Th-POK deficiency compromises IRS-1 expression and Akt-mTOR-SREBP signaling in the lactating mammary glands. Conversely, insulin induces Th-POK expression. Thus, Th-POK functions as an important feed-forward regulator of insulin signaling in mammary gland lactation. PMID:29420538
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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
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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
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Differential DNA lesion formation and repair in heterochromatin and euchromatin
Han, Chunhua; Srivastava, Amit Kumar; Cui, Tiantian; Wang, Qi-En; Wani, Altaf A.
2016-01-01
Discretely orchestrated chromatin condensation is important for chromosome protection from DNA damage. However, it is still unclear how different chromatin states affect the formation and repair of nucleotide excision repair (NER) substrates, e.g. ultraviolet (UV)-induced cyclobutane pyrimidine dimers (CPD) and the pyrimidine (6-4) pyrimidone photoproducts (6-4PP), as well as cisplatin-induced intrastrand crosslinks (Pt-GG). Here, by using immunofluorescence and chromatin immunoprecipitation assays, we have demonstrated that CPD, which cause minor distortion of DNA double helix, can be detected in both euchromatic and heterochromatic regions, while 6-4PP and Pt-GG, which cause major distortion of DNA helix, can exclusively be detected in euchromatin, indicating that the condensed chromatin environment specifically interferes with the formation of these DNA lesions. Mechanistic investigation revealed that the class III histone deacetylase SIRT1 is responsible for restricting the formation of 6-4PP and Pt-GG in cells, probably by facilitating the maintenance of highly condensed heterochromatin. In addition, we also showed that the repair of CPD in heterochromatin is slower than that in euchromatin, and DNA damage binding protein 2 (DDB2) can promote the removal of CPD from heterochromatic region. In summary, our data provide evidence for differential formation and repair of DNA lesions that are substrates of NER. Both the sensitivity of DNA to damage and the kinetics of repair can be affected by the underlying level of chromatin compaction. PMID:26717995
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Kushwaha, Amit K; Sangwan, Neelam S; Tripathi, Sandhya; Sangwan, Rajender S
2013-03-10
Tropinone reductases (TRs) are small proteins belonging to the SDR (short chain dehydrogenase/reductase) family of enzymes. TR-I and TR-II catalyze the conversion of tropinone into tropane alcohols (tropine and pseudotropine, respectively). The steps are intermediary enroute to biosynthesis of tropane esters of medicinal importance, hyoscyamine/scopolamine, and calystegins, respectively. Biosynthesis of tropane alkaloids has been proposed to occur in roots. However, in the present report, a tropine forming tropinone reductase (TR-I) cDNA was isolated from the aerial tissue (leaf) of a medicinal plant, Withania coagulans. The ORF was deduced to encode a polypeptide of 29.34 kDa. The complete cDNA (WcTRI) was expressed in E. coli and the recombinant His-tagged protein was purified for functional characterization. The enzyme had a narrow pH range of substantial activity with maxima at 6.6. Relatively superior thermostability of the enzyme (30% retention of activity at 60 °C) was catalytic novelty in consonance with the desert area restricted habitat of the plant. The in vitro reaction kinetics predominantly favoured the forward reaction. The enzyme had wide substrate specificity but did not cover the substrates of other well-known plant SDR related to menthol metabolism. To our knowledge, this pertains to be the first report on any gene and enzyme of secondary metabolism from the commercially and medicinally important vegetable rennet species. Copyright © 2012 Elsevier B.V. All rights reserved.
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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.
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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.
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ATP-independent reversal of a membrane protein aggregate by a chloroplast SRP
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
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Payne, Anthony M; Dodd, Stephen L; Leeuwenburgh, Christiaan
2003-12-01
The decline in muscle function is associated with an age-related decrease in muscle mass and an age-related decline in strength. However, decreased strength is not solely due to decreased muscle mass. The age-related decline in muscle-specific force (force/muscle cross-sectional area), a measure of intrinsic muscle function, also contributes to age-related strength decline, and the mechanisms by which this occurs are only partially known. Moreover, changes in the extracellular space could have a profound effect on skeletal muscle function. Life-long calorie restriction in rodents has shown to be a powerful anti-aging intervention. In this study, we examine whether calorie restriction is able to attenuate the loss of muscle function and elevations in extracellular space associated with aging. We hypothesize that calorie restriction attenuates the age-associated decline in specific force and increases in extracellular space. Measurements of in vitro contractile properties of the extensor digitorum longus (type II) and soleus (type I) muscles from 12-mo and 26- to 28-mo-old ad libitum-fed, as well as 27- to 28-mo-old life-long calorie-restricted male Fischer 344 rats, were performed. We found that calorie restriction attenuated the age-associated decline in muscle mass-to-body mass ratio (mg/g) and strength-to-body mass ratio (N/kg) in the extensor digitorum longus muscle (P < 0.05) but not in the soleus muscle (P > 0.05). Importantly, muscle-specific force (N/cm2) in the extensor digitorum longus, but not in the soleus muscle, of the old calorie-restricted rats was equal to that of the young 12-mo-old animals. Moreover, the age-associated increase in extracellular space was reduced in the fast-twitch extensor digitorum longus muscle (P < 0.05) but not in the soleus muscle with calorie restriction. We also found a significant correlation between the extracellular space and the muscle-specific force in the extensor digitorum longus (r = -0.58; P < 0.05) but not in the soleus muscle (r = -0.38; P > 0.05). Hence, this study shows a loss of muscle function with age and suggests that long-term calorie restriction is an effective intervention against the loss of muscle function with age.
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Bullas, L R; Colson, C; Neufeld, B
1980-01-01
With the use of four different phages, Salmonella strains representing 85 different serotypes were examined to determine their restriction-modification phenotype. They fell into one of three groups on this basis: group 1, those which lacked the common LT system; group 2, those in which only the LT system could be recognized; and group 3. those which possessed the LT system and at least one other system shown with some serotypes to be closely linked to serB. The specificity of the serB-linked restriction-modification system was unique for each serotype, but different strains of the same serotype expressed the same specificity. Two of the systems were shown to behave in genetic crosses as functional alleles of the S. typhimurium SB system. It is possible that these serB-linked restriction-modification systems constitute a large multiallelic series of genes extending throughout the Salmonella genus and Escherichia coli. We suggest that the division of the Salmonella into the three restriction-modification groups may be significant in defining a "biological grouping" of the different serotypes within the genus which may ultimately be useful in describing the Salmonella species. From the genetic relatedness between the genes of some of the Salmonella restriction-modification systems with those of the E. coli systems, we deduce that the restriction endonuclases produced by the Salmonella serB-linked systems are of type 1. Determination of the nucleotide sequences of the recognition sites of the restriction endonucleases of selected Salmonella systems should further our understanding of specificity with these enzymes. PMID:6243623
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Wooldridge, Amy L; Bischof, Robert J; Meeusen, Els N; Liu, Hong; Heinemann, Gary K; Hunter, Damien S; Giles, Lynne C; Kind, Karen L; Owens, Julie A; Clifton, Vicki L; Gatford, Kathryn L
2014-04-01
Prenatal and early childhood exposures are implicated as causes of allergy, but the effects of intrauterine growth restriction on immune function and allergy are poorly defined. We therefore evaluated effects of experimental restriction of fetal growth on immune function and allergic sensitization in adolescent sheep. Immune function (circulating total red and white blood cells, neutrophils, lymphocytes, monocytes, eosinophils, and basophils, and the antibody response to Clostridial vaccination) and responses to house dust mite (HDM) allergen and ovalbumin (OVA) antigen sensitization (specific total Ig, IgG1, and IgE antibodies, and cutaneous hypersensitivity) were investigated in adolescent sheep from placentally restricted (PR, n = 23) and control (n = 40) pregnancies. Increases in circulating HDM-specific IgE (P = 0.007) and OVA-specific IgE (P = 0.038) were greater in PR than control progeny. PR did not alter total Ig, IgG1, or IgM responses to either antigen. PR increased OVA-specific but not HDM-specific IgA responses in females only (P = 0.023). Multiple birth increased Ig responses to OVA in a sex-specific manner. PR decreased the proportion of positive cutaneous hypersensitivity responders to OVA at 24 h (P = 0.030) but had no effect on cutaneous responses to HDM. Acute wheal responses to intradermal histamine correlated positively with birth weight in singletons (P = 0.023). Intrauterine growth restriction may suppress inflammatory responses in skin downstream of IgE induction, without impairment in antibody responses to a nonpolysaccharide vaccine. Discord between cutaneous and IgE responses following sensitization suggests new mechanisms for prenatal allergy programming.
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Smiles, William J; Conceição, Miguel S; Telles, Guilherme D; Chacon-Mikahil, Mara P T; Cavaglieri, Cláudia R; Vechin, Felipe C; Libardi, Cleiton A; Hawley, John A; Camera, Donny M
2017-02-01
Autophagy is an intracellular degradative system sensitive to hypoxia and exercise-induced perturbations to cellular bioenergetics. We determined the effects of low-intensity endurance-based exercise performed with blood-flow restriction (BFR) on cell signaling adaptive responses regulating autophagy and substrate metabolism in human skeletal muscle. In a randomized cross-over design, nine young, healthy but physically inactive males completed three experimental trials separated by 1 week of recovery consisting of either a resistance exercise bout (REX: 4 × 10 leg press repetitions, 70% 1-RM), endurance exercise (END: 30 min cycling, 70% VO 2peak ), or low-intensity cycling with BFR (15 min, 40% VO 2peak ). A resting muscle biopsy was obtained from the vastus lateralis 2 weeks prior to the first exercise trial and 3 h after each exercise bout. END increased ULK1 Ser757 phosphorylation above rest and BFR (~37 to 51%, P < 0.05). Following REX, there were significant elevations compared to rest (~348%) and BFR (~973%) for p38γ MAPK Thr180/Tyr182 phosphorylation (P < 0.05). Parkin content was lower following BFR cycling compared to REX (~20%, P < 0.05). There were no exercise-induced changes in select markers of autophagy following BFR. Genes implicated in substrate metabolism (HK2 and PDK4) were increased above rest (~143 to 338%) and BFR cycling (~212 to 517%) with END (P < 0.001). A single bout of low-intensity cycling with BFR is insufficient to induce intracellular "stress" responses (e.g., high rates of substrate turnover and local hypoxia) necessary to activate skeletal muscle autophagy signaling.
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Federal Register 2010, 2011, 2012, 2013, 2014
2012-08-22
... Restrictions: Site-Specific Treatment Variance for Hazardous Selenium-Bearing Waste Treated by U.S. Ecology... program, to U.S. Ecology Nevada in Beatty, Nevada for the treatment of a hazardous selenium- bearing waste.... Ecology Nevada located in Beatty, Nevada. B. Table of Contents I. Background [[Page 50623
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Schloesser, Anke; Campbell, Graeme; Glüer, Claus-Christian; Rimbach, Gerald; Huebbe, Patricia
2015-02-01
Dietary restriction (DR) on a normal low-fat diet improves metabolic health and may prolong life span. However, it is still uncertain whether restriction of an energy-dense, high-fat diet would also be beneficial and mitigate age-related processes. In the present study, we determined biomarkers of metabolic health, energy metabolism, and cellular aging in obesity-prone mice subjected to 30% DR on a high-fat diet for 6 months. Dietary-restricted mice had significantly lower body weights, less adipose tissue, lower energy expenditure, and altered substrate oxidation compared to their ad libitum-fed counterparts. Hepatic major urinary proteins (Mup) expression, which is linked to glucose and energy metabolism, and biomarkers of metabolic health, including insulin, glucose, cholesterol, and leptin/adiponectin ratio, were likewise reduced in high-fat, dietary-restricted mice. Hallmarks of cellular senescence such as Lamp2a and Hsc70 that mediate chaperone-mediated autophagy were induced and mechanistic target of rapamycin (mTOR) signaling mitigated upon high-fat DR. In contrast to DR applied in low-fat diets, anti-oxidant gene expression, proteasome activity, as well as 5'-adenosine monophosphate-activated protein kinase (AMPK) activation were not changed, suggesting that high-fat DR may attenuate some processes associated with cellular aging without the induction of cellular stress response or energy deprivation.
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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.
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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
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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.
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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.
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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
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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.
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Design of N-acyl homoserine lactonase with high substrate specificity by a rational approach.
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.
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Code of Federal Regulations, 2010 CFR
2010-04-01
... decision and notified you of the restriction. Your request must: (a) Be in writing; (b) Specifically request a hearing to challenge the restriction; and (c) Be hand delivered to the BIA office or postmarked...
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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
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A Measure of the Broad Substrate Specificity of Enzymes Based on ‘Duplicate’ Catalytic Residues
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
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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
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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
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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.
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[Substrate specifity in Amoeba proteus].
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.
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Belle, Sebastian; Han, Fang; Condomines, Maud; Christensen, Olaf; Witzens-Harig, Mathias; Kasper, Bernd; Kleist, Christian; Terness, Peter; Moos, Marion; Cremer, Friedrich; Hose, Dirk; Ho, Anthony D; Goldschmidt, Hartmut; Klein, Bernard; Hundemer, Michael
2008-07-01
The aim of this study is the identification of HLA-A2 restricted T-cell epitopes in the conserved region of the immunoglobulin-G-heavy-chain (IgGH) that can be used for immunotherapy in multiple myeloma (MM) patients. After the IgGH gene sequence was scanned for HLA-A2 restricted T-cell epitopes with a high binding affinity to the MHC-I-complex, promising nona-peptides were synthesized. Peptide specific CD8+ T-cells were generated from peripheral blood mononuclear cells (PBMC) of healthy donors (HD) and patients with MM using peptide pulsed dendritic cells (DC) in vitro. The activation and cytotoxicity of CD8+ T-cells was analyzed by IFN-alpha ELISpot-assay and 51Chromium release-assay. HLA-A2 restriction was proven by blocking T-cell activation with anti-HLA-A2 antibodies. Two HLA-A2 restricted T-cell epitopes-TLVTVSSAS derived from the IgGH-framework-region 4 (FR4) and LMISRTPEV from the constant region (CR)-induced expansion of specific CD8+ T-cells from PBMC in two of three (TLVTVSSAS) and one of three (LMISRTPEV) HD respectively. Specific T-cells were induced from PBMC in two of six (TLVTVSSAS) and eight of 19 (LMISRTPEV) patients with MM. Specific CD8+ T-cells also lysed peptide-pulsed target cells in 51Chromium release-assay. LMISRTPEV specific CD8+ T-cells from MM patients lysed specifically the HLA-A2+ IgG myeloma cell line XG-6. We identified two HLA-A2 restricted T-cell epitopes-TLVTVSSAS and LMISRTPEV--which can yield an expansion of CD8+ T-cells with the ability to kill peptide-loaded target cells and HLA-A2+ IgG+ myeloma cells. We conclude that TLVTVSSAS and LMISRTPEV could be T-cell epitopes for immunotherapy in MM patients.
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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.
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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.
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Coupling Oxygen Consumption with Hydrocarbon Oxidation in Bacterial Multicomponent Monooxygenases.
Wang, Weixue; Liang, Alexandria D; Lippard, Stephen J
2015-09-15
A fundamental goal in catalysis is the coupling of multiple reactions to yield a desired product. Enzymes have evolved elegant approaches to address this grand challenge. A salient example is the biological conversion of methane to methanol catalyzed by soluble methane monooxygenase (sMMO), a member of the bacterial multicomponent monooxygenase (BMM) superfamily. sMMO is a dynamic protein complex of three components: a hydroxylase, a reductase, and a regulatory protein. The active site, a carboxylate-rich non-heme diiron center, is buried inside the 251 kDa hydroxylase component. The enzyme processes four substrates: O2, protons, electrons, and methane. To couple O2 activation to methane oxidation, timely control of substrate access to the active site is critical. Recent studies of sMMO, as well as its homologues in the BMM superfamily, have begun to unravel the mechanism. The emerging and unifying picture reveals that each substrate gains access to the active site along a specific pathway through the hydroxylase. Electrons and protons are delivered via a three-amino-acid pore located adjacent to the diiron center; O2 migrates via a series of hydrophobic cavities; and hydrocarbon substrates reach the active site through a channel or linked set of cavities. The gating of these pathways mediates entry of each substrate to the diiron active site in a timed sequence and is coordinated by dynamic interactions with the other component proteins. The result is coupling of dioxygen consumption with hydrocarbon oxidation, avoiding unproductive oxidation of the reductant rather than the desired hydrocarbon. To initiate catalysis, the reductase delivers two electrons to the diiron(III) center by binding over the pore of the hydroxylase. The regulatory component then displaces the reductase, docking onto the same surface of the hydroxylase. Formation of the hydroxylase-regulatory component complex (i) induces conformational changes of pore residues that may bring protons to the active site; (ii) connects hydrophobic cavities in the hydroxylase leading from the exterior to the diiron active site, providing a pathway for O2 and methane, in the case of sMMO, to the reduced diiron center for O2 activation and substrate hydroxylation; (iii) closes the pore, as well as a channel in the case of four-component BMM enzymes, restricting proton access to the diiron center during formation of "Fe2O2" intermediates required for hydrocarbon oxidation; and (iv) inhibits undesired electron transfer to the Fe2O2 intermediates by blocking reductase binding during O2 activation. This mechanism is quite different from that adopted by cytochromes P450, a large class of heme-containing monooxygenases that catalyze reactions very similar to those catalyzed by the BMM enzymes. Understanding the timed enzyme control of substrate access has implications for designing artificial catalysts. To achieve multiple turnovers and tight coupling, synthetic models must also control substrate access, a major challenge considering that nature requires large, multimeric, dynamic protein complexes to accomplish this feat.
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Hydrolytic properties and substrate specificity of the foot-and-mouth disease leader protease.
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.
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Liu, Shijia; Shao, Shangjin; Li, Linlin; Cheng, Zhi; Tian, Li; Gao, Peiji; Wang, Lushan
2015-12-11
Chitinases and chitosanases, referred to as chitinolytic enzymes, are two important categories of glycoside hydrolases (GH) that play a key role in degrading chitin and chitosan, two naturally abundant polysaccharides. Here, we investigate the active site architecture of the major chitosanase (GH8, GH46) and chitinase families (GH18, GH19). Both charged (Glu, His, Arg, Asp) and aromatic amino acids (Tyr, Trp, Phe) are observed with higher frequency within chitinolytic active sites as compared to elsewhere in the enzyme structure, indicating significant roles related to enzyme function. Hydrogen bonds between chitinolytic enzymes and the substrate C2 functional groups, i.e. amino groups and N-acetyl groups, drive substrate recognition, while non-specific CH-π interactions between aromatic residues and substrate mainly contribute to tighter binding and enhanced processivity evident in GH8 and GH18 enzymes. For different families of chitinolytic enzymes, the number, type, and position of substrate atoms bound in the active site vary, resulting in different substrate-binding specificities. The data presented here explain the synergistic action of multiple enzyme families at a molecular level and provide a more reasonable method for functional annotation, which can be further applied toward the practical engineering of chitinases and chitosanases. Copyright © 2015 Elsevier Ltd. All rights reserved.
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Reactive ion etched substrates and methods of making and using
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.
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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.
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Hudson, Quanah J.; Seidl, Christine I.M.; Kulinski, Tomasz M.; Huang, Ru; Warczok, Katarzyna E.; Bittner, Romana; Bartolomei, Marisa S.; Barlow, Denise P.
2011-01-01
A subset of imprinted genes in the mouse have been reported to show imprinted expression that is restricted to the placenta, a short-lived extra-embryonic organ. Notably these so-called 'placental-specific' imprinted genes are expressed from both parental alleles in embryo and adult tissues. The placenta is an embryonic-derived organ that is closely associated with maternal tissue and as a consequence, maternal contamination can be mistaken for maternal-specific imprinted expression. The complexity of the placenta, which arises from multiple embryonic lineages, poses additional problems in accurately assessing allele-specific repressive epigenetic modifications in genes that also show lineage-specific silencing in this organ. These problems require that extra evidence be obtained to support the imprinted status of genes whose imprinted expression is restricted to the placenta. We show here that the extra-embryonic visceral yolk sac (VYS), a nutritive membrane surrounding the developing embryo, shows a similar 'extra-embryonic-lineage-specific' pattern of imprinted expression. We present an improved enzymatic technique for separating the bilaminar VYS and show that this pattern of imprinted expression is restricted to the endoderm layer. Finally, we show that VYS 'extra-embryonic-lineage-specific' imprinted expression is regulated by DNA methylation in a similar manner as shown for genes showing multi-lineage imprinted expression in extra-embryonic, embryonic and adult tissues. These results show that the VYS is an improved model for studying the epigenetic mechanisms regulating extra-embryonic-lineage-specific imprinted expression. PMID:21354127
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Synthesis and evaluation of fluorogenic triglycerides as lipase assay substrates.
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.
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Regulation of Proteolysis by Human Deubiquitinating Enzymes
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
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Affective neural response to restricted interests in Autism Spectrum Disorders
Cascio, Carissa J.; Foss-Feig, Jennifer H.; Heacock, Jessica; Schauder, Kimberly B.; Loring, Whitney A.; Rogers, Baxter P.; Pryweller, Jennifer R.; Newsom, Cassandra R.; Cockhren, Jurnell; Cao, Aize; Bolton, Scott
2013-01-01
Background Restricted interests are a class of repetitive behavior in autism spectrum disorders (ASD) whose intensity and narrow focus often contribute to significant interference with daily functioning. While numerous neuroimaging studies have investigated executive circuits as putative neural substrates of repetitive behavior, recent work implicates affective neural circuits in restricted interests. We sought to explore the role of affective neural circuits and determine how restricted interests are distinguished from hobbies or interests in typical development. Methods We compared a group of children with ASD to a typically developing (TD) group of children with strong interests or hobbies, employing parent report, an operant behavioral task, and functional imaging with personalized stimuli based on individual interests. Results While performance on the operant task was similar between the two groups, parent report of intensity and interference of interests was significantly higher in the ASD group. Both the ASD and TD groups showed increased BOLD response in widespread affective neural regions to pictures of their own interest. When viewing pictures of other children's interests, the TD group showed a similar pattern, whereas BOLD response in the ASD group was much more limited. Increased BOLD response in the insula and anterior cingulate cortex distinguished the ASD from the TD group, and parent report of the intensity and interference with daily life of the child's restricted interest predicted insula response. Conclusions While affective neural network response and operant behavior are comparable in typical and restricted interests, the narrowness of focus that clinically distinguishes restricted interests in ASD is reflected in more interference in daily life and aberrantly enhanced insula and anterior cingulate response to individuals’ own interests in the ASD group. These results further support the involvement of affective neural networks in repetitive behaviors in ASD. PMID:24117668
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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
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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.
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46 CFR 182.405 - Fuel restrictions.
Code of Federal Regulations, 2014 CFR
2014-10-01
... 46 Shipping 7 2014-10-01 2014-10-01 false Fuel restrictions. 182.405 Section 182.405 Shipping...) MACHINERY INSTALLATION Specific Machinery Requirements § 182.405 Fuel restrictions. The use of alternative fuels, other than diesel fuel or gasoline, as fuel for an internal combustion engine will be reviewed on...
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46 CFR 182.405 - Fuel restrictions.
Code of Federal Regulations, 2011 CFR
2011-10-01
... 46 Shipping 7 2011-10-01 2011-10-01 false Fuel restrictions. 182.405 Section 182.405 Shipping...) MACHINERY INSTALLATION Specific Machinery Requirements § 182.405 Fuel restrictions. The use of alternative fuels, other than diesel fuel or gasoline, as fuel for an internal combustion engine will be reviewed on...
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46 CFR 182.405 - Fuel restrictions.
Code of Federal Regulations, 2013 CFR
2013-10-01
... 46 Shipping 7 2013-10-01 2013-10-01 false Fuel restrictions. 182.405 Section 182.405 Shipping...) MACHINERY INSTALLATION Specific Machinery Requirements § 182.405 Fuel restrictions. The use of alternative fuels, other than diesel fuel or gasoline, as fuel for an internal combustion engine will be reviewed on...
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46 CFR 182.405 - Fuel restrictions.
Code of Federal Regulations, 2010 CFR
2010-10-01
... 46 Shipping 7 2010-10-01 2010-10-01 false Fuel restrictions. 182.405 Section 182.405 Shipping...) MACHINERY INSTALLATION Specific Machinery Requirements § 182.405 Fuel restrictions. The use of alternative fuels, other than diesel fuel or gasoline, as fuel for an internal combustion engine will be reviewed on...
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46 CFR 182.405 - Fuel restrictions.
Code of Federal Regulations, 2012 CFR
2012-10-01
... 46 Shipping 7 2012-10-01 2012-10-01 false Fuel restrictions. 182.405 Section 182.405 Shipping...) MACHINERY INSTALLATION Specific Machinery Requirements § 182.405 Fuel restrictions. The use of alternative fuels, other than diesel fuel or gasoline, as fuel for an internal combustion engine will be reviewed on...
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Ding, Ke, E-mail: dk1118@yeah.net; Yang, Zhong; Xu, Jian-zhong, E-mail: xjzspine@163.com
The application of satellite cell-derived myoblasts in regenerative medicine has been restricted by the rapid loss of stemness during in vitro cell expansion using traditional culture systems. However, studies published in the past decade have highlighted the influence of substrate elasticity on stem cell fate and revealed that culture on a soft hydrogel substrate can promote self-renewal and prolong the regenerative potential of muscle stem cells. Whether hydrogel substrates have similar effects after long-term robust expansion remains to be determined. Herein we prepared an elastic chitosan/beta-glycerophosphate/collagen hydrogel mimicking the soft microenvironment of muscle tissues for use as the substrate formore » satellite cell culture and investigated its influence on long-term cell expansion. After 20 passages in culture, satellite cell-derived myoblasts cultured on our hydrogel substrate exhibited significant improvements in proliferation capability, cell viability, colony forming frequency, and potential for myogenic differentiation compared to those cultured on a routine rigid culture surface. Immunochemical staining and western blot analysis both confirmed that myoblasts cultured on the hydrogel substrate expressed higher levels of several differentiation-related markers, including Pax7, Pax3, and SSEA-1, and a lower level of MyoD compared to myoblasts cultured on rigid culture plates (all p<0.05). After transplantation into the tibialis anterior of nude mice, myoblasts that had been cultured on the hydrogel substrate demonstrated a significantly greater engraftment efficacy than those cultured on the traditional surface. Collectively, these results indicate that the elastic hydrogel substrate supported robust expansion of murine myoblasts and enhanced their engraftment in vivo. - Highlights: • An elastic hydrogel was designed to mimic the pliable muscle tissue microenvironment. • Myoblasts retained their stemness in long-term culture on the elastic hydrogels. • Myoblasts expanded on the elastic hydrogel exhibited enhanced in vivo engraftment.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Ahrenkiel, Phil
The primary objective of the project was to develop Ga0.82In0.18As (GaInAs) solar cells grown on epilayers of elemental Al. At this composition, GaInAs has a nearly optimal bandgap (1.16 eV) for a single-junction photovoltaic device. However, GaInAs lacks a convenient, lattice-matched substrate, restricting most investigations to metamorphic structures. The metal Al is, in fact, precisely lattice-matched to GaInAs in the orientation GaInAs (001)[100]||Al(001)[110]. At present, however, epi-ready Al substrates are not readily available commercially and are subject to oxidation. However, epitaxial Al buffer layers could enable control of defect generation, thermal and light management, and rapid epitaxial lift-off for ultrathinmore » devices.« less
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Recent Developments of Magnetoresistive Sensors for Industrial Applications
Jogschies, Lisa; Klaas, Daniel; Kruppe, Rahel; Rittinger, Johannes; Taptimthong, Piriya; Wienecke, Anja; Rissing, Lutz; Wurz, Marc Christopher
2015-01-01
The research and development in the field of magnetoresistive sensors has played an important role in the last few decades. Here, the authors give an introduction to the fundamentals of the anisotropic magnetoresistive (AMR) and the giant magnetoresistive (GMR) effect as well as an overview of various types of sensors in industrial applications. In addition, the authors present their recent work in this field, ranging from sensor systems fabricated on traditional substrate materials like silicon (Si), over new fabrication techniques for magnetoresistive sensors on flexible substrates for special applications, e.g., a flexible write head for component integrated data storage, micro-stamping of sensors on arbitrary surfaces or three dimensional sensing under extreme conditions (restricted mounting space in motor air gap, high temperatures during geothermal drilling). PMID:26569263
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NASA Astrophysics Data System (ADS)
Hoek, J.; Reysenbach, A.; Habicht, K.; Canfield, D. E.
2004-12-01
Sulfate-reducing bacteria fractionate sulfur isotopes during dissimilatory sulfate reduction, producing sulfide depleted in 34S. Although isotope fractionation during sulfate reduction of pure cultures has been extensively studied, most of the research to date has focused on mesophilic sulfate reducers, particularly for the species Desulfovibrio desulfuricans. Results from these studies show that: 1) fractionations range from 3-46‰ with an average around 18‰ , 2) when organic electron donors are utilized, the extent of fractionation is dependent on the rate of sulfate reduction, with decreasing fractionations observed with higher specific rates, 3) fractionations are suppressed with low sulfate concentrations, and when hydrogen is used as the electron donor. High specific sulfate-reduction rates are encountered when sulfate-reducing bacteria metabolize at their optimal temperature and under non-limiting substrate conditions. Changes in both temperature and substrate availability could shift fractionations from those expressed under optimal growth conditions. Sulfate reducers may frequently experience substrate limitation and sub-optimal growth temperatures in the environment. Therefore it is important to understand how sulfate-reducing bacteria fractionate sulfur isotopes under conditions that more closely resemble the restrictions imposed by the environment. In this study the fractionation of sulfur isotopes by Thermodesulfatator indicus was explored during sulfate reduction under a wide range of temperatures and with both hydrogen-saturating and hydrogen-limited conditions. T. indicus is a thermophilic (temperature optimum = 70° C) chemolithotrophic sulfate-reducing bacterium, which was recently isolated from a deep-sea hydrothermal vent on the Central Indian Ridge. This bacterium represents the type species of a new genus and to date is the most deeply branching sulfate-reducing bacterium known. T. indicus was grown in carbonate-buffered salt-water medium with H2 as the sole electron donor, and CO2 as primary carbon source. The fractionation of sulfur isotopes was measured in batch cultures and in a thermal gradient block over the full temperature range of growth (40-80° C). For experiments in the gradient block, cell-specific rates of sulfate reduction increased with increasing temperatures to 70° C after which sulfate-reduction rates rapidly decreased. The range of fractionations (1.5-10‰ ) was typical for growth with hydrogen as the electron donor. Fractionations decreased with increasing temperature from 40--60° C, and increased with increasing temperatures from 60-80° C. Growth under H2-limited conditions in a fed-batch culture revealed high fractionations of 24-37‰ . This is the first report of sulfur isotope fractionation under H2 limited growth and indicates that large fractionations are produced when H2 is supplied as a limiting substrate. Our results suggest that fractionation is controlled by the competition of forward and reverse enzymatic reaction rates during sulfate reduction and by sulfate transport into the cell.
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Lipoxygenase is involved in the control of potato tuber development.
Kolomiets, M V; Hannapel, D J; Chen, H; Tymeson, M; Gladon, R J
2001-03-01
Plant lipoxygenases (LOXs) are a functionally diverse class of dioxygenases implicated in physiological processes such as growth, senescence, and stress-related responses. LOXs incorporate oxygen into their fatty acid substrates and produce hydroperoxide fatty acids that are precursors of jasmonic acid and related compounds. Here, we report the involvement of the tuber-associated LOXs, designated the Lox1 class, in the control of tuber growth. RNA hybridization analysis showed that the accumulation of Lox1 class transcripts was restricted to developing tubers, stolons, and roots and that mRNA accumulation correlated positively with tuber initiation and growth. In situ hybridization showed that Lox1 class transcripts accumulated in the apical and subapical regions of the newly formed tuber, specifically in the vascular tissue of the perimedullary region, the site of the most active cell growth during tuber enlargement. Suppression mutants produced by expressing antisense coding sequence of a specific tuber LOX, designated POTLX-1, exhibited a significant reduction in LOX activity in stolons and tubers. The suppression of LOX activity correlated with reduced tuber yield, decreased average tuber size, and a disruption of tuber formation. Our results indicate that the pathway initiated by the expression of the Lox1 class genes of potato is involved in the regulation of tuber enlargement.
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The effect of substrate composition and storage time on urine specific gravity in dogs.
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.
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USDA-ARS?s Scientific Manuscript database
Computerized point-of-sale (POS) machine software that allows parents to place restrictions on their child’s school meal accounts is available. Parents could restrict specific foods (eg, chips), identify specific days the child can purchase extra foods, or set monetary limits. This descriptive study...
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Dedkov, V S
2009-01-01
The specificity of DNA-methyltransferase M.Bsc4I was defined in cellular lysate of Bacillus schlegelii 4. For this purpose, we used methylation sensitivity of restriction endonucleases, and also modeling of methylation. The modeling consisted in editing sequences of DNA using replacements of methylated bases and their complementary bases. The substratum DNA processed by M.Bsc4I also were used for studying sensitivity of some restriction endonucleases to methylation. Thus, it was shown that M.Bsc4I methylated 5'-Cm4CNNNNNNNGG-3' and the overlapped dcm-methylation blocked its activity. The offered approach can appear universal enough and simple for definition of specificity of DNA-methyltransferases.
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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.
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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.
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Acidotolerant Bacteria and Fungi as a Sink of Methanol-Derived Carbon in a Deciduous Forest Soil
Morawe, Mareen; Hoeke, Henrike; Wissenbach, Dirk K.; Lentendu, Guillaume; Wubet, Tesfaye; Kröber, Eileen; Kolb, Steffen
2017-01-01
Methanol is an abundant atmospheric volatile organic compound that is released from both living and decaying plant material. In forest and other aerated soils, methanol can be consumed by methanol-utilizing microorganisms that constitute a known terrestrial sink. However, the environmental factors that drive the biodiversity of such methanol-utilizers have been hardly resolved. Soil-derived isolates of methanol-utilizers can also often assimilate multicarbon compounds as alternative substrates. Here, we conducted a comparative DNA stable isotope probing experiment under methylotrophic (only [13C1]-methanol was supplemented) and combined substrate conditions ([12C1]-methanol and alternative multi-carbon [13Cu]-substrates were simultaneously supplemented) to (i) identify methanol-utilizing microorganisms of a deciduous forest soil (European beech dominated temperate forest in Germany), (ii) assess their substrate range in the soil environment, and (iii) evaluate their trophic links to other soil microorganisms. The applied multi-carbon substrates represented typical intermediates of organic matter degradation, such as acetate, plant-derived sugars (xylose and glucose), and a lignin-derived aromatic compound (vanillic acid). An experimentally induced pH shift was associated with substantial changes of the diversity of active methanol-utilizers suggesting that soil pH was a niche-defining factor of these microorganisms. The main bacterial methanol-utilizers were members of the Beijerinckiaceae (Bacteria) that played a central role in a detected methanol-based food web. A clear preference for methanol or multi-carbon substrates as carbon source of different Beijerinckiaceae-affiliated phylotypes was observed suggesting a restricted substrate range of the methylotrophic representatives. Apart from Bacteria, we also identified the yeasts Cryptococcus and Trichosporon as methanol-derived carbon-utilizing fungi suggesting that further research is needed to exclude or prove methylotrophy of these fungi. PMID:28790984
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Arabidopsis ATG4 cysteine proteases specificity toward ATG8 substrates
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
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Cloning and characterization of a new broadspecific β-glucosidase from Lactococcus sp. FSJ4.
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.
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Molecular mechanisms of substrate recognition and specificity of botulinum neurotoxin serotype F.
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.
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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.
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Aerobic biological treatment of leachates from municipal solid waste landfill.
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).
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Intapad, Suttira; Ojeda, Norma B.; Varney, Elliott; Royals, Thomas P.; Alexander, Barbara T.
2015-01-01
The renal endothelin system contributes to sex differences in blood pressure with males demonstrating greater endothelin type-A receptor-mediated responses relative to females. Intrauterine growth restriction programs hypertension and enhanced renal sensitivity to acute angiotensin II in male growth-restricted rats. Endothelin is reported to work synergistically with angiotensin II. Thus, this study tested the hypothesis that endothelin augments the blood pressure response to acute angiotensin II in male growth-restricted rats. Systemic and renal hemodynamics were determined in response to acute angiotensin II (100 nanogram/kilogram/minute for 30 minutes) with and without the endothelin type-A receptor antagonist, ABT 627(10 nanogram/kilogram/minute for 30 minutes), in rats pretreated with enalapril (250 milligram/Liter for one week) to normalize the endogenous renin angiotensin system. Endothelin type-A receptor blockade reduced angiotensin II-mediated increases in blood pressure in male control and male growth-restricted rats. Endothelin type-A receptor blockade also abolished hyper-responsiveness to acute angiotensin II in male growth-restricted rats. Yet, blood pressure remained significantly elevated above baseline following endothelin type-A receptor blockade suggesting that factors in addition to endothelin contribute to the basic angiotensin II-induced pressor response in male rats. We also determined sex-specific effects of endothelin on acute angiotensin II-mediated hemodynamic responses. Endothelin type-A receptor blockade did not reduce acute angiotensin II-mediated increases in blood pressure in female control or growth-restricted rats, intact or ovariectomized. Thus, these data suggest that endothelin type-A receptor blockade contributes to hypersensitivity to acute angiotensin II in male growth-restricted rats and further supports the sex-specific effect of endothelin on blood pressure. PMID:26459423
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Pawlica, Paulina; Dufour, Caroline; Berthoux, Lionel
2015-04-01
IFN-induced restriction factors can significantly affect the replicative capacity of retroviruses in mammals. TRIM5α (tripartite motif protein 5, isoform α) is a restriction factor that acts at early stages of the virus life cycle by intercepting and destabilizing incoming retroviral cores. Sensitivity to TRIM5α maps to the N-terminal domain of the retroviral capsid proteins. In several New World and Old World monkey species, independent events of retrotransposon-mediated insertion of the cyclophilin A (CypA)-coding sequence in the trim5 gene have given rise to TRIMCyp (also called TRIM5-CypA), a hybrid protein that is active against some lentiviruses in a species-specific fashion. In particular, TRIMCyp from the owl monkey (omkTRIMCyp) very efficiently inhibits human immunodeficiency virus type 1 (HIV-1). Previously, we showed that disrupting the integrity of microtubules (MTs) and of cytoplasmic dynein complexes partially rescued replication of retroviruses, including HIV-1, from restriction mediated by TRIM5α. Here, we showed that efficient restriction of HIV-1 by omkTRIMCyp was similarly dependent on the MT network and on dynein complexes, but in a context-dependent fashion. When omkTRIMCyp was expressed in human HeLa cells, restriction was partially counteracted by pharmacological agents targeting MTs or by small interfering RNA-mediated inhibition of dynein. The same drugs (nocodazole and paclitaxel) also rescued HIV-1 from restriction in cat CRFK cells, although to a lesser extent. Strikingly, neither nocodazole, paclitaxel nor depletion of the dynein heavy chain had a significant effect on the restriction of HIV-1 in an owl monkey cell line. These results suggested the existence of cell-specific functional interactions between MTs/dynein and TRIMCyp. © 2015 The Authors.
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Motor and cortico-striatal-thalamic connectivity alterations in intrauterine growth restriction.
Eixarch, Elisenda; Muñoz-Moreno, Emma; Bargallo, Nuria; Batalle, Dafnis; Gratacos, Eduard
2016-06-01
Intrauterine growth restriction is associated with short- and long-term neurodevelopmental problems. Structural brain changes underlying these alterations have been described with the use of different magnetic resonance-based methods that include changes in whole structural brain networks. However, evaluation of specific brain circuits and its correlation with related functions has not been investigated in intrauterine growth restriction. In this study, we aimed to investigate differences in tractography-related metrics in cortico-striatal-thalamic and motor networks in intrauterine growth restricted children and whether these parameters were related with their specific function in order to explore its potential use as an imaging biomarker of altered neurodevelopment. We included a group of 24 intrauterine growth restriction subjects and 27 control subjects that were scanned at 1 year old; we acquired T1-weighted and 30 directions diffusion magnetic resonance images. Each subject brain was segmented in 93 regions with the use of anatomical automatic labeling atlas, and deterministic tractography was performed. Brain regions included in motor and cortico-striatal-thalamic networks were defined based in functional and anatomic criteria. Within the streamlines that resulted from the whole brain tractography, those belonging to each specific circuit were selected and tractography-related metrics that included number of streamlines, fractional anisotropy, and integrity were calculated for each network. We evaluated differences between both groups and further explored the correlation of these parameters with the results of socioemotional, cognitive, and motor scales from Bayley Scale at 2 years of age. Reduced fractional anisotropy (cortico-striatal-thalamic, 0.319 ± 0.018 vs 0.315 ± 0.015; P = .010; motor, 0.322 ± 0.019 vs 0.319 ± 0.020; P = .019) and integrity cortico-striatal-thalamic (0.407 ± 0.040 vs 0.399 ± 0.034; P = .018; motor, 0.417 ± 0.044 vs 0.409 ± 0.046; P = .016) in both networks were observed in the intrauterine growth restriction group, with no differences in number of streamlines. More importantly, strong specific correlation was found between tractography-related metrics and its relative function in both networks in intrauterine growth restricted children. Motor network metrics were correlated specifically with motor scale results (fractional anisotropy: rho = 0.857; integrity: rho = 0.740); cortico-striatal-thalamic network metrics were correlated with cognitive (fractional anisotropy: rho = 0.793; integrity, rho = 0.762) and socioemotional scale (fractional anisotropy: rho = 0.850; integrity: rho = 0.877). These results support the existence of altered brain connectivity in intrauterine growth restriction demonstrated by altered connectivity in motor and cortico-striatal-thalamic networks, with reduced fractional anisotropy and integrity. The specific correlation between tractography-related metrics and neurodevelopmental outcomes in intrauterine growth restriction shows the potential to use this approach to develop imaging biomarkers to predict specific neurodevelopmental outcome in infants who are at risk because of intrauterine growth restriction and other prenatal diseases. Copyright © 2015 Elsevier Inc. All rights reserved.
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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.
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Shang, Shaobin; Siddiqui, Sarah; Bian, Yao; Zhao, Jie; Wang, Chyung-Ru
2016-01-01
MHC Ib-restricted CD8+ T cells have been implicated in host defense against Mycobacterium tuberculosis (Mtb) infection. However, the relative contribution of various MHC Ib-restricted T cell populations to anti-mycobacterial immunity remains elusive. In this study, we used mice that lack MHC Ia (Kb-/-Db-/-), MHC Ia/H2-M3 (Kb-/-Db-/-M3-/-), or β2m (β2m-/-) to study the role of M3-restricted and other MHC Ib-restricted T cells in immunity against Mtb. Unlike their dominant role in Listeria infection, we found that M3-restricted CD8+ T cells only represented a small proportion of the CD8+ T cells responding to Mtb infection. Non-M3, MHC Ib-restricted CD8+ T cells expanded preferentially in the lungs of Mtb-infected Kb-/-Db-/-M3-/- mice, exhibited polyfunctional capacities and conferred protection against Mtb. These MHC Ib-restricted CD8+ T cells recognized several Mtb-derived protein antigens at a higher frequency than MHC Ia-restricted CD8+ T cells. The presentation of Mtb antigens to MHC Ib-restricted CD8+ T cells was mostly β2m-dependent but TAP-independent. Interestingly, a large proportion of Mtb-specific MHC Ib-restricted CD8+ T cells in Kb-/-Db-/-M3-/- mice were Qa-2-restricted while no considerable numbers of MR1 or CD1-restricted Mtb-specific CD8+ T cells were detected. Our findings indicate that nonclassical CD8+ T cells other than the known M3, CD1, and MR1-restricted CD8+ T cells contribute to host immune responses against Mtb infection. Targeting these MHC Ib-restricted CD8+ T cells would facilitate the design of better Mtb vaccines with broader coverage across MHC haplotypes due to the limited polymorphism of MHC class Ib molecules. PMID:27272249
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NASA Astrophysics Data System (ADS)
Nooshabadi, Fatemeh; Yang, Hee-Jeong; Cheng, Yunfeng; Xie, Hexin; Rao, Jianghong; Cirillo, Jeffrey D.; Maitland, Kristen C.
2016-03-01
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains one of the most frequent causes of death worldwide. The slow growth rate of Mtb limits progress toward understanding tuberculosis including diagnosis of infections and evaluating therapeutic efficacy. Development of near-infrared (NIR) β-lactamase (BlaC)-specific fluorogenic substrate has made a significant breakthrough in the whole-animal imaging to detect Mtb infection. The reporter enzyme fluorescence (REF) system using a BlaC-specific fluorogenic substrate has improved the detection sensitivity in whole-animal optical imaging down to ~104 colony forming units (CFU) of bacteria, about 100-fold improvement over recombinant strains. However, improvement of detection sensitivity is strongly needed for clinical diagnosis of early stage infection at greater tissue depth. In order to improve detection sensitivity, we have integrated a fiber-based microendoscpe into a whole-animal imaging system to transmit the excitation light from the fiber bundle to the fluorescent target directly and measure fluorescent level using BlaC-specific REF substrate in the mouse lung. REF substrate, CNIR800, was delivered via aerosol route to the pulmonary infected mice with M. bovis BCG strain at 24 hours post-infection and groups of mice were imaged at 1-4 hours post-administration of the substrate using the integrated imaging system. In this study we evaluated the kinetics of CNIR800 substrate using REF technology using the integrated imaging system. Integration of these technologies has great promise for improved detection sensitivity allowing pre-clinical imaging for evaluation of new therapeutic agents.
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Junyong Zhu; G.S. Wang; X.J. Pan; Roland Gleisner
2009-01-01
Sieving methods have been almost exclusively used for feedstock size-reduction characterization in the biomass refining literature. This study demonstrates a methodology to properly characterize specific surface of biomass substrates through two dimensional measurement of each fiber of the substrate using a wet imaging technique. The methodology provides more...
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Bjerve, K S
1984-01-01
The specificity of the L-serine base-exchange enzyme towards the fatty acid composition of the phospholipid substrate was investigated with a rat liver microsomal fraction. The relative rates of L-serine incorporation into saturated-hexaenoic, saturated-pentaenoic, saturated-tetraenoic, saturated-trienoic, dienoic-dienoic, monoenoic-dienoic, saturated-dienoic and saturated-monoenoic + saturated-saturated phosphatidylserine molecular species were 42, 5, 23, 4, 5, 4, 5 and 11% respectively. This is similar to, but not identical with, the relative mass abundance of these molecular species in total liver cell phosphatidylserines. The results indicate that the substrate-specificity of the L-serine base-exchange enzyme can at least in part explain the observed fatty acid composition of rat liver phosphatidylserines. PMID:6430274
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Guilfoyle, Richard A.; Guo, Zhen
2001-01-01
A restriction site indexing method for selectively amplifying any fragment generated by a Class II restriction enzyme includes adaptors specific to fragment ends containing adaptor indexing sequences complementary to fragment indexing sequences near the termini of fragments generated by Class II enzyme cleavage. A method for combinatorial indexing facilitates amplification of restriction fragments whose sequence is not known.
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Guilfoyle, Richard A.; Guo, Zhen
1999-01-01
A restriction site indexing method for selectively amplifying any fragment generated by a Class II restriction enzyme includes adaptors specific to fragment ends containing adaptor indexing sequences complementary to fragment indexing sequences near the termini of fragments generated by Class II enzyme cleavage. A method for combinatorial indexing facilitates amplification of restriction fragments whose sequence is not known.
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Restrictions in Means for Suicide: An Effective Tool in Preventing Suicide: The Danish Experience
ERIC Educational Resources Information Center
Nordentoft, Merete; Qin, Ping; Helweg-Larsen, Karin
2007-01-01
Restriction of means for suicide is an important part of suicide preventive strategies in different countries. The effect on method-specific suicide rate and overall suicide rate of restrictions on availability of carbon monoxide, barbiturates, and dextropropoxyphene was examined. From 1970 to 2000, overall suicide mortality and method-specific…
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Tavladoraki, Paraskevi; Cervelli, Manuela; Antonangeli, Fabrizio; Minervini, Giovanni; Stano, Pasquale; Federico, Rodolfo; Mariottini, Paolo; Polticelli, Fabio
2011-04-01
Spermine oxidase (SMO) and acetylpolyamine oxidase (APAO) are FAD-dependent enzymes that are involved in the highly regulated pathways of polyamine biosynthesis and degradation. Polyamine content is strictly related to cell growth, and dysfunctions in polyamine metabolism have been linked with cancer. Specific inhibitors of SMO and APAO would allow analyzing the precise role of these enzymes in polyamine metabolism and related pathologies. However, none of the available polyamine oxidase inhibitors displays the desired characteristics of selective affinity and specificity. In addition, repeated efforts to obtain structural details at the atomic level on these two enzymes have all failed. In the present study, in an effort to better understand structure-function relationships, SMO enzyme-substrate complex has been probed through a combination of molecular modeling, site-directed mutagenesis and biochemical studies. Results obtained indicate that SMO binds spermine in a similar conformation as that observed in the yeast polyamine oxidase FMS1-spermine complex and demonstrate a major role for residues His82 and Lys367 in substrate binding and catalysis. In addition, the SMO enzyme-substrate complex highlights the presence of an active site pocket with highly polar characteristics, which may explain the different substrate specificity of SMO with respect to APAO and provide the basis for the design of specific inhibitors for SMO and APAO.
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Wlodarski, Tomasz; Kutner, Jan; Towpik, Joanna; Knizewski, Lukasz; Rychlewski, Leszek; Kudlicki, Andrzej; Rowicka, Maga; Dziembowski, Andrzej; Ginalski, Krzysztof
2011-01-01
Methylation is one of the most common chemical modifications of biologically active molecules and it occurs in all life forms. Its functional role is very diverse and involves many essential cellular processes, such as signal transduction, transcriptional control, biosynthesis, and metabolism. Here, we provide further insight into the enzymatic methylation in S. cerevisiae by conducting a comprehensive structural and functional survey of all the methyltransferases encoded in its genome. Using distant homology detection and fold recognition, we found that the S. cerevisiae methyltransferome comprises 86 MTases (53 well-known and 33 putative with unknown substrate specificity). Structural classification of their catalytic domains shows that these enzymes may adopt nine different folds, the most common being the Rossmann-like. We also analyzed the domain architecture of these proteins and identified several new domain contexts. Interestingly, we found that the majority of MTase genes are periodically expressed during yeast metabolic cycle. This finding, together with calculated isoelectric point, fold assignment and cellular localization, was used to develop a novel approach for predicting substrate specificity. Using this approach, we predicted the general substrates for 24 of 33 putative MTases and confirmed these predictions experimentally in both cases tested. Finally, we show that, in S. cerevisiae, methylation is carried out by 34 RNA MTases, 32 protein MTases, eight small molecule MTases, three lipid MTases, and nine MTases with still unknown substrate specificity.
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Ju, Xiaohui; Bowden, Mark E.; Engelhard, Mark H.
Three commercial cellulase preparations, Novozymes Cellic® Ctec2, Dupont Accellerase® 1500, and DSM Cytolase CL, were evaluated for their hydrolytic activity using a set of reference biomass substrates with controlled substrate characteristics. It was found that lignin remains a significant recalcitrance factor to all the preparations, although different enzyme preparations respond to the inhibitory effect of lignin differently. Also, different types of biomass lignin can inhibit cellulose enzymes in different manners. Enhancing enzyme activity toward biomass fiber swelling is an area significantly contributing to potential improvement in cellulose performance. While the degree of polymerization of cellulose in the reference substrates didmore » not present a major recalcitrance factor to Novozymes Cellic® Ctec2, cellulose crystallite has been shown to have a significant lower reactivity toward all enzyme mixtures. The presence of polysaccharide monooxygenases (PMOs) in Novozymes Ctec2 appears to enhance enzyme activity toward decrystallization of cellulose. This study demonstrated that reference substrates with controlled chemical and physical characteristics of structural features can be applied as an effective and practical strategy to identify cellulosic enzyme activities toward specific biomass recalcitrance factor(s) and provide specific targets for enzyme improvement.« less
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Specific Inhibition of β-Secretase Processing of the Alzheimer Disease Amyloid Precursor Protein.
Ben Halima, Saoussen; Mishra, Sabyashachi; Raja, K Muruga Poopathi; Willem, Michael; Baici, Antonio; Simons, Kai; Brüstle, Oliver; Koch, Philipp; Haass, Christian; Caflisch, Amedeo; Rajendran, Lawrence
2016-03-08
Development of disease-modifying therapeutics is urgently needed for treating Alzheimer disease (AD). AD is characterized by toxic β-amyloid (Aβ) peptides produced by β- and γ-secretase-mediated cleavage of the amyloid precursor protein (APP). β-secretase inhibitors reduce Aβ levels, but mechanism-based side effects arise because they also inhibit β-cleavage of non-amyloid substrates like Neuregulin. We report that β-secretase has a higher affinity for Neuregulin than it does for APP. Kinetic studies demonstrate that the affinities and catalytic efficiencies of β-secretase are higher toward non-amyloid substrates than toward APP. We show that non-amyloid substrates are processed by β-secretase in an endocytosis-independent manner. Exploiting this compartmentalization of substrates, we specifically target the endosomal β-secretase by an endosomally targeted β-secretase inhibitor, which blocked cleavage of APP but not non-amyloid substrates in many cell systems, including induced pluripotent stem cell (iPSC)-derived neurons. β-secretase inhibitors can be designed to specifically inhibit the Alzheimer process, enhancing their potential as AD therapeutics without undesired side effects. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.
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Different substrates and starter inocula govern microbial community structures in biogas reactors.
Satpathy, Preseela; Steinigeweg, Sven; Cypionka, Heribert; Engelen, Bert
2016-01-01
The influence of different starter inocula on the microbial communities in biogas batch reactors fed with fresh maize and maize silage as substrates was investigated. Molecular biological analysis by Denaturing Gradient Gel Electrophoresis (DGGE) of 16S rRNA gene fragments showed that each inoculum bore specific microbial communities with varying predominant phylotypes. Both, bacterial and archaeal DGGE profiles displayed three distinct communities that developed depending on the type of inoculum. Although maize and silage are similar substrates, different communities dominated the lactate-rich silage compared to lactate-free fresh maize. Cluster analysis of DGGE gels showed the communities of the same substrates to be stable with their respective inoculum. Bacteria-specific DGGE analysis revealed a rich diversity with Firmicutes being predominant. The other abundant phylotypes were Bacteroidetes and Synergistetes. Archaea-specific DGGE analysis displayed less diverse community structures, identifying members of the Methanosarcinales as the dominant methanogens present in all the three biogas digesters. In general, the source of inoculum played a significant role in shaping microbial communities. Adaptability of the inoculum to the substrates fed also influenced community compositions which further impacted the rates of biogas production.
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Roles of s3 site residues of nattokinase on its activity and substrate specificity.
Wu, Shuming; Feng, Chi; Zhong, Jin; Huan, Liandong
2007-09-01
Nattokinase (Subtilisin NAT, NK) is a bacterial serine protease with high fibrinolytic activity. To probe their roles on protease activity and substrate specificity, three residues of S3 site (Gly(100), Ser(101) and Leu(126)) were mutated by site-directed mutagenesis. Kinetics parameters of 20 mutants were measured using tetrapeptides as substrates, and their fibrinolytic activities were determined by fibrin plate method. Results of mutation analysis showed that Gly(100) and Ser(101) had reverse steric and electrostatic effects. Residues with bulky or positively charged side chains at position 100 decreased the substrate binding and catalytic activity drastically, while residues with the same characters at position 101 could obviously enhance protease and fibrinolytic activity of NK. Mutation of Leu(126) might impair the structure of the active cleft and drastically decreased the activity of NK. Kinetics studies of the mutants showed that S3 residues were crucial to keep protease activity while they moderately affected substrate specificity of NK. The present study provided some original insight into the P3-S3 interaction in NK and other subtilisins, as well as showed successful protein engineering cases to improve NK as a potential therapeutic agent.
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Ju, Xiaohui; Bowden, Mark; Engelhard, Mark; Zhang, Xiao
2014-05-01
Three commercial cellulase preparations, Novozymes Cellic(®) Ctec2, Dupont Accellerase(®) 1500, and DSM Cytolase CL, were evaluated for their hydrolytic activity using a set of reference biomass substrates with controlled substrate characteristics. It was found that lignin remains a significant recalcitrance factor to all the preparations, although different enzyme preparations respond to the inhibitory effect of lignin differently. Also, different types of biomass lignin can inhibit cellulase enzymes in different manners. Enhancing enzyme activity toward biomass fiber swelling is an area significantly contributing to potential improvement in cellulase performance. While the degree of polymerization of cellulose in the reference substrates did not present a major recalcitrance factor to Novozymes Cellic(®) Ctec2, cellulose crystallite has been shown to have a significant lower reactivity toward all enzyme mixtures. The presence of polysaccharide monooxygenases (PMOs) in Novozymes Ctec2 appears to enhance enzyme activity toward decrystallization of cellulose. This study demonstrated that reference substrates with controlled chemical and physical characteristics of structural features can be applied as an effective and practical strategy to identify cellulosic enzyme activities toward specific biomass recalcitrance factor(s) and provide specific targets for enzyme improvement.
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De Baetselier, P; Hamers-Casterman, C; Van der Loo, W; Hamers, R
1977-01-01
Rabbits capable of producing antibodies of restricted heterogeneity in response to Micrococcus lysodeikticus are equally capable of producing antibodies of restricted heterogeneity to bovine serum albumin. These antibodies are produced when animals are simultaneously injected with micrococcus and BSA and their specificity is restricted to a small number of epitopes. These results suggest that micrococcal vaccines can induce the restriction of heterogeneity in antibodies raised against totally unrelated antigens. Images Figure 4 Figure 5 Figure 2 Figure 6 Figure 7 Figure 8 PMID:71263
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Haack, S.K.; Garchow, H.; Klug, M.J.; Forney, L.J.
1995-01-01
We determined factors that affect responses of bacterial isolates and model bacterial communities to the 95 carbon substrates in Biolog microliter plates. For isolates and communities of three to six bacterial strains, substrate oxidation rates were typically nonlinear and were delayed by dilution of the inoculum. When inoculum density was controlled, patterns of positive and negative responses exhibited by microbial communities to each of the carbon sources were reproducible. Rates and extents of substrate oxidation by the communities were also reproducible but were not simply the sum of those exhibited by community members when tested separately. Replicates of the same model community clustered when analyzed by principal- components analysis (PCA), and model communities with different compositions were clearly separated un the first PCA axis, which accounted for >60% of the dataset variation. PCA discrimination among different model communities depended on the extent to which specific substrates were oxidized. However, the substrates interpreted by PCA to be most significant in distinguishing the communities changed with reading time, reflecting the nonlinearity of substrate oxidation rates. Although whole-community substrate utilization profiles were reproducible signatures for a given community, the extent of oxidation of specific substrates and the numbers or activities of microorganisms using those substrates in a given community were not correlated. Replicate soil samples varied significantly in the rate and extent of oxidation of seven tested substrates, suggesting microscale heterogeneity in composition of the soil microbial community.
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Dudek, Hanna M.; de Gonzalo, Gonzalo; Torres Pazmiño, Daniel E.; Stępniak, Piotr; Wyrwicz, Lucjan S.; Rychlewski, Leszek; Fraaije, Marco W.
2011-01-01
Baeyer-Villiger monooxygenases catalyze oxidations that are of interest for biocatalytic applications. Among these enzymes, phenylacetone monooxygenase (PAMO) from Thermobifida fusca is the only protein showing remarkable stability. While related enzymes often present a broad substrate scope, PAMO accepts only a limited number of substrates. Due to the absence of a substrate in the elucidated crystal structure of PAMO, the substrate binding site of this protein has not yet been defined. In this study, a structural model of cyclopentanone monooxygenase, which acts on a broad range of compounds, has been prepared and compared with the structure of PAMO. This revealed 15 amino acid positions in the active site of PAMO that may account for its relatively narrow substrate specificity. We designed and analyzed 30 single and multiple mutants in order to verify the role of these positions. Extensive substrate screening revealed several mutants that displayed increased activity and altered regio- or enantioselectivity in Baeyer-Villiger reactions and sulfoxidations. Further substrate profiling resulted in the identification of mutants with improved catalytic properties toward synthetically attractive compounds. Moreover, the thermostability of the mutants was not compromised in comparison to that of the wild-type enzyme. Our data demonstrate that the positions identified within the active site of PAMO, namely, V54, I67, Q152, and A435, contribute to the substrate specificity of this enzyme. These findings will aid in more dedicated and effective redesign of PAMO and related monooxygenases toward an expanded substrate scope. PMID:21724896
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Zimanyi, Christina M; Chen, Percival Yang-Ting; Kang, Gyunghoon; Funk, Michael A; Drennan, Catherine L
2016-01-01
Ribonucleotide reductase (RNR) converts ribonucleotides to deoxyribonucleotides, a reaction that is essential for DNA biosynthesis and repair. This enzyme is responsible for reducing all four ribonucleotide substrates, with specificity regulated by the binding of an effector to a distal allosteric site. In all characterized RNRs, the binding of effector dATP alters the active site to select for pyrimidines over purines, whereas effectors dGTP and TTP select for substrates ADP and GDP, respectively. Here, we have determined structures of Escherichia coli class Ia RNR with all four substrate/specificity effector-pairs bound (CDP/dATP, UDP/dATP, ADP/dGTP, GDP/TTP) that reveal the conformational rearrangements responsible for this remarkable allostery. These structures delineate how RNR ‘reads’ the base of each effector and communicates substrate preference to the active site by forming differential hydrogen bonds, thereby maintaining the proper balance of deoxynucleotides in the cell. DOI: http://dx.doi.org/10.7554/eLife.07141.001 PMID:26754917
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Wilusz, J; Shenk, T; Takagaki, Y; Manley, J L
1990-01-01
A 64-kilodalton (kDa) polypeptide that is cross-linked by UV light specifically to polyadenylation substrate RNAs containing a functional AAUAAA element has been identified previously. Fractionated HeLa nuclear components that can be combined to regenerate efficient and accurate polyadenylation in vitro have now been screened for the presence of the 64-kDa protein. None of the individual components contained an activity which could generate the 64-kDa species upon UV cross-linking in the presence of substrate RNA. It was necessary to mix two components, cleavage stimulation factor and specificity factor, to reconstitute 64-kDa protein-RNA cross-linking. The addition of cleavage factors to this mixture very efficiently reconstituted the AAUAAA-specific 64-kDa protein-RNA interaction. The 64-kDa protein, therefore, is present in highly purified, reconstituted polyadenylation reactions. However, it is necessary to form a multicomponent complex to efficiently cross-link the protein to a substrate RNA. Images PMID:2304466
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Aich, Sanjukta; Prasad, Lata; Delbaere, Louis T.J.
GTP-dependent phosphoenolpyruvate carboxykinase (PCK) is the key enzyme that controls the blood glucose level during fasting in higher animals. Here we report the first substrate-free structure of a GTP-dependent phosphoenolpyruvate (PEP) carboxykinase from a bacterium, Corynebacterium glutamicum (CgPCK). The protein crystallizes in space group P2{sub 1} with four molecules per asymmetric unit. The 2.3 {angstrom} resolution structure was solved by molecular replacement using the human cytosolic PCK (hcPCK) structure (PDB ID: 1KHF) as the starting model. The four molecules in the asymmetric unit pack as two dimers, and is an artifact of crystal packing. However, the P-loop and the guaninemore » binding loop of the substrate-free CgPCK structure have different conformations from the other published GTP-specific PCK structures, which all have bound substrates and/or metal ions. It appears that a change in the P-loop and guanine binding loop conformation is necessary for substrate binding in GTP-specific PCKs, as opposed to overall domain movement in ATP-specific PCKs.« less
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Butterfield, Cristina N; Tebo, Bradley M
2017-02-22
Manganese(ii) oxidation in the environment is thought to be driven by bacteria because enzymatic catalysis is many orders of magnitude faster than the abiotic processes. The heterologously purified Mn oxidase (Mnx) from marine Bacillus sp. PL-12 is made up of the multicopper oxidase (MCO) MnxG and two small Cu and heme-binding proteins of unknown function, MnxE and MnxF. Mnx binds Cu and oxidizes both Mn(ii) and Mn(iii), generating Mn(iv) oxide minerals that resemble those found on the Bacillus spore surface. Spectroscopic techniques have illuminated details about the metallo-cofactors of Mnx, but very little is known about their requirement for catalytic activity, and even less is known about the substrate specificity of Mnx. Here we quantify the canonical MCO Cu and persistent peripheral Cu bound to Mnx, and test Mnx oxidizing ability toward different substrates at varying pH. Mn(ii) appears to be the best substrate in terms of k cat , but its oxidation does not follow Michaelis-Menten kinetics, instead showing a sigmoidal cooperative behavior. Mnx also oxidizes Fe(ii) substrate, but in a Michaelis-Menten manner and with a decreased activity, as well as organic substrates. The reduced metals are more rapidly consumed than the larger organic substrates, suggesting the hypothesis that the Mnx substrate site is small and tuned for metal oxidation. Of biological relevance is the result that Mnx has the highest catalytic efficiency for Mn(ii) at the pH of sea water, especially when the protein is loaded with greater than the requisite four MCO copper atoms, suggesting that the protein has evolved specifically for Mn oxidation.
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Malyavantham, Kishore S.; Suresh, Lakshmanan
2018-01-01
Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies available for ANA screening, indirect immunofluorescence (IIF) using Human epithelial cells-2 (HEp-2) substrate remains the primary and recommended method because of its superior sensitivity. HEp-2 substrates can detect a multitude of patterns resulting from autoantibody binding to various protein and nucleic acid autoantigens distributed throughout the nucleus and cytoplasm of the cells. The great diversity of monospecific and mixed patterns resulting from positive reactions on HEp-2 substrate also complicate the interpretation and accuracy of reporting. One specific example which received utmost attention recently is the dense fine speckled 70 (DFS70) pattern resulting from autoantibodies that specifically bind to a protein called lens epithelium derived growth factor (LEDGF). Lack of clear association with a specific systemic autoimmune disease and high prevalence in healthy populations have made accurate interpretation of DFS70 pattern important. Accurate distinction of DFS70 pattern from disease-associated patterns using conventional HEp-2 substrate is challenging. Moreover, frequent co-occurrence of DFS70 pattern along with disease-associated patterns such as homogeneous, speckled, and mixed homogeneous-speckled patterns complicate the IIF interpretation. The goal of this paper is to demonstrate the utility of a novel engineered HEp-2 IIF substrate that retains all advantages of conventional HEp-2 substrate while simultaneously providing the ability to distinguish DFS70 pattern with high confidence in both monospecific and mixed ANA positive examples. The new substrate is further able to unmask disease-associated ANA patterns previously concealed by DFS70 pattern. PMID:29364249
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Yu, S; Ahmad, T; Kenne, L; Pedersén, M
1995-05-11
The alpha-1,4-glucan lyase (EC 4.2.2.-), purified from the red alga Gracilariopsis lemaneiformis, is a single polypeptide with a molecular mass of 116,654 Da as determined by matrix-assisted laser-desorption mass spectrometry. It degraded maltose, maltosaccharides, amylose, amylopectin and glycogen, forming 1,5-anhydro-D-fructose from the non-reducing end groups. The substrate specificity, mode of action, and cleavage mechanism of the enzyme were studied by using various naturally occurring and synthesized substrates. This enzyme was highly specific for the alpha-1,4-D-glucosidic bond. When a linear alpha-1,4-glucan was used as substrate, the enzyme split the substrate from the non-reducing end and released 1,5-anhydro-D-fructose successively until only one glucose unit was left. When a branched pentasaccharide of 6(2)-alpha-maltosylmaltotriose, obtained from glycogen by alpha-amylase limitation, was used as substrate, the glucose group in the 4-position of the 4,6-branched residue was not cleaved off. Using maltoheptaose as substrate and following the reaction with HPLC and 1H-NMR spectroscopy, it was found that the action mode of the lyase followed a multichain attack mechanism. 1H- and 13C-NMR spectroscopic studies on unlabelled and labelled amylose (1-2H, 2-2H, 1-13C) as substrates indicated that the lyase cleaved the C-(1')-O(4) bond forming a double bond between C-1' and C-2', thus forming the enol form of 1,5-anhydro-D-fructose. It also indicated that the catalytic process of the lyase involved proton exchanges among C-1, C-2, C-3 and the solvent.
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Matzel, Philipp; Krautschick, Lukas; Höhne, Matthias
2017-10-18
Imine reductases (IREDs) have emerged as promising enzymes for the asymmetric synthesis of secondary and tertiary amines starting from carbonyl substrates. Screening the substrate specificity of the reductive amination reaction is usually performed by time-consuming GC analytics. We found two highly active IREDs in our enzyme collection, IR-20 from Streptomyces tsukubaensis and IR-Sip from Streptomyces ipomoeae, that allowed a comprehensive substrate screening with a photometric NADPH assay. We screened 39 carbonyl substrates combined with 17 amines as nucleophiles. Activity data from 663 combinations provided a clear picture about substrate specificity and capabilities in the reductive amination of these enzymes. Besides aliphatic aldehydes, the IREDs accepted various cyclic (C 4 -C 8 ) and acyclic ketones, preferentially with methylamine. IR-Sip also accepted a range of primary and secondary amines as nucleophiles. In biocatalytic reactions, IR-Sip converted (R)-3-methylcyclohexanone with dimethylamine or pyrrolidine with high diastereoselectivity (>94-96 % de). The nucleophile acceptor spectrum depended on the carbonyl substrate employed. The conversion of well-accepted substrates could also be detected if crude lysates were employed as the enzyme source. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
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NASA Astrophysics Data System (ADS)
Kromer, R.; Danlos, Y.; Costil, S.
2018-04-01
Cold spraying enables a variety of metals dense coatings onto metal surfaces. Supersonic gas jet accelerates particles which undergo with the substrate plastic deformation. Different bonding mechanisms can be created depending on the materials. The particle-substrate contact time, contact temperature and contact area upon impact are the parameters influencing physicochemical and mechanical bonds. The resultant bonding arose from plastic deformation of the particle and substrate and temperature increasing at the interface. The objective was to create specific topography to enable metallic particle adhesion onto ceramic substrates. Ceramic did not demonstrate deformation during the impact which minimized the intimate bonds. Laser surface texturing was hence used as prior surface treatment to create specific topography and to enable mechanical anchoring. Particle compressive states were necessary to build up coating. The coating deposition efficiency and adhesion strength were evaluated. Textured surface is required to obtain strong adhesion of metallic coatings onto ceramic substrates. Consequently, cold spray coating parameters depend on the target material and a methodology was established with particle parameters (diameters, velocities, temperatures) and particle/substrate properties to adapt the surface topography. Laser surface texturing is a promising tool to increase the cold spraying applications.
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Baldauf, Hanna-Mari; Stegmann, Lena; Schwarz, Sarah-Marie; Ambiel, Ina; Trotard, Maud; Martin, Margarethe; Burggraf, Manja; Lenzi, Gina M; Lejk, Helena; Pan, Xiaoyu; Fregoso, Oliver I; Lim, Efrem S; Abraham, Libin; Nguyen, Laura A; Rutsch, Frank; König, Renate; Kim, Baek; Emerman, Michael; Fackler, Oliver T; Keppler, Oliver T
2017-03-07
Early after entry into monocytes, macrophages, dendritic cells, and resting CD4 T cells, HIV encounters a block, limiting reverse transcription (RT) of the incoming viral RNA genome. In this context, dNTP triphosphohydrolase SAM domain and HD domain-containing protein 1 (SAMHD1) has been identified as a restriction factor, lowering the concentration of dNTP substrates to limit RT. The accessory lentiviral protein X (Vpx) proteins from the major simian immunodeficiency virus of rhesus macaque, sooty mangabey, and HIV-2 (SIVsmm/SIVmac/HIV-2) lineage packaged into virions target SAMHD1 for proteasomal degradation, increase intracellular dNTP pools, and facilitate HIV cDNA synthesis. We find that virion-packaged Vpx proteins from a second SIV lineage, SIV of red-capped mangabeys or mandrills (SIVrcm/mnd-2), increased HIV infection in resting CD4 T cells, but not in macrophages, and, unexpectedly, acted in the absence of SAMHD1 degradation, dNTP pool elevation, or changes in SAMHD1 phosphorylation. Vpx rcm/mnd-2 virion incorporation resulted in a dramatic increase of HIV-1 RT intermediates and viral cDNA in infected resting CD4 T cells. These analyses also revealed a barrier limiting HIV-1 infection of resting CD4 T cells at the level of nuclear import. Single amino acid changes in the SAMHD1-degrading Vpx mac239 allowed it to enhance early postentry steps in a Vpx rcm/mnd-2-like fashion. Moreover, Vpx enhanced HIV-1 infection of SAMHD1-deficient resting CD4 T cells of a patient with Aicardi-Goutières syndrome. These results indicate that Vpx, in addition to SAMHD1, overcomes a previously unappreciated restriction for lentiviruses at the level of RT that acts independently of dNTP concentrations and is specific to resting CD4 T cells.
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Horz, Hans-Peter; Yimga, Merlin Tchawa; Liesack, Werner
2001-01-01
The diversity of methanotrophic bacteria associated with roots of submerged rice plants was assessed using cultivation-independent techniques. The research focused mainly on the retrieval of pmoA, which encodes the α subunit of the particulate methane monooxygenase. A novel methanotroph-specific community-profiling method was established using the terminal restriction fragment length polymorphism (T-RFLP) technique. The T-RFLP profiles clearly revealed a more complex root-associated methanotrophic community than did banding patterns obtained by pmoA-based denaturing gradient gel electrophoresis. The comparison of pmoA-based T-RFLP profiles obtained from rice roots and bulk soil of flooded rice microcosms suggested that there was a substantially higher abundance of type I methanotrophs on rice roots than in the bulk soil. These were affiliated to the genera Methylomonas, Methylobacter, Methylococcus, and to a novel type I methanotroph sublineage. By contrast, type II methanotrophs of the Methylocystis-Methylosinus group could be detected with high relative signal intensity in both soil and root compartments. Phylogenetic treeing analyses and a set of substrate-diagnostic amino acid residues provided evidence that a novel pmoA lineage was detected. This branched distinctly from all currently known methanotrophs. To examine whether the retrieval of pmoA provided a complete view of root-associated methanotroph diversity, we also assessed the diversity detectable by recovery of genes coding for subunits of soluble methane monooxygenase (mmoX) and methanol dehydrogenase (mxaF). In addition, both 16S rRNA and 16S ribosomal DNA (rDNA) were retrieved using a PCR primer set specific to type I methanotrophs. The overall methanotroph diversity detected by recovery of mmoX, mxaF, and 16S rRNA and 16S rDNA corresponded well to the diversity detectable by retrieval of pmoA. PMID:11526021
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Cahoon, Edgar B.; Shanklin, John; Lindqvist, Ylva; Schneider, Gunter
1999-03-30
Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity.
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Cahoon, Edgar B.; Shanklin, John; Lindgvist, Ylva; Schneider, Gunter
1998-01-06
Disclosed is a methods for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity.
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Chuah, Jo-Ann; Tomizawa, Satoshi; Yamada, Miwa; Tsuge, Takeharu; Doi, Yoshiharu
2013-01-01
Saturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase from Chromobacterium sp. strain USM2 (PhaCCs) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaCCs for 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity. In vitro activities for polymerization of 3HV and 3HHx monomers were consistent with in vivo substrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C4 and C5) or MCL (C6) substrates substantiates the fundamental classification of PHA synthases. PMID:23584780
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Exosites in the substrate specificity of blood coagulation reactions.
Bock, P E; Panizzi, P; Verhamme, I M A
2007-07-01
The specificity of blood coagulation proteinases for substrate, inhibitor, and effector recognition is mediated by exosites on the surfaces of the catalytic domains, physically separated from the catalytic site. Some thrombin ligands bind specifically to either exosite I or II, while others engage both exosites. The involvement of different, overlapping constellations of exosite residues enables binding of structurally diverse ligands. The flexibility of the thrombin structure is central to the mechanism of complex formation and the specificity of exosite interactions. Encounter complex formation is driven by electrostatic ligand-exosite interactions, followed by conformational rearrangement to a stable complex. Exosites on some zymogens are in low affinity proexosite states and are expressed concomitant with catalytic site activation. The requirement for exosite expression controls the specificity of assembly of catalytic complexes on the coagulation pathway, such as the membrane-bound factor Xa*factor Va (prothrombinase) complex, and prevents premature assembly. Substrate recognition by prothrombinase involves a two-step mechanism with initial docking of prothrombin to exosites, followed by a conformational change to engage the FXa catalytic site. Prothrombin and its activation intermediates bind prothrombinase in two alternative conformations determined by the zymogen to proteinase transition that are hypothesized to involve prothrombin (pro)exosite I interactions with FVa, which underpin the sequential activation pathway. The role of exosites as the major source of substrate specificity has stimulated development of exosite-targeted anticoagulants for treatment of thrombosis.
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Cofactor-dependent specificity of a DEAD-box protein.
Young, Crystal L; Khoshnevis, Sohail; Karbstein, Katrin
2013-07-16
DEAD-box proteins, a large class of RNA-dependent ATPases, regulate all aspects of gene expression and RNA metabolism. They can facilitate dissociation of RNA duplexes and remodeling of RNA-protein complexes, serve as ATP-dependent RNA-binding proteins, or even anneal duplexes. These proteins have highly conserved sequence elements that are contained within two RecA-like domains; consequently, their structures are nearly identical. Furthermore, crystal structures of DEAD-box proteins with bound RNA reveal interactions exclusively between the protein and the RNA backbone. Together, these findings suggest that DEAD-box proteins interact with their substrates in a nonspecific manner, which is confirmed in biochemical experiments. Nevertheless, this contrasts with the need to target these enzymes to specific substrates in vivo. Using the DEAD-box protein Rok1 and its cofactor Rrp5, which both function during maturation of the small ribosomal subunit, we show here that Rrp5 provides specificity to the otherwise nonspecific biochemical activities of the Rok1 DEAD-domain. This finding could reconcile the need for specific substrate binding of some DEAD-box proteins with their nonspecific binding surface and expands the potential roles of cofactors to specificity factors. Identification of helicase cofactors and their RNA substrates could therefore help define the undescribed roles of the 19 DEAD-box proteins that function in ribosome assembly.
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Olkowski, A A; Nain, S; Wojnarowicz, C; Laarveld, B; Alcorn, J; Ling, B B
2007-09-01
In order to explain the biochemical mechanisms associated with deteriorating heart function in broiler chickens, this study compared myocardial high energy phosphate substrates in leghorns, feed restricted (Broilers-Res) broilers, ad libitum fed broilers (Broilers-AL), and in broilers that developed heart failure and ascites. The profile of adenine nucleotide content in the heart tissue did not differ between leghorns and Broilers-Res, but there were significant differences among Broilers-Res, Broilers-AL, and broilers with ascites. During intensive growth periods, leghorns and Broilers-Res showed increasing trends in heart ATP levels, whereas in fast growing broilers the heart ATP declined (p<0.021). ATP:ADP and ATP:CrP ratios increased with age in both leghorn and Broilers-Res, declined in fast growing broilers, and were the lowest in broilers that developed heart failure. The changes in heart high energy phosphate profile in broilers suggest that the energy demand of the heart during a rapid growth phase may exceed the bird's metabolic capacity to supply adequate levels of high energy phosphate substrate. The insufficiency of energy substrate likely contributes to the declining heart rate. In some individuals this may lead to impaired heart pump function, and in more severe cases may progress to heart pump failure.
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Stabilization of magnetic skyrmions by RKKY interactions
NASA Astrophysics Data System (ADS)
Bezvershenko, Alla V.; Kolezhuk, Alexei K.; Ivanov, Boris A.
2018-02-01
We study the stabilization of an isolated magnetic skyrmion in a magnetic monolayer on a nonmagnetic conducting substrate via the Ruderman-Kittel-Kasuya-Yosida (RKKY) exchange interaction. Two different types of the substrate are considered, usual normal metal and single-layer graphene. While the full stability analysis for skyrmions in the presence of the RKKY coupling requires a separate effort that is outside the scope of this work, we are able to study the radial stability (stability of a skyrmion against collapse) using variational energy estimates obtained within first-order perturbation theory, with the unperturbed Hamiltonian describing the isotropic Heisenberg magnet, and the two perturbations being the RKKY exchange and the easy-axis anisotropy. We show that a proper treatment of the long-range nature of the RKKY interaction leads to a qualitatively different stabilization scenario compared to previous studies, where solitons were stabilized by the frustrated exchange coupling (leading to terms with the fourth power of the magnetization gradients) or by the Dzyaloshinskii-Moriya interaction (described by terms linear in the magnetization gradients). In the case of a metallic substrate, the skyrmion stabilization is possible under restrictive conditions on the Fermi surface parameters, while in the case of a graphene substrate the stabilization is naturally achieved in several geometries with a lattice-matching of graphene and magnetic layer.
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Tucci, Sara; Herebian, Diran; Sturm, Marga; Seibt, Annette; Spiekerkoetter, Ute
2012-01-01
Very long-chain acyl-CoA dehydrogenase (VLCAD)-deficiency is the most common long-chain fatty acid oxidation disorder presenting with heterogeneous phenotypes. Similar to many patients with VLCADD, VLCAD-deficient mice (VLCAD(-/-)) remain asymptomatic over a long period of time. In order to identify the involved compensatory mechanisms, wild-type and VLCAD(-/-) mice were fed one year either with a normal diet or with a diet in which medium-chain triglycerides (MCT) replaced long-chain triglycerides, as approved intervention in VLCADD. The expression of the mitochondrial long-chain acyl-CoA dehydrogenase (LCAD) and medium-chain acyl-CoA dehydrogenase (MCAD) was quantified at mRNA and protein level in heart, liver and skeletal muscle. The oxidation capacity of the different tissues was measured by LC-MS/MS using acyl-CoA substrates with a chain length of 8 to 20 carbons. Moreover, in white skeletal muscle the role of glycolysis and concomitant muscle fibre adaptation was investigated. In one year old VLCAD(-/-) mice MCAD and LCAD play an important role in order to compensate deficiency of VLCAD especially in the heart and in the liver. However, the white gastrocnemius muscle develops alternative compensatory mechanism based on a different substrate selection and increased glucose oxidation. Finally, the application of an MCT diet over one year has no effects on LCAD or MCAD expression. MCT results in the VLCAD(-/-) mice only in a very modest improvement of medium-chain acyl-CoA oxidation capacity restricted to cardiac tissue. In conclusion, VLCAD(-/-) mice develop tissue-specific strategies to compensate deficiency of VLCAD either by induction of other mitochondrial acyl-CoA dehydrogenases or by enhancement of glucose oxidation. In the muscle, there is evidence of a muscle fibre type adaptation with a predominance of glycolytic muscle fibres. Dietary modification as represented by an MCT-diet does not improve these strategies long-term.
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Li de La Sierra, I M; Gallay, J; Vincent, M; Bertrand, T; Briozzo, P; Bârzu, O; Gilles, A M
2000-12-26
The conformation and dynamics of the ATP binding site of cytidine monophosphate kinase from Escherichia coli (CMPK(coli)), which catalyzes specifically the phosphate exchange between ATP and CMP, was studied using the fluorescence properties of 3'-anthraniloyl-2'-deoxy-ADP, a specific ligand of the enzyme. The spectroscopic properties of the bound fluorescent nucleotide change strongly with respect to those in aqueous solution. These changes (red shift of the absorption and excitation spectra, large increase of the excited state lifetime) are compared to those observed in different solvents. These data, as well as acrylamide quenching experiments, suggest that the anthraniloyl moiety is protected from the aqueous solvent upon binding to the ATP binding site, irrespective of the presence of CMP or CDP. The protein-bound ADP analogue exhibits a restricted fast subnanosecond rotational motion, completely blocked by CMP binding. The energy-minimized models of CMPK(coli) complexed with 3'-anthraniloyl-2'-deoxy-ADP using the crystal structures of the ligand-free protein and of its complex with CDP (PDB codes and, respectively) were compared to the crystal structure of UMP/CMP kinase from Dictyostelium discoideum complexed with substrates (PDB code ). The key residues for ATP/ADP binding to CMPK(coli) were identified as R157 and I209, their side chains sandwiching the adenine ring. Moreover, the residues involved in the fixation of the phosphate groups are conserved in both proteins. In the model, the accessibility of the fluorescent ring to the solvent should be substantial if the LID conformation remained unchanged, by contrast to the fluorescence data. These results provide the first experimental arguments about an ATP-mediated induced-fit of the LID in CMPK(coli) modulated by CMP, leading to a closed conformation of the active site, protected from water.
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Helms, S. R.; Brattain, M. G.; Pretlow, T. G.; Kreisberg, J. I.
1977-01-01
Numerous investigators have found several substrates and inhibitors to be particularly suited for the demonstration of acid phosphatase of prostatic origin. There has been much controversy over the specificity or lack of specificity of several substrates and inhibitors. We have investigated acid phosphatase activities obtained from several kinds of purified cells. None of the substrates or inhibitors which we studied permitted us to discriminate "prostatic" acid phosphatase from acid phosphatase activities obtained from other kinds of cells. PMID:560800
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NASA Astrophysics Data System (ADS)
Woolley, Adam T.; Mathies, Richard A.
1994-11-01
Capillary electrophoresis arrays have been fabricated on planar glass substrates by photolithographic masking and chemical etching techniques. The photolithographically defined channel patterns were etched in a glass substrate, and then capillaries were formed by thermally bonding the etched substrate to a second glass slide. High-resolution electrophoretic separations of φX174 Hae III DNA restriction fragments have been performed with these chips using a hydroxyethyl cellulose sieving matrix in the channels. DNA fragments were fluorescently labeled with dye in the running buffer and detected with a laser-excited, confocal fluorescence system. The effects of variations in the electric field, procedures for injection, and sizes of separation and injection channels (ranging from 30 to 120 μm) have been explored. By use of channels with an effective length of only 3.5 cm, separations of φX174 Hae III DNA fragments from ≈70 to 1000 bp are complete in only 120 sec. We have also demonstrated high-speed sizing of PCR-amplified HLA-DQα alleles. This work establishes methods for high-speed, high-throughput DNA separations on capillary array electrophoresis chips.
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Interactions between Soil Habitat and Geographic Range Location Affect Plant Fitness
Stanton-Geddes, John; Shaw, Ruth G.; Tiffin, Peter
2012-01-01
Populations are often found on different habitats at different geographic locations. This habitat shift may be due to biased dispersal, physiological tolerances or biotic interactions. To explore how fitness of the native plant Chamaecrista fasciculata depends on habitat within, at and beyond its range edge, we planted seeds from five populations in two soil substrates at these geographic locations. We found that with reduced competition, lifetime fitness was always greater or equivalent in one habitat type, loam soils, though early-season survival was greater on sand soils. At the range edge, natural populations are typically found on sand soil habitats, which are also less competitive environments. Early-season survival and fitness differed among source populations, and when transplanted beyond the range edge, range edge populations had greater fitness than interior populations. Our results indicate that even when the optimal soil substrate for a species does not change with geographic range location, the realized niche of a species may be restricted to sub-optimal habitats at the range edge because of the combined effects of differences in abiotic and biotic effects (e.g. competitors) between substrates. PMID:22615745
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The effect of dissipation on the resistive admittance of an elastic medium.
Photiadis, Douglas M
2012-03-01
The effect of dissipation on the real part of the admittance of an elastic half-space is typically thought to be unimportant if the loss factor ζ of the elastic medium is small. However, dissipation induces losses in the near field of the source and, provided the size of the source is small enough, this phenomenon can be more important than elastic wave radiation. Such losses give rise to a fundamental limit in the quality factor of an oscillator attached to a substrate. Near field losses associated with strains in the elastic substrate can actually be larger than intrinsic losses in the oscillator itself if the internal friction of the substrate is larger than the internal friction of the oscillator. For a uniform stress applied to a disk of radius a, a monopole source, such phenomena become significant for k(L)a<ζ, while for higher order multipole sources of order l, near field losses are important for (k(L)a)(l+1)<ζ, a far less restrictive constraint. © 2012 Acoustical Society of America
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Probes of the catalytic site of cysteine dioxygenase.
Chai, Sergio C; Bruyere, John R; Maroney, Michael J
2006-06-09
The first major step of cysteine catabolism, the oxidation of cysteine to cysteine sulfinic acid, is catalyzed by cysteine dioxygenase (CDO). In the present work, we utilize recombinant rat liver CDO and cysteine derivatives to elucidate structural parameters involved in substrate recognition and x-ray absorption spectroscopy to probe the interaction of the active site iron center with cysteine. Kinetic studies using cysteine structural analogs show that most are inhibitors and that a terminal functional group bearing a negative charge (e.g. a carboxylate) is required for binding. The substrate-binding site has no stringent restrictions with respect to the size of the amino acid. Lack of the amino or carboxyl groups at the alpha-carbon does not prevent the molecules from interacting with the active site. In fact, cysteamine is shown to be a potent activator of the enzyme without being a substrate. CDO was also rendered inactive upon complexation with the metal-binding inhibitors azide and cyanide. Unlike many non-heme iron dioxygenases that employ alpha-keto acids as cofactors, CDO was shown to be the only dioxygenase known to be inhibited by alpha-ketoglutarate.
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NASA Astrophysics Data System (ADS)
El-Sorogy, Abdelbaset S.; Alharbi, Talal; Richiano, Sebastián
2018-05-01
Salinity is one the major stress factors that controls the biotic activities in marine environments. In general, the mixture with fresh-water has been mention as a great stress factor, but the opposite, i.e. high-salinity conditions, is less developed in the ichnological literature. Along the Al-Khafji coastline, Saudi Arabia, hard substrates (constituted by gastropods, bivalves and coral skeletons) contain diverse and abundant bioerosion traces and associated encrusters. Field and laboratory observations allowed the recognition of eight ichnospecies belong to the ichnogenera Gastrochaenolites, Entobia, Oichnus, Caulostrepsis and Trypanites, which can be attributed to various activities produced by bivalves, sponges, gastropods and annelids. The borings demonstrate two notable ichnological boring assemblages, namely, Entobia-dominated and Gastrochaenolites-dominated assemblages. The highly diversified bioerosion and encrustation in the studied hard organic substrate indicate a long exposition period of organic substrate with slow to moderate rate of deposition in a restricted (high-salinity) marine environment. This bioerosion study shows that high-salinity, at least for the study area, is not an important controlling factor for ichnology.
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Effects of fasting on maximum thermogenesis in temperature-acclimated rats
NASA Astrophysics Data System (ADS)
Wang, L. C. H.
1981-09-01
To further investigate the limiting effect of substrates on maximum thermogenesis in acute cold exposure, the present study examined the prevalence of this effect at different thermogenic capabilities consequent to cold- or warm-acclimation. Male Sprague-Dawley rats (n=11) were acclimated to 6, 16 and 26‡C, in succession, their thermogenic capabilities after each acclimation temperature were measured under helium-oxygen (21% oxygen, balance helium) at -10‡C after overnight fasting or feeding. Regardless of feeding conditions, both maximum and total heat production were significantly greater in 6>16>26‡C-acclimated conditions. In the fed state, the total heat production was significantly greater than that in the fasted state at all acclimating temperatures but the maximum thermogenesis was significant greater only in the 6 and 16‡C-acclimated states. The results indicate that the limiting effect of substrates on maximum and total thermogenesis is independent of the magnitude of thermogenic capability, suggesting a substrate-dependent component in restricting the effective expression of existing aerobic metabolic capability even under severe stress.
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Sideris, Dionisia P.; Petrakis, Nikos; Katrakili, Nitsa; Mikropoulou, Despina; Gallo, Angelo; Ciofi-Baffoni, Simone; Banci, Lucia; Bertini, Ivano
2009-01-01
Mia40 imports Cys-containing proteins into the mitochondrial intermembrane space (IMS) by ensuring their Cys-dependent oxidative folding. In this study, we show that the specific Cys of the substrate involved in docking with Mia40 is substrate dependent, the process being guided by an IMS-targeting signal (ITS) present in Mia40 substrates. The ITS is a 9-aa internal peptide that (a) is upstream or downstream of the docking Cys, (b) is sufficient for crossing the outer membrane and for targeting nonmitochondrial proteins, (c) forms an amphipathic helix with crucial hydrophobic residues on the side of the docking Cys and dispensable charged residues on the other side, and (d) fits complementary to the substrate cleft of Mia40 via hydrophobic interactions of micromolar affinity. We rationalize the dual function of Mia40 as a receptor and an oxidase in a two step–specific mechanism: an ITS-guided sliding step orients the substrate noncovalently, followed by docking of the substrate Cys now juxtaposed to pair with the Mia40 active Cys. PMID:20026652
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Mode of VAMP Substrate Recognition and Inhibition of Clostridium botulinum Neurotoxin F
DOE Office of Scientific and Technical Information (OSTI.GOV)
Agarwal, R.; Schmidt, J; Stafford, R
2009-01-01
Clostridium botulinum neurotoxins (BoNTs) cleave neuronal proteins responsible for neurotransmitter release, causing the neuroparalytic disease botulism. BoNT serotypes B, D, F and G cleave and inactivate vesicle-associated membrane protein (VAMP), each at a unique peptide bond. The specificity of BoNTs depends on the mode of substrate recognition. We have investigated the mechanism of substrate recognition of BoNT F by determining the crystal structures of its complex with two substrate-based inhibitors, VAMP 22-58/Gln58D-cysteine and 27-58/Gln58D-cysteine. The inhibitors bind to BoNT F in the canonical direction (as seen for BoNTs A and E substrates) but are positioned specifically via three major exositesmore » away from the active site. The cysteine sulfur of the inhibitors interacts with the zinc and exists as sulfinic acid in the inhibitor VAMP 27-58/Gln58D-cysteine. Arg133 and Arg171, which form part of two separate exosites, are crucial for substrate binding and catalysis.« less
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Substrate Specificity of Human Protein Arginine Methyltransferase 7 (PRMT7)
Feng, You; Hadjikyriacou, Andrea; Clarke, Steven G.
2014-01-01
Protein arginine methyltransferase 7 (PRMT7) methylates arginine residues on various protein substrates and is involved in DNA transcription, RNA splicing, DNA repair, cell differentiation, and metastasis. The substrate sequences it recognizes in vivo and the enzymatic mechanism behind it, however, remain to be explored. Here we characterize methylation catalyzed by a bacterially expressed GST-tagged human PRMT7 fusion protein with a broad range of peptide and protein substrates. After confirming its type III activity generating only ω-NG-monomethylarginine and its distinct substrate specificity for RXR motifs surrounded by basic residues, we performed site-directed mutagenesis studies on this enzyme, revealing that two acidic residues within the double E loop, Asp-147 and Glu-149, modulate the substrate preference. Furthermore, altering a single acidic residue, Glu-478, on the C-terminal domain to glutamine nearly abolished the activity of the enzyme. Additionally, we demonstrate that PRMT7 has unusual temperature dependence and salt tolerance. These results provide a biochemical foundation to understanding the broad biological functions of PRMT7 in health and disease. PMID:25294873
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Surface-enhanced hyper-Raman spectroscopy with a picosecond laser: gold and copper colloids
NASA Astrophysics Data System (ADS)
Lipscomb, Leigh Ann; Nie, Shuming; Feng, Sibo; Yu, Nai-Teng
1990-07-01
We have obtained surface-enhanced hyper-Raman scattering (SEHRS) spectra of crystal violet, rhodamine 6G and Ru(trpy) (BPE) 32+ adsorbed on gold and copper colloidal surfaces (where trpy=2,2',2″-terpyridine, BPE=trans-bis(4-pyridyl)ethylene). Our results demonstrate that the SEHRS effect is not intrinsically restricted to a Ag substrate and that surface enhancements at the emitted hyper-Raman photon frequencies are not required for observing SEHRS signals.
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Song, Bongkeun; Kerkhof, Lee J; Häggblom, Max M
2002-08-06
4-Chlorobenzoate and 4-bromobenzoate were readily degraded in denitrifying enrichment cultures established with river sediment, estuarine sediment or agricultural soil as inoculum. Stable denitrifying consortia were obtained and maintained by serial dilution and repeated feeding of substrates. Microbial community analyses were performed to characterize the 4-chlorobenzoate and 4-bromobenzoate degrading consortia with terminal restriction fragment length polymorphism (T-RFLP) and cloning of 16S rRNA genes from the cultures. Interestingly, two major terminal restriction fragments (T-RFs) in the 4-chlorobenzoate degrading consortia and one T-RF in the 4-bromobenzoate utilizing consortium were observed from T-RFLP analysis regardless of their geographical and ecological origins. The two T-RFs (clones 4CB1 and 4CB2) in 4-chlorobenzoate degrading consortia were identified as members of the beta-subunit of the Proteobacteria on the basis of 16S rRNA sequencing analysis. Phylogenetic analysis of 16S rRNA genes showed that clone 4CB1 was closely related to Thauera aromatica while clone 4CB2 was distantly related to the genera Limnobacter and Ralstonia. The 4-bromobenzoate utilizing consortium mainly consisted of one T-RF, which was identical to clone 4CB2 in spite of different enrichment substrate. This suggests that degradation of 4-chlorobenzoate and 4-bromobenzoate under denitrifying conditions was mediated by bacteria belonging to the beta-subunit of the Proteobacteria.
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Haggie, Peter M; Verkman, A S
2002-10-25
It has been proposed that enzymes in many metabolic pathways, including the tricarboxylic acid cycle in the mitochondrial matrix, are physically associated to facilitate substrate channeling and overcome diffusive barriers. We have used fluorescence recovery after photobleaching to measure the diffusional mobilities of chimeras consisting of green fluorescent protein (GFP) fused to the C terminus of four tricarboxylic acid cycle enzymes: malate dehydrogenase, citrate synthase, isocitrate dehydrogenase, and succinyl-CoA synthetase. The GFP-enzyme chimeras were localized selectively in the mitochondrial matrix in transfected Chinese hamster ovary (CHO) and COS7 cells. Laser photobleaching using a 0.7-microm diameter spot demonstrated restricted diffusion of the GFP-enzyme chimeras. Interestingly, all four chimeras had similar diffusional characteristics, approximately 45% of each chimera was mobile and had a diffusion coefficient of 4 x 10(-8) cm(2)/s. In contrast, unconjugated GFP in the mitochondrial matrix (targeted using COX8 leader sequence) diffused freely (nearly 100% mobility) with a greater diffusion coefficient of 20 x 10(-8) cm(2)/s. The mobility of the GFP-enzyme chimeras was insensitive to substrate source, ATP depletion, or inhibition of the adenine nucleotide translocase. These results indicate similar mobility characteristics of unrelated tricarboxylic acid cycle enzymes having different sizes and physical properties, providing biophysical evidence for a diffusible multienzyme complex in the mitochondrial matrix.
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Sun, Xun; Lu, You; Bish, Lawrence T; Calcedo, Roberto; Wilson, James M; Gao, Guangping
2010-06-01
Vectors based on several new adeno-associated viral (AAV) serotypes demonstrated strong hepatocyte tropism and transduction efficiency in both small- and large-animal models for liver-directed gene transfer. Efficiency of liver transduction by AAV vectors can be further improved in both murine and nonhuman primate (NHP) animals when the vector genomes are packaged in a self-complementary (sc) format. In an attempt to understand potential molecular mechanism(s) responsible for enhanced transduction efficiency of the sc vector in liver, we performed extensive molecular studies of genome structures of conventional single-stranded (ss) and sc AAV vectors from liver after AAV gene transfer in both mice and NHPs. These included treatment with exonucleases with specific substrate preferences, single-cutter restriction enzyme digestion and polarity-specific hybridization-based vector genome mapping, and bacteriophage phi29 DNA polymerase-mediated and double-stranded circular template-specific rescue of persisted circular genomes. In mouse liver, vector genomes of both genome formats seemed to persist primarily as episomal circular forms, but sc vectors converted into circular forms more rapidly and efficiently. However, the overall differences in vector genome abundance and structure in the liver between ss and sc vectors could not account for the remarkable differences in transduction. Molecular structures of persistent genomes of both ss and sc vectors were significantly more heterogeneous in macaque liver, with noticeable structural rearrangements that warrant further characterizations.
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Sun, Xun; Lu, You; Bish, Lawrence T.; Calcedo, Roberto; Wilson, James M.
2010-01-01
Abstract Vectors based on several new adeno-associated viral (AAV) serotypes demonstrated strong hepatocyte tropism and transduction efficiency in both small- and large-animal models for liver-directed gene transfer. Efficiency of liver transduction by AAV vectors can be further improved in both murine and nonhuman primate (NHP) animals when the vector genomes are packaged in a self-complementary (sc) format. In an attempt to understand potential molecular mechanism(s) responsible for enhanced transduction efficiency of the sc vector in liver, we performed extensive molecular studies of genome structures of conventional single-stranded (ss) and sc AAV vectors from liver after AAV gene transfer in both mice and NHPs. These included treatment with exonucleases with specific substrate preferences, single-cutter restriction enzyme digestion and polarity-specific hybridization-based vector genome mapping, and bacteriophage ϕ29 DNA polymerase-mediated and double-stranded circular template-specific rescue of persisted circular genomes. In mouse liver, vector genomes of both genome formats seemed to persist primarily as episomal circular forms, but sc vectors converted into circular forms more rapidly and efficiently. However, the overall differences in vector genome abundance and structure in the liver between ss and sc vectors could not account for the remarkable differences in transduction. Molecular structures of persistent genomes of both ss and sc vectors were significantly more heterogeneous in macaque liver, with noticeable structural rearrangements that warrant further characterizations. PMID:20113166
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Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; ...
2015-09-15
Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structuremore » consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.
Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structuremore » consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.« less
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DOE Office of Scientific and Technical Information (OSTI.GOV)
Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.
ABSTRACT Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consistingmore » of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation. IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.« less
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Hoppe-Seyler, T S; Jaeger, B; Bockelmann, W; Noordman, W H; Geis, A; Heller, K J
2003-09-01
ARDRA (Amplified Ribosomal-DNA Restriction Analysis) was used to differentiate among species and genera of Arthrobacter and Microbacteria. Species-specific restriction patterns of PCR-products were obtained with NciI for Arthrobacter citreus (DSM 20133T), A. sulfureus (DSM 20167T), A. globiformis (DSM 20124T) and A. nicotianae strains (DSM 20123T, MGE 10D, CA13, CA14, isolate 95293, 95294, and 95299), A. rhombi CCUG 38813T, and CCUG 38812, and Microbacterium barkeri strains (DSM 30123T, MGE 10D, CA12 and CA15, isolate 95292, and isolate 95207). All yellow pigmented coryneforme bacteria isolated from the smear of surface ripened cheeses were identified as either A. nicotianae or M. barkeri strains. Using pulsed field gel electrophoresis (PFGE) strain specific restriction pattern for all Arthrobacter species and Microbacteria tested were obtained with restriction enzymes AscI and SpeI.
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Abnormal eating behaviour in people with a specific phobia of vomiting (emetophobia).
Veale, David; Costa, Ana; Murphy, Philip; Ellison, Nell
2012-09-01
The aim of this study was to explore the eating behaviour in people with a specific phobia of vomiting (SPOV), and whether those identified as restricting their food had a greater degree of psychopathology and impairment than those who do not restrict their food. We recruited 94 participants with SPOV. They were divided into those who reported restricting their food (SPOV-R) (n = 32) because of fear of vomiting and those who did not restrict their food (SPOV-NR) (n = 62). People with SPOV frequently have abnormal eating behaviours to reduce the perceived risk of vomiting. Only 3.7% had a body mass index (BMI) of less than 17.5, and 8.5% had a BMI of less than 18.5. The SPOV-R group had significantly higher frequency of psychopathology and abnormal eating behaviours than the SPOV-NR group. Abnormal eating behaviour, BMI, and the degree of food restriction are important factors in the assessment of SPOV. Copyright © 2011 John Wiley & Sons, Ltd and Eating Disorders Association.
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Mechanistic Insights into Glucan Phosphatase Activity against Polyglucan Substrates*
Meekins, David A.; Raththagala, Madushi; Auger, Kyle D.; Turner, Benjamin D.; Santelia, Diana; Kötting, Oliver; Gentry, Matthew S.; Vander Kooi, Craig W.
2015-01-01
Glucan phosphatases are central to the regulation of starch and glycogen metabolism. Plants contain two known glucan phosphatases, Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), which dephosphorylate starch. Starch is water-insoluble and reversible phosphorylation solubilizes its outer surface allowing processive degradation. Vertebrates contain a single known glucan phosphatase, laforin, that dephosphorylates glycogen. In the absence of laforin, water-soluble glycogen becomes insoluble, leading to the neurodegenerative disorder Lafora Disease. Because of their essential role in starch and glycogen metabolism glucan phosphatases are of significant interest, yet a comparative analysis of their activities against diverse glucan substrates has not been established. We identify active site residues required for specific glucan dephosphorylation, defining a glucan phosphatase signature motif (CζAGΨGR) in the active site loop. We further explore the basis for phosphate position-specific activity of these enzymes and determine that their diverse phosphate position-specific activity is governed by the phosphatase domain. In addition, we find key differences in glucan phosphatase activity toward soluble and insoluble polyglucan substrates, resulting from the participation of ancillary glucan-binding domains. Together, these data provide fundamental insights into the specific activity of glucan phosphatases against diverse polyglucan substrates. PMID:26231210
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ERIC Educational Resources Information Center
Smith, Christopher J.; Lang, Colleen M.; Kryzak, Lauren; Reichenberg, Abraham; Hollander, Eric; Silverman, Jeremy M.
2009-01-01
Background: Clinical heterogeneity of autism likely hinders efforts to find genes associated with this complex psychiatric disorder. Some studies have produced promising results by restricting the sample according to the expression of specific familial factors or components of autism. Previous factor analyses of the restricted, repetitive…
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Purification and characterization of the restriction endonuclease RsrI, an isoschizomer of EcoRI.
Greene, P J; Ballard, B T; Stephenson, F; Kohr, W J; Rodriguez, H; Rosenberg, J M; Boyer, H W
1988-08-15
Rhodobacter sphaeroides strain 630 produces restriction enzyme RsrI which is an isoschizomer of EcoRI. We have purified this enzyme and initiated a comparison with the EcoRI endonuclease. The properties of RsrI are consistent with a reaction mechanism similar to that of EcoRI: the position of cleavage within the -GAATTC-site is identical, the MgCl2 optimum for the cleavage is identical, and the pH profile is similar. Methylation of the substrate sequence by the EcoRI methylase protects the site from cleavage by the RsrI endonuclease. RsrI cross-reacts strongly with anti-EcoRI serum indicating three-dimensional structural similarities. We have determined the sequence of 34 N terminal amino acids for RsrI and this sequence possesses significant similarity to the EcoRI N terminus.
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Weilenmann, Hans-Ueli; Engeli, Barbara; Bucheli-Witschel, Margarete; Egli, Thomas
2004-10-01
A Gram-negative, ethylenediaminetetraacetic acid (EDTA)-degrading bacterium (deposited at the German Culture Collection as strain DSM 9103) utilising EDTA as the only source of carbon, energy and nitrogen was isolated from a mixed EDTA-degrading population that was originally enriched in a column system from a mixture of activated sludge and soil. Chemotaxonomic analysis of quinones, polar lipids and fatty acids allowed allocation of the isolate to the alpha-subclass of Proteobacteria. 16S rDNA sequencing and phylogenetic analysis revealed highest similarity to the Mesorhizobium genus followed by the Aminobacter genus. However, the EDTA-degrading strain apparently forms a new branch within the Phyllobacteriaceae/Mesorhizobia family. Growth of the strain was rather slow not only on EDTA (micro(max) = 0.05 h(-1)) but also on other substrates. Classical substrate utilisation testing in batch culture suggested a quite restricted carbon source spectrum with only lactate, glutamate, and complexing agents chemically related to EDTA (nitrilotriacetate, iminodiacetate and ethylenediaminedisuccinate) supporting growth. However, when EDTA-limited continuous cultures of strain DSM 9103 were pulsed with fumarate, succinate, glucose or acetate, these substrates were assimilated immediately. Apparently, the strain can use a broader spectrum than indicated by traditional substrate testing techniques. The EDTA species CaEDTA and MgEDTA served as growth substrates of the strain because in the mineral medium employed EDTA was predicted to be mainly present in the form of these two complexes. The bacterium was not able to degrade Fe3+-complexed EDTA.
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Käppler, Andrea; Windrich, Frank; Löder, Martin G J; Malanin, Mikhail; Fischer, Dieter; Labrenz, Matthias; Eichhorn, Klaus-Jochen; Voit, Brigitte
2015-09-01
The presence of microplastics in aquatic ecosystems is a topical problem and leads to the need of appropriate and reliable analytical methods to distinctly identify and to quantify these particles in environmental samples. As an example transmission, Fourier transform infrared (FTIR) imaging can be used to analyze samples directly on filters without any visual presorting, when the environmental sample was afore extracted, purified, and filtered. However, this analytical approach is strongly restricted by the limited IR transparency of conventional filter materials. Within this study, we describe a novel silicon (Si) filter substrate produced by photolithographic microstructuring, which guarantees sufficient transparency for the broad mid-infrared region of 4000-600 cm(-1). This filter type features holes with a diameter of 10 μm and exhibits adequate mechanical stability. Furthermore, it will be shown that our Si filter substrate allows a distinct identification of the most common microplastics, polyethylene (PE), and polypropylene (PP), in the characteristic fingerprint region (1400-600 cm(-1)). Moreover, using the Si filter substrate, a differentiation of microparticles of polyesters having quite similar chemical structure, like polyethylene terephthalate (PET) and polybutylene terephthalate (PBT), is now possible, which facilitates a visualization of their distribution within a microplastic sample by FTIR imaging. Finally, this Si filter can also be used as substrate for Raman microscopy-a second complementary spectroscopic technique-to identify microplastic samples.
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Kollamaram, Gayathri; Hopkins, Simon C; Glowacki, Bartek A; Croker, Denise M; Walker, Gavin M
2018-03-30
Drop-on-demand inkjet printing is a potential enabling technology both for continuous manufacturing of pharmaceuticals and for personalized medicine, but its use is often restricted to low-viscosity solutions and nano-suspensions. In the present study, a robust electromagnetic (valvejet) inkjet technology has been successfully applied to deposit prototype dosage forms from solutions with a wide range of viscosities, and from suspensions with particle sizes exceeding 2 μm. A detailed solid-state study of paracetamol, printed from a solution ink on hydroxypropyl methylcellulose (HPMC), revealed that the morphology of the substrate and its chemical interactions can have a considerable influence on polymorphic selectivity. Paracetamol ink crystallized exclusively into form II when printed on a smooth polyethylene terephthalate substrate, and exclusively into form I when in sufficient proximity to the rough surface of the HPMC substrate to be influenced by confinement in pores and chemical interactions. The relative standard deviation in the strength of the dosage forms was <4% in all cases, for doses as low as 0.8 mg, demonstrating the accuracy and reproducibility associated with electromagnetic inkjet technology. Good adhesion of indomethacin on HPMC was achieved using a suspension ink with hydroxypropyl cellulose, but not on an alternative polyethylene terephthalate substrate, emphasising the need to tailor the binder to the substrate. Future work will focus on lower-dose drugs, for which dosing flexibility and fixed dose combinations are of particular interest. Copyright © 2018 Elsevier B.V. All rights reserved.
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F-box proteins involved in cancer-associated drug resistance.
Gong, Jian; Zhou, Yuqian; Liu, Deliang; Huo, Jirong
2018-06-01
The ubiquitin proteasome system (UPS) regulated human biological processes through the appropriate and efficient proteolysis of cellular proteins. F-box proteins are the vital components of SKP1-CUL1-FBP (SCF)-type E3 ubiquitin ligases that determine substrate specificity. As F-box proteins have the ability to control the degradation of several crucial protein targets associated with drug resistance, the dysregulation of these proteins may lead to induction of chemoresistance in cancer cells. Chemotherapy is one of the most conventional therapeutic approaches of treatment of patients with cancer. However, its exclusive application in clinical settings is restricted due to the development of chemoresistance, which typically results treatment failure. Therefore, overcoming drug resistance is considered as one of the most critical issues that researchers and clinician associated with oncology face. The present review serves to provide a comprehensive overview of F-box proteins and their possible targets as well as their correlation with the chemoresistance and chemosensitization of cancer cells. The article also presents an integrated representation of the complex regulatory mechanisms responsible for chemoresistance, which may lay the foundation to explore sensible candidate drugs for therapeutic intervention.
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Expanding the product profile of a microbial alkane biosynthetic pathway.
Harger, Matthew; Zheng, Lei; Moon, Austin; Ager, Casey; An, Ju Hye; Choe, Chris; Lai, Yi-Ling; Mo, Benjamin; Zong, David; Smith, Matthew D; Egbert, Robert G; Mills, Jeremy H; Baker, David; Pultz, Ingrid Swanson; Siegel, Justin B
2013-01-18
Microbially produced alkanes are a new class of biofuels that closely match the chemical composition of petroleum-based fuels. Alkanes can be generated from the fatty acid biosynthetic pathway by the reduction of acyl-ACPs followed by decarbonylation of the resulting aldehydes. A current limitation of this pathway is the restricted product profile, which consists of n-alkanes of 13, 15, and 17 carbons in length. To expand the product profile, we incorporated a new part, FabH2 from Bacillus subtilis , an enzyme known to have a broader specificity profile for fatty acid initiation than the native FabH of Escherichia coli . When provided with the appropriate substrate, the addition of FabH2 resulted in an altered alkane product profile in which significant levels of n-alkanes of 14 and 16 carbons in length are produced. The production of even chain length alkanes represents initial steps toward the expansion of this recently discovered microbial alkane production pathway to synthesize complex fuels. This work was conceived and performed as part of the 2011 University of Washington international Genetically Engineered Machines (iGEM) project.
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Greenwood, Edward JD; Matheson, Nicholas J; Wals, Kim; van den Boomen, Dick JH; Antrobus, Robin; Williamson, James C; Lehner, Paul J
2016-01-01
Viruses manipulate host factors to enhance their replication and evade cellular restriction. We used multiplex tandem mass tag (TMT)-based whole cell proteomics to perform a comprehensive time course analysis of >6500 viral and cellular proteins during HIV infection. To enable specific functional predictions, we categorized cellular proteins regulated by HIV according to their patterns of temporal expression. We focussed on proteins depleted with similar kinetics to APOBEC3C, and found the viral accessory protein Vif to be necessary and sufficient for CUL5-dependent proteasomal degradation of all members of the B56 family of regulatory subunits of the key cellular phosphatase PP2A (PPP2R5A-E). Quantitative phosphoproteomic analysis of HIV-infected cells confirmed Vif-dependent hyperphosphorylation of >200 cellular proteins, particularly substrates of the aurora kinases. The ability of Vif to target PPP2R5 subunits is found in primate and non-primate lentiviral lineages, and remodeling of the cellular phosphoproteome is therefore a second ancient and conserved Vif function. DOI: http://dx.doi.org/10.7554/eLife.18296.001 PMID:27690223
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Vertical nanopillars for highly localized fluorescence imaging
Xie, Chong; Hanson, Lindsey; Cui, Yi; Cui, Bianxiao
2011-01-01
Observing individual molecules in a complex environment by fluorescence microscopy is becoming increasingly important in biological and medical research, for which critical reduction of observation volume is required. Here, we demonstrate the use of vertically aligned silicon dioxide nanopillars to achieve below-the-diffraction-limit observation volume in vitro and inside live cells. With a diameter much smaller than the wavelength of visible light, a transparent silicon dioxide nanopillar embedded in a nontransparent substrate restricts the propagation of light and affords evanescence wave excitation along its vertical surface. This effect creates highly confined illumination volume that selectively excites fluorescence molecules in the vicinity of the nanopillar. We show that this nanopillar illumination can be used for in vitro single-molecule detection at high fluorophore concentrations. In addition, we demonstrate that vertical nanopillars interface tightly with live cells and function as highly localized light sources inside the cell. Furthermore, specific chemical modification of the nanopillar surface makes it possible to locally recruit proteins of interest and simultaneously observe their behavior within the complex, crowded environment of the cell. PMID:21368157
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Tanaka, Shiho; Endo, Haruka; Adegawa, Satomi; Iizuka, Ami; Imamura, Kazuhiro; Kikuta, Shingo; Sato, Ryoichi
2017-12-01
Because Bombyx mori ABC transporter C2 (BmABCC2) has 1000-fold higher potential than B. mori cadherin-like protein as a receptor for Bacillus thuringiensis Cry1Aa toxin (Tanaka et al., 2013), the gate-opening ability of the latent pore under six extracellular loops (ECLs) of BmABCC2 was expected to be the reason for its higher potential (Heckel, 2012). In this study, cell swelling assays in Sf9 cells showed that BmABCC2 mutants lacking substrate-excreting activity retained receptor activity, indicating that the gate-opening activity of BmABCC2 is not responsible for Cry1Aa toxicity. The analysis of 29 BmABCC2 mutants demonstrated that 770 DYWL 773 of ECL 4 comprise a putative binding site to Cry1Aa. This suggests that specific toxicity of Cry1Aa toxin to a restricted range of lepidopteran insects is dependent on conservation and variation in the amino acid residues around 770 DYWL 773 of ECL 4 in the ABCC2. Copyright © 2017 Elsevier Ltd. All rights reserved.
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Diffusion and retention are major determinants of protein targeting to the inner nuclear membrane
Ungricht, Rosemarie; Klann, Michael; Horvath, Peter
2015-01-01
Newly synthesized membrane proteins are constantly sorted from the endoplasmic reticulum (ER) to various membranous compartments. How proteins specifically enrich at the inner nuclear membrane (INM) is not well understood. We have established a visual in vitro assay to measure kinetics and investigate requirements of protein targeting to the INM. Using human LBR, SUN2, and LAP2β as model substrates, we show that INM targeting is energy-dependent but distinct from import of soluble cargo. Accumulation of proteins at the INM relies on both a highly interconnected ER network, which is affected by energy depletion, and an efficient immobilization step at the INM. Nucleoporin depletions suggest that translocation through nuclear pore complexes (NPCs) is rate-limiting and restricted by the central NPC scaffold. Our experimental data combined with mathematical modeling support a diffusion-retention–based mechanism of INM targeting. We experimentally confirmed the sufficiency of diffusion and retention using an artificial reporter lacking natural sorting signals that recapitulates the energy dependence of the process in vivo. PMID:26056139
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Pal Sharma, C; Goldmann, Wolfgang H
2004-01-01
Actin-binding protein (ABP-280; filamin) is a phosphoprotein present in the periphery of the cytoplasm where it can cross-link actin filaments, associate with lipid membranes, and bind to membrane surface receptors. Given its function and localization in the cell, we decided to investigate the possibility of whether it serves as substrate for p56lck, a lymphocyte-specific member of the src family of protein tyrosine kinases associated with cell surface glycoproteins. The interaction of p56lck with membrane glycoproteins is important for cell development and functional activation. Here, we show that purified p56lck interacts and catalyzes in vitro kinase reactions. Tyrosine phosphorylation by p56lck is restricted to a single peptide of labeled ABP-280 shown by protease digest. The addition of phorbol ester to cells results in the inhibition of phosphorylation of ABP-280 by p56lck. These results show a decrease in phosphorylation suggesting conformationally induced regulation. Dynamic light scattering confirmed increased actin filament cross-linking due to phosphorylation of ABP-280 by p56lck.
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Cahoon, E.B.; Shanklin, J.; Lindgvist, Y.; Schneider, G.
1998-01-06
Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity. 1 fig.
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Cahoon, E.B.; Shanklin, J.; Lindqvist, Y.; Schneider, G.
1999-03-30
Disclosed is a method for modifying the chain length and double bond positional specificities of a soluble plant fatty acid desaturase. More specifically, the method involves modifying amino acid contact residues in the substrate binding channel of the soluble fatty acid desaturase which contact the fatty acid. Specifically disclosed is the modification of an acyl-ACP desaturase. Amino acid contact residues which lie within the substrate binding channel are identified, and subsequently replaced with different residues to effect the modification of activity. 2 figs.
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NASA Astrophysics Data System (ADS)
Dan, Cai; Lie, Liu; Jin-Chuan, Ju; Xue-Long, Zhao; Hong-Yu, Zhou; Xiao, Wang
2016-04-01
The carbon nanotube (CNT)-based materials can be used as vacuum device cathodes. Owing to the excellent field emission properties of CNT, it has great potentials in the applications of an explosive field emission cathode. The falling off of CNT from the substrate, which frequently appears in experiments, restricts its application. In addition, the onset time of vacuum breakdown limits the performance of the high-power explosive-emission-cathode-based diode. In this paper, the characteristics of the CNT, electric field strength, contact resistance and the kind of substrate material are varied to study the parameter effects on the onset time of vacuum breakdown and failure mechanism of the CNT by using the finite element method. Project supported by the National Natural Science Foundation of China (Grant Nos. 11305263 and 61401484).
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Modeling Weather Impact on Airport Arrival Miles-in-Trail Restrictions
NASA Technical Reports Server (NTRS)
Wang, Yao; Grabbe, Shon
2013-01-01
When the demand for either a region of airspace or an airport approaches or exceeds the available capacity, miles-in-trail (MIT) restrictions are the most frequently issued traffic management initiatives (TMIs) that are used to mitigate these imbalances. Miles-intrail operations require aircraft in a traffic stream to meet a specific inter-aircraft separation in exchange for maintaining a safe and orderly flow within the stream. This stream of aircraft can be departing an airport, over a common fix, through a sector, on a specific route or arriving at an airport. This study begins by providing a high-level overview of the distribution and causes of arrival MIT restrictions for the top ten airports in the United States. This is followed by an in-depth analysis of the frequency, duration and cause of MIT restrictions impacting the Hartsfield-Jackson Atlanta International Airport (ATL) from 2009 through 2011. Then, machine-learning methods for predicting (1) situations in which MIT restrictions for ATL arrivals are implemented under low demand scenarios, and (2) days in which a large number of MIT restrictions are required to properly manage and control ATL arrivals are presented. More specifically, these predictions were accomplished by using an ensemble of decision trees with Bootstrap aggregation (BDT) and supervised machine learning was used to train the BDT binary classification models. The models were subsequently validated using data cross validation methods. When predicting the occurrence of arrival MIT restrictions under low demand situations, the model was able to achieve over all accuracy rates ranging from 84% to 90%, with false alarm ratios ranging from 10% to 15%. In the second set of studies designed to predict days on which a high number of MIT restrictions were required, overall accuracy rates of 80% were achieved with false alarm ratios of 20%. Overall, the predictions proposed by the model give better MIT usage information than what has been currently provided under current day operations. Traffic flow managers can use these predictions to identify potential MIT restrictions to eliminate (e.g., those occurring during low arrival demand periods), and to determine the days in which a significant number of restrictions may be required
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ERIC Educational Resources Information Center
Andrepont, Emmy; Cullen, Karen W.; Taylor, Wendell C.
2011-01-01
Background: Computerized point-of-sale (POS) machine software that allows parents to place restrictions on their child's school meal accounts is available. Parents could restrict specific foods (e.g., chips), identify specific days the child can purchase extra foods, or set monetary limits. This descriptive study examines the use of parental…
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Crystal structure of isoflavone reductase from alfalfa (Medicago sativa L.).
Wang, Xiaoqiang; He, Xianzhi; Lin, Jianqiao; Shao, Hui; Chang, Zhenzhan; Dixon, Richard A
2006-05-19
Isoflavonoids play important roles in plant defense and exhibit a range of mammalian health-promoting activities. Isoflavone reductase (IFR) specifically recognizes isoflavones and catalyzes a stereospecific NADPH-dependent reduction to (3R)-isoflavanone. The crystal structure of Medicago sativa IFR with deletion of residues 39-47 has been determined at 1.6A resolution. Structural analysis, molecular modeling and docking, and comparison with the structures of other NADPH-dependent enzymes, defined the putative binding sites for co-factor and substrate and potential key residues for enzyme activity and substrate specificity. Further mutagenesis has confirmed the role of Lys144 as a catalytic residue. This study provides a structural basis for understanding the enzymatic mechanism and substrate specificity of IFRs as well as the functions of IFR-like proteins.
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Shiota, Hiroto; Kanzaki, Hiroshi; Hatanaka, Tadashi; Nitoda, Teruhiko
2013-06-28
TMG-chitotriomycin (1) produced by the actinomycete Streptomyces annulatus NBRC13369 was examined as a probe for the prediction of substrate specificity of β-N-acetylhexosaminidases (HexNAcases). According to the results of inhibition assays, 14 GH20 HexNAcases from various organisms were divided into 1-sensitive and 1-insensitive enzymes. Three representatives of each group were investigated for their substrate specificity. The 1-sensitive HexNAcases hydrolyzed N-acetylchitooligosaccharides but not N-glycan-type oligosaccharides, whereas the 1-insensitive enzymes hydrolyzed N-glycan-type oligosaccharides but not N-acetylchitooligosaccharides, indicating that TMG-chitotriomycin can be used as a molecular probe to distinguish between chitin-degrading HexNAcases and glycoconjugate-processing HexNAcases. Copyright © 2013 Elsevier Ltd. All rights reserved.